The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking

*Jose Luis Aleixandre-Tudo and Wessel du Toit*

## **Abstract**

Phenolic compounds are bioactive substances present in a large number of food products including wine. The importance of these compounds in wine is due to their large effect on the organoleptic attributes of wine. Phenolic compounds play a crucial role in the colour as well as mouthfeel properties of wines. UV-visible spectroscopy appears as a suitable technique for the evaluation of phenolic compounds' properties and content. The ability of the phenolic ring to absorb UV light and the fact that some of the phenolic substances are coloured compounds, i.e. show absorption features in the visible region, make UV-visible spectroscopy a suitable technique to investigate and quantify grape and wine phenolic compounds. A number of analytical techniques are currently used for phenolic quantification. These include both simpler approaches (spectrophotometric determinations) as well as more complex methodologies such liquid chromatography analysis. Moreover, a number of spectroscopy applications have also been recently reported and are becoming popular within the wine industry. This chapter reviews information on the UV-visible spectral properties of phenolic compounds, changes occurring during wine ageing and also discusses the current UV-visible based analytical techniques used for the quantification of phenolic compounds in grapes and wine.

**Keywords:** UV-visible, spectrophotometry, phenolic compounds, anthocyanins, tannins, liquid chromatography, spectroscopy, chemometrics, fluorescence

## **1. Introduction**

Phenolic compounds are bioactive molecules that are involved in some of the most relevant wine organoleptic attributes. Phenolic substances have been reported as being responsible for wine colour, mouthfeel perception and flavour. The appropriate management of the phenolic accumulation in the berry, extraction during the skin contact phase as well as the evolution during ageing in barrels or bottles will ensure a desired phenolic content and composition that will lead to a good quality wine [1]. Furthermore, the ability of phenolic molecules to act as antioxidant has placed this group of compounds in the spotlight of a considerable amount of research. Phenolic compounds have been reported as effective antioxidants and their preventive role against inflammatory, neurodegenerative, cardiovascular

diseases or even against cancer has been widely acknowledged [2]. The quantification of phenolic compounds is thus of high importance and UV-visible spectroscopy has proven to be one of the most suitable and reliable techniques to quantify these substances during the winemaking process.

The accumulation of the amino acid phenylalanine is the first step towards the biosynthesis of phenolic compounds. Phenolic substances or polyphenols are thus secondary metabolites that contain at least one aromatic ring and one or several hydroxyl groups. Two main families of phenolic compounds are generally classified as the non-flavonoids and the flavonoids. Phenolic acids, including hydroxycinnamic and hydroxybenzoic acids and stilbens are part of the structurally less complex non-flavonoid group (**Figure 1**). Flavonoids share a common C3-C6-C3 structure and contain flavonols, anthocyanins and flavanols, with the latter also known as proanthocyanidins or more widely as tannins [3]. The biosynthesis and accumulation of these key substances is due to a number of plant biological functions which include growth, plant reproduction and plant protection roles against environmental signals as well as biotic and abiotic stresses [4].

Phenolic compounds are released from the solid parts of the berries into the must during the winemaking process. The contact period refers to the period of time that the must is in contact with the skins and seeds and generally coincides with the alcoholic fermentation. The presence or absence of the solid parts during the winemaking process will determine the phenolic content and composition. In white winemaking the skin contact period is limited to a minimum and the levels of phenolic compounds found in wines are thus lower than in red wines (where the fermentation takes place in the presence of skins and seeds). Due to its location in the flesh, hydroxycinnamic acids are therefore the main phenolic compounds found in white wines. On the contrary, red wines contain high levels of tannins, anthocyanins and flavonol compounds that are extracted from the solid parts of the berries during the aforementioned skin contact phase [5].

Among the subclasses of phenolic compounds found in grapes, two of the subfamilies are mostly of importance to wine production. Anthocyanins are coloured compounds responsible for the red wine colour attributes. The state of the anthocyanins and the wine medium conditions have a major impact on the final wine colour. Anthocyanins are found in red grapes and wines in five mono-glucoside forms. The 3-glucoside forms of delphinidin, cyanidin, petunidin, peonidin and malvidin are present in *Vitis vinifera* cultivars (**Figure 2**). Monomeric anthocyanins are highly reactive substances involved in a large number of reactions and interactions. Simple anthocyanins are acylated with a number of grape components such as acetic acid, *p-*coumaric or caffeic acid, they are also able to combine with themselves through intra- and intermolecular copigmentation interactions [6]. During the winemaking and ageing processes further reactions to form pyranoanthocyanins have also been documented, in combination with several associations with tannins, some of them through acetaldehyde mediated reactions. Anthocyanin interactions and reactions lead to a number of complex pigments with increased

**27**

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

stability during wine ageing. These combinations also entail a modification of the anthocyanin coloration, phenomena that gives rise to the large variety of red and brown based colours found in red wines [7]. Additionally, tannin-anthocyanin interactions give rise to a decrease in the ability of tannins to elicit astringency [8]. Proanthocyanidins or tannins are the most abundant class of phenolic compounds. Tannins are polymeric compounds of varying size and structure, containing a combination of five flavanol monomers. The polymerisation of catechin, epicatechin, gallocatechin, epigallocatechin and catechin gallate subunits gives rise to larger and more polymerised tannin compounds (**Figure 3**) with varying ability to elicit astringency and bitterness [3, 9]. The reactivity of the hydroxyl groups towards salivary proteins creates a macromolecular complex that precipitates from solution and leads to a puckering and drying sensation, also known as astringency. Small molecular weight tannins are initially bitter and they became more astringent as the molecular size increases [9]. Young wines, initially more astringent, contain tannins that have been polymerising and have therefore and increased ability to react with salivary proteins. During the ageing process several phenomena explain why the wines become softer (less astringent). When a certain molecular size is reached the tannin molecule may become insoluble, thus precipitating from solution and lowering the tannin content of the wines. Moreover, as molecules grow in size, its conformation might hinder the tannin protein interactions which will also lead to decreased astringency intensity. It is also possible that large tannin molecules cleavages give rise to smaller and less astringent tannins. Finally, the tannin-anthocyanin combinations that take place during the ageing process may also be involved in the decrease of the astringency intensity experienced in older wines [1, 10].

*Chemical structure of the main anthocyanins found in grapes and wines.*

The use of UV and visible light for the quantification of chemical compounds is a widely used technique [1]. Due to their biochemical and molecular properties, phenolic compounds are highly suitable to be quantified with UV-visible light. The ability of the phenolic ring to absorb UV light is exploited to quantify these compounds [11]. In addition to this, visible light can also provide valuable information due to the coloured nature of some of the phenolic compounds (e.g. red anthocyanins or yellow flavonols). The UV-visible spectra of a wine is thus attributed to the electronic transitions occurring within the hydroxyl groups of the phenolic molecules, with different transitions corresponding to the different phenolic subclasses [12]. A number of UV-visible applications have been exploited to quantify phenolic compounds. Among these the use of UV-visible spectrophotometry to estimate the content of phenolic compounds stands out as the most widely used approach. A number of methods have been optimised for the different phenolic subclasses, making nowadays the efficient estimation of

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

**Figure 2.**

### **Figure 1.** *Chemical structure of the non-flavonoid group of phenolic compounds found in grapes and wines.*

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

**Figure 2.**

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

environmental signals as well as biotic and abiotic stresses [4].

during the aforementioned skin contact phase [5].

substances during the winemaking process.

diseases or even against cancer has been widely acknowledged [2]. The quantification of phenolic compounds is thus of high importance and UV-visible spectroscopy has proven to be one of the most suitable and reliable techniques to quantify these

The accumulation of the amino acid phenylalanine is the first step towards the biosynthesis of phenolic compounds. Phenolic substances or polyphenols are thus secondary metabolites that contain at least one aromatic ring and one or several hydroxyl groups. Two main families of phenolic compounds are generally classified as the non-flavonoids and the flavonoids. Phenolic acids, including hydroxycinnamic and hydroxybenzoic acids and stilbens are part of the structurally less complex non-flavonoid group (**Figure 1**). Flavonoids share a common C3-C6-C3 structure and contain flavonols, anthocyanins and flavanols, with the latter also known as proanthocyanidins or more widely as tannins [3]. The biosynthesis and accumulation of these key substances is due to a number of plant biological functions which include growth, plant reproduction and plant protection roles against

Phenolic compounds are released from the solid parts of the berries into the must during the winemaking process. The contact period refers to the period of time that the must is in contact with the skins and seeds and generally coincides with the alcoholic fermentation. The presence or absence of the solid parts during the winemaking process will determine the phenolic content and composition. In white winemaking the skin contact period is limited to a minimum and the levels of phenolic compounds found in wines are thus lower than in red wines (where the fermentation takes place in the presence of skins and seeds). Due to its location in the flesh, hydroxycinnamic acids are therefore the main phenolic compounds found in white wines. On the contrary, red wines contain high levels of tannins, anthocyanins and flavonol compounds that are extracted from the solid parts of the berries

Among the subclasses of phenolic compounds found in grapes, two of the subfamilies are mostly of importance to wine production. Anthocyanins are coloured compounds responsible for the red wine colour attributes. The state of the anthocyanins and the wine medium conditions have a major impact on the final wine colour. Anthocyanins are found in red grapes and wines in five mono-glucoside forms. The 3-glucoside forms of delphinidin, cyanidin, petunidin, peonidin and malvidin are present in *Vitis vinifera* cultivars (**Figure 2**). Monomeric anthocyanins are highly reactive substances involved in a large number of reactions and interactions. Simple anthocyanins are acylated with a number of grape components such as acetic acid, *p-*coumaric or caffeic acid, they are also able to combine with themselves through intra- and intermolecular copigmentation interactions [6]. During the winemaking and ageing processes further reactions to form pyranoanthocyanins have also been documented, in combination with several associations with tannins, some of them through acetaldehyde mediated reactions. Anthocyanin interactions and reactions lead to a number of complex pigments with increased

*Chemical structure of the non-flavonoid group of phenolic compounds found in grapes and wines.*

**26**

**Figure 1.**

*Chemical structure of the main anthocyanins found in grapes and wines.*

stability during wine ageing. These combinations also entail a modification of the anthocyanin coloration, phenomena that gives rise to the large variety of red and brown based colours found in red wines [7]. Additionally, tannin-anthocyanin interactions give rise to a decrease in the ability of tannins to elicit astringency [8].

Proanthocyanidins or tannins are the most abundant class of phenolic compounds. Tannins are polymeric compounds of varying size and structure, containing a combination of five flavanol monomers. The polymerisation of catechin, epicatechin, gallocatechin, epigallocatechin and catechin gallate subunits gives rise to larger and more polymerised tannin compounds (**Figure 3**) with varying ability to elicit astringency and bitterness [3, 9]. The reactivity of the hydroxyl groups towards salivary proteins creates a macromolecular complex that precipitates from solution and leads to a puckering and drying sensation, also known as astringency. Small molecular weight tannins are initially bitter and they became more astringent as the molecular size increases [9]. Young wines, initially more astringent, contain tannins that have been polymerising and have therefore and increased ability to react with salivary proteins. During the ageing process several phenomena explain why the wines become softer (less astringent). When a certain molecular size is reached the tannin molecule may become insoluble, thus precipitating from solution and lowering the tannin content of the wines. Moreover, as molecules grow in size, its conformation might hinder the tannin protein interactions which will also lead to decreased astringency intensity. It is also possible that large tannin molecules cleavages give rise to smaller and less astringent tannins. Finally, the tannin-anthocyanin combinations that take place during the ageing process may also be involved in the decrease of the astringency intensity experienced in older wines [1, 10].

The use of UV and visible light for the quantification of chemical compounds is a widely used technique [1]. Due to their biochemical and molecular properties, phenolic compounds are highly suitable to be quantified with UV-visible light. The ability of the phenolic ring to absorb UV light is exploited to quantify these compounds [11]. In addition to this, visible light can also provide valuable information due to the coloured nature of some of the phenolic compounds (e.g. red anthocyanins or yellow flavonols). The UV-visible spectra of a wine is thus attributed to the electronic transitions occurring within the hydroxyl groups of the phenolic molecules, with different transitions corresponding to the different phenolic subclasses [12]. A number of UV-visible applications have been exploited to quantify phenolic compounds. Among these the use of UV-visible spectrophotometry to estimate the content of phenolic compounds stands out as the most widely used approach. A number of methods have been optimised for the different phenolic subclasses, making nowadays the efficient estimation of

**Figure 3.**

*Chemical structure of flavan-3-ol compounds found in grapes and wines.*

phenolic content using a simple UV-visible spectrophotometer possible [1]. However, UV-visible spectroscopy is also used in more advanced separation techniques, such as liquid chromatography, that allows for the quantification of individual phenolic compounds [13]. The quantification of phenolic compounds is thus achieved through the UV-visible signal given by the individually separated phenolic compounds. On the other hand, fluorescence spectroscopy also makes use of the UV-visible spectral features of the excited substances. After the excitation process a coloured fluorophore is quantified based on its absorption intensity projected in the visible region [14]. Finally, UV-visible spectroscopy combined with chemometrics is also included in the techniques used for phenolic compounds' quantification [15]. In this case the spectral properties are used to predict the phenolic content of a given grape phenolic extract or wine [16]. This approach makes use of partial least squares regression analysis to correlate spectral information with reference data (phenolic levels). If successfully performed, a validated calibration can provide accurate predictions of phenolic content by only measuring the UV-visible spectral properties of wines.

This manuscript, in its different sections, reports therefore the current status of the different analytical techniques available for the quantification of phenolic content in grapes and wines. Moreover, the UV-visible spectral features observed in wines during the winemaking process, from the early stages of fermentation and through the ageing process are also reported and discussed.

**29**

**Figure 4.**

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

Among other analytical techniques, UV-visible spectroscopy appears to be suitable for the quantification of phenolic compounds. This is due to two main reasons. First of all, phenolic substances have the ability to strongly absorb UV light [11] and secondly, certain compounds due to the coloured nature can lead to absorption features in the visible range [17]. Polyphenols are biological compounds containing π conjugated systems with hydroxyl-phenolic groups. The π type molecular orbitals electronic transitions provide the UV-visible spectrum of this group of compounds. UV-visible spectroscopy is used in winemaking to quantify different sub-groups within the phenolic family [18]. The most common procedures for phenolic analysis are reported to quantify anthocyanins, phenolic acids (including hydroxycinnamates and hydroxybenzoates), stilbenes, flavonols and flavanols or tannins.

The main absorption feature of the flavanol monomers is a strong absorption band around 280 nm (**Figure 4c**). These colourless compounds do not show absorption features in the visible region of the electromagnetic spectrum. The flavanol monomers may contain a galloyl molecule attached to the flavan-3-ol structure. A galloylated flavanol has been reported to have higher absorption intensity, when compared to its non-galloylated form, it also shows a shoulder at 310 nm, characteristic of the galloyl group (see gallic acid as example in **Figure 4d**) [11]. For flavanol polymers or tannins the absorption features remain the same despite the degree of polymerisation (number of monomers) of the proanthocyanidin structure with a

Anthocyanin compounds co-exist under different forms and its colour intensity and tonality depends on the proportion of the different molecular structures

*UV-vis spectral properties of individual phenolic compounds. (a) Malvidin-3-glucoside, (b) malvidin-3-pcoumarylglucoside, (c) catechin, (d) gallic acid, (e) caftaric acid, (f) coutaric acid, (g) rutin, (h) quercetin.*

**2. UV-visible features of wines during winemaking and ageing**

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

predominant absorption band at 280 nm.

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

## **2. UV-visible features of wines during winemaking and ageing**

Among other analytical techniques, UV-visible spectroscopy appears to be suitable for the quantification of phenolic compounds. This is due to two main reasons. First of all, phenolic substances have the ability to strongly absorb UV light [11] and secondly, certain compounds due to the coloured nature can lead to absorption features in the visible range [17]. Polyphenols are biological compounds containing π conjugated systems with hydroxyl-phenolic groups. The π type molecular orbitals electronic transitions provide the UV-visible spectrum of this group of compounds. UV-visible spectroscopy is used in winemaking to quantify different sub-groups within the phenolic family [18]. The most common procedures for phenolic analysis are reported to quantify anthocyanins, phenolic acids (including hydroxycinnamates and hydroxybenzoates), stilbenes, flavonols and flavanols or tannins.

The main absorption feature of the flavanol monomers is a strong absorption band around 280 nm (**Figure 4c**). These colourless compounds do not show absorption features in the visible region of the electromagnetic spectrum. The flavanol monomers may contain a galloyl molecule attached to the flavan-3-ol structure. A galloylated flavanol has been reported to have higher absorption intensity, when compared to its non-galloylated form, it also shows a shoulder at 310 nm, characteristic of the galloyl group (see gallic acid as example in **Figure 4d**) [11]. For flavanol polymers or tannins the absorption features remain the same despite the degree of polymerisation (number of monomers) of the proanthocyanidin structure with a predominant absorption band at 280 nm.

Anthocyanin compounds co-exist under different forms and its colour intensity and tonality depends on the proportion of the different molecular structures

#### **Figure 4.**

*UV-vis spectral properties of individual phenolic compounds. (a) Malvidin-3-glucoside, (b) malvidin-3-pcoumarylglucoside, (c) catechin, (d) gallic acid, (e) caftaric acid, (f) coutaric acid, (g) rutin, (h) quercetin.*

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

phenolic content using a simple UV-visible spectrophotometer possible [1]. However, UV-visible spectroscopy is also used in more advanced separation techniques, such as liquid chromatography, that allows for the quantification of individual phenolic compounds [13]. The quantification of phenolic compounds is thus achieved through the UV-visible signal given by the individually separated phenolic compounds. On the other hand, fluorescence spectroscopy also makes use of the UV-visible spectral features of the excited substances. After the excitation process a coloured fluorophore is quantified based on its absorption intensity projected in the visible region [14]. Finally, UV-visible spectroscopy combined with chemometrics is also included in the techniques used for phenolic compounds' quantification [15]. In this case the spectral properties are used to predict the phenolic content of a given grape phenolic extract or wine [16]. This approach makes use of partial least squares regression analysis to correlate spectral information with reference data (phenolic levels). If successfully performed, a validated calibration can provide accurate predictions of phenolic

content by only measuring the UV-visible spectral properties of wines.

through the ageing process are also reported and discussed.

*Chemical structure of flavan-3-ol compounds found in grapes and wines.*

This manuscript, in its different sections, reports therefore the current status of the different analytical techniques available for the quantification of phenolic content in grapes and wines. Moreover, the UV-visible spectral features observed in wines during the winemaking process, from the early stages of fermentation and

**28**

**Figure 3.**

present at the time of evaluation. The main absorption features of this phenolic subfamily are given by an intense absorption band at 280 nm, common to all phenolic substances, and by a characteristic absorption intensity around 520 nm characteristic of red colouring substances (**Figure 4a**). In addition, the anthocyanins are found in grapes and wines acylated with a number of other wine components, including some phenolic acids such as caffeic or *p*-coumaric acids [3]. In this case the anthocyanin molecule will also show a characteristic broad band around 320 nm (see malvidin-3-*p*-coumarylglucoside in **Figure 4b**). Anthocyanins are highly reactive phenolic compounds strongly influenced by the pH conditions and by the presence of SO2 [19]. Lower pH values increase the proportion of anthocyanins present in the red flavylium form, leading to increased colour intensity, through an hyperchromic effect in the visible region. The opposite behaviour is thus observed if the wine's pH increases to higher values, leading to a decrease of the absorption intensity at 520 nm (hypochromic effect). On the other hand, the ability of anthocyanins to exist in its red forms is highly dependent on the SO2 content. Sulphur dioxide has the ability to interact and combine with the anthocyanin molecule in position 4 of the central phenolic ring, causing the decolouration of the chromophore, leading to a colourless flavilium sulphonate [6]. The protective role of sulphur dioxide is due to its ability as antioxidant. In the case of the anthocyanins, SO2 protects the non-coloured anthocyanin in solution until, due to the reversible nature of this reactions, the red anthocyanin chromophore is liberated.

Phenolic acids in grapes and wines include both hydroxycinnamic and hydoxybenzoic acids. Hydroxybenzoic acids, such as gallic acid, show a single intense absorption band at 280 nm, common to all phenolic substances (**Figure 4d**). On the other hand, hydroxycinnamic acids show an absorption band around 320 nm, characteristic of this group of compounds (**Figure 4e** (caftaric acid) and f (coutaric acid)). Finally, the flavonol group show also particular UV-visible absorption features with an additional absorption band around 360 nm (**Figure 4g** (rutin) and h (quercetin)). This absorption band together with the 280 nm absorption features define the UV-visible spectra of the flavonol group.

Grape phenolic compounds are released into the must after the crushing operation. Phenolic compounds are initially located in the solid parts of the berries. Seeds, skins and to a lesser extent, stems, are the main sources of phenolic compounds found in wines. During crushing the juice contained in the berries comes in contact with skins and seeds. Subsequently, during the maceration step this contact will lead to the diffusion of the phenolic substances into the must. While tannins are found in both skins and seed tissues, the anthocyanins are only located in the skins (also found in the flesh of a few tenturier cultivars). Hydroxycinnamic acids are, on the contrary, the only group of phenolic compounds that is found in high levels in the flesh, whereas hydroxybenzoic acids (seeds), stilbenes (skins) and flavonols (skins) are found mainly in the solid parts of the grape berries [4]. The winemaking strategy i.e. presence or absence of skin contact, length and conditions of the skin contact and grape characteristics will define the pool of phenolic compounds that will be present in the wine after the fermentation. Due to this phenolic extraction, important changes in the UV-visible spectral feature take place.

**Figure 5** shows the average UV-visible spectral features of 13 different red wines during the first 15 days of the fermentation that included cultivars such as Cabernet Sauvignon, Shiraz, Grenache or Pinotage. Three main absorption bands are observed in the UV-visible spectral features. The first and more prominent band is observed around 280 nm. Following this, broad high intensity absorption properties are also observed around 320 nm. Finally, a third intense absorption band is identified in the visible region around 520 nm. As can be observed in **Figure 5**, right after crushing (Day 0) low absorption intensity bands are observed in the 280 and 320 regions,

**31**

content material.

**Figure 5.**

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

whereas no absorption is observed at the visible anthocyanin absorbing 520 nm region. This can be explained by the instant release of some of the phenolic compounds located in the flesh such as the hydroxycinnamic acids. As fermentation progresses a hyperchromic effect is rapidly observed during the first days after crushing. The absorption band around 280 nm rapidly increases until Day 9 of fermentation. From then on, an increase is still identified but to a lesser extent than that initially observed. A different behaviour is observed for the absorption features around 320 and 520 nm. For these two regions, the intense hyperchromism is observed until Day 9 with no subsequent significant increase until the completion of fermentation.

*UV-visible spectral features (250–600 nm) of red wines during fermentation.*

Anthocyanins are water soluble compounds that are extracted during the early stages of fermentation. Alongside with the anthocyanins, the extraction of other skin-localised phenolics, such as flavonols and flavanols or tannins also takes place. However, as alcohol content increases, seed phenolics, mainly flavanols and tannins, are released into the must. The later extraction of seed flavanols and tannins requires the hydrolysis of the lipidic layer around the seed as well as the hydration of the seed tissue itself. Seed tannins have been defined as more astringent and bitter tannins while skin tannins have been described as softer or less reactive towards proteins [10]. The flavanol content in terms of individual composition (procyanidins or prodelphinidins), galloylated subunits and mean degree of polymerisation will provide the intensity and sub qualities of the bitterness and astringency perception of wines [9]. The intense absorption band at 280 nm is due to the extraction of flavonols, hydroxycinnamic acids, flavanols and the UV absorption part of the anthocyanins. The band observed around 320 nm is purely ascribed to the hydroxycinnamic acids. Finally, the band observed at 520 nm is due to the anthocyanin extraction during fermentation. The further increase in the absorption intensity at 280 nm after Day 9 may be due to further extraction of seed tannin

Phenolic compounds are highly reactive and a large number of interactions and reactions can take place during wine ageing and storage. Some of these phenolic reactions benefit from the presence of oxygen during the barrel ageing period. This is the case for some of the direct tannin-anthocyanin complexes as well as the indirectly acetaldehyde mediated tannin-anthocyanin reactions. On the other hand, the absence or shortage of oxygen during the bottle ageing period will stimulate tannin polymerisation reactions and also some direct tannin-anthocyanin combinations. **Figure 6** shows the average UV-visible spectra of a number of commercial red wines after the fermentation process was completed as well as after a year of barrel ageing (12 months after fermentation is completed), followed by a year of bottle ageing (24 months of fermentation completion). In this case an average spectra of a large number of wines including Cabernet Sauvignon, Pinotage, Shiraz, Merlot,

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

**Figure 5.** *UV-visible spectral features (250–600 nm) of red wines during fermentation.*

whereas no absorption is observed at the visible anthocyanin absorbing 520 nm region. This can be explained by the instant release of some of the phenolic compounds located in the flesh such as the hydroxycinnamic acids. As fermentation progresses a hyperchromic effect is rapidly observed during the first days after crushing. The absorption band around 280 nm rapidly increases until Day 9 of fermentation. From then on, an increase is still identified but to a lesser extent than that initially observed. A different behaviour is observed for the absorption features around 320 and 520 nm. For these two regions, the intense hyperchromism is observed until Day 9 with no subsequent significant increase until the completion of fermentation.

Anthocyanins are water soluble compounds that are extracted during the early stages of fermentation. Alongside with the anthocyanins, the extraction of other skin-localised phenolics, such as flavonols and flavanols or tannins also takes place. However, as alcohol content increases, seed phenolics, mainly flavanols and tannins, are released into the must. The later extraction of seed flavanols and tannins requires the hydrolysis of the lipidic layer around the seed as well as the hydration of the seed tissue itself. Seed tannins have been defined as more astringent and bitter tannins while skin tannins have been described as softer or less reactive towards proteins [10]. The flavanol content in terms of individual composition (procyanidins or prodelphinidins), galloylated subunits and mean degree of polymerisation will provide the intensity and sub qualities of the bitterness and astringency perception of wines [9]. The intense absorption band at 280 nm is due to the extraction of flavonols, hydroxycinnamic acids, flavanols and the UV absorption part of the anthocyanins. The band observed around 320 nm is purely ascribed to the hydroxycinnamic acids. Finally, the band observed at 520 nm is due to the anthocyanin extraction during fermentation. The further increase in the absorption intensity at 280 nm after Day 9 may be due to further extraction of seed tannin content material.

