Metabolomics: New Insights into Biology

**3**

**Chapter 1**

**Abstract**

Biosurfactants from Marine

biological properties, and potential biotechnological applications.

natural tensioactive, biotechnology

**1. Introduction**

**Keywords:** bacteria, surfactants, aquatic environments, biodegradability,

In aquatic ecosystems, marine microorganisms have developed unique metabolic and physiological capabilities to adapt to diverse habitats which cover a wide range of thermal, pressure, salinity, pH, and nutrient conditions [1]. In this way, the symbiotic association and the interaction of biological systems with bacteria have induced the production of a variety of bioactive compounds such as biosurfactants (BSs), antibiotics, enzymes, vitamins, [2] and more than 10,000 metabolites with a broad spectrum of biological activities that have been isolated from marine microbes. By evolution, bacteria have adapted themselves to feeding on waterimmiscible materials by producing and using surface-active products that help them in aqueous phase to adsorb, emulsify, wet, and disperse or solubilize waterimmiscible materials [3]. In this context, surface active metabolites as BSs have gained much attention because of their biodegradability, low toxicity, and ability to be produced from renewable and cheaper substrates, thus getting an important ecological role due to their structural, functional diversity, and the potential multidisciplinary applications in industrial and environmental fields. These features in

*Rosanna Floris, Carmen Rizzo and Angelina Lo Giudice*

The marine biosphere represents a yet underexploited natural source of bioactive compounds, mainly of microbial origin. Among them, biosurfactants (BSs) are functional molecules, which are attracting a great interest due to their biocompatibility, versatility, and applications in several biotechnological fields. BSs are surface active amphipathic compounds, containing both a hydrophilic and a hydrophobic moiety, which are grouped in low (glycolipids and lipopeptides) or high molecular weight (polymeric complexes) compounds. A number of environmental factors such as pH, salinity, temperature, and nutrient availability can affect microbial BS production. Marine microorganisms with different phylogenetic affiliations and isolated from several marine habitats (e.g., seawater, sediments, and higher organisms) worldwide (spanning from the Mediterranean Sea to Antarctica) have been reported as surfactant producers. However, most of the marine microbial world remains still unexplored. The present chapter aims at giving a general overview on the recent advances about BSs of marine origin, in order to enhance the knowledge inherent their production, chemical characterization and identification, interesting

Microorganisms

#### **Chapter 1**

## Biosurfactants from Marine Microorganisms

*Rosanna Floris, Carmen Rizzo and Angelina Lo Giudice*

#### **Abstract**

The marine biosphere represents a yet underexploited natural source of bioactive compounds, mainly of microbial origin. Among them, biosurfactants (BSs) are functional molecules, which are attracting a great interest due to their biocompatibility, versatility, and applications in several biotechnological fields. BSs are surface active amphipathic compounds, containing both a hydrophilic and a hydrophobic moiety, which are grouped in low (glycolipids and lipopeptides) or high molecular weight (polymeric complexes) compounds. A number of environmental factors such as pH, salinity, temperature, and nutrient availability can affect microbial BS production. Marine microorganisms with different phylogenetic affiliations and isolated from several marine habitats (e.g., seawater, sediments, and higher organisms) worldwide (spanning from the Mediterranean Sea to Antarctica) have been reported as surfactant producers. However, most of the marine microbial world remains still unexplored. The present chapter aims at giving a general overview on the recent advances about BSs of marine origin, in order to enhance the knowledge inherent their production, chemical characterization and identification, interesting biological properties, and potential biotechnological applications.

**Keywords:** bacteria, surfactants, aquatic environments, biodegradability, natural tensioactive, biotechnology

#### **1. Introduction**

In aquatic ecosystems, marine microorganisms have developed unique metabolic and physiological capabilities to adapt to diverse habitats which cover a wide range of thermal, pressure, salinity, pH, and nutrient conditions [1]. In this way, the symbiotic association and the interaction of biological systems with bacteria have induced the production of a variety of bioactive compounds such as biosurfactants (BSs), antibiotics, enzymes, vitamins, [2] and more than 10,000 metabolites with a broad spectrum of biological activities that have been isolated from marine microbes. By evolution, bacteria have adapted themselves to feeding on waterimmiscible materials by producing and using surface-active products that help them in aqueous phase to adsorb, emulsify, wet, and disperse or solubilize waterimmiscible materials [3]. In this context, surface active metabolites as BSs have gained much attention because of their biodegradability, low toxicity, and ability to be produced from renewable and cheaper substrates, thus getting an important ecological role due to their structural, functional diversity, and the potential multidisciplinary applications in industrial and environmental fields. These features in