Phenolic compounds are highly reactive and a large number of interactions and reactions can take place during wine ageing and storage. Some of these phenolic reactions benefit from the presence of oxygen during the barrel ageing period. This is the case for some of the direct tannin-anthocyanin complexes as well as the indirectly acetaldehyde mediated tannin-anthocyanin reactions. On the other hand, the absence or shortage of oxygen during the bottle ageing period will stimulate tannin polymerisation reactions and also some direct tannin-anthocyanin combinations. **Figure 6** shows the average UV-visible spectra of a number of commercial red wines after the fermentation process was completed as well as after a year of barrel ageing (12 months after fermentation is completed), followed by a year of bottle ageing (24 months of fermentation completion). In this case an average spectra of a large number of wines including Cabernet Sauvignon, Pinotage, Shiraz, Merlot,

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

reactions, the red anthocyanin chromophore is liberated.

define the UV-visible spectra of the flavonol group.

important changes in the UV-visible spectral feature take place.

present at the time of evaluation. The main absorption features of this phenolic subfamily are given by an intense absorption band at 280 nm, common to all phenolic substances, and by a characteristic absorption intensity around 520 nm characteristic of red colouring substances (**Figure 4a**). In addition, the anthocyanins are found in grapes and wines acylated with a number of other wine components, including some phenolic acids such as caffeic or *p*-coumaric acids [3]. In this case the anthocyanin molecule will also show a characteristic broad band around 320 nm (see malvidin-3-*p*-coumarylglucoside in **Figure 4b**). Anthocyanins are highly reactive phenolic compounds strongly influenced by the pH conditions and by the presence of SO2 [19]. Lower pH values increase the proportion of anthocyanins present in the red flavylium form, leading to increased colour intensity, through an hyperchromic effect in the visible region. The opposite behaviour is thus observed if the wine's pH increases to higher values, leading to a decrease of the absorption intensity at 520 nm (hypochromic effect). On the other hand, the ability of anthocyanins to exist in its red forms is highly dependent on the SO2 content. Sulphur dioxide has the ability to interact and combine with the anthocyanin molecule in position 4 of the central phenolic ring, causing the decolouration of the chromophore, leading to a colourless flavilium sulphonate [6]. The protective role of sulphur dioxide is due to its ability as antioxidant. In the case of the anthocyanins, SO2 protects the non-coloured anthocyanin in solution until, due to the reversible nature of this

Phenolic acids in grapes and wines include both hydroxycinnamic and hydoxybenzoic acids. Hydroxybenzoic acids, such as gallic acid, show a single intense absorption band at 280 nm, common to all phenolic substances (**Figure 4d**). On the other hand, hydroxycinnamic acids show an absorption band around 320 nm, characteristic of this group of compounds (**Figure 4e** (caftaric acid) and f (coutaric acid)). Finally, the flavonol group show also particular UV-visible absorption features with an additional absorption band around 360 nm (**Figure 4g** (rutin) and h (quercetin)). This absorption band together with the 280 nm absorption features

Grape phenolic compounds are released into the must after the crushing opera-

**Figure 5** shows the average UV-visible spectral features of 13 different red wines during the first 15 days of the fermentation that included cultivars such as Cabernet Sauvignon, Shiraz, Grenache or Pinotage. Three main absorption bands are observed in the UV-visible spectral features. The first and more prominent band is observed around 280 nm. Following this, broad high intensity absorption properties are also observed around 320 nm. Finally, a third intense absorption band is identified in the visible region around 520 nm. As can be observed in **Figure 5**, right after crushing (Day 0) low absorption intensity bands are observed in the 280 and 320 regions,

tion. Phenolic compounds are initially located in the solid parts of the berries. Seeds, skins and to a lesser extent, stems, are the main sources of phenolic compounds found in wines. During crushing the juice contained in the berries comes in contact with skins and seeds. Subsequently, during the maceration step this contact will lead to the diffusion of the phenolic substances into the must. While tannins are found in both skins and seed tissues, the anthocyanins are only located in the skins (also found in the flesh of a few tenturier cultivars). Hydroxycinnamic acids are, on the contrary, the only group of phenolic compounds that is found in high levels in the flesh, whereas hydroxybenzoic acids (seeds), stilbenes (skins) and flavonols (skins) are found mainly in the solid parts of the grape berries [4]. The winemaking strategy i.e. presence or absence of skin contact, length and conditions of the skin contact and grape characteristics will define the pool of phenolic compounds that will be present in the wine after the fermentation. Due to this phenolic extraction,

**30**

**Figure 6.**

*UV-visible spectral features of wines after malolactic fermentation completion (AMLF) and at 12 months of barrel ageing (12M), followed by 12 months of bottle ageing (24M).*

Ruby Cabernet, Petit Verdot, Cinsault, Malbec, Grenache, Pinot Noir and Cabernet Franc was evaluated. The most important features are observed at 280 and 520 nm, whereas the broad band at 320 nm remained constant over the ageing period. It is also important to mention that the bigger decrease in absorption intensity was observed at the 520 nm region which corresponds to the visible absorption part of the anthocyanins.

After reaching maximum levels during the fermentation process, anthocyanin content starts decreasing. Anthocyanins are involved in a large number of phenomena, such as degradation, oxidation, reabsorption into grape and yeast cell walls, precipitation with tartaric salts, interaction with SO2 or reaction with tannins, among others [7]. Despite this, red wines still maintain an intense colour during ageing which is due to the transformation of anthocyanins into longer term stable polymeric pigments. Anthocyanins give rise to a number of pigments from acylation with diverse grape components, intra and intermolecular copigmentation reactions and interactions, occurring early during the process, to more complex reaction leading to pyranoanthocyanin or tannin-anthocyanin complexes formation [7]. The limited decrease observed around 280 nm is attributed to a larger extent to the decrease of the UV absorption ability of the anthocyanins and to a lesser extent to a decrease of tannin compounds through precipitation. Tannins are also highly reactive substances with high affinity for proteins and polysaccharides, which can lead to tannins precipitation. In addition, the polymerisation ability of these compounds may result in insoluble larger molecules that also precipitate from solution, thus reducing its content in wine. Finally, the absorption band around 320 nm remains stable during ageing, indicating stability of this region absorbing compounds during barrel and bottle ageing.

## **3. Spectrophotometric methods for phenolic analysis**

#### **3.1 Total phenolic content**

**Total phenolic index (TPI).** The measurement of UV-visible absorption light to quantify phenolic compounds was first proposed in the late 1950's. The absorbance at 280 nm was selected as the best indicator of the phenolic content in wine due to the ability of phenolic substances, and more specifically the phenolic ring, to absorb UV light [20]. A simple wine or grape extract dilution is used to quantify the total phenolic content or total phenolic index (TPI). The TPI corresponds to the A280 nm times the dilution factor. The dilution factor might change depending on the sample under evaluation, as well as the path length of the cuvette. Dilution factors of 100

**33**

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

and 50 have been reported for red wines. Depending on the extraction methods dilution factors between 50 and 20 for grape extracts have been proposed. In the case of white and rose wines, with lower phenolic levels, the dilution factor needs to be adjusted. In this case dilution factors from 5 to 20 have been used. The TPI can also be expressed as gallic acid equivalents when used as a standard. This method has been reported to be simple, fast and reliable, although overestimation of the total phenolic content occurs due to the ability of other grape component that also absorb UV-light. A value of 4 units, that can be subtracted from the index, has been proposed to account for the interferences caused by these other UV absorbing material. Additionally, some other phenolic compounds such as cinnamic acids or chalcones do not show absorption features at 280 nm, however due to its low

content in wines the expected differences are considered negligible [21].

content between these two methods comparable [1].

**3.2 Total anthocyanin content**

i.e. malvidin-3-glucoside.

**Folin-Ciocalteu.** The Folin-Ciocalteu assay for total phenolic content relies on the ability of the Folin-Ciocalteu reagent to strongly react with phenolic compounds. A mixture of two acids, namely phosphotungstic (H3PW12O40) and phosphomolybdic (H3PMO12O40) acids, react with mono and dihydroxylated phenolic substances due to their high ability to donate electrons. This reaction creates a blue coloured complex that is quantified at 750 nm [22]. After a simple wine dilution, the Folin-Ciocalteu reagent is added. A 20% NaCO3 solution is then added to the mixture with some additional distilled water. The sample is then incubated for 30 min before absorbance measurement can be performed. Moreover, it is of crucial importance to maintain the order of the additions to ensure that the reaction takes place under alkaline conditions. In order to preserve accuracy, the A750 nm needs to be around 0.3 A.U. If this is not achieved, a different wine dilution needs to be performed. A blank with distilled water, to account for background interferences, is also included [23]. The results are commonly reported as gallic acid equivalents. Although the method is very often used, the ability of some other wine component to also donate electrons leads to potential overestimation of the phenolic content. This compromises the comparison of different samples containing varying phenolic and wine composition. In addition, the comparison of the Folin-Ciocalteu with the TPI is also possible by multiplying the A750 nm times the dilution factor times 20. A strong correlation between the two methods has been reported, thus making the total phenolic

**Hydrochloric acid method.** The estimation of the total concentration of anthocyanins in wine or grape extracts is possible due to the characteristic absorption band of this group of compounds around 520 nm. The coloration of anthocyanins are highly influenced by pH, with lower pH values leading to a higher proportion of anthocyanins in the red flavilium ion form. This property is thus exploited in this method to quantify the total anthocyanin content. Due to its ability to decrease pH, the method makes use of hydrochloric acid (HCl) i.e. the sample is diluted with a 1 M HCl solution. After a waiting period, to allow the free monomeric forms of the anthocyanin to be transformed into their red coloured forms, the A520 nm is measured [24]. The waiting period was initially reported to be longer than 3 hours but shorter than 24 hours, however later research confirmed that a waiting period of 1 hour is sufficient [25]. The values can be reported as A.U. or as malvidin-3-glucoside equivalents by making use of the molar extinction coefficient (commonly used 28,000 L/cm\*mol) and the molecular weight (MW = 529 g/mol) of the major anthocyanin found in grapes and wines

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

#### *The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

and 50 have been reported for red wines. Depending on the extraction methods dilution factors between 50 and 20 for grape extracts have been proposed. In the case of white and rose wines, with lower phenolic levels, the dilution factor needs to be adjusted. In this case dilution factors from 5 to 20 have been used. The TPI can also be expressed as gallic acid equivalents when used as a standard. This method has been reported to be simple, fast and reliable, although overestimation of the total phenolic content occurs due to the ability of other grape component that also absorb UV-light. A value of 4 units, that can be subtracted from the index, has been proposed to account for the interferences caused by these other UV absorbing material. Additionally, some other phenolic compounds such as cinnamic acids or chalcones do not show absorption features at 280 nm, however due to its low content in wines the expected differences are considered negligible [21].

**Folin-Ciocalteu.** The Folin-Ciocalteu assay for total phenolic content relies on the ability of the Folin-Ciocalteu reagent to strongly react with phenolic compounds. A mixture of two acids, namely phosphotungstic (H3PW12O40) and phosphomolybdic (H3PMO12O40) acids, react with mono and dihydroxylated phenolic substances due to their high ability to donate electrons. This reaction creates a blue coloured complex that is quantified at 750 nm [22]. After a simple wine dilution, the Folin-Ciocalteu reagent is added. A 20% NaCO3 solution is then added to the mixture with some additional distilled water. The sample is then incubated for 30 min before absorbance measurement can be performed. Moreover, it is of crucial importance to maintain the order of the additions to ensure that the reaction takes place under alkaline conditions. In order to preserve accuracy, the A750 nm needs to be around 0.3 A.U. If this is not achieved, a different wine dilution needs to be performed. A blank with distilled water, to account for background interferences, is also included [23]. The results are commonly reported as gallic acid equivalents. Although the method is very often used, the ability of some other wine component to also donate electrons leads to potential overestimation of the phenolic content. This compromises the comparison of different samples containing varying phenolic and wine composition. In addition, the comparison of the Folin-Ciocalteu with the TPI is also possible by multiplying the A750 nm times the dilution factor times 20. A strong correlation between the two methods has been reported, thus making the total phenolic content between these two methods comparable [1].

## **3.2 Total anthocyanin content**

**Hydrochloric acid method.** The estimation of the total concentration of anthocyanins in wine or grape extracts is possible due to the characteristic absorption band of this group of compounds around 520 nm. The coloration of anthocyanins are highly influenced by pH, with lower pH values leading to a higher proportion of anthocyanins in the red flavilium ion form. This property is thus exploited in this method to quantify the total anthocyanin content. Due to its ability to decrease pH, the method makes use of hydrochloric acid (HCl) i.e. the sample is diluted with a 1 M HCl solution. After a waiting period, to allow the free monomeric forms of the anthocyanin to be transformed into their red coloured forms, the A520 nm is measured [24]. The waiting period was initially reported to be longer than 3 hours but shorter than 24 hours, however later research confirmed that a waiting period of 1 hour is sufficient [25]. The values can be reported as A.U. or as malvidin-3-glucoside equivalents by making use of the molar extinction coefficient (commonly used 28,000 L/cm\*mol) and the molecular weight (MW = 529 g/mol) of the major anthocyanin found in grapes and wines i.e. malvidin-3-glucoside.

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

*barrel ageing (12M), followed by 12 months of bottle ageing (24M).*

Ruby Cabernet, Petit Verdot, Cinsault, Malbec, Grenache, Pinot Noir and Cabernet Franc was evaluated. The most important features are observed at 280 and 520 nm, whereas the broad band at 320 nm remained constant over the ageing period. It is also important to mention that the bigger decrease in absorption intensity was observed at the 520 nm region which corresponds to the visible absorption part of

*UV-visible spectral features of wines after malolactic fermentation completion (AMLF) and at 12 months of* 

After reaching maximum levels during the fermentation process, anthocyanin content starts decreasing. Anthocyanins are involved in a large number of phenomena, such as degradation, oxidation, reabsorption into grape and yeast cell walls, precipitation with tartaric salts, interaction with SO2 or reaction with tannins, among others [7]. Despite this, red wines still maintain an intense colour during ageing which is due to the transformation of anthocyanins into longer term stable polymeric pigments. Anthocyanins give rise to a number of pigments from acylation with diverse grape components, intra and intermolecular copigmentation reactions and interactions, occurring early during the process, to more complex reaction leading to pyranoanthocyanin or tannin-anthocyanin complexes formation [7]. The limited decrease observed around 280 nm is attributed to a larger extent to the decrease of the UV absorption ability of the anthocyanins and to a lesser extent to a decrease of tannin compounds through precipitation. Tannins are also highly reactive substances with high affinity for proteins and polysaccharides, which can lead to tannins precipitation. In addition, the polymerisation ability of these compounds may result in insoluble larger molecules that also precipitate from solution, thus reducing its content in wine. Finally, the absorption band around 320 nm remains stable during ageing, indicating stability of this region absorbing

**32**

the anthocyanins.

**Figure 6.**

compounds during barrel and bottle ageing.

**3.1 Total phenolic content**

**3. Spectrophotometric methods for phenolic analysis**

**Total phenolic index (TPI).** The measurement of UV-visible absorption light to quantify phenolic compounds was first proposed in the late 1950's. The absorbance at 280 nm was selected as the best indicator of the phenolic content in wine due to the ability of phenolic substances, and more specifically the phenolic ring, to absorb UV light [20]. A simple wine or grape extract dilution is used to quantify the total phenolic content or total phenolic index (TPI). The TPI corresponds to the A280 nm times the dilution factor. The dilution factor might change depending on the sample under evaluation, as well as the path length of the cuvette. Dilution factors of 100

**Bisulphite bleaching method.** Another property of the anthocyanins is in this case used to quantify this group of compounds. Sulphur dioxide is able to combine with the anthocyanin in the position 4 of the central phenolic ring, giving rise to a non-coloured flavene sulphonate. The decolouration ability of SO2 is thus used to estimate the total content of free anthocyanins in the wine. The method also makes use of HCl with the aim of transforming the anthocyanins to their red coloured flavilium form. Two test samples are in this case compared. The control sample, with no SO2 addition is compared against a treatment sample where the anthocyanins have been bleached by the SO2 addition. After a waiting period, the A520 nm of both samples are compared and the total anthocyanin content calculated [26]. However, the ability of SO2 to react and bleach some pigmented forms might lead to an overestimation of the total content [27].

**pH differential method.** Another method that exploits the effect of pH on the anthocyanin coloration was reported by Giusti and Worldstad [17]. This methodology compares a red flavilium form sample at pH 1 against a sample where the anthocyanins are transformed to its non-coloured hemiketal from at pH 4.5. Instead of measuring the anthocyanin content of both pH 1 and pH 4.5 samples at a fixed wavenumber (520 nm), the method measures the Amax observed around the 520 nm absorption band, which may not coincide with 520 nm. In addition, the method also includes the measurement of the A700 nm that is subtracted from the Amax, with the aim of accounting for possible light scattering caused by other sample components. By doing this the method ensures that the recorded absorption values only correspond to the anthocyanin content in the samples. The results are reported as malvidin-3-glucoside equivalents, by also using the molar extinction coefficient and the molecular weight of this anthocyanin. In addition, the method also allows for the calculation of additional indices by using the ability of sulphur dioxide to combine and bleach anthocyanins. A more complete picture of the anthocyanin content and composition is thus obtained after the inclusion of the pigment degradation, polymeric colour and browning indexes. In this case the method makes use of the absorbance at 420 nm to account for the polymeric anthocyanin material with colour properties closer to this region of the visible spectrum (orange colouration). The polymeric pigment colour is calculated as the proportion between the colour observed in the bleached samples at 420 nm and the Amax around 520 nm and that measured at the same wavelengths in the non-bleached samples. In order to ensure accuracy, measurements need to be taken between 15 min and 1 hour in line with what was reported earlier to avoid increased absorption properties at longer times [17].

**Modified Somers assay.** This methodology is based in the original method reported by Somers and Evans [21]. More recently a modified protocol, adapted to a high throughput format, using a microplate reader spectrophotometer was reported for both grape extract and wine samples [28]. The method presents a number of parameters and provides a broad overview of the status of the anthocyanin's equilibria in the sample. The method relies on the effect of hydrochloric acid, acetaldehyde and sulphur dioxide on the anthocyanins. Sulphur dioxide is added with the aim of calculating the levels of non-blanchable pigments, which includes more stable pigments such as tannin-anthocyanin complexes as well as pyranoanthocyanins. Moreover, acetaldehyde is used to negate the bleaching effect of SO2 on anthocyanins and thus measure the total content of coloured anthocyanins. Finally, hydrochloric acid is added to account for those free anthocyanins that were not bleached or were derived from copigmentation complexes. The main advantage of the method relies on the fact that the pH adjustment, crucial to accurately estimate the state of the anthocyanins, is done by adjusting the pH of a buffer solution [28]. In the original protocol the pH of the samples was individually adjusted, with a

**35**

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

considerable extension of the time of analysis. This method provides information on the wine "chemical age", which provides an estimation of the extent that the polymeric pigments has displaced the monomeric anthocyanins. Additional parameters report on the percentage of anthocyanins in its flavilium red form (% of ionisation), SO2 resistant pigments (polymeric pigments), colour intensity, hue as

**Copigmentation assay.** Anthocyanins interact with other wine components including other phenolic substances to form pigmented molecules through weak hydrophobic forces. The sandwich-like structure is composed of copigment molecules in between the anthocyanins [29]. The newly formed structure places the sugar moieties of the anthocyanin towards the external part of the complex, thus protecting the copigmented pigment from decolouration by water. These interactions account for a large part of the colour of young red wines with its contribution to wine colour decreasing over time, due to the weak nature of the copigmented structure [29]. Two main effects are characteristic of these complexes, which includes an increased absorption intensity in the visible absorption region of the anthocyanins (hyperchromic effect) accompanied by a shift into the absorption maxima towards higher wavelengths (blue colouration) through a bathochromic effect. The copigmentation assay was developed by Boulton and it is the only available method for the quantification of the colour due to copigmentation in red wines. The method relies on the ability of the anthocyanin complexes to avoid decolouration by water at constant pH i.e. measures the decolouration of the anthocyanins

**Colour density.** Coloured anthocyanins and anthocyanin derived pigments are responsible for the colour properties of red wines. During the early stages of winemaking the colour properties of wines are mainly due to less complex monomeric forms of anthocyanins, however as the wine ages and anthocyanins start interacting with other wine components, more stable pigmented polymeric forms are responsible for the colour properties of red wines. The wine colour density was initially measured through the addition of the absorption values at 420 and 520 nm,

which corresponds to the yellow and red colorations of wine [30]. Using this information, the hue of a wine samples was defined as the ratio between these two absorption values (A420 nm/A520 nm). More recently the absorption at 620 nm, which accounts for the blue wine colouration, was also added to the colour density parameter [31]. The method relies on a simple measurement (without dilution) and provides an estimation of the colour intensity of the wine. The results are often reported as %yellow, %red and %blue providing thus a more complete interpretation of wine colour properties. On the other hand, the A420 nm or A440 nm are commonly used to measure the colour properties of white wines including the

**CIElab colour space.** Wine colour can also be measured through the information contained in the visible spectra of wines. Three colour components result from the integration of the visible absorption features. The Commission International de l'eclairage [33] proposed a method that uses three chromatic coordinates X, Y and Z to determine the chromatic characteristics of wines (also applicable to other beverages). The method aims to simulate the perception that real observers have for the colour properties of a sample. The calculation of the CIElab coordinates is based on measurement conditions given by a spectrophotometer with illuminant D65 and observed placed at 10°. The colour of a wine is thus described by the intensity of the wine colour (chromaticism), the luminosity of the wine and the colour itself based on the

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

due to the dissociation of the copigmented forms [29].

brownish wine colour (browning index) [32].

well as total phenolic content.

**3.3 Colour measurements**

#### *The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

considerable extension of the time of analysis. This method provides information on the wine "chemical age", which provides an estimation of the extent that the polymeric pigments has displaced the monomeric anthocyanins. Additional parameters report on the percentage of anthocyanins in its flavilium red form (% of ionisation), SO2 resistant pigments (polymeric pigments), colour intensity, hue as well as total phenolic content.

**Copigmentation assay.** Anthocyanins interact with other wine components including other phenolic substances to form pigmented molecules through weak hydrophobic forces. The sandwich-like structure is composed of copigment molecules in between the anthocyanins [29]. The newly formed structure places the sugar moieties of the anthocyanin towards the external part of the complex, thus protecting the copigmented pigment from decolouration by water. These interactions account for a large part of the colour of young red wines with its contribution to wine colour decreasing over time, due to the weak nature of the copigmented structure [29]. Two main effects are characteristic of these complexes, which includes an increased absorption intensity in the visible absorption region of the anthocyanins (hyperchromic effect) accompanied by a shift into the absorption maxima towards higher wavelengths (blue colouration) through a bathochromic effect. The copigmentation assay was developed by Boulton and it is the only available method for the quantification of the colour due to copigmentation in red wines. The method relies on the ability of the anthocyanin complexes to avoid decolouration by water at constant pH i.e. measures the decolouration of the anthocyanins due to the dissociation of the copigmented forms [29].

### **3.3 Colour measurements**

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

an overestimation of the total content [27].

**Bisulphite bleaching method.** Another property of the anthocyanins is in this case used to quantify this group of compounds. Sulphur dioxide is able to combine with the anthocyanin in the position 4 of the central phenolic ring, giving rise to a non-coloured flavene sulphonate. The decolouration ability of SO2 is thus used to estimate the total content of free anthocyanins in the wine. The method also makes use of HCl with the aim of transforming the anthocyanins to their red coloured flavilium form. Two test samples are in this case compared. The control sample, with no SO2 addition is compared against a treatment sample where the anthocyanins have been bleached by the SO2 addition. After a waiting period, the A520 nm of both samples are compared and the total anthocyanin content calculated [26]. However, the ability of SO2 to react and bleach some pigmented forms might lead to

**pH differential method.** Another method that exploits the effect of pH on the anthocyanin coloration was reported by Giusti and Worldstad [17]. This methodology compares a red flavilium form sample at pH 1 against a sample where the anthocyanins are transformed to its non-coloured hemiketal from at pH 4.5. Instead of measuring the anthocyanin content of both pH 1 and pH 4.5 samples at a fixed wavenumber (520 nm), the method measures the Amax observed around the 520 nm absorption band, which may not coincide with 520 nm. In addition, the method also includes the measurement of the A700 nm that is subtracted from the Amax, with the aim of accounting for possible light scattering caused by other sample components. By doing this the method ensures that the recorded absorption values only correspond to the anthocyanin content in the samples. The results are reported as malvidin-3-glucoside equivalents, by also using the molar extinction coefficient and the molecular weight of this anthocyanin. In addition, the method also allows for the calculation of additional indices by using the ability of sulphur dioxide to combine and bleach anthocyanins. A more complete picture of the anthocyanin content and composition is thus obtained after the inclusion of the pigment degradation, polymeric colour and browning indexes. In this case the method makes use of the absorbance at 420 nm to account for the polymeric anthocyanin material with colour properties closer to this region of the visible spectrum (orange colouration). The polymeric pigment colour is calculated as the proportion between the colour observed in the bleached samples at 420 nm and the Amax around 520 nm and that measured at the same wavelengths in the non-bleached samples. In order to ensure accuracy, measurements need to be taken between 15 min and 1 hour in line with what was reported earlier to avoid increased absorption properties at longer

**Modified Somers assay.** This methodology is based in the original method reported by Somers and Evans [21]. More recently a modified protocol, adapted to a high throughput format, using a microplate reader spectrophotometer was reported for both grape extract and wine samples [28]. The method presents a number of parameters and provides a broad overview of the status of the anthocyanin's equilibria in the sample. The method relies on the effect of hydrochloric acid, acetaldehyde and sulphur dioxide on the anthocyanins. Sulphur dioxide is added with the aim of calculating the levels of non-blanchable pigments, which includes more stable pigments such as tannin-anthocyanin complexes as well as pyranoanthocyanins. Moreover, acetaldehyde is used to negate the bleaching effect of SO2 on anthocyanins and thus measure the total content of coloured anthocyanins. Finally, hydrochloric acid is added to account for those free anthocyanins that were not bleached or were derived from copigmentation complexes. The main advantage of the method relies on the fact that the pH adjustment, crucial to accurately estimate the state of the anthocyanins, is done by adjusting the pH of a buffer solution [28]. In the original protocol the pH of the samples was individually adjusted, with a

**34**

times [17].

**Colour density.** Coloured anthocyanins and anthocyanin derived pigments are responsible for the colour properties of red wines. During the early stages of winemaking the colour properties of wines are mainly due to less complex monomeric forms of anthocyanins, however as the wine ages and anthocyanins start interacting with other wine components, more stable pigmented polymeric forms are responsible for the colour properties of red wines. The wine colour density was initially measured through the addition of the absorption values at 420 and 520 nm, which corresponds to the yellow and red colorations of wine [30]. Using this information, the hue of a wine samples was defined as the ratio between these two absorption values (A420 nm/A520 nm). More recently the absorption at 620 nm, which accounts for the blue wine colouration, was also added to the colour density parameter [31]. The method relies on a simple measurement (without dilution) and provides an estimation of the colour intensity of the wine. The results are often reported as %yellow, %red and %blue providing thus a more complete interpretation of wine colour properties. On the other hand, the A420 nm or A440 nm are commonly used to measure the colour properties of white wines including the brownish wine colour (browning index) [32].

**CIElab colour space.** Wine colour can also be measured through the information contained in the visible spectra of wines. Three colour components result from the integration of the visible absorption features. The Commission International de l'eclairage [33] proposed a method that uses three chromatic coordinates X, Y and Z to determine the chromatic characteristics of wines (also applicable to other beverages). The method aims to simulate the perception that real observers have for the colour properties of a sample. The calculation of the CIElab coordinates is based on measurement conditions given by a spectrophotometer with illuminant D65 and observed placed at 10°. The colour of a wine is thus described by the intensity of the wine colour (chromaticism), the luminosity of the wine and the colour itself based on the

red, yellow, green and blue components (tonality). The colorimetric measurements are defined by the chromatic coordinates red/green component (a\*) (a\* > 0 red, a\* < 0 green), blue/yellow component (b\*) (b\* > 0 yellow, b\* < 0 blue), clarity (L\*) (L\* = 0 black and L\* = 100 colourless) and its complementary magnitudes tone (H\*) and chroma (C\*). The ability to compare the colorimetric differences between two colours (ΔE\*) makes it possible to directly compare the colour properties of wines. Moreover, it has been established that a colour difference higher than 2.7 indicates that the colour of two samples can be perceived different by the human eye [34].

#### **3.4 Total tannin content**

**Acid hydrolysis.** Due to the complex nature of proanthocyanidins or tannins the determination of these compounds is a difficult undertaking and has been challenging researchers for a long time. However, a number of methods, albeit with certain limitations, have been reported and will be discussed. The acid hydrolysis method is based on the transformation of proanthocyanidins in carbocations that are partially converted into anthocyanidins when exposed to heating under acidic conditions (Bate-Smith reaction). The total tannin content is thus estimated by using the red coloration of the resulting anthocyanin compounds at 550 nm and expressing it in cyaniding-3-glucoside equivalents. Although the method is widely used, a number of limitations have also been reported. First of all, the tannin concentration seems to be overestimated with higher values for tannins reported than those for total phenolic content. Moreover, it is also common to observe an increase in the total tannin content of wine during ageing and finally the method does not provide any information on the structure of the tannins [35].

**Methylcellulose precipitable (MCP) tannins assay.** This method falls under the precipitation based methods category as it uses the tannin precipitation ability of a methylcellulose polymer to estimate the total tannin content of grape extracts and wines. As mentioned the method relies on tannin-MCP interactions in the presence of ammonium sulphate, giving rise to an insoluble polymer-tannin complex that precipitates and is further separated by centrifugation [36]. This method has also been lately adapted and validated into a high throughput format leading to a considerable reduction of the analytical time [28]. A control sample without MCP addition (absence of tannin precipitation) is compared against a treated sample where the tannins have been removed after precipitation with MCP. The absorption difference measured at 280 nm is then used to quantify the total tannin content of a sample. The total tannin content is in this case estimated as epicatechin equivalents. In addition, one of the main benefits of precipitation based methods is that a theoretic positive correlation with astringency intensity is foreseen [37–39]. The hypothesis is based on the assumption that the method simulates the phenomena that naturally occurs in wine when it becomes in contact with the salivary proteins. An insoluble macromolecular complex is then formed that precipitates from solution causing the drying and puckering sensation known as astringency.