addition to their stability at extremes of temperatures, pH, and salinity make them commercially optimal alternatives to their chemically synthesized counterparts. For this reason, different authors have tested BSs of marine origin for environmental applications such as bioremediation processes, dispersion of oil spills, and enhancement of oil recovery. Furthermore, some BSs play an essential role for the survival of microbial producers against other competing or dangerous microorganisms by acting as biocide agents [4], and some others have shown the ability to stimulate plant and animal defense responses against microbes [5]. By considering that, these aspects have been explored especially for terrestrial bacteria, in the light of the above said characteristics, BSs from marine bacteria have been getting attention more and more as new suitable alternatives to chemical surfactants of petroleum origin in the food, cosmetic, health care industries [6], synthetic medicines and can be safe and effective therapeutic agents in medicine.

The purpose of this chapter is to provide a comprehensive overview on the recent advances about different types of marine BSs, to highlight the recent studies on the new sources of production, and to focus on the state of art of the screening methodologies for the identification, characterization, and potential biotechnological applications.

#### **2. Biosurfactants**

BSs are secondary metabolism bacterial products which exhibit surface and emulsifying activities thanks to the hydrophobic part of the molecule and the hydrophilic water soluble end. They are produced extracellularly or as a part of the cell membrane by a variety of yeasts, bacteria, and filamentous fungi from various substrates as sugars, oils, alkanes, and wastes [7]. The name *surfactant* derives from their surface chemical action, as they tend to interact at the boundary level between two phases in a heterogeneous system by forming a film which can change the properties (wettability and surface energy) of the original surface. They are mainly classified in BSs acting by reducing surface tension at the airwater interface (biosurfactants), and BSs that reduce the interfacial tensions between immiscible liquids or at the solid-liquid interface (bioemulsifiers) [8]. The investigations on BS production from microorganisms are more and more frequent and are subdivided into three main steps: (1) the potential BS producer isolation; (2) the screening for BS production; and (3) the extraction and purification step, sometimes improved with the chemical characterization of molecules. According to Ref. [6], the producer isolation and detection are crucial phases in the research for new BSs, which have to be strongly related to the aim of the investigation. While the terrestrial sources have been extensively explored, the marine environments have been focused as potential optimal source only in recent decades. It has been reported that BS-producing bacteria are widely distributed in both contaminated and undisturbed water or soil [9, 10]. Indeed, the most exploited source of microbial BS producers is represented by sediment and water samples, with different types and levels of contamination [11–13]. As a matter of fact, it is retained that microbial degraders of insoluble substrates, that is, hydrocarbons or oils, are stimulated to produce secondary metabolites that are able to enhance the cellular uptake and degradation of such compounds. In some cases, enrichment cultures have been performed with natural samples in order to favor the isolation of potential producers [14, 15]. Despite the application of this principle has gained optimal results in the discovery of new marine producers and BSs [14, 16], some authors assessed that the BS production is not strictly linked

**5**

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

ments represent.

to the hydrocarbon uptake [17, 18]. Only recently, innovative marine sources of isolation has been proposed, and researchers have focused their studies on biological matrices, that is, filter-feeding organisms as host of microbial communities specialized in the production of secondary metabolites with functional roles. As reviewed in [6], BS producers with optimal potentialities have been isolated from polychaetes [19, 20], sponges [21–25], sea pens [26], cnidarians [27, 28], and fish [29]. A common characteristic of BSs is to relax or decrease surface tension and this increases solubility so that BSs may interact with the interfaces and affect the adhesion and the detachment of bacteria [4]. All these properties confer to the BS antibacterial, antifungal, and antiviral activities [30], in addition to the pollutants removal potential. For these reasons, particular growing interest at global level is centered into new and unexplored sources of BSs as marine and deep-sea environ-