**Bovine serum albumin (BSA) tannin assay.** This precipitation based method exploits the ability of proteins to combine and precipitate tannins. The precipitation is achieved through the incorporation of bovine serum albumin protein. The precipitated protein-tannin complexes are then redissolved and quantified at 510 nm after the addition of ferric chloride [40, 41]. The accuracy of the method is based on obtaining the appropriate wine dilution as concentrated or very diluted samples tend to underestimate the tannin content. The BSA tannin assay, as part of the precipitation based methods for tannin analysis, has also been found to positively correlate with astringency intensities given by sensorial evaluation [37–39]. The total tannin content is in this assay calculated as catechin equivalents. In addition,

**37**

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

amount of tannins when tested under the same conditions [39, 43].

of phenolic substances as well as the identification of novel compounds.

**4. UV-visible role in liquid chromatography**

the method also allows for the determination of additional parameters related to the anthocyanin and polymeric pigment fraction. Specifically, the method makes use of SO2 to obtain information on the nature of the polymeric pigments by dividing them into small (SPP) (pigments that do not precipitate with BSA) and large polymeric pigments (LPP) that do precipitate with the protein. On the other hand, the comparison of both precipitations based methods has shown that MCP tannin values are on average three time higher than those found for BSA. However, a strong correlation (0.8) between the values obtained with the two methods has also been reported [42], whereas no correlation was observed between these two methods and the tannin content obtained with the acid hydrolysis method [37]. Finally, despite the differences in absolute values, attributable to the differences in both procedures, it has also been stated that both precipitants (BSA and MCP) precipitate the same

High liquid pressure chromatography (HPLC) is a suitable method to quantify individual phenolic compounds in grape extracts and wines. HPLC instruments make use of a diode array detector that allows for the quantification of phenolic substances at different wavelength within the UV-visible regions. The benefit of using diode array detectors in liquid chromatography is beyond using retention times for peak identification as it adds qualitative information by the incorporation of the UV-visible spectral features of a specific peak or compound [44]. It is thus nowadays possible to obtain a number of individual phenolic compounds by direct injection of wine samples without any sample pre-treatment. Based on its spectral features, phenolic compounds will be quantified at their absorption maxima, i.e. sub-families of phenolics are quantified at 280 nm for flavanol monomers and polymers and some phenolic acids, 320 nm for hydroxicinnamic acids, 360 nm for flavonols and finally 520 nm for anthocyanins. Although a considerable number of individual phenolics can be quantified using HPLC, the majority of the methods are not able to separate larger molecular structures such as polymeric phenols and pigments [13]. These two groups of compounds are commonly identified as broad absorption bands at later elution times at 280 nm for the polymeric phenols and at 520 nm for the polymeric pigments. Furthermore, in a previous study, the composition of the broad absorption band observed at 520 nm theoretically attributed to polymeric pigment material was investigated and confirmed [13]. Additionally, the polymeric pigments peak was also found to correlate with the spectrophotometric measurements of phenolic compounds and with wine age. In terms of polymeric phenols, it is believed that the phenolic compounds forming part of this broad absorption band correspond to a large extent to proanthocyanidins or tannins of high degree of polymerisation. The strong correlation (0.83) observed for a significant number of wines between the polymeric phenol peak area and the total tannin content, obtained with the MCP tannins assay, confirmed this [16]. HPLC methods for quantification of phenolic substances can also incorporate mass spectrometers. Mass spectrometry provides information about the molecular weight of the compounds and it is used to discern the identity of unknown compounds. The identification of phenolic compounds in chromatographic techniques using DAD is limited by co-elution (impure UV-visible spectra) or by similarities in the UV-visible properties of phenolic compounds belonging to the same phenolic family. These factors combined with similar elution times of some of the phenolic substances complicates the accurate quantification of chromatographic peaks. The use of mass-spectrometry provides thus a valid tool to confirm the identity

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

the method also allows for the determination of additional parameters related to the anthocyanin and polymeric pigment fraction. Specifically, the method makes use of SO2 to obtain information on the nature of the polymeric pigments by dividing them into small (SPP) (pigments that do not precipitate with BSA) and large polymeric pigments (LPP) that do precipitate with the protein. On the other hand, the comparison of both precipitations based methods has shown that MCP tannin values are on average three time higher than those found for BSA. However, a strong correlation (0.8) between the values obtained with the two methods has also been reported [42], whereas no correlation was observed between these two methods and the tannin content obtained with the acid hydrolysis method [37]. Finally, despite the differences in absolute values, attributable to the differences in both procedures, it has also been stated that both precipitants (BSA and MCP) precipitate the same amount of tannins when tested under the same conditions [39, 43].

## **4. UV-visible role in liquid chromatography**

High liquid pressure chromatography (HPLC) is a suitable method to quantify individual phenolic compounds in grape extracts and wines. HPLC instruments make use of a diode array detector that allows for the quantification of phenolic substances at different wavelength within the UV-visible regions. The benefit of using diode array detectors in liquid chromatography is beyond using retention times for peak identification as it adds qualitative information by the incorporation of the UV-visible spectral features of a specific peak or compound [44]. It is thus nowadays possible to obtain a number of individual phenolic compounds by direct injection of wine samples without any sample pre-treatment. Based on its spectral features, phenolic compounds will be quantified at their absorption maxima, i.e. sub-families of phenolics are quantified at 280 nm for flavanol monomers and polymers and some phenolic acids, 320 nm for hydroxicinnamic acids, 360 nm for flavonols and finally 520 nm for anthocyanins. Although a considerable number of individual phenolics can be quantified using HPLC, the majority of the methods are not able to separate larger molecular structures such as polymeric phenols and pigments [13]. These two groups of compounds are commonly identified as broad absorption bands at later elution times at 280 nm for the polymeric phenols and at 520 nm for the polymeric pigments. Furthermore, in a previous study, the composition of the broad absorption band observed at 520 nm theoretically attributed to polymeric pigment material was investigated and confirmed [13]. Additionally, the polymeric pigments peak was also found to correlate with the spectrophotometric measurements of phenolic compounds and with wine age. In terms of polymeric phenols, it is believed that the phenolic compounds forming part of this broad absorption band correspond to a large extent to proanthocyanidins or tannins of high degree of polymerisation. The strong correlation (0.83) observed for a significant number of wines between the polymeric phenol peak area and the total tannin content, obtained with the MCP tannins assay, confirmed this [16]. HPLC methods for quantification of phenolic substances can also incorporate mass spectrometers. Mass spectrometry provides information about the molecular weight of the compounds and it is used to discern the identity of unknown compounds. The identification of phenolic compounds in chromatographic techniques using DAD is limited by co-elution (impure UV-visible spectra) or by similarities in the UV-visible properties of phenolic compounds belonging to the same phenolic family. These factors combined with similar elution times of some of the phenolic substances complicates the accurate quantification of chromatographic peaks. The use of mass-spectrometry provides thus a valid tool to confirm the identity of phenolic substances as well as the identification of novel compounds.

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

of two samples can be perceived different by the human eye [34].

information on the structure of the tannins [35].

**3.4 Total tannin content**

red, yellow, green and blue components (tonality). The colorimetric measurements are defined by the chromatic coordinates red/green component (a\*) (a\* > 0 red, a\* < 0 green), blue/yellow component (b\*) (b\* > 0 yellow, b\* < 0 blue), clarity (L\*) (L\* = 0 black and L\* = 100 colourless) and its complementary magnitudes tone (H\*) and chroma (C\*). The ability to compare the colorimetric differences between two colours (ΔE\*) makes it possible to directly compare the colour properties of wines. Moreover, it has been established that a colour difference higher than 2.7 indicates that the colour

**Acid hydrolysis.** Due to the complex nature of proanthocyanidins or tannins the determination of these compounds is a difficult undertaking and has been challenging researchers for a long time. However, a number of methods, albeit with certain limitations, have been reported and will be discussed. The acid hydrolysis method is based on the transformation of proanthocyanidins in carbocations that are partially converted into anthocyanidins when exposed to heating under acidic conditions (Bate-Smith reaction). The total tannin content is thus estimated by using the red coloration of the resulting anthocyanin compounds at 550 nm and expressing it in cyaniding-3-glucoside equivalents. Although the method is widely used, a number of limitations have also been reported. First of all, the tannin concentration seems to be overestimated with higher values for tannins reported than those for total phenolic content. Moreover, it is also common to observe an increase in the total tannin content of wine during ageing and finally the method does not provide any

**Methylcellulose precipitable (MCP) tannins assay.** This method falls under the precipitation based methods category as it uses the tannin precipitation ability of a methylcellulose polymer to estimate the total tannin content of grape extracts and wines. As mentioned the method relies on tannin-MCP interactions in the presence of ammonium sulphate, giving rise to an insoluble polymer-tannin complex that precipitates and is further separated by centrifugation [36]. This method has also been lately adapted and validated into a high throughput format leading to a considerable reduction of the analytical time [28]. A control sample without MCP addition (absence of tannin precipitation) is compared against a treated sample where the tannins have been removed after precipitation with MCP. The absorption difference measured at 280 nm is then used to quantify the total tannin content of a sample. The total tannin content is in this case estimated as epicatechin equivalents. In addition, one of the main benefits of precipitation based methods is that a theoretic positive correlation with astringency intensity is foreseen [37–39]. The hypothesis is based on the assumption that the method simulates the phenomena that naturally occurs in wine when it becomes in contact with the salivary proteins. An insoluble macromolecular complex is then formed that precipitates from solu-

tion causing the drying and puckering sensation known as astringency.

**Bovine serum albumin (BSA) tannin assay.** This precipitation based method exploits the ability of proteins to combine and precipitate tannins. The precipitation is achieved through the incorporation of bovine serum albumin protein. The precipitated protein-tannin complexes are then redissolved and quantified at 510 nm after the addition of ferric chloride [40, 41]. The accuracy of the method is based on obtaining the appropriate wine dilution as concentrated or very diluted samples tend to underestimate the tannin content. The BSA tannin assay, as part of the precipitation based methods for tannin analysis, has also been found to positively correlate with astringency intensities given by sensorial evaluation [37–39]. The total tannin content is in this assay calculated as catechin equivalents. In addition,

**36**

## **5. Fluorescence spectroscopy**

An interesting and more recent technique to quantify phenolic compounds makes use of the ability of this group of substances to emit fluorescence light after the excitation/emission process. Fluorescence spectroscopy is able to measure the analyte concentration through its fluorescence properties, being thus suitable to measure compounds in solution, such as phenolics found in grape extracts or wines [14]. If phenolic compounds are excited at the appropriate light intensity and wavelength, generally through UV light exposure, the energy change occurring at electronic level will cause a light emission in the visible region of the electromagnetic spectrum [45]. Phenolic molecules are initially at ground levels at low energy state until light exposure elevate the vibrational levels to an elevated high energy state. After a period of time (in the order of milliseconds) the excited molecule while returning to its non-excited electronic state emits light (so-called fluorescence) at higher wavelengths than those absorbed during the excitation process. During the excitation/emission sequence both the absorbed and emitted light can be measured, with higher emission intensity corresponding to higher concentration of the analyte. Fluorescence spectroscopy has been commonly applied to the quantification of phenolic compounds in combination with liquid chromatography techniques. The main benefit of these applications rely on the increased sensitivity and selectivity of the method [45]. Additionally, fluorescence spectroscopy has been defined as a fast, non-destructive, easy to perform technique that can also be used for process monitoring purposes due to the versatility of the fluorescence spectrometers. Excitation emission spectral (EEM) properties might potentially be correlated with reference analytical data to establish regression calibrations for the quantification of phenolic compounds in a similar manner than what is reported for UV-visible or infrared spectroscopy calibrations.

## **6. UV-visible spectroscopy with chemometrics**

The UV-visible spectra can alternatively be used in combination with powerful chemometric analysis to obtain spectroscopic calibrations for the prediction of phenolic content in grapes as well as in wines during the winemaking process [15]. In this case the totality or parts of the UV-visible spectra are correlated through multivariate regression approaches with reference phenolic data. After the spectral and phenolic content acquisition of a significant number of samples and in the case that strong correlations are found between the spectral data and the phenolic levels, a reliable prediction calibration can be obtained after the corresponding calibration and validation procedure. The advantage of these spectroscopy calibrations relies on the possibility of estimating the total content of phenolic substances through a simple spectral measurement, therefore avoiding the tedious reference method procedures. The main advantage of the spectroscopy calibrations is due to the rapidness, simplicity, reliability and cost-effectiveness ascribed to these techniques. Moreover, due to the multi-parametric nature of this approach a single spectral measurement is able to provide the levels of a number of phenolic compounds. Spectroscopic applications are also highly suitable to perform online measurement during the process of winemaking, allowing for improved process control strategies, through process monitoring, in line with a process analytical technologies (PAT) approach [46].

The first indication of the use of UV-visible spectroscopy calibrations to quantify some of the most important phenolic parameters was reported in 1995 [47]. In this first approach the total tannin and anthocyanin content was predicted using

**39**

samples [50].

were observed [49].

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

a limited number of samples. The UV-visible spectra was collected from 200 to 650 nm at 6 nm intervals. Errors in prediction (root mean standard error of prediction (RMSEP)) of 0.35 g/L (14% RMSEP%) and 29 mg/L (8%) where reported for tannins and anthocyanins respectively. Despite the relative small sample set and the limitations of the analytical reference method investigated (mainly due to nonspecificity for phenolic compounds of the employed procedures) this publication reported for the first time the suitability of UV-visible spectroscopy to quantify phenolic content in wines through partial least squares (PLS) regression analysis. In a further study the UV-visible spectral properties of a large dataset (400) were used to quantify phenolic content of samples collected at different stages of the winemaking process. The sample set included samples from a variety of different regions and cultivars. Spectral data was collected over the 230–900 nm at 0.17 nm intervals. The parameters derived from the BSA tannin assay including the anthocyanin and total phenolic related parameters were in this case evaluated. RMSEP of 87 mg/L (20% RMSEP%) for total anthocyanin content; 0.37 (26.4%), 0.46 (76.7%) and 0.48 (24%) A.U. for small, large and total polymeric pigments, respectively; 66 mg/L (30.1%) for tannin content; 99 (17.2%) mg/L for non-tannin phenols and 130 mg/L (16.4%) for total phenols were reported [48]. Later on, the same phenolic parameters were again investigated. A 100 samples of Cabernet Sauvignon and 100 samples of Shiraz were collected during the fermentation process over a single vintage to provide calibration that can be used for the prediction of phenolic content in must. In this case an adaptation of the BSA tannin assay was used for phenolic analysis. UV-visible spectral properties were collected in the 200–900 nm range. The results showed calibrations able to predict the phenolic content of Cabernet Sauvignon samples, but not for Shiraz, suggesting cultivar specificity of the predicted calibrations. Standard errors in cross validation (RMSECV) of 102.22 mg/L (23.8% RMSECV%) and 211.38 mg/L (25.6%) were reported for total tannin and iron reactive phenolic content, respectively for Cabernet Sauvignon samples. In terms of anthocyanin measurements, error in cross validation of 101 mg/L (43.3% RMSECV%), 0.46 A.U. (26.1%) and 0.48 A.U. (41.4%) for total anthocyanins, small and large polymeric pigments

Due to its characteristic absorption band at 280 nm the UV spectral properties of wines have also been used for the determination of phenolic content and more specifically for total tannin content. The MCP tannin levels of a significant number of samples from a variety of different locations, cultivars and during different steps of the winemaking process were successfully predicted with the use of multiple linear regression (MLR) and partial least square regression (PLS). In this case spectral properties were collected between the 230 and 350 nm range of the UV part of the electromagnetic spectrum. Errors in cross validation (RMSECV) of 0.2 g/L (9.3% RMSECV%) were reported for MLR models using the above-mentioned UV region. Moreover, the authors also reported calibrations but in this case using only a limited number of key wavelengths. Further calibrations were investigated using the UV absorption values at 250, 270, 280, 290 and 315 nm. The external validation calibrations showed errors in prediction (RMSEP) of 0.18 g/L (9.2%) which confirmed the suitability of the UV region spectral properties to quantify tannin content in wine

In a more recent study the ability of UV-visible spectroscopy to predict tannin content in finished wines was reported. In this case two precipitation based methods, namely MCP and BSA tannin assays, were used to generate the spectral data. A large number of samples containing a varying number of cultivars from different regions as well as vintages were included in the model optimization procedure. UV-vis spectra was measured in the 260–610 nm region at 2 nm intervals. The best

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

#### *The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

a limited number of samples. The UV-visible spectra was collected from 200 to 650 nm at 6 nm intervals. Errors in prediction (root mean standard error of prediction (RMSEP)) of 0.35 g/L (14% RMSEP%) and 29 mg/L (8%) where reported for tannins and anthocyanins respectively. Despite the relative small sample set and the limitations of the analytical reference method investigated (mainly due to nonspecificity for phenolic compounds of the employed procedures) this publication reported for the first time the suitability of UV-visible spectroscopy to quantify phenolic content in wines through partial least squares (PLS) regression analysis.

In a further study the UV-visible spectral properties of a large dataset (400) were used to quantify phenolic content of samples collected at different stages of the winemaking process. The sample set included samples from a variety of different regions and cultivars. Spectral data was collected over the 230–900 nm at 0.17 nm intervals. The parameters derived from the BSA tannin assay including the anthocyanin and total phenolic related parameters were in this case evaluated. RMSEP of 87 mg/L (20% RMSEP%) for total anthocyanin content; 0.37 (26.4%), 0.46 (76.7%) and 0.48 (24%) A.U. for small, large and total polymeric pigments, respectively; 66 mg/L (30.1%) for tannin content; 99 (17.2%) mg/L for non-tannin phenols and 130 mg/L (16.4%) for total phenols were reported [48]. Later on, the same phenolic parameters were again investigated. A 100 samples of Cabernet Sauvignon and 100 samples of Shiraz were collected during the fermentation process over a single vintage to provide calibration that can be used for the prediction of phenolic content in must. In this case an adaptation of the BSA tannin assay was used for phenolic analysis. UV-visible spectral properties were collected in the 200–900 nm range. The results showed calibrations able to predict the phenolic content of Cabernet Sauvignon samples, but not for Shiraz, suggesting cultivar specificity of the predicted calibrations. Standard errors in cross validation (RMSECV) of 102.22 mg/L (23.8% RMSECV%) and 211.38 mg/L (25.6%) were reported for total tannin and iron reactive phenolic content, respectively for Cabernet Sauvignon samples. In terms of anthocyanin measurements, error in cross validation of 101 mg/L (43.3% RMSECV%), 0.46 A.U. (26.1%) and 0.48 A.U. (41.4%) for total anthocyanins, small and large polymeric pigments were observed [49].

Due to its characteristic absorption band at 280 nm the UV spectral properties of wines have also been used for the determination of phenolic content and more specifically for total tannin content. The MCP tannin levels of a significant number of samples from a variety of different locations, cultivars and during different steps of the winemaking process were successfully predicted with the use of multiple linear regression (MLR) and partial least square regression (PLS). In this case spectral properties were collected between the 230 and 350 nm range of the UV part of the electromagnetic spectrum. Errors in cross validation (RMSECV) of 0.2 g/L (9.3% RMSECV%) were reported for MLR models using the above-mentioned UV region. Moreover, the authors also reported calibrations but in this case using only a limited number of key wavelengths. Further calibrations were investigated using the UV absorption values at 250, 270, 280, 290 and 315 nm. The external validation calibrations showed errors in prediction (RMSEP) of 0.18 g/L (9.2%) which confirmed the suitability of the UV region spectral properties to quantify tannin content in wine samples [50].

In a more recent study the ability of UV-visible spectroscopy to predict tannin content in finished wines was reported. In this case two precipitation based methods, namely MCP and BSA tannin assays, were used to generate the spectral data. A large number of samples containing a varying number of cultivars from different regions as well as vintages were included in the model optimization procedure. UV-vis spectra was measured in the 260–610 nm region at 2 nm intervals. The best

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

An interesting and more recent technique to quantify phenolic compounds makes use of the ability of this group of substances to emit fluorescence light after the excitation/emission process. Fluorescence spectroscopy is able to measure the analyte concentration through its fluorescence properties, being thus suitable to measure compounds in solution, such as phenolics found in grape extracts or wines [14]. If phenolic compounds are excited at the appropriate light intensity and wavelength, generally through UV light exposure, the energy change occurring at electronic level will cause a light emission in the visible region of the electromagnetic spectrum [45]. Phenolic molecules are initially at ground levels at low energy state until light exposure elevate the vibrational levels to an elevated high energy state. After a period of time (in the order of milliseconds) the excited molecule while returning to its non-excited electronic state emits light (so-called fluorescence) at higher wavelengths than those absorbed during the excitation process. During the excitation/emission sequence both the absorbed and emitted light can be measured, with higher emission intensity corresponding to higher concentration of the analyte. Fluorescence spectroscopy has been commonly applied to the quantification of phenolic compounds in combination with liquid chromatography techniques. The main benefit of these applications rely on the increased sensitivity and selectivity of the method [45]. Additionally, fluorescence spectroscopy has been defined as a fast, non-destructive, easy to perform technique that can also be used for process monitoring purposes due to the versatility of the fluorescence spectrometers. Excitation emission spectral (EEM) properties might potentially be correlated with reference analytical data to establish regression calibrations for the quantification of phenolic compounds in a similar manner than what is reported for

**5. Fluorescence spectroscopy**

UV-visible or infrared spectroscopy calibrations.

**6. UV-visible spectroscopy with chemometrics**

The UV-visible spectra can alternatively be used in combination with powerful chemometric analysis to obtain spectroscopic calibrations for the prediction of phenolic content in grapes as well as in wines during the winemaking process [15]. In this case the totality or parts of the UV-visible spectra are correlated through multivariate regression approaches with reference phenolic data. After the spectral and phenolic content acquisition of a significant number of samples and in the case that strong correlations are found between the spectral data and the phenolic levels, a reliable prediction calibration can be obtained after the corresponding calibration and validation procedure. The advantage of these spectroscopy calibrations relies on the possibility of estimating the total content of phenolic substances through a simple spectral measurement, therefore avoiding the tedious reference method procedures. The main advantage of the spectroscopy calibrations is due to the rapidness, simplicity, reliability and cost-effectiveness ascribed to these techniques. Moreover, due to the multi-parametric nature of this approach a single spectral measurement is able to provide the levels of a number of phenolic compounds. Spectroscopic applications are also highly suitable to perform online measurement during the process of winemaking, allowing for improved process control strategies, through process monitoring, in line with a process analytical technologies

The first indication of the use of UV-visible spectroscopy calibrations to quantify some of the most important phenolic parameters was reported in 1995 [47]. In this first approach the total tannin and anthocyanin content was predicted using

**38**

(PAT) approach [46].

calibrations were found for both reference methods where the spectral properties in the UV region were used as spectral data (260–310 nm). A RMSEP of 0.16 g/L (9.9% RMSEP%) and 0.08 g/L (13.3%) were reported for MCP and BSA tannin content, respectively. In agreement with previous studies, accurate calibrations were also observed when a reduced number of wavelength were used as spectral data. Models optimised using the absorption values at 270, 280, 290, 300 and 314 nm lead to errors in prediction of 0.18 g/L (11.2% RMSEP%) and 0.11 g/L (18.3%) for MCP and BSA tannin content, respectively. Also in agreement with previous findings, cultivar and vintage specificity issues influenced to a certain extent the accuracy of the calibrations [42].

In a more recent study PLS calibrations based on UV-visible spectral data for the quantification of phenolic content in grapes, fermenting samples and wines have been reported. A large number of fermenting samples from 13 different vinifications over two consecutive vintages were included into the calibrations. Moreover, a number of finished wines from varying vintages and from a number of cultivars were also included. PLS validation calibrations showed prediction errors of 209 mg/L (14.3% RMSEP%), 14 mg/L (3.2%), 1.6 (3.2%) and 2.6 (14.7%) for total tannin content (MCP tannin assay), total anthocyanins, total phenolic content and colour density, respectively. In addition, individual phenolic compounds quantified using a HPLC method to generate the reference data were also reported, including flavanol monomers and the dimer B1, phenolic acids, flavonols as well as monomeric and acylated anthocyanins. Calibration for the estimation of polymeric phenol and pigment content were also reported. On the other hand, the same study reported PLS calibrations for determination of phenolic content in grapes extracts obtained through two extraction protocols. A phenolic extraction in high solvent content and after the entire berries being finely blended lead to successful calibrations for total tannin content, anthocyanins levels, total phenol index and colour density. The RMSEP reported was 0.22 mg/g (7% RMSEP%), 0.034 mg/g (3.1%), 0.17 (1.32%) and 0.72 (6.61%), for the above mentioned parameters, respectively. In addition, an alternative method with phenolic extraction performed under winelike ethanol levels from hand crushed grapes was also reported. Validation errors of 0.12 mg/g (10.7% RMSEP%), 0.03 mg/g (8.33%), 0.42 (1%) and 6.2 (20%) for total tannins, total anthocyanins, total phenolic index and colour density, respectively were reported [16].

## **7. Conclusions**

The role of UV-visible spectroscopy in wine science appears to be of high importance. A number of applications can be used to quantify the levels of phenolic compounds in grape extracts and wines. Apart from the conventional routine spectrophotometric methods for phenolic analysis, more advanced analytical techniques such as liquid chromatography can be also used to quantify individual phenolic substances using UV-visible spectroscopy. Moreover, fluorescence spectroscopy, making use of the ability of phenolic molecules to emit fluorescence light, appears to be a promising technique that can also be used to quantify phenolic content at different stages of the winemaking process and under different conditions. Finally, UV-visible spectroscopy calibrations are also a valid alternative as they allow for the efficient measurement of phenolics in grape extracts as well as wines during fermentation and ageing. These new developments in phenolic monitoring during the winemaking process opens exciting new possibilities for wine producers in their bid to obtain wines of a certain composition and style in a more controlled manner.

**41**

**Author details**

South Africa

provided the original work is properly cited.

Jose Luis Aleixandre-Tudo\* and Wessel du Toit

\*Address all correspondence to: joaltu@sun.ac.za

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Viticulture and Oenology, Stellenbosch University, Matieland,

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

## **Author details**

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

extent the accuracy of the calibrations [42].

calibrations were found for both reference methods where the spectral properties in the UV region were used as spectral data (260–310 nm). A RMSEP of 0.16 g/L (9.9% RMSEP%) and 0.08 g/L (13.3%) were reported for MCP and BSA tannin content, respectively. In agreement with previous studies, accurate calibrations were also observed when a reduced number of wavelength were used as spectral data. Models optimised using the absorption values at 270, 280, 290, 300 and 314 nm lead to errors in prediction of 0.18 g/L (11.2% RMSEP%) and 0.11 g/L (18.3%) for MCP and BSA tannin content, respectively. Also in agreement with previous findings, cultivar and vintage specificity issues influenced to a certain

In a more recent study PLS calibrations based on UV-visible spectral data for the quantification of phenolic content in grapes, fermenting samples and wines have been reported. A large number of fermenting samples from 13 different vinifications over two consecutive vintages were included into the calibrations. Moreover, a number of finished wines from varying vintages and from a number of cultivars were also included. PLS validation calibrations showed prediction errors of 209 mg/L (14.3% RMSEP%), 14 mg/L (3.2%), 1.6 (3.2%) and 2.6 (14.7%) for total tannin content (MCP tannin assay), total anthocyanins, total phenolic content and colour density, respectively. In addition, individual phenolic compounds quantified using a HPLC method to generate the reference data were also reported, including flavanol monomers and the dimer B1, phenolic acids, flavonols as well as monomeric and acylated anthocyanins. Calibration for the estimation of polymeric phenol and pigment content were also reported. On the other hand, the same study reported PLS calibrations for determination of phenolic content in grapes extracts obtained through two extraction protocols. A phenolic extraction in high solvent content and after the entire berries being finely blended lead to successful calibrations for total tannin content, anthocyanins levels, total phenol index and colour density. The RMSEP reported was 0.22 mg/g (7% RMSEP%), 0.034 mg/g (3.1%), 0.17 (1.32%) and 0.72 (6.61%), for the above mentioned parameters, respectively. In addition, an alternative method with phenolic extraction performed under winelike ethanol levels from hand crushed grapes was also reported. Validation errors of 0.12 mg/g (10.7% RMSEP%), 0.03 mg/g (8.33%), 0.42 (1%) and 6.2 (20%) for total tannins, total anthocyanins, total phenolic index and colour density, respectively

The role of UV-visible spectroscopy in wine science appears to be of high importance. A number of applications can be used to quantify the levels of phenolic compounds in grape extracts and wines. Apart from the conventional routine spectrophotometric methods for phenolic analysis, more advanced analytical techniques such as liquid chromatography can be also used to quantify individual phenolic substances using UV-visible spectroscopy. Moreover, fluorescence spectroscopy, making use of the ability of phenolic molecules to emit fluorescence light, appears to be a promising technique that can also be used to quantify phenolic content at different stages of the winemaking process and under different conditions. Finally, UV-visible spectroscopy calibrations are also a valid alternative as they allow for the efficient measurement of phenolics in grape extracts as well as wines during fermentation and ageing. These new developments in phenolic monitoring during the winemaking process opens exciting new possibilities for wine producers in their bid to obtain wines of a

certain composition and style in a more controlled manner.

**40**

were reported [16].

**7. Conclusions**

Jose Luis Aleixandre-Tudo\* and Wessel du Toit Department of Viticulture and Oenology, Stellenbosch University, Matieland, South Africa

\*Address all correspondence to: joaltu@sun.ac.za

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Aleixandre-Tudo JL, Buica A, Nieuwoudt H, Aleixandre JL, du Toit W. Spectrophotometric analysis of phenolic compounds in grapes and wines. Journal of Agricultural and Food Chemistry. 2017;**65**:4009-4026

[2] Aleixandre R, Aleixandre-Tudó JL, Bolaños-Pizarro JL, Aleixandre-Benavent M. Mapping the scientific research on wine and health (2001−2011). Journal of Agricultural and Food Chemistry. 2013;**61**:11871-11880

[3] Cheynier V, Schneider R, Salmon J, Fulcrand H. Chemistry of wine. In: Comprehensive Natural Products II. The Netherlands: Elsevier Ltd.; 2010. pp. 1119-1172

[4] Teixeira A, Eiras-Dias J, Castellarin SD, Gerós H. Berry phenolics of grapevine under challenging environments. International Journal of Molecular Sciences. 2013;**14**:18711-18739

[5] Casassa LF, Harbertson JF. Extraction, evolution, and sensory impact of phenolic compounds during red wine maceration. Annual Review of Food Science and Technology. 2014

[6] He F, Liang NN, Mu L, Pan QH, Wang J, Reeves MJ, Duan CQ. Anthocyanins and their variation in red wines. I. Monomeric anthocyanins and their color expression. Molecules. 2012;**17**:1571-1601

[7] He F, Liang NN, Mu L, Pan QH, Wang J, Reeves MJ, Duan CQ. Anthocyanins and their variation in red wines. II. Anthocyanin derived pigments and their color evolution. Molecules. 2012;**17**:1483-1519

[8] Gomez-Plaza M, Cano-Lopez E. A review on micro-oxygenation of red wines: Claims, benefits and the

underlying chemistry. Food Chemistry. 2011;**125**:1131-1140

[9] McRae JM, Kennedy JA. Wine and grape tannin interactions with salivary proteins and their impact on astringency: A review of current research. Molecules. 2011;**16**:2348-2364

[10] Smith PA, Mcrae JM, Bindon KA. Impact of winemaking practices on the concentration and composition of tannins in red wine. Australian Journal of Grape and Wine Research. 2015;**21**:601-614

[11] Boulet JC, Ducasse MA, Cheynier V. Ultraviolet spectroscopy study of phenolic substances and other major compounds in red wines: Relationship between astringency and the concentration of phenolic substances. Australian Journal of Grape and Wine Research. 2017;**23**:193-199

[12] Sanna R, Piras C, Marincola FC, Lecca V, Maurichi S, Scano P. Multivariate statistical analysis of the UV-vis profiles of wine polyphenolic extracts during vinification. The Journal of Agricultural Science. 2014;**6**:152-162

[13] Peng Z, Iland PG, Oberholster A, Sefton MA, Waters EJ. Analysis of pigmented polymers in red wine by reverse phase HPLC. Australian Journal of Grape and Wine Research. 2002;**8**:70-75

[14] Airado-Rodríguez D, Durán-Merás I, Galeano-Díaz T, Wold JP. Front-face fluorescence spectroscopy: A new tool for control in the wine industry. Journal of Food Composition and Analysis. 2011;**24**:257-264

[15] Cozzolino D. The role of visible and infrared spectroscopy combined with chemometrics to measure phenolic compounds in grape and wine samples. Molecules. 2015;**20**:726-737

**43**

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

[24] Mazza G, Fukumoto L, Delaquis P, Girard B, Ewert B. Anthocyanins, phenolics, and color of cabernet franc, merlot, and pinot noir wines from British Columbia. Journal of Agricultural and Food Chemistry. 1999;**47**:4009-4017

[25] Bindon KA, Kassara S, Cynkar WU, Robinson EMC, Scrimgeour N, Smith PA. Comparison of extraction protocols to determine differences in wineextractable tannin and anthocyanin in *Vitis vinifera* L. cv. Shiraz and cabernet sauvignon grapes. Journal of Agricultural and Food Chemistry.

[26] Ribéreau-Gayon E, Stonestreet P. Le dosage des anthocyannes dans Je vin rouge. Bulletin de la Société Chimique

[27] De Beer D, Harbertson JF, Kilmartin PA, Roginsky V, Barsukova T, Adams DO, Waterhouse AL. Phenolics: A comparison of diverse analytical methods. American Journal of Enology and Viticulture. 2004;**55**:389-400

[28] Mercurio MD, Dambergs RG, Herderich MJ, Smith PA. High throughput analysis of red wine and grape phenolics—Adaptation and validation of methyl cellulose precipitable tannin assay and modified Somers color assay to a rapid 96 well plate format. Journal of Agricultural and Food Chemistry. 2007;**55**:4651-4657

[29] Boulton RB. The copigmentation of anthocynins and its role in the color of red wine: Comments on a critical review. American Journal of Enology and Viticulture. 2001;**52**:67-87

[30] Sudraud P. Interpretation des courbes de'absorption des vin rouges. Annals of Agricultural Sciences.

[31] Glories Y. La couleur des vins rouges, 2eme partie, Connaiss. La Vigne

Du Vin. 1984;**18**:253-271

n.d.;**7**:203-208

de France. 1965;**9**:2649-2652

2014;**62**:4558-4570

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

[16] Aleixandre-Tudo JL, Nieuwoudt H, Olivieri A, Aleixandre JL, du Toit W. Phenolic profiling of grapes, fermenting samples and wines using UV-visible spectroscopy with chemometrics. Food Control.

R. Characterization and measurement

spectroscopy. Current Prvtocols in Food

[18] Hassane E, Gierschner J, Duroux J, Trouillas P. UV/visible spectra of natural polyphenols: A time-dependent density functional theory study. Food

[19] Bueno JM, Ramos-Escudero F, Sáez-Plaza P, Muñoz AM, Navas MJ, Asuero AG. Analysis and antioxidant capacity of anthocyanin pigments. Part II: Chemical structure, color, and intake of anthocyanins. Critical Reviews in Analytical Chemistry.

[20] Flanzy C, Poux M. Les possibilités de la microvinification, application à l'étude de la macération. Annales de Technologie Agricole. 1958:390-394

[21] Somers TC, Evans ME. Wine quality: Correlations with colour density and anthocyanin equilibria in a group of young red wines. Journal of the Science of Food and Agriculture.

[22] Singleton VL, Rossi JA Jr. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture. 1965;**16**:144-158

[23] Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology.

of anthocyanins by UV-visible

2018;**85**:11-22

[17] Giusti M, Wrolstad

Analytical. 2001:1-13

Chemistry. 2012;**131**:79-89

2012;**42**:102-125

1974;**25**:1369-1379

1998;**299**:152-178

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

[16] Aleixandre-Tudo JL, Nieuwoudt H, Olivieri A, Aleixandre JL, du Toit W. Phenolic profiling of grapes, fermenting samples and wines using UV-visible spectroscopy with chemometrics. Food Control. 2018;**85**:11-22

[17] Giusti M, Wrolstad R. Characterization and measurement of anthocyanins by UV-visible spectroscopy. Current Prvtocols in Food Analytical. 2001:1-13

[18] Hassane E, Gierschner J, Duroux J, Trouillas P. UV/visible spectra of natural polyphenols: A time-dependent density functional theory study. Food Chemistry. 2012;**131**:79-89

[19] Bueno JM, Ramos-Escudero F, Sáez-Plaza P, Muñoz AM, Navas MJ, Asuero AG. Analysis and antioxidant capacity of anthocyanin pigments. Part II: Chemical structure, color, and intake of anthocyanins. Critical Reviews in Analytical Chemistry. 2012;**42**:102-125

[20] Flanzy C, Poux M. Les possibilités de la microvinification, application à l'étude de la macération. Annales de Technologie Agricole. 1958:390-394

[21] Somers TC, Evans ME. Wine quality: Correlations with colour density and anthocyanin equilibria in a group of young red wines. Journal of the Science of Food and Agriculture. 1974;**25**:1369-1379

[22] Singleton VL, Rossi JA Jr. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture. 1965;**16**:144-158

[23] Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology. 1998;**299**:152-178

[24] Mazza G, Fukumoto L, Delaquis P, Girard B, Ewert B. Anthocyanins, phenolics, and color of cabernet franc, merlot, and pinot noir wines from British Columbia. Journal of Agricultural and Food Chemistry. 1999;**47**:4009-4017

[25] Bindon KA, Kassara S, Cynkar WU, Robinson EMC, Scrimgeour N, Smith PA. Comparison of extraction protocols to determine differences in wineextractable tannin and anthocyanin in *Vitis vinifera* L. cv. Shiraz and cabernet sauvignon grapes. Journal of Agricultural and Food Chemistry. 2014;**62**:4558-4570

[26] Ribéreau-Gayon E, Stonestreet P. Le dosage des anthocyannes dans Je vin rouge. Bulletin de la Société Chimique de France. 1965;**9**:2649-2652

[27] De Beer D, Harbertson JF, Kilmartin PA, Roginsky V, Barsukova T, Adams DO, Waterhouse AL. Phenolics: A comparison of diverse analytical methods. American Journal of Enology and Viticulture. 2004;**55**:389-400

[28] Mercurio MD, Dambergs RG, Herderich MJ, Smith PA. High throughput analysis of red wine and grape phenolics—Adaptation and validation of methyl cellulose precipitable tannin assay and modified Somers color assay to a rapid 96 well plate format. Journal of Agricultural and Food Chemistry. 2007;**55**:4651-4657

[29] Boulton RB. The copigmentation of anthocynins and its role in the color of red wine: Comments on a critical review. American Journal of Enology and Viticulture. 2001;**52**:67-87

[30] Sudraud P. Interpretation des courbes de'absorption des vin rouges. Annals of Agricultural Sciences. n.d.;**7**:203-208

[31] Glories Y. La couleur des vins rouges, 2eme partie, Connaiss. La Vigne Du Vin. 1984;**18**:253-271

**42**

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

underlying chemistry. Food Chemistry.

[9] McRae JM, Kennedy JA. Wine and grape tannin interactions with salivary proteins and their impact on astringency: A review of current research. Molecules. 2011;**16**:2348-2364

[10] Smith PA, Mcrae JM, Bindon KA. Impact of winemaking practices on the concentration and composition of tannins in red wine. Australian Journal of Grape and Wine Research.

[11] Boulet JC, Ducasse MA, Cheynier V. Ultraviolet spectroscopy study of phenolic substances and other major compounds in red wines: Relationship

concentration of phenolic substances. Australian Journal of Grape and Wine

P. Multivariate statistical analysis of the UV-vis profiles of wine polyphenolic extracts during vinification. The Journal of Agricultural Science. 2014;**6**:152-162

between astringency and the

Research. 2017;**23**:193-199

[12] Sanna R, Piras C, Marincola FC, Lecca V, Maurichi S, Scano

[13] Peng Z, Iland PG, Oberholster A, Sefton MA, Waters EJ. Analysis of pigmented polymers in red wine by reverse phase HPLC. Australian Journal of Grape and Wine Research.

[14] Airado-Rodríguez D, Durán-Merás I, Galeano-Díaz T, Wold JP. Front-face fluorescence spectroscopy: A new tool for control in the wine industry. Journal of Food Composition and Analysis.

[15] Cozzolino D. The role of visible and infrared spectroscopy combined with chemometrics to measure phenolic compounds in grape and wine samples.

Molecules. 2015;**20**:726-737

2002;**8**:70-75

2011;**24**:257-264

2011;**125**:1131-1140

2015;**21**:601-614

[1] Aleixandre-Tudo JL, Buica A, Nieuwoudt H, Aleixandre JL, du Toit W. Spectrophotometric analysis of phenolic compounds in grapes and wines. Journal of Agricultural and Food

**References**

Chemistry. 2017;**65**:4009-4026

[2] Aleixandre R, Aleixandre-Tudó JL, Bolaños-Pizarro JL, Aleixandre-Benavent M. Mapping the scientific research on wine and health (2001−2011). Journal of Agricultural and Food Chemistry.

[3] Cheynier V, Schneider R, Salmon J, Fulcrand H. Chemistry of wine. In: Comprehensive Natural Products II. The Netherlands: Elsevier Ltd.; 2010.

[4] Teixeira A, Eiras-Dias J, Castellarin

JF. Extraction, evolution, and sensory impact of phenolic compounds during red wine maceration. Annual Review of Food Science and Technology. 2014

CQ. Anthocyanins and their variation in red wines. I. Monomeric anthocyanins and their color expression. Molecules.

SD, Gerós H. Berry phenolics of grapevine under challenging environments. International Journal of Molecular Sciences.

2013;**14**:18711-18739

2012;**17**:1571-1601

[5] Casassa LF, Harbertson

[6] He F, Liang NN, Mu L, Pan QH, Wang J, Reeves MJ, Duan

[7] He F, Liang NN, Mu L, Pan QH, Wang J, Reeves MJ, Duan

CQ. Anthocyanins and their variation in red wines. II. Anthocyanin derived pigments and their color evolution. Molecules. 2012;**17**:1483-1519

[8] Gomez-Plaza M, Cano-Lopez E. A review on micro-oxygenation of red wines: Claims, benefits and the

2013;**61**:11871-11880

pp. 1119-1172

[32] Singleton VL, Kramling TE. Browning of white wines and an accelerated test for browning capacity. American Journal of Enology and Viticulture. 1976;**27**:4-7

[33] Commission Internationale de l'Eclairage. CIE Recommendations on Uniform Color Spaces, Color-Difference Equations, Psychometric Color Terms; CIE Public. Commission Internationale de l'Eclairage; 1978

[34] Martinez JA, Melgosa M, Perez MM, Hita E, Negueruela AII, Martínez JA, Pérez MM. Note. Visual and instrumental color evaluation in red wines. Food Science and Technology International. 2001;**7**:439-444

[35] Ribéreau-Gayón P, Stonestreet E. Dógase des tannins du vin rouges et détermination du leur structure. Chemia Analityczna. 1966

[36] Sarneckis CJ, Dambergs RG, Jones P, Mercurio M, Herderich MJ, Smith PA. Quantification of condensed tannins by precipitation with methyl cellulose: Development and validation of an optimised tool for grape and wine analysis. Australian Journal of Grape and Wine Research. 2006;**12**:39-49

[37] Cáceres-Mella A, Peña-Neira Á, Narváez-Bastias J, Jara-Campos C, López-Solís R, Canals JM. Comparison of analytical methods for measuring proanthocyanidins in wines and their relationship with perceived astringency. International Journal of Food Science and Technology. 2013;**48**:2588-2594

[38] Kennedy JA, Ferrier J, Harbertson JF, Peyrot Des C. Gachons, analysis of tannins in red wine using multiple methods: Correlation with perceived astringency. American Journal of Enology and Viticulture. 2006;**57**:481-485

[39] Mercurio MD, Smith PA. Tannin quantification in red grapes and wine: Comparison of polysaccharide- and protein-based tannin precipitation techniques and their ability to model wine astringency. Journal of Agricultural and Food Chemistry. 2008;**56**:5528-5537

[40] Harbertson JF, Yuan C, Mireles MS, Hanlin RL, Downey MO. Glucose, fructose and sucrose increase the solubility of protein-tannin complexes and at high concentration, glucose and sucrose interfere with bisulphite bleaching of wine pigments. Food Chemistry. 2013;**138**:556-563

[41] Harbertson JF, Mireles M, Yu Y. Improvement of BSA tannin precipitation assay by reformulation of resuspension buffer. American Journal of Enology and Viticulture. 2015;**66**:95-99

[42] Aleixandre-Tudo JL, Nieuwoudt H, Aleixandre JL, Du Toit WJ. Robust ultraviolet-visible (UV-vis) partial least-squares (PLS) models for tannin quantification in red wine. Journal of Agricultural and Food Chemistry. 2015;**63**:1088-1098

[43] Harbertson JF, Downey MO. Technical brief investigating differences in tannin levels determined by methylcellulose and protein precipitation. American Journal of Enology and Viticulture. 2009;**60**:246-249

[44] Lamuala-Raventós RM, Waterhouse A. A direct HPLC separation of wine phenolics. American Journal of Enology and Viticulture. 1994;**45**:1-5

[45] Cabrera-Bañegil M, Hurtado-Sánchez M d C, Galeano-Díaz T, Durán-Merás I. Front-face fluorescence spectroscopy combined with secondorder multivariate algorithms for the quantification of polyphenols in red wine samples. Food Chemistry. 2017;**220**:168-176

**45**

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking*

*DOI: http://dx.doi.org/10.5772/intechopen.79550*

[46] Aleixandre-Tudo JL, Nieuwoudt H, Aleixandre JL, du Toit W. Chemometric compositional analysis of phenolic compounds in fermenting samples and wines using different infrared spectroscopy techniques. Talanta.

[47] Garcia-Jares C, Medina B, García-Jares C, Medina B. Prediction of some physico-chemical parameters in red wines from ultraviolet-visible spectra using a partial least-squares model in latent variables. The Analyst.

[48] Skogerson K, Downey M, Mazza M, Boulton R. Rapid determination of phenolic components in red wines from UV-visible spectra and the method of partial least squares. American Journal of Enology and Viticulture.

2018;**176**:526-536

1995;**120**:1891-1896

2007;**58**:318-325

2012;**66**:656-664

[49] Beaver CW, Harbertson JF. Comparison of multivariate regression methods for the analysis of phenolics in wine made from two vitis vinifera cultivars. American Journal of Enology and Viticulture. 2016;**67**:56-64

[50] Dambergs RG, Mercurio MD, Kassara S, Cozzolino D, Smith PA. Rapid measurement of methyl cellulose precipitable tannins using ultraviolet spectroscopy with chemometrics: Application to red wine and inter-laboratory calibration transfer. Applied Spectroscopy.

*The Role of UV-Visible Spectroscopy for Phenolic Compounds Quantification in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.79550*

[46] Aleixandre-Tudo JL, Nieuwoudt H, Aleixandre JL, du Toit W. Chemometric compositional analysis of phenolic compounds in fermenting samples and wines using different infrared spectroscopy techniques. Talanta. 2018;**176**:526-536

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

Comparison of polysaccharide- and protein-based tannin precipitation techniques and their ability to model wine astringency. Journal of Agricultural and Food Chemistry.

[40] Harbertson JF, Yuan C, Mireles MS, Hanlin RL, Downey MO. Glucose, fructose and sucrose increase the solubility of protein-tannin complexes and at high concentration, glucose and sucrose interfere with bisulphite bleaching of wine pigments. Food Chemistry. 2013;**138**:556-563

[41] Harbertson JF, Mireles M, Yu Y. Improvement of BSA tannin precipitation assay by reformulation of resuspension buffer. American Journal of Enology and Viticulture.

[42] Aleixandre-Tudo JL, Nieuwoudt H, Aleixandre JL, Du Toit WJ. Robust ultraviolet-visible (UV-vis) partial least-squares (PLS) models for tannin quantification in red wine. Journal of Agricultural and Food Chemistry.

2008;**56**:5528-5537

2015;**66**:95-99

2015;**63**:1088-1098

2009;**60**:246-249

2017;**220**:168-176

[43] Harbertson JF, Downey MO. Technical brief investigating differences in tannin levels

and Viticulture. 1994;**45**:1-5

[45] Cabrera-Bañegil M, Hurtado-Sánchez M d C, Galeano-Díaz T, Durán-Merás I. Front-face fluorescence spectroscopy combined with secondorder multivariate algorithms for the quantification of polyphenols in red wine samples. Food Chemistry.

determined by methylcellulose and protein precipitation. American Journal of Enology and Viticulture.

[44] Lamuala-Raventós RM, Waterhouse A. A direct HPLC separation of wine phenolics. American Journal of Enology

[32] Singleton VL, Kramling

Viticulture. 1976;**27**:4-7

de l'Eclairage; 1978

TE. Browning of white wines and an accelerated test for browning capacity. American Journal of Enology and

[33] Commission Internationale de l'Eclairage. CIE Recommendations on Uniform Color Spaces, Color-Difference Equations, Psychometric Color Terms; CIE Public. Commission Internationale

[34] Martinez JA, Melgosa M, Perez MM, Hita E, Negueruela AII, Martínez JA, Pérez MM. Note. Visual and instrumental color evaluation in red wines. Food Science and Technology International. 2001;**7**:439-444

[35] Ribéreau-Gayón P, Stonestreet E. Dógase des tannins du vin rouges et détermination du leur structure.

[36] Sarneckis CJ, Dambergs RG, Jones P, Mercurio M, Herderich MJ, Smith PA. Quantification of condensed tannins by precipitation with methyl cellulose: Development and validation of an optimised tool for grape and wine analysis. Australian Journal of Grape and Wine Research. 2006;**12**:39-49

[37] Cáceres-Mella A, Peña-Neira Á, Narváez-Bastias J, Jara-Campos C, López-Solís R, Canals JM. Comparison of analytical methods for measuring proanthocyanidins in wines and their relationship with perceived astringency. International Journal of Food Science and Technology. 2013;**48**:2588-2594

[38] Kennedy JA, Ferrier J, Harbertson JF, Peyrot Des C. Gachons, analysis of tannins in red wine using multiple methods: Correlation with perceived astringency. American Journal of Enology

and Viticulture. 2006;**57**:481-485

[39] Mercurio MD, Smith PA. Tannin quantification in red grapes and wine:

Chemia Analityczna. 1966

**44**

[47] Garcia-Jares C, Medina B, García-Jares C, Medina B. Prediction of some physico-chemical parameters in red wines from ultraviolet-visible spectra using a partial least-squares model in latent variables. The Analyst. 1995;**120**:1891-1896

[48] Skogerson K, Downey M, Mazza M, Boulton R. Rapid determination of phenolic components in red wines from UV-visible spectra and the method of partial least squares. American Journal of Enology and Viticulture. 2007;**58**:318-325

[49] Beaver CW, Harbertson JF. Comparison of multivariate regression methods for the analysis of phenolics in wine made from two vitis vinifera cultivars. American Journal of Enology and Viticulture. 2016;**67**:56-64

[50] Dambergs RG, Mercurio MD, Kassara S, Cozzolino D, Smith PA. Rapid measurement of methyl cellulose precipitable tannins using ultraviolet spectroscopy with chemometrics: Application to red wine and inter-laboratory calibration transfer. Applied Spectroscopy. 2012;**66**:656-664

**47**

**Chapter 4**

**Abstract**

**1. Introduction**

*Nicoleta-Maricica Maftei*

Probiotic, Prebiotic and Synbiotic

The health benefits imparted by probiotics and prebiotics as well as synbiotics have been the subject of extensive research in the past few decades. What is the real role of probiotics strains, prebiotics and synbiotics in influencing a health? To battle the increase in health care costs, in recent years has been developed a preventive approach to medicine with the development of new probiotics and prebiotics or symbiotic products. Many studies suggest that probiotics, prebiotics and synbiotics supplementation may be beneficial in prevention and management of nutritional and health. While these studies show promising beneficial effects, the long-term risks or health benefits of prebiotics, probiotics and synbiotics supplementation are not clear. In this chapter review the literature regarding available information and summarises the current knowledge on the effects of probiotics, prebiotics, and synbiotics on human health and explore recent trends and developments in this field.

Probiotic, prebiotic and synbiotic are words of the modern era, bookmark "for life" and is in use to define bacterial association with beneficial effects on human health. In the world of highly processed food, both at the industrial and nutritional level clear consideration are paid to the composition and safety of the intake products. The nutrition quality is essential for human health because of the food poisoning, obesity, allergy, cardiovascular diseases, and cancer, that is consider the plague of the twenty-first century. Worldwide, many research reports underline the health advantages of using probiotics, prebiotics and also, synbiotics in human consumption [1]. In early 1990s, Metchnikoff [2] defined probiotics in a scientific context as the microorganisms that alter of floral/microbial diversity in human bodies and replaces the harmful microbes with useful ones. However, Tissier detected that the microbial population of a particular type of bacteria in stool samples of infected diarrheic children was significantly lower comparing to healthy children [3]. He suggested that patients with diarrhoea (infantile diarrhoea) should oral administration of live organisms (bifidobacteria) and in this way a healthy gut flora was restored. Havenaar and Huis in't Veld [4] have given the modern definition of probiotic: as a viable mono or mixed culture of bacteria which, when applied to animal or man, affects the host beneficially by improving the properties of the indigenous flora. In 2002, Food and Agriculture Organisation of the United Nations (FAO) and World Health Organisation (WHO) defined probiotics as being "live strains of strictly selected microorganisms which, when administered in adequate

Products in Human Health

**Keywords:** probiotics, prebiotics, synbiotics, health, functional food

## **Chapter 4**

## Probiotic, Prebiotic and Synbiotic Products in Human Health

*Nicoleta-Maricica Maftei*

## **Abstract**

The health benefits imparted by probiotics and prebiotics as well as synbiotics have been the subject of extensive research in the past few decades. What is the real role of probiotics strains, prebiotics and synbiotics in influencing a health? To battle the increase in health care costs, in recent years has been developed a preventive approach to medicine with the development of new probiotics and prebiotics or symbiotic products. Many studies suggest that probiotics, prebiotics and synbiotics supplementation may be beneficial in prevention and management of nutritional and health. While these studies show promising beneficial effects, the long-term risks or health benefits of prebiotics, probiotics and synbiotics supplementation are not clear. In this chapter review the literature regarding available information and summarises the current knowledge on the effects of probiotics, prebiotics, and synbiotics on human health and explore recent trends and developments in this field.

**Keywords:** probiotics, prebiotics, synbiotics, health, functional food

## **1. Introduction**

Probiotic, prebiotic and synbiotic are words of the modern era, bookmark "for life" and is in use to define bacterial association with beneficial effects on human health. In the world of highly processed food, both at the industrial and nutritional level clear consideration are paid to the composition and safety of the intake products. The nutrition quality is essential for human health because of the food poisoning, obesity, allergy, cardiovascular diseases, and cancer, that is consider the plague of the twenty-first century. Worldwide, many research reports underline the health advantages of using probiotics, prebiotics and also, synbiotics in human consumption [1]. In early 1990s, Metchnikoff [2] defined probiotics in a scientific context as the microorganisms that alter of floral/microbial diversity in human bodies and replaces the harmful microbes with useful ones. However, Tissier detected that the microbial population of a particular type of bacteria in stool samples of infected diarrheic children was significantly lower comparing to healthy children [3]. He suggested that patients with diarrhoea (infantile diarrhoea) should oral administration of live organisms (bifidobacteria) and in this way a healthy gut flora was restored. Havenaar and Huis in't Veld [4] have given the modern definition of probiotic: as a viable mono or mixed culture of bacteria which, when applied to animal or man, affects the host beneficially by improving the properties of the indigenous flora. In 2002, Food and Agriculture Organisation of the United Nations (FAO) and World Health Organisation (WHO) defined probiotics as being "live strains of strictly selected microorganisms which, when administered in adequate

amounts, confer a health benefit on the host" [5]. The definition was preserved also, by the International Scientific Association for Probiotics and Prebiotics (ISAPP) in 2013 [6]. The vast majority of results of the clinical research underline the positive effect of the probiotics on the gastrointestinal diseases, such as: irritable bowel syndrome, gastrointestinal disorders, elimination of *Helicobacter*, inflammatory bowel disease, diarrhoeas, and allergic diseases, like as atopic dermatitis. Also, numerous clinical reports have demonstrated the efficiency of the probiotics for the treatment of diseases such as obesity, insulin resistance syndrome, type 2 diabetes, and nonalcoholic fatty liver disease. Increasing the body's immunity (immunomodulation) was the positive effect of probiotics on human health. Majority of scientific reports also show the benefits of the prophylactic use of probiotics in different types of cancer and side effects associated with cancer [1].