**2.1 BS classification/types, census of BS marine microbial producers**

Surfactants are generally classified according to the nature of the charge on polar moiety: anionic (negatively charged), nonionic (polymerization products), cationic (positively charged), and amphoteric (both negatively and positively charged). They can be grouped into two categories: (a) low molecular mass molecules (rhamnolipids, sophorolipids, trehalose lipids, lipopeptides phospholipids, fatty acids, and neutral lipids) [31], which lower the surface and interfacial tensions and (b) high molecular polymer mass (high molecular weight polysaccharide, polysaccharide-protein complexes, lipopolysaccharides, and lipoproteins called emulsans), which bind tightly to surfaces [32]. Various microbial species produce different BSs [33]. The most known ones are glycolipids, a class of molecules made of mono-, di-, tri- and tetra-saccharides which include glucose, mannose, galactose, glucuronic acid, rhamnose, and galactose sulfate in combination with long-chain aliphatic acids or hydroxyaliphatic acids. Among glycolipids, rhamnolipids, and trehalolipids, sophorolipids are the most studied disaccharides. Rhamnolipids are known to be produced by the *Pseudomonas* genus [31, 34], while trehalolipids are generally produced by many members of the genera *Mycobacterium*, *Nocardia* and *Corynebacterium*, *Rhodococcus erythropolis*, and *Arthrobacter* sp. [35]. Finally, sophorolipids represent a group of BSs by *Candida* spp. [31]. Another class of surfactant compounds which received considerable attention is the fatty acids such as the phospholipids derived from alkane substrates. Indeed, they represent the major components of microbial membranes which are mainly produced by hydrocarbondegrading bacteria as *Acinetobacter* sp. *Rhodococcus erythropolis*, *Thiobacillus thiooxidans, Capnocytophaga* sp*., Penicillium spiculisporum,* and *Corynebacterium* sp*.,* or yeasts [32]. Moreover, a number of microorganisms (e.g., *Candida lipolytica* and *Saccharomyces cerevisiae*) with different taxonomic affiliations produce exocellular polymeric surfactants called bioemulsans, composed of polysaccharides, protein, lipopolysaccharides, and lipoproteins, among which the bioemulsans produced by different species of *Acinetobacter* are the most studied [36]. Different authors described microbial BSs from marine sources (sediments, corals, sponges, sea, and hot water springs) which include several lipopeptide antibiotics with potent surface-active properties [22, 37–41]. Some authors [22] detected carbohydrate, protein, and lipid contents (20 μg/0.1 ml, 35 μg/0.1 ml, and 573 μg/0.1 ml, respectively) in the analyzed BS, suggesting a chemical structure typical of a lipopeptidic compound for the sponge-associated marine actinomycetes *Nocardiopsis alba* MSA10. Further chemical analyses [Fourier-transformed infrared spectrophotometer analysis (FT-IR), nuclear magnetic resonance (NMR), and gas chromatography

#### *Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

*Metabolomics - New Insights into Biology and Medicine*

be safe and effective therapeutic agents in medicine.

cal applications.

**2. Biosurfactants**

addition to their stability at extremes of temperatures, pH, and salinity make them commercially optimal alternatives to their chemically synthesized counterparts. For this reason, different authors have tested BSs of marine origin for environmental applications such as bioremediation processes, dispersion of oil spills, and enhancement of oil recovery. Furthermore, some BSs play an essential role for the survival of microbial producers against other competing or dangerous microorganisms by acting as biocide agents [4], and some others have shown the ability to stimulate plant and animal defense responses against microbes [5]. By considering that, these aspects have been explored especially for terrestrial bacteria, in the light of the above said characteristics, BSs from marine bacteria have been getting attention more and more as new suitable alternatives to chemical surfactants of petroleum origin in the food, cosmetic, health care industries [6], synthetic medicines and can

The purpose of this chapter is to provide a comprehensive overview on the recent advances about different types of marine BSs, to highlight the recent studies on the new sources of production, and to focus on the state of art of the screening methodologies for the identification, characterization, and potential biotechnologi-