In 1995, Gibson and Roberfroid defined prebiotics were by as non-digested food components that, through the stimulation of growth and/or activity of a single type or a limited amount of microorganisms which residing in the gastrointestinal tract, improve the health condition of a host [7]. Instead, in 2004, prebiotics were described as selectively fermented compounds permitting precise changes in the composition and/or activity of the gastrointestinal tract microorganisms, these changes being useful for the host's health and wellbeing [8]. Recently, in 2007, FAO/WHO experts, designated prebiotics as a nonviable food constituent that confers a health advantage on the host linked to the microbiota modulation [9]. However, in the literature it is specified that prebiotics can be used as a probiotics substitute or as a supplementary support for them. Instead, numerous prebiotics can improve the growth of indigenous gut bacteria and have tremendous potential for changing the gut microbiota, but these variations occur just at the level of individual strains. Worldwide, numerous scientific studies underline the positively effects of the prebiotics for human health.

For the simultaneous use of probiotics and prebiotics high potential is attributed. In 1995, Gibson and Roberfroid introduced the term "synbiotic" to describe union between probiotics and prebiotics synergistically acting of health [7]. Synbiotic is a designated compound that introduced in the gastrointestinal tract can careful stimulates the growth and/or activates the metabolism of physiological intestinal microbiota, thus conferring beneficial result to the host's health [10]. As the word "synbiotic" is a synergy, the term can be attributed only to the products where a prebiotic compound selectively improves a probiotic microorganism [11]. The main aim of this type of combination is the improvement of probiotic microorganism's survival in the gastrointestinal tract. Therefore, synbiotic have both probiotic and prebiotic assets and were designed in order to solve the probiotics survival in the gastrointestinal tract [12]. An adequate combination of both components (prebiotic and probiotic) in a single product should guarantee a superior effect, compared to the action of the probiotic or prebiotic alone [13, 14].

Besides basic role of the nutrition consisting in the supply of necessary nutrients for growth and development of the organism, some additional aspects are becoming increasingly important, including the maintenance of health and counteracting diseases. The introduction of probiotics, prebiotics, or synbiotics into human diet is favourable for the intestinal microbiota and the human health. They may be consumed in the form of dairy products, raw vegetables and fruit or fermented pickles. Another source of probiotics, prebiotics, or synbiotics may be pharmaceutical formulas and functional food. Although probiotics, prebiotics and synbiotics have considerable potential in nutritional and clinical applications, considerable researches are required for the implementation of probiotics into human health, nutrition and regulation of different abnormalities. The screening of probiotics, prebiotics and synbiotics and their amounts is essential in gaining a therapeutic effect in health. However, further research focused on discovering new probiotic strains,

**49**

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

ating lactic acid bacteria to their alimentary tracts" [16].

the assortment of probiotics and prebiotics for synbiotics, dose setting, safety of use, and clinical trials is necessary. Also, the health benefits should be established in properly scheduled clinical trials conducted by independent research centres.

This chapter is an attempt to emphasise the possible benefaction of probiotics, prebiotics and synbiotics for improving human health and regulation of common

Gut bacterial colonisation starts since at birth when new-borns are exposed to a nonsterile climate. Henceforth, it changes and transforms over a lifetime, depends on a complex and dynamic interaction between the diet, genome, and lifestyle of the host, as well as antibiotic consumption. Remarkable bacterial colonisation of age-specific changes described in gut microbiota configuration include a decrease in the Bacteroidetes/Firmicutes ratio and a reduction in bifidobacteria in people aged over 60 years, when the immune system starts to decline [15]. Normally, the composition of the intestinal microflora is considered to be constantly throughout

Since the beginning of the twentieth century the interest in lactic acid fermentation was expressed by the Russian scientist and immunologist, Ilia Miecznikow, that worked at Pasteur Institute, Paris. In the book "Studies on Optimism" he affirmed that "with various foods undergoing lactic acid fermentation and consumed raw (sour milk, kefir, sauerkraut, pickles) humans introduced huge amounts of prolifer-

The microorganisms that are used as probiotics can belong to different types, such as bacteria, yeast and mould. Selected probiotic bacteria strains can be as

a.*Lactobacillus: acidophilus*, *sporogenes, plantarum, rhamnosus, delbrueckii, reuteri,* 

b.*Bifidobacterium*: *bifidum, infantis, adolescentis, longum, thermophilum, breve, lactis;*

The literature mentions as probiotics the following yeast and mould strains:

a.Yeast: *Saccharomyces cerevisiae, Saccharomyces bourlardii*, *Candida pintolopesii,* 

*fermentum, brevis, casei, farciminis, paracasei, gasseri, crispatus*;

c.*Streptococcus*: *lactis, cremoris, thermophilis, diacetylactis*;

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

metabolic disorders or abnormalities.

**2. Probiotics**

adulthood period.

**2.1 Probiotic strains**

d.*Leuconostoc mesenteroides*;

f. *Propionibacterium* spp*.*;

*and Sacaromyces boulardii*

g.*Enterococcus*—*Enterococcus faecium*;

b.Moulds: *Aspergillus niger, A. oryzae* [17].

e.*Pediococcus* spp.;

following:

#### *Probiotic, Prebiotic and Synbiotic Products in Human Health DOI: http://dx.doi.org/10.5772/intechopen.81553*

the assortment of probiotics and prebiotics for synbiotics, dose setting, safety of use, and clinical trials is necessary. Also, the health benefits should be established in properly scheduled clinical trials conducted by independent research centres.

This chapter is an attempt to emphasise the possible benefaction of probiotics, prebiotics and synbiotics for improving human health and regulation of common metabolic disorders or abnormalities.

## **2. Probiotics**

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

cancer and side effects associated with cancer [1].

amounts, confer a health benefit on the host" [5]. The definition was preserved also, by the International Scientific Association for Probiotics and Prebiotics (ISAPP) in 2013 [6]. The vast majority of results of the clinical research underline the positive effect of the probiotics on the gastrointestinal diseases, such as: irritable bowel syndrome, gastrointestinal disorders, elimination of *Helicobacter*, inflammatory bowel disease, diarrhoeas, and allergic diseases, like as atopic dermatitis. Also, numerous clinical reports have demonstrated the efficiency of the probiotics for the treatment of diseases such as obesity, insulin resistance syndrome, type 2 diabetes, and nonalcoholic fatty liver disease. Increasing the body's immunity (immunomodulation) was the positive effect of probiotics on human health. Majority of scientific reports also show the benefits of the prophylactic use of probiotics in different types of

In 1995, Gibson and Roberfroid defined prebiotics were by as non-digested food components that, through the stimulation of growth and/or activity of a single type or a limited amount of microorganisms which residing in the gastrointestinal tract, improve the health condition of a host [7]. Instead, in 2004, prebiotics were described as selectively fermented compounds permitting precise changes in the composition and/or activity of the gastrointestinal tract microorganisms, these changes being useful for the host's health and wellbeing [8]. Recently, in 2007, FAO/WHO experts, designated prebiotics as a nonviable food constituent that confers a health advantage on the host linked to the microbiota modulation [9]. However, in the literature it is specified that prebiotics can be used as a probiotics substitute or as a supplementary support for them. Instead, numerous prebiotics can improve the growth of indigenous gut bacteria and have tremendous potential for changing the gut microbiota, but these variations occur just at the level of individual strains. Worldwide, numerous scientific studies underline the positively effects of the prebiotics for human health. For the simultaneous use of probiotics and prebiotics high potential is attributed. In 1995, Gibson and Roberfroid introduced the term "synbiotic" to describe union between probiotics and prebiotics synergistically acting of health [7]. Synbiotic is a designated compound that introduced in the gastrointestinal tract can careful stimulates the growth and/or activates the metabolism of physiological intestinal microbiota, thus conferring beneficial result to the host's health [10]. As the word "synbiotic" is a synergy, the term can be attributed only to the products where a prebiotic compound selectively improves a probiotic microorganism [11]. The main aim of this type of combination is the improvement of probiotic microorganism's survival in the gastrointestinal tract. Therefore, synbiotic have both probiotic and prebiotic assets and were designed in order to solve the probiotics survival in the gastrointestinal tract [12]. An adequate combination of both components (prebiotic and probiotic) in a single product should guarantee a superior

effect, compared to the action of the probiotic or prebiotic alone [13, 14].

Besides basic role of the nutrition consisting in the supply of necessary nutrients for growth and development of the organism, some additional aspects are becoming increasingly important, including the maintenance of health and counteracting diseases. The introduction of probiotics, prebiotics, or synbiotics into human diet is favourable for the intestinal microbiota and the human health. They may be consumed in the form of dairy products, raw vegetables and fruit or fermented pickles. Another source of probiotics, prebiotics, or synbiotics may be pharmaceutical formulas and functional food. Although probiotics, prebiotics and synbiotics have considerable potential in nutritional and clinical applications, considerable researches are required for the implementation of probiotics into human health, nutrition and regulation of different abnormalities. The screening of probiotics, prebiotics and synbiotics and their amounts is essential in gaining a therapeutic effect in health. However, further research focused on discovering new probiotic strains,

**48**

Gut bacterial colonisation starts since at birth when new-borns are exposed to a nonsterile climate. Henceforth, it changes and transforms over a lifetime, depends on a complex and dynamic interaction between the diet, genome, and lifestyle of the host, as well as antibiotic consumption. Remarkable bacterial colonisation of age-specific changes described in gut microbiota configuration include a decrease in the Bacteroidetes/Firmicutes ratio and a reduction in bifidobacteria in people aged over 60 years, when the immune system starts to decline [15]. Normally, the composition of the intestinal microflora is considered to be constantly throughout adulthood period.

Since the beginning of the twentieth century the interest in lactic acid fermentation was expressed by the Russian scientist and immunologist, Ilia Miecznikow, that worked at Pasteur Institute, Paris. In the book "Studies on Optimism" he affirmed that "with various foods undergoing lactic acid fermentation and consumed raw (sour milk, kefir, sauerkraut, pickles) humans introduced huge amounts of proliferating lactic acid bacteria to their alimentary tracts" [16].

## **2.1 Probiotic strains**

The microorganisms that are used as probiotics can belong to different types, such as bacteria, yeast and mould. Selected probiotic bacteria strains can be as following:


The literature mentions as probiotics the following yeast and mould strains:


The type of the microbes used as probiotics increased due to the increase in the research concerning the health but as well as by the increase of the newly discovered and identified microbes, which could be used as probiotics in different food and beverages with huge impact on human body.

With the development of better culturing methodologies, more affordable genome and metagenome sequencing, the probiotic research is in a fulminant era, one which permits designing adapted probiotics that address specific consumer needs and issues. Also, the data of the conformation and role of the human gut microbiome accelerated by massively parallel sequencing, has extended the range of microorganisms with possible human benefits, although many of these are still at the very early stage of research.

These organisms are sometimes referred to as next-generation probiotics (NGPs), but may also be termed live biotherapeutic products (LBPs). NPGs obviously follow to the standard classification of a probiotic, but mainly referring to those microorganisms that have not been used as agents to promote health till now, and which are more likely to be delivered under a drug regulatory framework. Next-generation probiotics fit well within the US Food and Drug Administration (FDA) definition of a live biotherapeutic products: "a biological product" that: comprises live microoorganisms, such as bacteria; it is not a vaccine; is applicable to the prevention, treatment, or cure of a disease or condition of human beings [18].

Examples of current NGP: *Faecalibacterium* spp*., Akkermansia* spp*., Bacteroides fragilis* strain ZY-312, *Bacteroides xylanisolvens* DSM 23964, *Clostridium butyricum* MIYAIRI 588, *Faecalibacterium prausnitzii* and other*.*

Probiotics are subject to regulations in the general food law worldwide, conforming to they should be safe for human and animal health. In the Unite State of America, microorganisms that are used for human consumption should have the Generally Regarded As Safe (GRAS) status, regulated by the Food and Drug Administration (FDA). Rather, in Europe, European Food Safety Authority (EFSA) introduced the term of Qualified Presumption of Safety (QPS). The term of QPS it is a concept which involves some additional criteria of the safety assessment of bacterial supplements, including the history of safe usage and absence of the risk of acquired resistance to antibiotics [19, 20]. Until this moment mechanism of action of probiotics has not been clearly understood, but research results are those obtained from animal models and in vitro experiments. From a medical point of view it is considered that action mode of probiotics may improve the barrier functions of the gut mucosa because several strains of *Lactobacillus* spp. and *Bifidobacterium* spp. as well as structural compounds, and microbial-produced metabolites are able to stimulate epithelial cell signalling pathways. Thomas and Versalovic [21] reported that the Nuclear FactorKappa-Light-Chain-Enhancer of activated B cells (NF-kB) pathway is controlled by probiotics at many different levels with effects seen on I Kappa B protein (IKB) degradation and ubiquitination, proteosome function [22] and nuclear-cytoplasmic movement of RelA through a PPAR-gamma dependent pathway. Also, it is known that probiotics can modulate the immune system functions for instance, *L. acidophilus* has been found to modulate toll-like receptors and the proteoglycan recognition proteins of enterocytes. This thing leads to activation of dendric cells and lymphocytes T-helper 1 responds. After stimulation of lymphocytes T-helper 1 cytokines can suppress lymphocyte T-helper 2 responses which provoke the atopic issues [23]. Another possible mechanism of action of probiotics may be their ability to suppress the growth of pathogenic bacteria by producing broad-spectrum bacteriocins [24]. After the latest research on probiotics we can conclude that molecular and genetic research allowed the determination of the beneficial effect of probiotics, involving four mechanisms:

**51**

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

1.Antagonism through the production of antimicrobial compounds [25];

2.Pathogens competition for adhesion to the epithelium and for nutrients [26];

The first two mechanisms are directly related with their effect on other microorganisms. Nevertheless, all four mechanisms, from medical point of view, play an important role in the infections prophylaxis and treatment and also, for maintenance a balanced host's intestinal microbiota [1]. The capability of probiotic strains to co-aggregate, as one of their mechanisms of action, can contribute to the development of a protective barrier preventing pathogenic bacteria from the colonisation of the gut epithelium [29]. Probiotics bacteria are able to adhere to epithelial cells, inhibiting the pathogens. This mechanism plays an important effect on the host's health condition. Also, the adhesion of probiotic microorganisms to epithelial cells can start a signalling cascade, leading to immunological modulation. Otherwise, the discharge of some soluble compounds may cause a direct or indirect (through

Probiotics may have an significant role in: chronic inflammation of the alimentary tract or of a part thereof, the prevention and treatment of contagious diseases, lactose intolerance and lactose digestion, cholesterol reduction, cardiovascular health, urogenital disease, allergic disease, oral health, gastrointestinal disease,

*2.2.1 Probiotics in prevention and treatment of acute diarrhoea and diarrhoea* 

*Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium longum, Bifidobacterium breve* there were some of the most widely studied probiotics for treatment of acute diarrhoea. In a recent meta-analysis of 21 studies (4780 patients), the administration of *S. boulardii* decreased the risk of antibiotic-induced diarrhoea in both children and adults from 19 to 8.5%, with a relative risk of 0.47. In another meta-analysis of 82 randomised clinical trials using diverse species (usually

Diarrhoea induced by antibiotics is a very common complication in the hospital setting, representing a percentage by 13–60% and disease caused by Clostridium difficile is also a significant cause of nosocomial diarrhoea and colitis that prolongs the hospital stay by 3–7 days and increases the risk of new nosocomial infections with 20–65%, costs, and mortality (2- or 3-fold depending on reports) [31]. The

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

3.Immunomodulation of the host [27];

4.Inhibition of bacterial toxin production [28].

epithelial cells) activation of immunological cells [30].

roles of the probiotics used to treat these patients are:

1. restoration intestinal microflora;

3. compete with pathogenic bacteria;

2.increase immune response;

4. remove their toxins.

**2.2 Probiotics in human health**

*associated with antibiotics*

obesity but and an possible role in the elimination of cancer cells.

*Probiotic, Prebiotic and Synbiotic Products in Human Health DOI: http://dx.doi.org/10.5772/intechopen.81553*

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

beverages with huge impact on human body.

MIYAIRI 588, *Faecalibacterium prausnitzii* and other*.*

the very early stage of research.

The type of the microbes used as probiotics increased due to the increase in the research concerning the health but as well as by the increase of the newly discovered and identified microbes, which could be used as probiotics in different food and

With the development of better culturing methodologies, more affordable genome and metagenome sequencing, the probiotic research is in a fulminant era, one which permits designing adapted probiotics that address specific consumer needs and issues. Also, the data of the conformation and role of the human gut microbiome accelerated by massively parallel sequencing, has extended the range of microorganisms with possible human benefits, although many of these are still at

These organisms are sometimes referred to as next-generation probiotics (NGPs), but may also be termed live biotherapeutic products (LBPs). NPGs obviously follow to the standard classification of a probiotic, but mainly referring to those microorganisms that have not been used as agents to promote health till now, and which are more likely to be delivered under a drug regulatory framework. Next-generation probiotics fit well within the US Food and Drug Administration (FDA) definition of a live biotherapeutic products: "a biological product" that: comprises live microoorganisms, such as bacteria; it is not a vaccine; is applicable to the prevention, treatment, or cure of a disease or condition of human beings [18]. Examples of current NGP: *Faecalibacterium* spp*., Akkermansia* spp*., Bacteroides fragilis* strain ZY-312, *Bacteroides xylanisolvens* DSM 23964, *Clostridium butyricum*

Probiotics are subject to regulations in the general food law worldwide, conforming to they should be safe for human and animal health. In the Unite State of America, microorganisms that are used for human consumption should have the Generally Regarded As Safe (GRAS) status, regulated by the Food and Drug Administration (FDA). Rather, in Europe, European Food Safety Authority (EFSA) introduced the term of Qualified Presumption of Safety (QPS). The term of QPS it is a concept which involves some additional criteria of the safety assessment of bacterial supplements, including the history of safe usage and absence of the risk of acquired resistance to antibiotics [19, 20]. Until this moment mechanism of action of probiotics has not been clearly understood, but research results are those obtained from animal models and in vitro experiments. From a medical point of view it is considered that action mode of probiotics may improve the barrier functions of the gut mucosa because several strains of *Lactobacillus* spp. and *Bifidobacterium* spp. as well as structural compounds, and microbial-produced metabolites are able to stimulate epithelial cell signalling pathways. Thomas and Versalovic [21] reported that the Nuclear FactorKappa-Light-Chain-Enhancer of activated B cells (NF-kB) pathway is controlled by probiotics at many different levels with effects seen on I Kappa B protein (IKB) degradation and ubiquitination, proteosome function [22] and nuclear-cytoplasmic movement of RelA through a PPAR-gamma dependent pathway. Also, it is known that probiotics can modulate the immune system functions for instance, *L. acidophilus* has been found to modulate toll-like receptors and the proteoglycan recognition proteins of enterocytes. This thing leads to activation of dendric cells and lymphocytes T-helper 1 responds. After stimulation of lymphocytes T-helper 1 cytokines can suppress lymphocyte T-helper 2 responses which provoke the atopic issues [23]. Another possible mechanism of action of probiotics may be their ability to suppress the growth of pathogenic bacteria by producing broad-spectrum bacteriocins [24]. After the latest research on probiotics we can conclude that molecular and genetic research allowed the determination of the beneficial effect of probiotics, involving four

**50**

mechanisms:


The first two mechanisms are directly related with their effect on other microorganisms. Nevertheless, all four mechanisms, from medical point of view, play an important role in the infections prophylaxis and treatment and also, for maintenance a balanced host's intestinal microbiota [1]. The capability of probiotic strains to co-aggregate, as one of their mechanisms of action, can contribute to the development of a protective barrier preventing pathogenic bacteria from the colonisation of the gut epithelium [29]. Probiotics bacteria are able to adhere to epithelial cells, inhibiting the pathogens. This mechanism plays an important effect on the host's health condition. Also, the adhesion of probiotic microorganisms to epithelial cells can start a signalling cascade, leading to immunological modulation. Otherwise, the discharge of some soluble compounds may cause a direct or indirect (through epithelial cells) activation of immunological cells [30].

Probiotics may have an significant role in: chronic inflammation of the alimentary tract or of a part thereof, the prevention and treatment of contagious diseases, lactose intolerance and lactose digestion, cholesterol reduction, cardiovascular health, urogenital disease, allergic disease, oral health, gastrointestinal disease, obesity but and an possible role in the elimination of cancer cells.

#### **2.2 Probiotics in human health**

## *2.2.1 Probiotics in prevention and treatment of acute diarrhoea and diarrhoea associated with antibiotics*

Diarrhoea induced by antibiotics is a very common complication in the hospital setting, representing a percentage by 13–60% and disease caused by Clostridium difficile is also a significant cause of nosocomial diarrhoea and colitis that prolongs the hospital stay by 3–7 days and increases the risk of new nosocomial infections with 20–65%, costs, and mortality (2- or 3-fold depending on reports) [31]. The roles of the probiotics used to treat these patients are:


*Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium longum, Bifidobacterium breve* there were some of the most widely studied probiotics for treatment of acute diarrhoea. In a recent meta-analysis of 21 studies (4780 patients), the administration of *S. boulardii* decreased the risk of antibiotic-induced diarrhoea in both children and adults from 19 to 8.5%, with a relative risk of 0.47. In another meta-analysis of 82 randomised clinical trials using diverse species (usually

*Lactobacillus* spp*.*, alone or combined with bifidobacteria, enterococci, or *S. boulardii*), a reduced risk of antibiotic-induced diarrhoea was also established, with a relative risk of 0.58 [31].

Floch et al. [32] reported that for the primary prevention of disease caused by *C. difficile* in patients treated with antibiotics, probiotics also decrease the incidence of such disease, especially when strains of *S. boulardii*, and possibly other *Lactobacillus*, such as GG, are administered. Instead, a recent meta-analysis settled that only four probiotic strains (not including *Lactobacillus* GG) have been shown to significantly decrease the incidence of diarrhoea induced by *C. difficile*, such as follows: *S. boulardii* (2 × 1010 CFU/day), *L. casei* DN114001 (probiotic drink twice daily), a mixture of *L. acidophilus* and *Bifidobacterium bifidum* (2 × 1010 CFU/day), and a mixture of *L. acidophilus*, *L. casei*, and *L. rhamnosus*) [33].

For patients that intake antibiotics to eradicate *Helicobacter pylori*, studies have been conducted based on probiotics adding in order to increase eradication rates and also to prevent side effects such as antibiotic-induced diarrhoea. Numerous meta-analyses showed that the addition of probiotics may increase the efficacy of eradication with an odds ratio (OR) ranging from 1.2 to 2 times compare to the control group. Although additional studies are needed, it appears that the most effective strains are *L. acidophilus* (1.25 × 109 CFU) (OR: 1.24), milk fermented with *L. casei* DN-114001 (2 packs daily) (OR: 1.47), yogurt with *Lactobacillus gasseri* (OR: 1.19) (2 packs daily), and *Bifidobacterium infantis* (2 × 109 CFU) (OR: 1.21) [31]. Also, in supplementary clinical researches where antibiotics are used, probiotics appear to decrease the incidence of diarrhoea (with an OR ranging from 0.16 to 0.47) [34].

Also, the efficiency of probiotic strains in the next therapy's: nosocomial, nonnosocomial, and viral diarrheas have been studied. The conclusion was as follows: it turns out that probiotics may increase the amount of IgA antibodies, which leads to the decrease number of a viral infection [35].

#### *2.2.2 Probiotics in in diseases of the gastrointestinal apparatus*

Inflammatory bowel disease (IBD) is a recurrent chronic condition in which an abnormal interaction exists between intestinal flora and the host. Patients with IBD have an increased risk of colorectal cancer [31]. Due to the growing area of disease spreading and ageing societies, the use of probiotic bacteria for human health is becoming increasingly important. The consumption of pre-processed food (fast food), often containing excessive amounts of fat and insufficient amounts of raw fruits and vegetables, is another factor of harmful modification of human intestinal microbiota. It seems that the system of intestinal microorganisms and its desirable modification with probiotic formulas and products may protect people against enteral problems, and improve health [1]. *L. plantarum* is a probiotic that has been used with good results in the management of some symptoms in patients with IBD. It has been reported that the DSM 9843 strain significantly reduced flatulence, and the LPO 1 and 299 V strains significantly reduced abdominal pain [36]. In many studies it has been reported that probiotics may be helpful in the treatment of inflammatory enteral conditions, Crohn's disease, ulcerative colitis, and non-specific ileitis. The aetiology of those diseases is not completely understood, but it is evident that they are associated with recurrent infections or chronic inflammations of the intestine. Using a complex probiotic, such as: VSL#3, which contains 4 lactobacilli strains—*L. acidophilus*, *L. casei*, *Lactobacillus delbrueckii* sp. *bulgaricus*, and *Lactobacillus plantarum;* 3 bifido bacterial strains: *B. longum*, *B. infantis*, and *Bifidobacterium breve;* and *Streptococcus salivarius* sp. *thermophilus*, revealed to decrease activity of pouchitis (a non-specific inflammation of the ileal pouch) in ulcerative colitis (UC) and after ileal anastomosis [37]. The frequent

**53**

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

reported less satisfactory results than in UC [32].

*2.2.3 Probiotics in liver disease*

doses recommended in pouchitis are 2–4 sachets daily (each sachet contains 450,000 million live bacteria 4.5 × 1011 CFU; but there are also capsules containing 112,000 millions of bacteria) [31]. However, in additional research was described lower improvements in the reduction of disease in association with conventional treatment in patients with UC, and minor to moderate contribution, with the use of probiotic VSL#3, *Escherichia coli* Nissle, *Lactobacillus* GG, or milk fermented with bifidobacteria and/or lactobacilli (whether or not compared to placebo or other treatments, such as mesalazine) [38]. In trials with probiotics on remission induction or maintenance in Crohn's disease (using several strains such as *Lactobacillus* GG, VSL3, *L. johnsonii* LA1, *Escherichia coli* Nissle 1917, *S. boulardii*) have been

In a 2007 [39] demonstrated that administering probiotics may improve the rate of eradication and reduce the incidence of adverse events in case of infection with *Helicobacter pylori*. Zhang et al. [40] informed that the using the probiotics for standard eradication therapy in patients infected with *H. pylori* may increase the rate of eradication of the microorganism by approximately 13% and decrease the overall rate of adverse effects by approximately 41%, based on the patient's age, gender or probiotics dose. The probiotic used to improve the results of eradication therapies was *Lactobacillus reuteri*. In these therapies which demonstrated an ability to inhibit the colonisation of the human gastric mucosa with *H. pylori*, in addition to an ability to produce reuterin, a broad-spectrum antibiotic active against *H. pylori*, DSM 17648 strain of *L. reuteri* seemed especially effective for eradication therapies [36]. Also, *S. boulardii* seemed to significantly increase the rate of eradication, although

under the desired success level (80% versus 71% in the control group) [36].

the genetic characteristics of patients) influence therapeutic response.

reduction of endotoxin level in rats exposed to alcohol [41].

Few studies are, so far available, and, consequently much clinical evaluation is needed in the future of the most effective strains and of how host factors (such as

The researchers reported that probiotics can be useful in treating hepatic diseases due to their potential ability to modulate alterations in the gut microbiota, intestinal permeability, and immune and inflammatory responses. More studies based on murine and *in vitro* models show the role of probiotics in several liver diseases [41]. From medical point of view the pathogenic mechanism involved in liver damage secondary to alcohol abuse is endotoxemia. Researchers reported that through using *L. plantarum* encapsulated alginate beads induce a dose-dependent

Domingo [36] suggests that non-alcoholic fatty liver disease (NAFLD) comprises a varied range of pathological circumstances, from simple steatosis to cirrhosis, through steatohepatitis and fibrosis. It is known that probiotics (VSL#3) can modulate the intestinal flora, influencing the bowel-liver axis and improving NAFLD. Xu et al. [42] reported in a study that compared two types of probiotics (*L. acidophilus* and *B. longum*), neither improved intestinal permeability, but *B. longum* probiotic attenuated hepatic fat accumulation. However there are few human stud-

Hepatitis viruses, especially B and C, are known to cause long-term hepatocellular injury. As in other hepatic diseases, the plasma level of endotoxin increases in these patients because of changes in the gut microbiota [41]. Several studies evaluated the effects of probiotics in patients with hepatitis B virus (HBV) and hepatitis C virus (HCV). A research study achieved with *Bifidobacterium adolescentis* SPM0212 lead to increased expression of myxovirus (Mx) resistance A, an interferon (IFN)-inducible antiviral effector. Further, the extracellular surface

ies on the efficacy of probiotics in the prevention or treatment of NAFLD.

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

and a mixture of *L. acidophilus*, *L. casei*, and *L. rhamnosus*) [33].

relative risk of 0.58 [31].

strains are *L. acidophilus* (1.25 × 109

(2 packs daily), and *Bifidobacterium infantis* (2 × 109

*2.2.2 Probiotics in in diseases of the gastrointestinal apparatus*

the decrease number of a viral infection [35].

*Lactobacillus* spp*.*, alone or combined with bifidobacteria, enterococci, or *S. boulardii*), a reduced risk of antibiotic-induced diarrhoea was also established, with a

Floch et al. [32] reported that for the primary prevention of disease caused by *C. difficile* in patients treated with antibiotics, probiotics also decrease the incidence of such disease, especially when strains of *S. boulardii*, and possibly other *Lactobacillus*, such as GG, are administered. Instead, a recent meta-analysis settled that only four probiotic strains (not including *Lactobacillus* GG) have been shown to significantly decrease the incidence of diarrhoea induced by *C. difficile*, such as follows: *S. boulardii* (2 × 1010 CFU/day), *L. casei* DN114001 (probiotic drink twice daily), a mixture of *L. acidophilus* and *Bifidobacterium bifidum* (2 × 1010 CFU/day),

For patients that intake antibiotics to eradicate *Helicobacter pylori*, studies have been conducted based on probiotics adding in order to increase eradication rates and also to prevent side effects such as antibiotic-induced diarrhoea. Numerous meta-analyses showed that the addition of probiotics may increase the efficacy of eradication with an odds ratio (OR) ranging from 1.2 to 2 times compare to the control group. Although additional studies are needed, it appears that the most effective

DN-114001 (2 packs daily) (OR: 1.47), yogurt with *Lactobacillus gasseri* (OR: 1.19)

supplementary clinical researches where antibiotics are used, probiotics appear to decrease the incidence of diarrhoea (with an OR ranging from 0.16 to 0.47) [34]. Also, the efficiency of probiotic strains in the next therapy's: nosocomial, nonnosocomial, and viral diarrheas have been studied. The conclusion was as follows: it turns out that probiotics may increase the amount of IgA antibodies, which leads to

Inflammatory bowel disease (IBD) is a recurrent chronic condition in which an abnormal interaction exists between intestinal flora and the host. Patients with IBD have an increased risk of colorectal cancer [31]. Due to the growing area of disease spreading and ageing societies, the use of probiotic bacteria for human health is becoming increasingly important. The consumption of pre-processed food (fast food), often containing excessive amounts of fat and insufficient amounts of raw fruits and vegetables, is another factor of harmful modification of human intestinal microbiota. It seems that the system of intestinal microorganisms and its desirable modification with probiotic formulas and products may protect people against enteral problems, and improve health [1]. *L. plantarum* is a probiotic that has been used with good results in the management of some symptoms in patients with IBD. It has been reported that the DSM 9843 strain significantly reduced flatulence, and the LPO 1 and 299 V strains significantly reduced abdominal pain [36]. In many studies it has been reported that probiotics may be helpful in the treatment of inflammatory enteral conditions, Crohn's disease, ulcerative colitis, and non-specific ileitis. The aetiology of those diseases is not completely understood, but it is evident that they are associated with recurrent infections or chronic inflammations of the intestine. Using a complex probiotic, such as: VSL#3, which contains 4 lactobacilli strains—*L. acidophilus*, *L. casei*, *Lactobacillus delbrueckii* sp. *bulgaricus*, and *Lactobacillus plantarum;* 3 bifido bacterial strains: *B. longum*, *B. infantis*, and *Bifidobacterium breve;* and *Streptococcus salivarius* sp. *thermophilus*, revealed to decrease activity of pouchitis (a non-specific inflammation of the ileal pouch) in ulcerative colitis (UC) and after ileal anastomosis [37]. The frequent

CFU) (OR: 1.24), milk fermented with *L. casei*

CFU) (OR: 1.21) [31]. Also, in

**52**

doses recommended in pouchitis are 2–4 sachets daily (each sachet contains 450,000 million live bacteria 4.5 × 1011 CFU; but there are also capsules containing 112,000 millions of bacteria) [31]. However, in additional research was described lower improvements in the reduction of disease in association with conventional treatment in patients with UC, and minor to moderate contribution, with the use of probiotic VSL#3, *Escherichia coli* Nissle, *Lactobacillus* GG, or milk fermented with bifidobacteria and/or lactobacilli (whether or not compared to placebo or other treatments, such as mesalazine) [38]. In trials with probiotics on remission induction or maintenance in Crohn's disease (using several strains such as *Lactobacillus* GG, VSL3, *L. johnsonii* LA1, *Escherichia coli* Nissle 1917, *S. boulardii*) have been reported less satisfactory results than in UC [32].

In a 2007 [39] demonstrated that administering probiotics may improve the rate of eradication and reduce the incidence of adverse events in case of infection with *Helicobacter pylori*. Zhang et al. [40] informed that the using the probiotics for standard eradication therapy in patients infected with *H. pylori* may increase the rate of eradication of the microorganism by approximately 13% and decrease the overall rate of adverse effects by approximately 41%, based on the patient's age, gender or probiotics dose. The probiotic used to improve the results of eradication therapies was *Lactobacillus reuteri*. In these therapies which demonstrated an ability to inhibit the colonisation of the human gastric mucosa with *H. pylori*, in addition to an ability to produce reuterin, a broad-spectrum antibiotic active against *H. pylori*, DSM 17648 strain of *L. reuteri* seemed especially effective for eradication therapies [36]. Also, *S. boulardii* seemed to significantly increase the rate of eradication, although under the desired success level (80% versus 71% in the control group) [36].

Few studies are, so far available, and, consequently much clinical evaluation is needed in the future of the most effective strains and of how host factors (such as the genetic characteristics of patients) influence therapeutic response.

### *2.2.3 Probiotics in liver disease*

The researchers reported that probiotics can be useful in treating hepatic diseases due to their potential ability to modulate alterations in the gut microbiota, intestinal permeability, and immune and inflammatory responses. More studies based on murine and *in vitro* models show the role of probiotics in several liver diseases [41]. From medical point of view the pathogenic mechanism involved in liver damage secondary to alcohol abuse is endotoxemia. Researchers reported that through using *L. plantarum* encapsulated alginate beads induce a dose-dependent reduction of endotoxin level in rats exposed to alcohol [41].

Domingo [36] suggests that non-alcoholic fatty liver disease (NAFLD) comprises a varied range of pathological circumstances, from simple steatosis to cirrhosis, through steatohepatitis and fibrosis. It is known that probiotics (VSL#3) can modulate the intestinal flora, influencing the bowel-liver axis and improving NAFLD. Xu et al. [42] reported in a study that compared two types of probiotics (*L. acidophilus* and *B. longum*), neither improved intestinal permeability, but *B. longum* probiotic attenuated hepatic fat accumulation. However there are few human studies on the efficacy of probiotics in the prevention or treatment of NAFLD.

Hepatitis viruses, especially B and C, are known to cause long-term hepatocellular injury. As in other hepatic diseases, the plasma level of endotoxin increases in these patients because of changes in the gut microbiota [41]. Several studies evaluated the effects of probiotics in patients with hepatitis B virus (HBV) and hepatitis C virus (HCV). A research study achieved with *Bifidobacterium adolescentis* SPM0212 lead to increased expression of myxovirus (Mx) resistance A, an interferon (IFN)-inducible antiviral effector. Further, the extracellular surface

antigen of HBV level decreased depend by the dose up to 50% and gene expression was inhibited by 40% in hepatoma *cell* line HepG2.2.15 [43].

Also, in the literature have been reported studies regarding treatment with probiotic of patients with cirrhosis. Zhang et al. [44] used a cirrhotic-rat model with modified gut microbiota. In this research it was observed that the effects on total bilirubin (BT) and the ratio between aerobic and anaerobic bacteria were similar in healthy and cirrhotic rats. After administration of norfloxacin and probiotics to modify the gut microbiota, BT, liver function and endotoxemia were estimated. Cirrhotic rats showed a higher population of *Enterobacteriaceae* compared to healthy rats. It was concluded that treatment with bifidobacteria decreased the amount of *Enterobacteriaceae* and endotoxin level and increased the amount of *Lactobacillus* compared with healthy rats [44]. There are limited studies suggesting the role of probiotics for hepatocellular carcinoma. Chávez-Tapia et al. [41] reported in his article that clinical data the next probiotics—*L. rhamnosus LC705* and *Propionibacterium freudenreichii subsp. Shermanii,* maybe reduce the biologically effective dose of aflatoxin exposure. Similar data from murine models with *L. rhamnosus GG* were reported. Particularly after aflatoxin exposure, lower expression of c-myc, cyclin D1, bcl-2 and rasp-21/g3pdh were found [41].

In recent years also, several studies have shown that probiotics have beneficial effects and after liver transplantation. In a research by [45] patients who suffered for liver transplant were allocated to groups that received one of three treatments: live *L. plantarum* 299 strain and prebiotics (fibre), heat-killed lactobacilli and fibre, or selective bowel decontamination. Also, all patients received early enteral feeding. Patients who intake live lactobacilli and fibre developed a lower amount of bacterial infections (e.g. 13%) when compared to patients that underwent selective bowel decontamination: 48% [45]. However, probiotic bacteria such as *Saccharomyces cerevisiae* and *Lactobacillus* have been associated in some studies to the development of sepsis but the use of probiotics in patients who underwent a liver transplant requires at this point, a much more careful analysis of their safety [41].

#### *2.2.4 Probiotics in urogenital and vaginal disease*

According to the Centers for Disease Control and Prevention (CDCP), more than 1 billion women around the world suffer from non-sexually transmitted urogenital infections, such as bacterial vaginitis (BV), urinary tract infection (UTI) and several other yeast infections [46]. The dominant microflora in a healthy human vagina is comprised from a variety of *Lactobacillus* species with crucial role in protecting women from genital infections. A slight change in lactobacilli concentration can result in microbial disproportion in the vagina, causing a quantitative and qualitative modification from normally occurring lactobacilli to a mixed microflora controlled by anaerobic bacteria such as *Gardnerella vaginalis*, *Bacteroides* spp*.*, *Prevotella* spp*.*, and *Mobiluncus* species [47]. Commane et al. [48] reported in a research study the importance of probiotics in a woman's urogenital wellbeing. It was confirmed that by supplementing with probiotics (*L. rhamnosus* GR-1 and *Lactobacillus reuteri)* it can stimulate the colonisation of beneficial microbiota and may improve the vaginal health. Daily oral consumption of probiotics such as *L. rhamnosus* and *L. fermentum* exhibited the modification of the vaginal flora [48]. It is well-known that there is an association between abnormal vaginal microbial flora and an increased incidence of urinary tract infection (UTI). When administered twice daily orally the only strains clinically shown to have an effect are *L. rhamnosus* GR-1 and *L. reuteri*, these strains reduce recurrences of UTI and restored a normal lactobacilli dominated vaginal flora in patients [49]. The smallest imbalance in the microbial composition greatly influences the health of the vaginal microenvironment, potentially leading to compromised state

**55**

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

of BV and UTI. The primary solution for compromised state would be balancing the

Cholesterol is a precursor in many biochemical processes of the body and plays

The human mouth harbours diverse microbiomes in the human body such as viruses, fungi, protozoa, archaea and bacteria and they cause different diseases. From a dental point of view the bacteria cause two common diseases: dental caries and the periodontal (gum) diseases. The most used probiotics for oral health are species of *Lactobacillus* and *Bifidobacterium*. In a double-blind, placebo-controlled trial the consumption of *Streptococcus salivarius* K12 decreased the occurrence of plaque and also, reduced the concentration of *Streptococcus mutans* [55]. It is known that *Streptococcus uberis* and *S. oralis* also can inhibit the periodontal pathogens [56]. Additionally, the halitosis and the volatile sulphur compounds synthesis could be

Bowen [57] declared that the evidence for periodontitis is less than dental caries, but the use of probiotics to manage the oral microflora appears tobe an effective method to control oral conditions [57]. Many more studies are needed to understand the mechanism by which these probiotics colonise and affect the oral cavity. Is

Daliria and Lee [58] supposed that lactose is an important nutrient in all mammalian neonates, almost all of them have the capability to metabolise lactose to glucose and galactose. It is known that in humans, lactase activity decreases during mid-childhood [58]. Medical research reports that lactose intolerance is determined

a vital role in many functions, like as production of steroidal hormones, while extreme cholesterol in the blood can lead to arterial clogging and increases the risk of heart disease and/or stroke. Patients with hypercholesterolemia showed the risk of heart attacks three times higher, compared to patients with normal blood lipid values [51]. The scientific literature reported some probiotic strains with hypocholerolemic effects, such as: *L. bulgaricus, L. reuteri, and B. coagulans*. Also, clinical research in humans with *L. acidophilus* L1 milk, revealed a significant reduction in serum cholesterol. Further, a clinical trial on 32 hypercholesterolemic patients that consumed low-fat yogurt with *B. longum* BL1 displayed a significant decline in triglycerides, total serum and LDL cholesterol. Also, HDL cholesterol was increased with 14.5% [52]. Thirty-two hyperc-holesterolemic men and women were intake *L. acidophilus* CHO-220 and inulin, during a randomised, double-blind, placebo-controlled, and parallel-designed trial. This research study demonstrated that plasma total cholesterol and low-density lipoprotein (LDL)-cholesterol reduced by 7.84 and 9.27%, respectively, after 12 weeks [53]. Worldwide, it is known that coronary heart disease (CHD) is one of the major causes of adult's death. The main coronary arteries supplying the heart are no longer able to provide sufficient blood and oxygen to the myocardium, mainly because of the accumulation of plaques in the intimae of arteries [54]. Ranjbar et al. [54] concluded that in recent years, several foods enriched with probiotics were produced industrially. These foods have recently been subject to more research for their beneficial effects on the gut microflora and links to their systemic effects on the lowering of lipids known to be risk factors for CHD.

number of *Lactobacillus* spp. via the supplementation of probiotics [50].

*2.2.5 Probiotics in cardiovascular diseases and lipid metabolism*

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

*2.2.6 Probiotics in oral health*

prevented by probiotics consumption.

*2.2.7 Probiotics in lactose intolerance*

needed to better understand how they improve oral health.

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

was inhibited by 40% in hepatoma *cell* line HepG2.2.15 [43].

sion of c-myc, cyclin D1, bcl-2 and rasp-21/g3pdh were found [41].

requires at this point, a much more careful analysis of their safety [41].

*2.2.4 Probiotics in urogenital and vaginal disease*

In recent years also, several studies have shown that probiotics have beneficial effects and after liver transplantation. In a research by [45] patients who suffered for liver transplant were allocated to groups that received one of three treatments: live *L. plantarum* 299 strain and prebiotics (fibre), heat-killed lactobacilli and fibre, or selective bowel decontamination. Also, all patients received early enteral feeding. Patients who intake live lactobacilli and fibre developed a lower amount of bacterial infections (e.g. 13%) when compared to patients that underwent selective bowel decontamination: 48% [45]. However, probiotic bacteria such as *Saccharomyces cerevisiae* and *Lactobacillus* have been associated in some studies to the development of sepsis but the use of probiotics in patients who underwent a liver transplant

According to the Centers for Disease Control and Prevention (CDCP), more than 1 billion women around the world suffer from non-sexually transmitted urogenital infections, such as bacterial vaginitis (BV), urinary tract infection (UTI) and several other yeast infections [46]. The dominant microflora in a healthy human vagina is comprised from a variety of *Lactobacillus* species with crucial role in protecting women from genital infections. A slight change in lactobacilli concentration can result in microbial disproportion in the vagina, causing a quantitative and qualitative modification from normally occurring lactobacilli to a mixed microflora controlled by anaerobic bacteria such as *Gardnerella vaginalis*, *Bacteroides* spp*.*, *Prevotella* spp*.*, and *Mobiluncus* species [47]. Commane et al. [48] reported in a research study the importance of probiotics in a woman's urogenital wellbeing. It was confirmed that by supplementing with probiotics (*L. rhamnosus* GR-1 and *Lactobacillus reuteri)* it can stimulate the colonisation of beneficial microbiota and may improve the vaginal health. Daily oral consumption of probiotics such as *L. rhamnosus* and *L. fermentum* exhibited the modification of the vaginal flora [48]. It is well-known that there is an association between abnormal vaginal microbial flora and an increased incidence of urinary tract infection (UTI). When administered twice daily orally the only strains clinically shown to have an effect are *L. rhamnosus* GR-1 and *L. reuteri*, these strains reduce recurrences of UTI and restored a normal lactobacilli dominated vaginal flora in patients [49]. The smallest imbalance in the microbial composition greatly influences the health of the vaginal microenvironment, potentially leading to compromised state

antigen of HBV level decreased depend by the dose up to 50% and gene expression

Also, in the literature have been reported studies regarding treatment with probiotic of patients with cirrhosis. Zhang et al. [44] used a cirrhotic-rat model with modified gut microbiota. In this research it was observed that the effects on total bilirubin (BT) and the ratio between aerobic and anaerobic bacteria were similar in healthy and cirrhotic rats. After administration of norfloxacin and probiotics to modify the gut microbiota, BT, liver function and endotoxemia were estimated. Cirrhotic rats showed a higher population of *Enterobacteriaceae* compared to healthy rats. It was concluded that treatment with bifidobacteria decreased the amount of *Enterobacteriaceae* and endotoxin level and increased the amount of *Lactobacillus* compared with healthy rats [44]. There are limited studies suggesting the role of probiotics for hepatocellular carcinoma. Chávez-Tapia et al. [41] reported in his article that clinical data the next probiotics—*L. rhamnosus LC705* and *Propionibacterium freudenreichii subsp. Shermanii,* maybe reduce the biologically effective dose of aflatoxin exposure. Similar data from murine models with *L. rhamnosus GG* were reported. Particularly after aflatoxin exposure, lower expres-

**54**

of BV and UTI. The primary solution for compromised state would be balancing the number of *Lactobacillus* spp. via the supplementation of probiotics [50].

## *2.2.5 Probiotics in cardiovascular diseases and lipid metabolism*

Cholesterol is a precursor in many biochemical processes of the body and plays a vital role in many functions, like as production of steroidal hormones, while extreme cholesterol in the blood can lead to arterial clogging and increases the risk of heart disease and/or stroke. Patients with hypercholesterolemia showed the risk of heart attacks three times higher, compared to patients with normal blood lipid values [51]. The scientific literature reported some probiotic strains with hypocholerolemic effects, such as: *L. bulgaricus, L. reuteri, and B. coagulans*. Also, clinical research in humans with *L. acidophilus* L1 milk, revealed a significant reduction in serum cholesterol. Further, a clinical trial on 32 hypercholesterolemic patients that consumed low-fat yogurt with *B. longum* BL1 displayed a significant decline in triglycerides, total serum and LDL cholesterol. Also, HDL cholesterol was increased with 14.5% [52]. Thirty-two hyperc-holesterolemic men and women were intake *L. acidophilus* CHO-220 and inulin, during a randomised, double-blind, placebo-controlled, and parallel-designed trial. This research study demonstrated that plasma total cholesterol and low-density lipoprotein (LDL)-cholesterol reduced by 7.84 and 9.27%, respectively, after 12 weeks [53]. Worldwide, it is known that coronary heart disease (CHD) is one of the major causes of adult's death. The main coronary arteries supplying the heart are no longer able to provide sufficient blood and oxygen to the myocardium, mainly because of the accumulation of plaques in the intimae of arteries [54]. Ranjbar et al. [54] concluded that in recent years, several foods enriched with probiotics were produced industrially. These foods have recently been subject to more research for their beneficial effects on the gut microflora and links to their systemic effects on the lowering of lipids known to be risk factors for CHD.

## *2.2.6 Probiotics in oral health*

The human mouth harbours diverse microbiomes in the human body such as viruses, fungi, protozoa, archaea and bacteria and they cause different diseases. From a dental point of view the bacteria cause two common diseases: dental caries and the periodontal (gum) diseases. The most used probiotics for oral health are species of *Lactobacillus* and *Bifidobacterium*. In a double-blind, placebo-controlled trial the consumption of *Streptococcus salivarius* K12 decreased the occurrence of plaque and also, reduced the concentration of *Streptococcus mutans* [55]. It is known that *Streptococcus uberis* and *S. oralis* also can inhibit the periodontal pathogens [56]. Additionally, the halitosis and the volatile sulphur compounds synthesis could be prevented by probiotics consumption.

Bowen [57] declared that the evidence for periodontitis is less than dental caries, but the use of probiotics to manage the oral microflora appears tobe an effective method to control oral conditions [57]. Many more studies are needed to understand the mechanism by which these probiotics colonise and affect the oral cavity. Is needed to better understand how they improve oral health.

## *2.2.7 Probiotics in lactose intolerance*

Daliria and Lee [58] supposed that lactose is an important nutrient in all mammalian neonates, almost all of them have the capability to metabolise lactose to glucose and galactose. It is known that in humans, lactase activity decreases during mid-childhood [58]. Medical research reports that lactose intolerance is determined by blood glucose concentrations, and breath hydrogen test following ingestion of a lactose load [58] and symptoms include: abdominal distress like diarrhoea, bloating, abdominal pain and flatulence. The researchers noticed that treatment with probiotics (such as *Lactobacillus bulgaricus* and *Streptococcus thermophiles*) relieves symptoms of lactose intolerance. It is also observed that consumption of milk containing *Bifidobacterium longum* and *L. acidophilus* cause significantly less hydrogen production and flatulence. In researches where was used a combination of *Lactobacillus casei shirota* and *Bifidobacterium breve* Yakult has shown better effect on patients and improved the symptoms of lactose intolerance significantly [59].

### *2.2.8 Probiotics in cancer*

Kerry et al. [50] declared that as per World Health Organisation (WHO) cancer fact sheet this is a dreadful disease affecting peoples all over the globe. Approximately 14 million new cases and 8.2 million cancer-related deaths added till 2012. The global cancer deaths are from Asian, African, and American continents (more than 70%) [60]. *In vitro* studies, probiotic strains, *Lactobacillus fermentum* NCIMB-5221 and -8829, revealed the highly potential in destroying the colorectal cancer cells and promoting normal epithelial colon cell growth by producing the SCFAs (ferulic acids). This probiotics were compared to other probiotics (*L. acidophilus* ATCC 314 and *L. rhamnosus* ATCC 51303) known with tumorigenic properties [61]. Also, *L. acidophilus* is known to prolong the induction of colon tumours. It was demonstrated that feeding milk and colostrum fermented with *L. acidophilus* resulted in 16–41% reduction in tumour proliferation [62]. Also, the other probiotic *L. bulgaricus* has also been reported to induce antitumor activity against sarcoma-180 and solid Ehrlich ascites tumours [63]. Probiotics could play a significant role in neutralising cancer but research is limited only to *in vitro* tests.

## **3. Prebiotics**

Like probiotics, prebiotics is also being widely explored for their utility in the various field of applied science, more specifically as nutrients and supplements [50]. Food and Agriculture Organisation (FAO)/WHO defines prebiotics as a nonviable food component that confer health benefit(s) on the host associated with modulation of the microbiota [62].

Sources of prebiotics are as follows: breast milk, soybeans, inulin from diverse sources (Jerusalem artichoke, chicory roots), raw oat, wheat bran, barley bran, yacon roots, non-digestible carbohydrates (non-digestible oligosaccharides). From prebiotics, only bifidogenic, non-digestible oligosaccharides, especially inulin, and its hydrolysis products, such as oligofructose, and (trans) galactooligosaccharides (GOS), achieve all the criteria for prebiotics term [64]. Prebiotics can be obtained naturally from sources like vegetables, fruits, and grains consumed in our daily life but are also artificially prebiotic products such as: lactulose, galactooligosaccharides, fructooligosaccharides.

Kuo [65] reported that an ideal prebiotic should be:

• resistant to the actions of acids in the stomach, bile salts and other hydrolysing enzymes in the intestine;

**57**

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

Prebiotics not only serve as an energy source because their presence of prebiotics in the diet may lead to numerous health benefits. Several health benefits are reported in scientific literature, such as inhibition of the development of pathogens, reducing the prevalence and duration of diarrhoea, increases the absorption of minerals, mostly of magnesium and calcium, exerting protective effects to prevent colon cancer and providing relief from inflammation and other symptoms associ-

Several studies demonstrated that the colorectal carcinoma was less present at people who consume a lot of vegetables and fruits. The inulin and oligofructose from fruits and vegetables could suppress the disease [66]. When it comes to the advantages of prebiotics, it can be mention the reduction of the blood LDL (lowdensity lipoprotein) level, stimulation of the immunological system, increased the calcium absorbability, preservation of adequate intestinal pH value, low caloric value, and alleviation of symptoms of peptic ulcers and vaginal mycosis [67]. Other benefits of inulin and oligofructose on human health could be the prevention of carcinogenesis, as well as the support of lactose intolerance or dental caries treatment [68]. Also, prebiotics are useful in combating pathogenic microorganisms, such as *Salmonella enteritidis* and *Escherichia coli*, and reduce odour compounds and [69] confirmed a positive effect of fructooligosaccharides (FOS) on protection against *Salmonella typhimurium* and *Listeria monocytogenes* infections. Pokusaeva et al. [70] said that prebiotics are also implicated in enhancing the bioavailability and uptake of minerals, lowering of some risk factors for cardiovascular disease,

Prebiotics have been reported to play a beneficial role in controlling the IBD. A major reduction in the number of bacteriodetes in faeces was reported in patients with chronic pouchitis treated with 24 g per day of inulin [71]. In another study, 10 Crohn's Disease patients receiving 15 g of FOS demonstrated a reduced disease activity index [72]. In another randomised study involving 103 Crohn's Disease patients who received FOS 15 g/day these showed no clinical improvement however, though no change in IL-12 was observed it was able to reduce IL-6 of lamina propria

Kerry et al. [50] suggests that even with their enormous nutritional and medicinal benefits, research concerning screening new versatile prebiotics is quite deficient. Therefore, the research should be focused on identifying new healthy supplements, while screening novel prebiotic strains should be a major concern.

Due to the expansion of microbial research were discovered synbiotics as a combination of probiotics and prebiotics products which provide the survival and the implantation of the live microorganism dietary supplements in the gut [73]. The synergistic welfares are more proficiently promoted when both the probiotic and prebiotic act together in the living system. It is known that the symbiotic association between prebiotics and probiotics significantly improve the human health [50]. From the medical point of view the term of synbiotic product positively influence the host through improving the survival and implantation of live microbial dietary supplements in to the gastrointestinal tract and stimulating the growth and/or activating the metabolism of health promoting bacteria [62]. Since the word synbiotics suggests synergism, this term should be reserved for products in which the prebiotic compound(s) positively influence the probiotic organism(s) [74]. Markowiak and Śliżewska [1] suggests that when develop a synbiotic product, the most important aspect that have taken into account, is the selection of an appropriate probiotic and

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

ated with intestinal bowel disorders.

and promoting satiety and weight loss.

dendritic cells [1].