BSs are secondary metabolism bacterial products which exhibit surface and emulsifying activities thanks to the hydrophobic part of the molecule and the hydrophilic water soluble end. They are produced extracellularly or as a part of the cell membrane by a variety of yeasts, bacteria, and filamentous fungi from various substrates as sugars, oils, alkanes, and wastes [7]. The name *surfactant* derives from their surface chemical action, as they tend to interact at the boundary level between two phases in a heterogeneous system by forming a film which can change the properties (wettability and surface energy) of the original surface. They are mainly classified in BSs acting by reducing surface tension at the airwater interface (biosurfactants), and BSs that reduce the interfacial tensions between immiscible liquids or at the solid-liquid interface (bioemulsifiers) [8]. The investigations on BS production from microorganisms are more and more frequent and are subdivided into three main steps: (1) the potential BS producer isolation; (2) the screening for BS production; and (3) the extraction and purification step, sometimes improved with the chemical characterization of molecules. According to Ref. [6], the producer isolation and detection are crucial phases in the research for new BSs, which have to be strongly related to the aim of the investigation. While the terrestrial sources have been extensively explored, the marine environments have been focused as potential optimal source only in recent decades. It has been reported that BS-producing bacteria are widely distributed in both contaminated and undisturbed water or soil [9, 10]. Indeed, the most exploited source of microbial BS producers is represented by sediment and water samples, with different types and levels of contamination [11–13]. As a matter of fact, it is retained that microbial degraders of insoluble substrates, that is, hydrocarbons or oils, are stimulated to produce secondary metabolites that are able to enhance the cellular uptake and degradation of such compounds. In some cases, enrichment cultures have been performed with natural samples in order to favor the isolation of potential producers [14, 15]. Despite the application of this principle has gained optimal results in the discovery of new marine producers and BSs [14, 16], some authors assessed that the BS production is not strictly linked

**4**

to the hydrocarbon uptake [17, 18]. Only recently, innovative marine sources of isolation has been proposed, and researchers have focused their studies on biological matrices, that is, filter-feeding organisms as host of microbial communities specialized in the production of secondary metabolites with functional roles. As reviewed in [6], BS producers with optimal potentialities have been isolated from polychaetes [19, 20], sponges [21–25], sea pens [26], cnidarians [27, 28], and fish [29]. A common characteristic of BSs is to relax or decrease surface tension and this increases solubility so that BSs may interact with the interfaces and affect the adhesion and the detachment of bacteria [4]. All these properties confer to the BS antibacterial, antifungal, and antiviral activities [30], in addition to the pollutants removal potential. For these reasons, particular growing interest at global level is centered into new and unexplored sources of BSs as marine and deep-sea environments represent.

#### **2.1 BS classification/types, census of BS marine microbial producers**

Surfactants are generally classified according to the nature of the charge on polar moiety: anionic (negatively charged), nonionic (polymerization products), cationic (positively charged), and amphoteric (both negatively and positively charged). They can be grouped into two categories: (a) low molecular mass molecules (rhamnolipids, sophorolipids, trehalose lipids, lipopeptides phospholipids, fatty acids, and neutral lipids) [31], which lower the surface and interfacial tensions and (b) high molecular polymer mass (high molecular weight polysaccharide, polysaccharide-protein complexes, lipopolysaccharides, and lipoproteins called emulsans), which bind tightly to surfaces [32]. Various microbial species produce different BSs [33]. The most known ones are glycolipids, a class of molecules made of mono-, di-, tri- and tetra-saccharides which include glucose, mannose, galactose, glucuronic acid, rhamnose, and galactose sulfate in combination with long-chain aliphatic acids or hydroxyaliphatic acids. Among glycolipids, rhamnolipids, and trehalolipids, sophorolipids are the most studied disaccharides. Rhamnolipids are known to be produced by the *Pseudomonas* genus [31, 34], while trehalolipids are generally produced by many members of the genera *Mycobacterium*, *Nocardia* and *Corynebacterium*, *Rhodococcus erythropolis*, and *Arthrobacter* sp. [35]. Finally, sophorolipids represent a group of BSs by *Candida* spp. [31]. Another class of surfactant compounds which received considerable attention is the fatty acids such as the phospholipids derived from alkane substrates. Indeed, they represent the major components of microbial membranes which are mainly produced by hydrocarbondegrading bacteria as *Acinetobacter* sp. *Rhodococcus erythropolis*, *Thiobacillus thiooxidans, Capnocytophaga* sp*., Penicillium spiculisporum,* and *Corynebacterium* sp*.,* or yeasts [32]. Moreover, a number of microorganisms (e.g., *Candida lipolytica* and *Saccharomyces cerevisiae*) with different taxonomic affiliations produce exocellular polymeric surfactants called bioemulsans, composed of polysaccharides, protein, lipopolysaccharides, and lipoproteins, among which the bioemulsans produced by different species of *Acinetobacter* are the most studied [36]. Different authors described microbial BSs from marine sources (sediments, corals, sponges, sea, and hot water springs) which include several lipopeptide antibiotics with potent surface-active properties [22, 37–41]. Some authors [22] detected carbohydrate, protein, and lipid contents (20 μg/0.1 ml, 35 μg/0.1 ml, and 573 μg/0.1 ml, respectively) in the analyzed BS, suggesting a chemical structure typical of a lipopeptidic compound for the sponge-associated marine actinomycetes *Nocardiopsis alba* MSA10. Further chemical analyses [Fourier-transformed infrared spectrophotometer analysis (FT-IR), nuclear magnetic resonance (NMR), and gas chromatography