**4. Synbiotics**


*Frontiers and New Trends in the Science of Fermented Food and Beverages*

*2.2.8 Probiotics in cancer*

**3. Prebiotics**

modulation of the microbiota [62].

rides, fructooligosaccharides.

enzymes in the intestine;

Kuo [65] reported that an ideal prebiotic should be:

• not be absorbed in the upper gastrointestinal tract;

• be easily fermentable by the beneficial intestinal microflora.

by blood glucose concentrations, and breath hydrogen test following ingestion of a lactose load [58] and symptoms include: abdominal distress like diarrhoea, bloating, abdominal pain and flatulence. The researchers noticed that treatment with probiotics (such as *Lactobacillus bulgaricus* and *Streptococcus thermophiles*) relieves symptoms of lactose intolerance. It is also observed that consumption of milk containing *Bifidobacterium longum* and *L. acidophilus* cause significantly less hydrogen production and flatulence. In researches where was used a combination of *Lactobacillus casei shirota* and *Bifidobacterium breve* Yakult has shown better effect on patients and improved the symptoms of lactose intolerance significantly [59].

Kerry et al. [50] declared that as per World Health Organisation (WHO) cancer fact sheet this is a dreadful disease affecting peoples all over the globe. Approximately 14 million new cases and 8.2 million cancer-related deaths added till 2012. The global cancer deaths are from Asian, African, and American continents (more than 70%) [60]. *In vitro* studies, probiotic strains, *Lactobacillus fermentum* NCIMB-5221 and -8829, revealed the highly potential in destroying the colorectal cancer cells and promoting normal epithelial colon cell growth by producing the

SCFAs (ferulic acids). This probiotics were compared to other probiotics

(*L. acidophilus* ATCC 314 and *L. rhamnosus* ATCC 51303) known with tumorigenic properties [61]. Also, *L. acidophilus* is known to prolong the induction of colon tumours. It was demonstrated that feeding milk and colostrum fermented with *L. acidophilus* resulted in 16–41% reduction in tumour proliferation [62]. Also, the other probiotic *L. bulgaricus* has also been reported to induce antitumor activity against sarcoma-180 and solid Ehrlich ascites tumours [63]. Probiotics could play a significant role in neutralising cancer but research is limited only to *in vitro* tests.

Like probiotics, prebiotics is also being widely explored for their utility in the various field of applied science, more specifically as nutrients and supplements [50]. Food and Agriculture Organisation (FAO)/WHO defines prebiotics as a nonviable food component that confer health benefit(s) on the host associated with

Sources of prebiotics are as follows: breast milk, soybeans, inulin from diverse sources (Jerusalem artichoke, chicory roots), raw oat, wheat bran, barley bran, yacon roots, non-digestible carbohydrates (non-digestible oligosaccharides). From prebiotics, only bifidogenic, non-digestible oligosaccharides, especially inulin, and its hydrolysis products, such as oligofructose, and (trans) galactooligosaccharides (GOS), achieve all the criteria for prebiotics term [64]. Prebiotics can be obtained naturally from sources like vegetables, fruits, and grains consumed in our daily life but are also artificially prebiotic products such as: lactulose, galactooligosaccha-

• resistant to the actions of acids in the stomach, bile salts and other hydrolysing

**56**

Prebiotics not only serve as an energy source because their presence of prebiotics in the diet may lead to numerous health benefits. Several health benefits are reported in scientific literature, such as inhibition of the development of pathogens, reducing the prevalence and duration of diarrhoea, increases the absorption of minerals, mostly of magnesium and calcium, exerting protective effects to prevent colon cancer and providing relief from inflammation and other symptoms associated with intestinal bowel disorders.

Several studies demonstrated that the colorectal carcinoma was less present at people who consume a lot of vegetables and fruits. The inulin and oligofructose from fruits and vegetables could suppress the disease [66]. When it comes to the advantages of prebiotics, it can be mention the reduction of the blood LDL (lowdensity lipoprotein) level, stimulation of the immunological system, increased the calcium absorbability, preservation of adequate intestinal pH value, low caloric value, and alleviation of symptoms of peptic ulcers and vaginal mycosis [67]. Other benefits of inulin and oligofructose on human health could be the prevention of carcinogenesis, as well as the support of lactose intolerance or dental caries treatment [68]. Also, prebiotics are useful in combating pathogenic microorganisms, such as *Salmonella enteritidis* and *Escherichia coli*, and reduce odour compounds and [69] confirmed a positive effect of fructooligosaccharides (FOS) on protection against *Salmonella typhimurium* and *Listeria monocytogenes* infections. Pokusaeva et al. [70] said that prebiotics are also implicated in enhancing the bioavailability and uptake of minerals, lowering of some risk factors for cardiovascular disease, and promoting satiety and weight loss.

Prebiotics have been reported to play a beneficial role in controlling the IBD. A major reduction in the number of bacteriodetes in faeces was reported in patients with chronic pouchitis treated with 24 g per day of inulin [71]. In another study, 10 Crohn's Disease patients receiving 15 g of FOS demonstrated a reduced disease activity index [72]. In another randomised study involving 103 Crohn's Disease patients who received FOS 15 g/day these showed no clinical improvement however, though no change in IL-12 was observed it was able to reduce IL-6 of lamina propria dendritic cells [1].

Kerry et al. [50] suggests that even with their enormous nutritional and medicinal benefits, research concerning screening new versatile prebiotics is quite deficient. Therefore, the research should be focused on identifying new healthy supplements, while screening novel prebiotic strains should be a major concern.

## **4. Synbiotics**

Due to the expansion of microbial research were discovered synbiotics as a combination of probiotics and prebiotics products which provide the survival and the implantation of the live microorganism dietary supplements in the gut [73]. The synergistic welfares are more proficiently promoted when both the probiotic and prebiotic act together in the living system. It is known that the symbiotic association between prebiotics and probiotics significantly improve the human health [50]. From the medical point of view the term of synbiotic product positively influence the host through improving the survival and implantation of live microbial dietary supplements in to the gastrointestinal tract and stimulating the growth and/or activating the metabolism of health promoting bacteria [62]. Since the word synbiotics suggests synergism, this term should be reserved for products in which the prebiotic compound(s) positively influence the probiotic organism(s) [74]. Markowiak and Śliżewska [1] suggests that when develop a synbiotic product, the most important aspect that have taken into account, is the selection of an appropriate probiotic and

#### *Frontiers and New Trends in the Science of Fermented Food and Beverages*

prebiotic, that can act separately on the host's health. The prebiotic compounds should selectively stimulate the growth of probiotics, with beneficial effect on human health and not to be able to stimulate the other microorganisms.

*Lactobacillus* spp., *Bifidobacteria* spp., *S. boulardii*, *B. coagulans* are one of the probiotic strains that are used in synbiotic formulations, whereas the prebiotics used are as follows: oligosaccharides (fructooligosaccharide (FOS), GOS and xyloseoligosaccharide (XOS)), and inulin (from natural sources like chicory and yacon roots) [62]. Synbiotics consumption by humans includes the following beneficial effects:


In adult subjects with non-alcoholic steatohepatisis (NASH) in a randomised study what used of a synbiotic product which contained five probiotics namely: *Lactobacillus plantarum*, *L. delbrueckii* spp. *bulgaricus*, *L. acidophilus*, *L. rhamnosus*, *Bifidobacterium bifidum* and inulin as a prebiotic has been demonstrated a significant reduction of intrahepatic triacylglycerol (IHTG) within 6 months [1]. Fifty-two adults participated for 28 weeks in a research trial based on the effects of the synbiotic product. The synbiotic comprised a mix of probiotic strains: *Lactobacillus casei, L. rhamnosus, Streptococcus thermophilus, Bifidobacterium breve, L. acidophilus, B. longum, L. bulgaricus* and fructooligosccharides, as prebiotic. The authors stated that consumption of the synbiotic product resulted in the inhibition of nuclear factor-kB (NF-kB) and a condensed production of tumour necrosis factor α (TNF-*α*) [76].

Moreover, synbiotics seems to be quite attractive for improving the immune system. A significant decrease in the levels of C-reactive protein and also increase the glutathione levels was obtained through combination of *B. coagulans* with inulin, in diet for 6 weeks [77].

Recently, commercial interest in functional foods based on synbiotics has improved due to the awareness of the welfares for gut health, disease prevention and therapy. Investigates in this scientific zone is presently concentrated on designing new functional foods, as well as on screening new strains with capability to inhabit the human gut, along with their aptitude to metabolise new prebiotics [50]. Trials and investigation *in vitro* and *in vivo* demonstrated that the beneficial effects of using probiotics, prebiotics, and synbiotics in health are much more active than their unitary use known till present. Nevertheless, more investigates concerning the designing new mixtures of probiotics, prebiotics and synbiotics are imperative necessary for achieve further opportunities of improving nutritional and clinical health.

## **5. Conclusion**

The use of probiotics, prebiotics, and synbiotics in health is emerging as a promising therapy which is generally safe in different disease. Probiotics, probiotics and synbiotics have systemic effects on the urogenital disease, liver disease, oral health and immune system. There are many published reports on the use of probiotics in

**59**

**Author details**

România

Nicoleta-Maricica Maftei

health and clinical nutrition.

Author declares no conflict of interest.

**Conflict of interest**

provided the original work is properly cited.

\*Address all correspondence to: nicoleta.aron@ugal.ro

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

humans but information on prebiotics and synbiotics is quite a few. It seems that we will see and in the coming years further studies on combinations of probiotics and prebiotics, and further development of synbiotics. It is possible that future studies may explain the mechanisms of actions of those components, which may confer a beneficial effect on human health. However, the health claims made needs to be substantiated and firmly established by properly designed large scale clinical trials on human body. Therefore, current focus is on evaluating new strains of probiotics, a new prebiotics and new synbiotics products and their applicability in biomedical/clinical research, paving a new direction for exploration and exploitation of probiotics, prebiotics and synbiotics aimed at improving human health. There is a need for more randomised, placebo-controlled clinical trials with adequate statistical power. I encourage researchers to submit possible publications in peerreviewed journals of all clinical trials, whether the outcome is positive, negative or adverse, because the scientific and medical world needs it relevant information on the dose–response effects, efficacy, and safety of probiotic, prebiotic and synbiotic products. At present, the available information on current probiotics, prebiotics and synbiotics provides convincing safety records. I believe it is highly likely that in the near future, the vast amount of research on the beneficial impact of the probiotics, prebiotics and synbiotics on human wellbeing will suppose discovery and development of innovative products derived from our microbiota. Further, these may belong to uncommon and formerly uncharacterized microorganisms with rare assets, or perhaps could be microorganisms formerly known as pathogens or pathobionts. These progresses will represent new trends but also significant challenges for scientific and medical research, for industrial exploitation and for human

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Faculty of Medicine and Pharmacy, "Dunărea de Jos" University of Galati, Galați,

*Probiotic, Prebiotic and Synbiotic Products in Human Health DOI: http://dx.doi.org/10.5772/intechopen.81553*

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

prebiotic, that can act separately on the host's health. The prebiotic compounds should selectively stimulate the growth of probiotics, with beneficial effect on human health and not to be able to stimulate the other microorganisms.

*Lactobacillus* spp., *Bifidobacteria* spp., *S. boulardii*, *B. coagulans* are one of the probiotic strains that are used in synbiotic formulations, whereas the prebiotics used are as follows: oligosaccharides (fructooligosaccharide (FOS), GOS and xyloseoligosaccharide (XOS)), and inulin (from natural sources like chicory and yacon roots) [62]. Synbiotics consumption by humans includes the following beneficial

• Increased levels of lactobacilli and bifidobacteria and balanced gut microbiota.

• Prevention of bacterial translocation and reduced incidences of nosocomial

In adult subjects with non-alcoholic steatohepatisis (NASH) in a randomised study what used of a synbiotic product which contained five probiotics namely: *Lactobacillus plantarum*, *L. delbrueckii* spp. *bulgaricus*, *L. acidophilus*, *L. rhamnosus*, *Bifidobacterium bifidum* and inulin as a prebiotic has been demonstrated a significant reduction of intrahepatic triacylglycerol (IHTG) within 6 months [1]. Fifty-two adults participated for 28 weeks in a research trial based on the effects of the synbiotic product. The synbiotic comprised a mix of probiotic strains: *Lactobacillus casei, L. rhamnosus, Streptococcus thermophilus, Bifidobacterium breve, L. acidophilus, B. longum, L. bulgaricus* and fructooligosccharides, as prebiotic. The authors stated that consumption of the synbiotic product resulted in the inhibition of nuclear factor-kB (NF-kB) and a condensed production of tumour necrosis factor α (TNF-*α*) [76]. Moreover, synbiotics seems to be quite attractive for improving the immune system. A significant decrease in the levels of C-reactive protein and also increase the glutathione levels was obtained through combination of *B. coagulans* with inulin, in

Recently, commercial interest in functional foods based on synbiotics has improved due to the awareness of the welfares for gut health, disease prevention and therapy. Investigates in this scientific zone is presently concentrated on designing new functional foods, as well as on screening new strains with capability to inhabit the human gut, along with their aptitude to metabolise new prebiotics [50]. Trials and investigation *in vitro* and *in vivo* demonstrated that the beneficial effects of using probiotics, prebiotics, and synbiotics in health are much more active than their unitary use known till present. Nevertheless, more investigates concerning the designing new mixtures of probiotics, prebiotics and synbiotics are imperative necessary for achieve further opportunities of improving nutritional and clinical

The use of probiotics, prebiotics, and synbiotics in health is emerging as a promising therapy which is generally safe in different disease. Probiotics, probiotics and synbiotics have systemic effects on the urogenital disease, liver disease, oral health and immune system. There are many published reports on the use of probiotics in

**58**

health.

**5. Conclusion**

effects:

infections in surgical patients.

diet for 6 weeks [77].

• Improvement of liver function in cirrhotic patients.

• Improvement of immunomodulating ability [75].

humans but information on prebiotics and synbiotics is quite a few. It seems that we will see and in the coming years further studies on combinations of probiotics and prebiotics, and further development of synbiotics. It is possible that future studies may explain the mechanisms of actions of those components, which may confer a beneficial effect on human health. However, the health claims made needs to be substantiated and firmly established by properly designed large scale clinical trials on human body. Therefore, current focus is on evaluating new strains of probiotics, a new prebiotics and new synbiotics products and their applicability in biomedical/clinical research, paving a new direction for exploration and exploitation of probiotics, prebiotics and synbiotics aimed at improving human health. There is a need for more randomised, placebo-controlled clinical trials with adequate statistical power. I encourage researchers to submit possible publications in peerreviewed journals of all clinical trials, whether the outcome is positive, negative or adverse, because the scientific and medical world needs it relevant information on the dose–response effects, efficacy, and safety of probiotic, prebiotic and synbiotic products. At present, the available information on current probiotics, prebiotics and synbiotics provides convincing safety records. I believe it is highly likely that in the near future, the vast amount of research on the beneficial impact of the probiotics, prebiotics and synbiotics on human wellbeing will suppose discovery and development of innovative products derived from our microbiota. Further, these may belong to uncommon and formerly uncharacterized microorganisms with rare assets, or perhaps could be microorganisms formerly known as pathogens or pathobionts. These progresses will represent new trends but also significant challenges for scientific and medical research, for industrial exploitation and for human health and clinical nutrition.

## **Conflict of interest**

Author declares no conflict of interest.

## **Author details**

Nicoleta-Maricica Maftei Faculty of Medicine and Pharmacy, "Dunărea de Jos" University of Galati, Galați, România

\*Address all correspondence to: nicoleta.aron@ugal.ro

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Markowiak P, Śliżewska K. Effects of probiotics, prebiotics, and synbiotics on human health. Nutrients. 2017;**9**(1021):1-30. DOI: 10.3390/ nu9091021

[2] Metchnikoff E, Mitchell PC, editors. Essais Optimistes. London: Heinemann; 1907

[3] Tissier H. Tritement des infections intestinales par la methode de translormation de la flore bacterienne de lintestin. Comptes Rendus Social Biology. 1906;**60**:359-361 (in French)

[4] Havenaar R, Huis in't Veld JHJ. Probiotics: A general view. In: Wood BJB, editor. The Lactic Acid Bacteria in Health and Disease. London: Elsevier Applied Science; 1992. pp. 151-170

[5] Food and Agriculture Organization (FAO). Guidelines for the Evaluation of Probiotics in Food; Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food. London, ON, Canada: FAO; 2002

[6] Hill C, Guarner F, Rei G, Gibson GR, Merenstein DJ, Pot B, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews Gastroenterology & Hepatology. 2014;**11**:506-514. DOI: 10.1038/ nrgastro.2014.66

[7] Gibson RG, Roberfroid MB. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. The Journal of Nutrition. 1995;**125**:1401-1412. DOI: 10.1093/ jn/125.6.1401

[8] Gibson GR, Probert HM, van Loo J, Rastall RA, Roberfroid M. Dietary

modulation of the human colonic microbiota: Updating the concept of the prebiotics. Nutrition Research Reviews. 2004;**17**:259-275. DOI: 10.1079/ NRR200479

[9] Food and Agriculture Organization. FAO Technical Meeting on Prebiotics: Food Quality and Standards Service (AGNS), Food and Agriculture Organization of the United Nations (FAO). Rome, Italy: FAO Technical Meeting Report; FAO; 2007. pp. 15-16

[10] Skalkam ML, Wiese M, Nielsen DS, van Zanten G. In Vitro Screening and Evaluation of Synbiotics. Copenhagen, Denmark: University of Copenhagen; 2016 (Chapter 33). pp. 477-486

[11] Cencic A, Chingwaru W. The role of functional foods, nutraceuticals, and food supplements in intestinal health. Nutrients. 2010;**2**:611-625. DOI: 10.3390/nu2060611

[12] Rioux KP, Madsen KL, Fedorak RN. The role of enteric microflora in inflammatory bowel disease: Human and animal studies with probiotics and prebiotics. Gastroenterology Clinics of North America. 2005;**34**:465-482. DOI: 10.3390/nu2060611

[13] Bengmark S. Bioecological control of the gastrointestinal tract: The role of flora and supplemented probiotics and synbiotics. Gastroenterology Clinics of North America. 2005;**34**:413-436. DOI: 10.1016/j.gtc.2005.05.002

[14] Panesar PS, Kaur G, Panesar R, Bera MB. Synbiotics: Potential Dietary Supplements in Functional Foods. Berkshire, UK: IFIS; 2009. DOI: 10.1136/ gut.2005.074971

**61**

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

intestinalinjury. PLoS One. 2013;**8**(5):

[23] Cosmi L, Maggi L, Santarlasci V, Liotta F, Annunziato F. T helper cells plasticity in inflammation. Cytometry. Part A. 2014;**85**(1):36-42. DOI: 10.1002/

[24] Hardy H, Harris J, Lyon E, Beal J, Foey A. Probiotics, prebiotics and immunomodulation of gut mucosal defences: Homeostasis andimmunopathology. Nutrients. 2013;**5**:1869-1912. DOI: 10.3390/

[25] Vandenbergh PA. Lactic acid bacteria, their metabolic products and interference with microbial growth. FEMS Microbiology Reviews. 1993;**12**:221-238. DOI: 10.1111/j.1574-

[26] Guillot JF. Probiotic feed additives. Journal of Veterinary Pharmacology and Therapeutics. 2003;**26**:52-55. DOI:

[27] Isolauri E, Sutas Y, Kankaanpaa P, Arvilommi H, Salminen S. Probiotics: Effects on immunity. American Journal of Clinical Nutrition. 2001;**73**:444-450.

[28] Brandao RL, Castro IM, Bambirra EA, Amaral SC, Fietto LG, Tropia MJM. Intracellular signal triggered by cholera toxin in *Saccharomyces boulardii* and *Saccharomyces cerevisiae*. Applied and Environmental Microbiology.

[29] Schachtsiek M, Hammes WP, Hertel C. Characterization of *Lactobacillus coryniformis* DSM 20001T surface protein CPF mediating coaggregation

pathogens. Applied and Environmental Microbiology. 2004;**70**:7078-7085. DOI: 10.1128/AEM.70.12.7078-7085.2004

with and aggregation among

e65108. DOI: 10.1371/journal.

pone.0065108

cyto.a.22348

nu5061869

6976.1993.tb00020.x

10.1046/J.1365-2885.26

DOI: 10.1093/ajcn/73.2.444s

1998;**64**:564-568

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

[16] Miecznikow E. O naturze ludzkiej—

(Translation F. Wermiński). Warszawa, Poland: Wydawnictwo Biblioteka

Medicine. 2016;**8**:1-11. DOI: 10.1186/

Zarys Filozofii Optymistycznej

[17] Amara AA, Shibl A. Role of probiotics in health improvement, infection control and disease treatment and management. Saudi Pharmaceutical Journal. 2015;**23**:107-114. DOI: 10.1016/j.

[18] O'Toole PW, Marchesi JR, Hill C. Next-generation probiotics: The spectrum from probiotics to live biotherapeutics. Nature Microbiology. 2017;**2**:1-6 (article number: 17057). DOI: 10.1038/ nmicrobiol.2017.57 Available from: www.nature.com/ naturemicrobiology

[19] Anadón A, Martínez-Larrańaga MR, Martínez MA. Probiotics for animal nutrition in the European Union. Regulation and safety

assessment. Regulatory Toxicology and Pharmacology. 2006;**45**:91-95. DOI:

[20] Gaggia F, Mattarelli P, Biavati B. Probiotics and prebiotics in animal feeding for safe food production. International Journal of Food

Microbiology. 2010;**141**:S15-S28. DOI: 10.1016/j.ijfoodmicro.2010.02.031

[21] Thomas CM, Versalovic J. Probiotics-host communication: Modulationof signaling pathways in the intestine. Gut Microbes. 2010;**1**(3): 148-163. DOI: 10.4161/gmic.1.3.11712

[22] Shiou R, Yu Y, Guo Y, He SM, Andrew CHM, Hoenig J, et al. Synergistic protection of combined probiotic con-ditioned media against neonatal necrotizing enterocolitis-like

10.1016/j.yrtph.2006.02.004

s13073-016-0307-y

Naukowa; 1907

jsps.2013.07.001

[15] Lloyd-Price J, Abu-Ali G, Huttenhower C. The healthy human microbiome. Genome *Probiotic, Prebiotic and Synbiotic Products in Human Health DOI: http://dx.doi.org/10.5772/intechopen.81553*

Medicine. 2016;**8**:1-11. DOI: 10.1186/ s13073-016-0307-y

[16] Miecznikow E. O naturze ludzkiej— Zarys Filozofii Optymistycznej (Translation F. Wermiński). Warszawa, Poland: Wydawnictwo Biblioteka Naukowa; 1907

[17] Amara AA, Shibl A. Role of probiotics in health improvement, infection control and disease treatment and management. Saudi Pharmaceutical Journal. 2015;**23**:107-114. DOI: 10.1016/j. jsps.2013.07.001

[18] O'Toole PW, Marchesi JR, Hill C. Next-generation probiotics: The spectrum from probiotics to live biotherapeutics. Nature Microbiology. 2017;**2**:1-6 (article number: 17057). DOI: 10.1038/ nmicrobiol.2017.57 Available from: www.nature.com/ naturemicrobiology

[19] Anadón A, Martínez-Larrańaga MR, Martínez MA. Probiotics for animal nutrition in the European Union. Regulation and safety assessment. Regulatory Toxicology and Pharmacology. 2006;**45**:91-95. DOI: 10.1016/j.yrtph.2006.02.004

[20] Gaggia F, Mattarelli P, Biavati B. Probiotics and prebiotics in animal feeding for safe food production. International Journal of Food Microbiology. 2010;**141**:S15-S28. DOI: 10.1016/j.ijfoodmicro.2010.02.031

[21] Thomas CM, Versalovic J. Probiotics-host communication: Modulationof signaling pathways in the intestine. Gut Microbes. 2010;**1**(3): 148-163. DOI: 10.4161/gmic.1.3.11712

[22] Shiou R, Yu Y, Guo Y, He SM, Andrew CHM, Hoenig J, et al. Synergistic protection of combined probiotic con-ditioned media against neonatal necrotizing enterocolitis-like intestinalinjury. PLoS One. 2013;**8**(5): e65108. DOI: 10.1371/journal. pone.0065108

[23] Cosmi L, Maggi L, Santarlasci V, Liotta F, Annunziato F. T helper cells plasticity in inflammation. Cytometry. Part A. 2014;**85**(1):36-42. DOI: 10.1002/ cyto.a.22348

[24] Hardy H, Harris J, Lyon E, Beal J, Foey A. Probiotics, prebiotics and immunomodulation of gut mucosal defences: Homeostasis andimmunopathology. Nutrients. 2013;**5**:1869-1912. DOI: 10.3390/ nu5061869

[25] Vandenbergh PA. Lactic acid bacteria, their metabolic products and interference with microbial growth. FEMS Microbiology Reviews. 1993;**12**:221-238. DOI: 10.1111/j.1574- 6976.1993.tb00020.x

[26] Guillot JF. Probiotic feed additives. Journal of Veterinary Pharmacology and Therapeutics. 2003;**26**:52-55. DOI: 10.1046/J.1365-2885.26

[27] Isolauri E, Sutas Y, Kankaanpaa P, Arvilommi H, Salminen S. Probiotics: Effects on immunity. American Journal of Clinical Nutrition. 2001;**73**:444-450. DOI: 10.1093/ajcn/73.2.444s

[28] Brandao RL, Castro IM, Bambirra EA, Amaral SC, Fietto LG, Tropia MJM. Intracellular signal triggered by cholera toxin in *Saccharomyces boulardii* and *Saccharomyces cerevisiae*. Applied and Environmental Microbiology. 1998;**64**:564-568

[29] Schachtsiek M, Hammes WP, Hertel C. Characterization of *Lactobacillus coryniformis* DSM 20001T surface protein CPF mediating coaggregation with and aggregation among pathogens. Applied and Environmental Microbiology. 2004;**70**:7078-7085. DOI: 10.1128/AEM.70.12.7078-7085.2004

**60**

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

modulation of the human colonic microbiota: Updating the concept of the prebiotics. Nutrition Research Reviews. 2004;**17**:259-275. DOI: 10.1079/

[9] Food and Agriculture Organization. FAO Technical Meeting on Prebiotics: Food Quality and Standards Service (AGNS), Food and Agriculture Organization of the United Nations (FAO). Rome, Italy: FAO Technical Meeting Report; FAO; 2007. pp. 15-16

[10] Skalkam ML, Wiese M, Nielsen DS, van Zanten G. In Vitro Screening and Evaluation of Synbiotics. Copenhagen, Denmark: University of Copenhagen; 2016 (Chapter 33). pp. 477-486

[11] Cencic A, Chingwaru W. The role of functional foods, nutraceuticals, and food supplements in intestinal health. Nutrients. 2010;**2**:611-625. DOI:

[12] Rioux KP, Madsen KL, Fedorak RN.

[13] Bengmark S. Bioecological control of the gastrointestinal tract: The role of flora and supplemented probiotics and synbiotics. Gastroenterology Clinics of North America. 2005;**34**:413-436. DOI:

[14] Panesar PS, Kaur G, Panesar R, Bera MB. Synbiotics: Potential Dietary Supplements in Functional Foods. Berkshire, UK: IFIS; 2009. DOI: 10.1136/

The role of enteric microflora in inflammatory bowel disease: Human and animal studies with probiotics and prebiotics. Gastroenterology Clinics of North America. 2005;**34**:465-482. DOI:

10.3390/nu2060611

10.3390/nu2060611

10.1016/j.gtc.2005.05.002

[15] Lloyd-Price J, Abu-Ali G, Huttenhower C. The healthy human microbiome. Genome

gut.2005.074971

NRR200479

[1] Markowiak P, Śliżewska K. Effects

synbiotics on human health. Nutrients. 2017;**9**(1021):1-30. DOI: 10.3390/

[2] Metchnikoff E, Mitchell PC, editors. Essais Optimistes. London: Heinemann;

[3] Tissier H. Tritement des infections

translormation de la flore bacterienne de lintestin. Comptes Rendus Social Biology. 1906;**60**:359-361 (in French)

[4] Havenaar R, Huis in't Veld JHJ. Probiotics: A general view. In: Wood BJB, editor. The Lactic Acid Bacteria in Health and Disease. London: Elsevier Applied Science; 1992. pp. 151-170

[5] Food and Agriculture Organization (FAO). Guidelines for the Evaluation of Probiotics in Food; Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food. London, ON,

[6] Hill C, Guarner F, Rei G, Gibson GR, Merenstein DJ, Pot B, et al. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews Gastroenterology & Hepatology. 2014;**11**:506-514. DOI: 10.1038/

[7] Gibson RG, Roberfroid MB. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. The Journal of Nutrition. 1995;**125**:1401-1412. DOI: 10.1093/

[8] Gibson GR, Probert HM, van Loo J, Rastall RA, Roberfroid M. Dietary

Canada: FAO; 2002

nrgastro.2014.66

jn/125.6.1401

intestinales par la methode de

of probiotics, prebiotics, and

nu9091021

**References**

1907

[30] Oelschlaeger A. Mechanisms of probiotic actions—A review. International Journal of Medical Microbiology. 2010;**300**:57-62. DOI: 10.1016/j.ijmm.2009.08.005

[31] Olveira G, González-Moleroa I. An update on probiotics, prebiotics and symbiotics in clinical nutrition. Endocrinología y Nutrición. 2016;**63**(9):482-494. DOI: 10.1016/j. endoen.2016.10.011

[32] Floch MH, Walker WA, Sanders ME, Nieuwdorp M, Kim AS, Brenner DA, et al. Recommendations for probiotic use-2015update: Proceedings and consensus opinion. Journal of Clinical Gastroenterology. 2015;**49**(Suppl. 1):S69-S73. DOI: 10.1016/j.iccn.2010.07.001

[33] McFarland LV. Probiotics for the primary and secondary preven-tion of *C. difficile* infections: A meta-analysis and systematic review. Antibiotics. 2015;**4**:160-178. DOI: 10.3390/ antibiotics4020160

[34] Dang Y, Reinhardt JD, Zhou X, Zhang G. The effect of probioticssupplementation on *Helicobacter pylori* eradication rates andside effects during eradication therapy: A meta-analysis. PLoS One. 2014;**9**:e111030. DOI: 10.1371/journal. pone.0111030

[35] Parvez S, Malik KA, Ah Kang S, Kim HY. Probiotics and their fermented food products are beneficial for health. Journal of Applied Microbiology. 2006;**100**:1171-1185. DOI: 10.1111/j.1365-2672.2006.02963.x

[36] Domingo JJS. Review of the role of probiotics in gastrointestinal diseases in adults. Gastroenterología y Hepatología. 2017;**40**(6):417-429. DOI: 10.1016/j. gastre.2016.12.001

[37] Guslandi M. Role of probiotics in Crohn's disease and in pouch-itis. Journal of Clinical Gastroenterology. 2015;**49**(1):S46-S59. DOI: 10.1097/ MCG.0000000000000351

[38] Chibbar R, Dieleman LA. Probiotics in the management of ulcerative colitis. Journal of Clinical Gastroenterology. 2015;**49**(1):S50-S55. DOI: 10.1097/ MCG.0000000000000368

[39] Tong JL, Ran ZH, Shen J, Zhang CX, Xiao SD. Meta-analysis: The effect of supplementation with probiotics on eradication rates and adverse events during *Helicobacter pylori* eradication therapy. Alimentary Pharmacology & Therapeutics. 2007;**25**:155-168. DOI: 10.1111/j.1365-2036.2006.03179.x

[40] Zhang MM, Qian W, Qin YY, He J, Zhou YH. Probiotics in *Helicobacter pylori* eradication therapy: A systematic review and meta-analysis. World Journal of Gastroenterology. 2015;**21**:4345-4357. DOI: 10.3748/wjg.v21.i14.4345

[41] Chávez-Tapia NC, González-Rodríguez L, Jeong MS, López-Ramírez Y, Barbero-Becerra V, Juárez-Hernández E, et al. Current evidence on the use of probiotics in liver diseases. Journal of Functional Foods. 2015;**17**:137-151. DOI: 10.1016/j.jff.2015.05.009

[42] Xu RY, Wan YP, Fang QY, Lu W, Cai W. Supplementation with probiotics modifies gut flora and attenuates liver fat accumulation in rat nonalcoholic fatty liver disease model. Journal of Clinical Biochemistry and Nutrition. 2012;**50**(1):72-77. DOI: 10.3164/ jcbn.11-38

[43] Lee DK, Kang JY, Shin HS, Park IH, Ha NJ. Antiviral activity of Bifidobacterium adolescentis SPM0212 against hepatitis B virus. Archives of Pharmacal Research. 2013;**36**(12): 1525-1532. DOI: 10.1007/ s12272-013-0141-3

[44] Zhang W, Gu Y, Chen Y, Deng H, Chen L, Che S, et al. Intestinal flora

**63**

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

2012;**3**(10):WMC003796. DOI: 10.9754/

[52] Homayouni A, Payahoo L, Azizi A. Effects of probiotics on lipid profile: A review. American Journal of Food Technology. 2012;**7**(5):251-265. DOI:

[53] Ooi LG, Liong MT. Cholesterollowering effects of probiotics

andprebiotics: A review of *in vivo* and *in vitro* findings. International Journal of Molecular Sciences. 2010;**11**:2499-2522.

journal.wmc.2012.003796

10.3923/ajft.2012.251.265

DOI: 10.3390/ijms11062499

[54] Ranjbar F, Akbarzadeh F, Homayouni A. Probiotics usage in heart disease and psychiatry, chapter 61, in book: Probiotics, Prebiotics, and Synbiotics Bioactive Foods in Health Promotion: Probiotics and Prebiotics, Edited by R. Ross Watson and V. R. Preedy. Elsevier Inc., Academic Press,

London, UK, 2016, pp.807-811

jmm.0.056663-0

2013;**87**:5-9

[55] Burton J, Drummond B, Chilcott C, Tagg J, Thomson W, Hale J, et al. Influence of the probiotic *Streptococcus salivarius* strain M18 on indices of dental health in children: A randomized double-blind,placebo-controlled trial. Journal of Medical Microbiology. 2013;**62**:875-884. DOI: 10.1099/

[56] Hillma J, McDonell E, Hillman C, Zahradnik R, Soni M. Safety assessment of ProBiora3, a probiotic mouthwash: Subchronic toxicity study in rats. International Journal of Toxicology. 2009;**28**:357-367. DOI:

[57] Bowen DM. Probiotics and oral health. Journal of Dental Hygiene.

[58] Daliria EBM, Lee BH. New perspectives on probiotics in health and disease. Food Science and Human Wellness. 2015;**4**(2):56-65. DOI: 10.1016/j.fshw.2015.06.002

10.1177/1091581809340705

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

imbalance results in altered bacterial translocation and liver function in rats with experimental cirrhosis. European Journal of Gastroenterology & Hepatology. 2010;**22**(12):1481-1486. DOI: 10.1097/MEG.0b013e32833eb8b0

[45] Rayes N, Seehofer D, Hansen S, Boucsein K, Müller AR, Serk S, et al. Early enteral supply of lactobacillus and fiber versus selective bowel decontamination: A controlled trial in liver transplant recipients. Transplantation. 2002;**74**(1):123-128

[46] Waigankar SS, Patel V. Role of probiotics in urogenital healthcare. Journal of Midlife Health. 2011;**2**:5-10.

[47] Petricevic L, Domig K, Nierscher F, Sandhofer M, Fidesser M, Krondorfer I, et al. Characterisation of the vaginal *Lactobacillus* microbiota associated with preterm delivery. Scientific Reports. 2014;**4**:1-6 (article number: 5136). DOI:

[48] Commane D, Hughes R, Shortt C, Rowland I. The potential mechanisms involved in the anti-carcinogenic action of probiotics. Mutation Research. 2005;**591**:276-289. DOI: 10.1016/j.

[49] Reid G, Bruce A. Probiotics to prevent urinary tract infections: The rationale and evidence. World Journal of Urology. 2006;**24**:28-32. DOI: 10.1007/

[50] Kerry GR, Patra JK, Gouda S, Park Y, Shin HS, Das G. Benefaction of probiotics for human health: A review. Journal of Food and Drug Analysis. 2018;**26**(3):927-939. DOI: 10.1016/j.

DOI: 10.4103/0976-7800.83253

10.1038/srep05136

mrfmmm.2005.02.027

s00345-005-0043-1

jfda.2018.01.002

[51] Ghosh AR. Appraisal of probiotics and prebiotics in gastrointestinal infections.

Webmed Central Gastroenterology.

12134110

*Probiotic, Prebiotic and Synbiotic Products in Human Health DOI: http://dx.doi.org/10.5772/intechopen.81553*

imbalance results in altered bacterial translocation and liver function in rats with experimental cirrhosis. European Journal of Gastroenterology & Hepatology. 2010;**22**(12):1481-1486. DOI: 10.1097/MEG.0b013e32833eb8b0

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

Journal of Clinical Gastroenterology. 2015;**49**(1):S46-S59. DOI: 10.1097/

[38] Chibbar R, Dieleman LA. Probiotics in the management of ulcerative colitis. Journal of Clinical Gastroenterology. 2015;**49**(1):S50-S55. DOI: 10.1097/ MCG.0000000000000368

[39] Tong JL, Ran ZH, Shen J, Zhang CX, Xiao SD. Meta-analysis: The effect of supplementation with probiotics on eradication rates and adverse events during *Helicobacter pylori* eradication therapy. Alimentary Pharmacology & Therapeutics. 2007;**25**:155-168. DOI: 10.1111/j.1365-2036.2006.03179.x

[40] Zhang MM, Qian W, Qin YY, He J, Zhou YH. Probiotics in *Helicobacter pylori* eradication therapy: A systematic review and meta-analysis. World Journal of Gastroenterology. 2015;**21**:4345-4357.

DOI: 10.3748/wjg.v21.i14.4345

10.1016/j.jff.2015.05.009

jcbn.11-38

[41] Chávez-Tapia NC, González-Rodríguez L, Jeong MS, López-Ramírez Y, Barbero-Becerra V, Juárez-Hernández E, et al. Current evidence on the use of probiotics in liver diseases. Journal of Functional Foods. 2015;**17**:137-151. DOI:

[42] Xu RY, Wan YP, Fang QY, Lu W, Cai W. Supplementation with probiotics modifies gut flora and attenuates liver fat accumulation in rat nonalcoholic fatty liver disease model. Journal of Clinical Biochemistry and Nutrition. 2012;**50**(1):72-77. DOI: 10.3164/

[43] Lee DK, Kang JY, Shin HS, Park IH, Ha NJ. Antiviral activity of

1525-1532. DOI: 10.1007/ s12272-013-0141-3

Bifidobacterium adolescentis SPM0212 against hepatitis B virus. Archives of Pharmacal Research. 2013;**36**(12):

[44] Zhang W, Gu Y, Chen Y, Deng H, Chen L, Che S, et al. Intestinal flora

MCG.0000000000000351

[30] Oelschlaeger A. Mechanisms of probiotic actions—A review. International Journal of Medical Microbiology. 2010;**300**:57-62. DOI:

[31] Olveira G, González-Moleroa I. An update on probiotics, prebiotics and symbiotics in clinical nutrition.

[32] Floch MH, Walker WA, Sanders ME, Nieuwdorp M, Kim AS, Brenner DA, et al. Recommendations for probiotic use-2015update: Proceedings and consensus opinion. Journal of Clinical Gastroenterology. 2015;**49**(Suppl. 1):S69-S73. DOI: 10.1016/j.iccn.2010.07.001

[33] McFarland LV. Probiotics for the primary and secondary preven-tion of *C. difficile* infections: A meta-analysis and systematic review. Antibiotics. 2015;**4**:160-178. DOI: 10.3390/

10.1016/j.ijmm.2009.08.005

Endocrinología y Nutrición. 2016;**63**(9):482-494. DOI: 10.1016/j.

endoen.2016.10.011

antibiotics4020160

pone.0111030

gastre.2016.12.001

[34] Dang Y, Reinhardt JD, Zhou X, Zhang G. The effect of probioticssupplementation on *Helicobacter pylori* eradication rates andside effects during eradication therapy: A meta-analysis. PLoS One. 2014;**9**:e111030. DOI: 10.1371/journal.

[35] Parvez S, Malik KA, Ah Kang S, Kim HY. Probiotics and their

for health. Journal of Applied

fermented food products are beneficial

Microbiology. 2006;**100**:1171-1185. DOI: 10.1111/j.1365-2672.2006.02963.x

[36] Domingo JJS. Review of the role of probiotics in gastrointestinal diseases in adults. Gastroenterología y Hepatología. 2017;**40**(6):417-429. DOI: 10.1016/j.

[37] Guslandi M. Role of probiotics in Crohn's disease and in pouch-itis.

**62**

[45] Rayes N, Seehofer D, Hansen S, Boucsein K, Müller AR, Serk S, et al. Early enteral supply of lactobacillus and fiber versus selective bowel decontamination: A controlled trial in liver transplant recipients. Transplantation. 2002;**74**(1):123-128 12134110

[46] Waigankar SS, Patel V. Role of probiotics in urogenital healthcare. Journal of Midlife Health. 2011;**2**:5-10. DOI: 10.4103/0976-7800.83253

[47] Petricevic L, Domig K, Nierscher F, Sandhofer M, Fidesser M, Krondorfer I, et al. Characterisation of the vaginal *Lactobacillus* microbiota associated with preterm delivery. Scientific Reports. 2014;**4**:1-6 (article number: 5136). DOI: 10.1038/srep05136

[48] Commane D, Hughes R, Shortt C, Rowland I. The potential mechanisms involved in the anti-carcinogenic action of probiotics. Mutation Research. 2005;**591**:276-289. DOI: 10.1016/j. mrfmmm.2005.02.027

[49] Reid G, Bruce A. Probiotics to prevent urinary tract infections: The rationale and evidence. World Journal of Urology. 2006;**24**:28-32. DOI: 10.1007/ s00345-005-0043-1

[50] Kerry GR, Patra JK, Gouda S, Park Y, Shin HS, Das G. Benefaction of probiotics for human health: A review. Journal of Food and Drug Analysis. 2018;**26**(3):927-939. DOI: 10.1016/j. jfda.2018.01.002

[51] Ghosh AR. Appraisal of probiotics and prebiotics in gastrointestinal infections. Webmed Central Gastroenterology. 2012;**3**(10):WMC003796. DOI: 10.9754/ journal.wmc.2012.003796

[52] Homayouni A, Payahoo L, Azizi A. Effects of probiotics on lipid profile: A review. American Journal of Food Technology. 2012;**7**(5):251-265. DOI: 10.3923/ajft.2012.251.265

[53] Ooi LG, Liong MT. Cholesterollowering effects of probiotics andprebiotics: A review of *in vivo* and *in vitro* findings. International Journal of Molecular Sciences. 2010;**11**:2499-2522. DOI: 10.3390/ijms11062499

[54] Ranjbar F, Akbarzadeh F, Homayouni A. Probiotics usage in heart disease and psychiatry, chapter 61, in book: Probiotics, Prebiotics, and Synbiotics Bioactive Foods in Health Promotion: Probiotics and Prebiotics, Edited by R. Ross Watson and V. R. Preedy. Elsevier Inc., Academic Press, London, UK, 2016, pp.807-811

[55] Burton J, Drummond B, Chilcott C, Tagg J, Thomson W, Hale J, et al. Influence of the probiotic *Streptococcus salivarius* strain M18 on indices of dental health in children: A randomized double-blind,placebo-controlled trial. Journal of Medical Microbiology. 2013;**62**:875-884. DOI: 10.1099/ jmm.0.056663-0

[56] Hillma J, McDonell E, Hillman C, Zahradnik R, Soni M. Safety assessment of ProBiora3, a probiotic mouthwash: Subchronic toxicity study in rats. International Journal of Toxicology. 2009;**28**:357-367. DOI: 10.1177/1091581809340705

[57] Bowen DM. Probiotics and oral health. Journal of Dental Hygiene. 2013;**87**:5-9

[58] Daliria EBM, Lee BH. New perspectives on probiotics in health and disease. Food Science and Human Wellness. 2015;**4**(2):56-65. DOI: 10.1016/j.fshw.2015.06.002

[59] Vonk RJ, Reckman GA, Harmsen HJ, Priebe MG. Probiotics and lactose intolerance. In: Rigobelo EC, editor. Probiotics. Rijeka, Crotatia: InTech; 2012. DOI: 10.5772/51424

[60] Vidya S, Thiruneelakandan G. Probiotic potentials of lactobacillus and its anti-cancer activity. International Journal of Current Research. 2015;**7**:20680-20684

[61] Kahouli I, Malhotra M, Alaoui-Jamali MA, Prakash S. *In-vitro* characterization of the anti-cancer activity of the probiotic bacterium *Lactobacillus fermentum* NCIMB 5221 and potential against colorectal cancer cells. Journal of Cancer Science and Therapy. 2015;**7**:224-235. DOI: 10.4172/1948-5956.1000354

[62] Pandey KR, Naik SR, Vakil BV. Probiotics, prebiotics and synbiotics—A review. Journal of Food Science and Technology. 2015;**52**(12):7577-7587. DOI: 10.1007/ s13197-015-1921-1

[63] Lee JH, Nam SH, Seo WT, Yun HD, Hong SY, Kim MK, et al. The production of surfactin during the fermentation of *cheonggukjang* by potential probiotic B*acillus subtilis* CSY191 and the resultant growth suppression of MCF-7 human breast cancer cells. Food Chemistry. 2012;**131**(4):1347-1354. DOI: 10.1016/j. foodchem.2011.09.133

[64] Pokusaeva K, Fitzgerald GF, van Sinderen D. Carbohydrate metabolism in Bifidobacteria. Genesis Nutrition. 2011;**6**(3):285-306. DOI: 10.1007/ s12263-010-0206-6

[65] Kuo SM. The interplay between fiber and the intestinal microbiome in the inflammatory response. Advances in Nutrition: Journal of Internal Medicine. 2013;**4**(1):16-28. DOI: 10.3945/ an.112.003046

[66] Mojka K. Probiotyki, prebiotyki i synbiotyki—Charakterystyka i funkcje. Problemy Higieny i Epidemiologii. 2014;**95**:541-549

[67] Socha P, Stolarczyk M, Socha J. Wpływ probiotyków i prebiotyków na gospodarke˛ lipidowa˛. Pediatria Współczesna Gastroenterologia Hepatologia I Żywienie Dziecka. 2002;**4**:85-88

[68] Jakubczyk E, Kosikowska M. Nowa generacja mlecznych produktów fermentowanych z udziałem probiotyków i prebiotyków, produkty synbiotyczne. Przegląd Mleczarski. 2000;**12**:397-400

[69] Buddington KK, Danohoo JB, Buddington RK. Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumour inducers. The Journal of Nutrition. 2002;**132**:472- 477. DOI: 10.1093/jn/132.3.472

[70] Pokusaeva K, Fitzgerald GF, Sinderen D. Carbohydrate metabolism in bifidobacteria. Genes & Nutrition. 2011;**6**:285-306. DOI: 10.1007/ s12263-010-0206-6

[71] Langen LV, Mirjam AC, Dieleman LA. Prebiotics in chronic intestinal inflammation. Inflammatory Bowel Diseases. 2009;**15**(3):454-462. DOI: 10.1002/ibd.20737

[72] Lindsay JO, Whelan K, Stagg AJ, Gobin P, HO A-H, Rayment N, et al. Clinical, microbiological, and immunological effects of fructooligosaccharide in patients with Crohn's disease. Gut. 2006;**55**(3):348-355. DOI: 10.1136/gut.2005.074971

[73] Tufarelli V, Laudadio V. An overview on the functional food concept: Prospectives and applied researches in probiotics, prebiotics and synbiotics. Journal of Experimental Biology and Agricultural Sciences. 2016;**4**:274-278. DOI: 10.18006/2016.4(3S).273.278

**65**

*Probiotic, Prebiotic and Synbiotic Products in Human Health*

*DOI: http://dx.doi.org/10.5772/intechopen.81553*

[74] Cencic A, Chingwaru W. The role of functional foods, nutraceuticals, and food supplements in intestinal health. Nutrients. 2010;**2**(6):611-625. DOI:

[75] Zhang MM, Cheng JQ, Lu YR, Yi ZH, Yang P, Wu XT. Use of pre-, pro-and synbiotics in patients with acute pancreatitis: A metaanalysis. World Journal of Gastroenterology. 2010;**16**(31):3970. DOI: 10.3748/wjg.v16.

[76] Eslamparast T, Poustchi H, Zamani F, Sharafkhah M, Malekzadeh

R, Hetmatdoost A. Synbiotic

10.3945/ajcn.113.068890

2006;**43**(3):235-240

supplementation in nonalcoholic fatty liver disease: A randomized, doubleblind, placebo-controlled pilot study. The American Journal of Clinical Nutrition. 2014;**99**:535-542. DOI:

[77] Panda AK, Rao SVR, Raju MV, Sharma SR. Dietary supplementation of Lactobacillus sporogenes on performance and serum biochemicolipid profile of broiler chickens. The Journal of Poultry Science.

10.3390/nu2060611

i31.3970

*Probiotic, Prebiotic and Synbiotic Products in Human Health DOI: http://dx.doi.org/10.5772/intechopen.81553*

[74] Cencic A, Chingwaru W. The role of functional foods, nutraceuticals, and food supplements in intestinal health. Nutrients. 2010;**2**(6):611-625. DOI: 10.3390/nu2060611

*Frontiers and New Trends in the Science of Fermented Food and Beverages*

[66] Mojka K. Probiotyki, prebiotyki i synbiotyki—Charakterystyka i funkcje. Problemy Higieny i Epidemiologii.

[67] Socha P, Stolarczyk M, Socha J. Wpływ probiotyków i prebiotyków na gospodarke˛ lipidowa˛. Pediatria Współczesna Gastroenterologia Hepatologia I Żywienie Dziecka.

[68] Jakubczyk E, Kosikowska M. Nowa generacja mlecznych produktów

[69] Buddington KK, Danohoo JB, Buddington RK. Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumour inducers. The Journal of Nutrition. 2002;**132**:472-

477. DOI: 10.1093/jn/132.3.472

[70] Pokusaeva K, Fitzgerald GF, Sinderen D. Carbohydrate metabolism in bifidobacteria. Genes & Nutrition.

2011;**6**:285-306. DOI: 10.1007/

[71] Langen LV, Mirjam AC, Dieleman LA. Prebiotics in chronic intestinal inflammation. Inflammatory Bowel Diseases. 2009;**15**(3):454-462. DOI:

[72] Lindsay JO, Whelan K, Stagg AJ, Gobin P, HO A-H, Rayment N, et al. Clinical, microbiological, and immunological effects of fructooligosaccharide in patients with Crohn's disease. Gut. 2006;**55**(3):348-355. DOI:

[73] Tufarelli V, Laudadio V. An overview

on the functional food concept: Prospectives and applied researches in probiotics, prebiotics and synbiotics. Journal of Experimental Biology and Agricultural Sciences. 2016;**4**:274-278. DOI: 10.18006/2016.4(3S).273.278

s12263-010-0206-6

10.1002/ibd.20737

10.1136/gut.2005.074971

probiotyków i prebiotyków, produkty synbiotyczne. Przegląd Mleczarski.

fermentowanych z udziałem

2014;**95**:541-549

2002;**4**:85-88

2000;**12**:397-400

[59] Vonk RJ, Reckman GA, Harmsen HJ, Priebe MG. Probiotics and lactose intolerance. In: Rigobelo EC, editor. Probiotics. Rijeka, Crotatia: InTech;

[60] Vidya S, Thiruneelakandan G. Probiotic potentials of lactobacillus and its anti-cancer activity. International

Journal of Current Research.

[61] Kahouli I, Malhotra M, Alaoui-Jamali MA, Prakash S. *In-vitro* characterization of the anti-cancer activity of the probiotic bacterium *Lactobacillus fermentum* NCIMB 5221 and potential against colorectal cancer cells. Journal of Cancer Science and Therapy. 2015;**7**:224-235. DOI: 10.4172/1948-5956.1000354

[62] Pandey KR, Naik SR, Vakil BV. Probiotics, prebiotics and synbiotics—A review. Journal of Food Science and Technology. 2015;**52**(12):7577-7587. DOI: 10.1007/

[63] Lee JH, Nam SH, Seo WT, Yun HD, Hong SY, Kim MK, et al. The production of surfactin during the fermentation of *cheonggukjang* by potential probiotic B*acillus subtilis* CSY191 and the resultant growth suppression of MCF-7 human breast cancer cells. Food Chemistry. 2012;**131**(4):1347-1354. DOI: 10.1016/j.

[64] Pokusaeva K, Fitzgerald GF, van Sinderen D. Carbohydrate metabolism in Bifidobacteria. Genesis Nutrition. 2011;**6**(3):285-306. DOI: 10.1007/

[65] Kuo SM. The interplay between fiber and the intestinal microbiome in the inflammatory response. Advances in Nutrition: Journal of Internal Medicine.

2013;**4**(1):16-28. DOI: 10.3945/

s13197-015-1921-1

foodchem.2011.09.133

s12263-010-0206-6

an.112.003046

2015;**7**:20680-20684

2012. DOI: 10.5772/51424

**64**

[75] Zhang MM, Cheng JQ, Lu YR, Yi ZH, Yang P, Wu XT. Use of pre-, pro-and synbiotics in patients with acute pancreatitis: A metaanalysis. World Journal of Gastroenterology. 2010;**16**(31):3970. DOI: 10.3748/wjg.v16. i31.3970

[76] Eslamparast T, Poustchi H, Zamani F, Sharafkhah M, Malekzadeh R, Hetmatdoost A. Synbiotic supplementation in nonalcoholic fatty liver disease: A randomized, doubleblind, placebo-controlled pilot study. The American Journal of Clinical Nutrition. 2014;**99**:535-542. DOI: 10.3945/ajcn.113.068890

[77] Panda AK, Rao SVR, Raju MV, Sharma SR. Dietary supplementation of Lactobacillus sporogenes on performance and serum biochemicolipid profile of broiler chickens. The Journal of Poultry Science. 2006;**43**(3):235-240

**67**

**Chapter 5**

Values

**Abstract**

**1. Introduction**

fermented food condiments among others.

good sources of proteins and vitamins [1, 4].

African Fermented Food

Condiments: Microbiology

Impacts on Their Nutritional

*Nurudeen Ayoade Olasupo and Princewill Chimezie Okorie*

Fermented food flavoring condiments are products usually derived from the fermentative activities of microorganisms on vegetable proteins of legumes or oil seeds. Africa is a continent that is endowed with many fermented food condiments. These condiments, apart from their flavoring properties, serve as a cheap source of plant protein to the populace, especially the rural dweller whose staple foods are mainly carbohydrate based. The production dynamics of these condiments vary from country to country. However, the microbial interplay during their production and their nutritional qualities appear to be same. This chapter seeks to evaluate the range of substrates employed in the production of fermented condiments of African origin, the microbial interplay in their production and their nutritional values.

**Keywords:** microbiology, nutrition, fermentation, African fermented condiments

Fermented foods constitute a significant component of African diets. There are many fermented foods known in Africa. These foods are classified into five major categories based on the substrate from which they are derived [1] and they include

Condiment is defined as a spice, sauce or other food preparation that is added to food to impart a particular flavor or enhance its taste (example salt). Fermented food flavoring condiments are products usually derived from the fermentative activities of microorganisms on vegetable proteins of legumes or oil seeds origin [2, 3]. They include *iru* from Africa locust bean, *ugba* from African oil bean seed and *ogiri* from melon seeds among others. These fermented food condiments are known to be

The use of fermented vegetable proteins as seasonings is wide spread in Africa, especially among the rural dwellers. In West Africa, some of the common fermented vegetable condiments include *iru or dawadawa* from locust bean (*Parkia biglobosa*) (**Figure 1**), *ogiri* from melon seeds (*Citrullus vulgaris*) (**Figure 2**), *daddawa* from soybean (*Glycine max*), *soumbala* from soybean (*Glycine max*) (**Figure 3**), *ugba* from African oil bean seed (*Pentaclethra macrophylla*) (**Figure 4**) and *owoh* from

## **Chapter 5**