mass spectrometry (GC/MS)] were carried out by other scientists [21, 23, 28], and lipopeptidic BSs were detected for *Bacillus* spp. strains, in line with previous data on the neighboring cluster *Bacillus* as prominent lipopeptide producers. Interestingly, the lipopeptide identified in [23] was firstly demonstrated from *Brevibacterium* stain MSA13 (so-called *brevifactin*) and showed a different structure respect to that observed for other lipopeptides. Indeed, it was composed of a hydrophobic moiety of octadecanoic acid methyl ester and a peptide part contained a short sequence of four amino acids including pro-leu-gly-gly (**Figure 1**). Similarly, the possible production of novel BSs by *P. rettgeri*, *Psychrobacter* sp., and *B. anthracis* isolates was suggested by the analysis of FTIR spectra [28].

In the marine context, the group of glycolipids has been widely studied because they are produced by a broad spectrum of bacteria isolated from various marine matrices, both animals (Annelida, sea pen *Pteroeides*, and fish gut) and contaminated soils (Arctic and Antarctic sediments) [14, 19, 20, 24, 26, 29, 42]. A glycolipopeptide BS produced by the coral-associated *Bacillus* sp. E34 was identified by means of FT-IR analysis [27].

A list of various studied BS types with the respective microbial producers and the source of isolation is shown in **Table 1**.

Finally, other studies drew their attention on a group of heterogeneous high molecular weight bioactive compounds not strictly defined as biosurfactants but endowed with interfacial properties, called exopolysaccharides (EPSs), often originated from marine both prokaryotes and eukaryotes (cyanobacteria and microalgae) and extreme environments [43].

#### **Figure 1.**

*MS spectra of octadecanoic acid methyl ester (a), probable structure of octadecanoic acid methyl ester with peptide moiety (b), crystalline appearance of the recovered crystals of lipopeptide (MSA13) examined under a light microscope at 40× [23].*

**7**

**Figure 2.**

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

**3. Natural roles and biotechnological applications**

*ND: not determined.*

**Table 1.**

phase and their bioavailability for uptake by cells.

Biosurfactants have various functions which are often unique for the physiology and ecology of their microbial producers. As stated above, one of the most interesting roles from the environmental point of view is represented by the different strategies adopted by microorganisms to enhance the bioavailability and the access to hydrophobic compounds as carbon source [44]. A mechanism proposed by which hydrocarbons became incorporated within the hydrophobic core of the BS micelles is shown in **Figure 2**. This process studied with alkanes as model substrates and referred to as "micelle solubilization" [45], favors their dispersion into the aqueous

*Overview of various studied BS types with the respective marine producers and the source of isolation.*

The potential application of BSs in hydrocarbon bioremediation has been investigated for marine microorganisms from different origins and allowed to obtain interesting results. In this case, the use of contaminated samples for isolation of potential BS producers is extremely encouraged. Strains affiliated to *Rhodococcus* spp. were reported as capable of reduce surface tension in the presence of oily substrates, and the extracted BSs have been proved optimal enhancer

*Mechanism of hydrocarbon solubilization so called "micelle solubilization" within biosurfactant micelles:* 

*biosurfactants at the interface between the aqueous and hydrocarbon phases [45].*

**Table 1.**

*Metabolomics - New Insights into Biology and Medicine*

suggested by the analysis of FTIR spectra [28].

the source of isolation is shown in **Table 1**.

microalgae) and extreme environments [43].

means of FT-IR analysis [27].

mass spectrometry (GC/MS)] were carried out by other scientists [21, 23, 28], and lipopeptidic BSs were detected for *Bacillus* spp. strains, in line with previous data on the neighboring cluster *Bacillus* as prominent lipopeptide producers. Interestingly, the lipopeptide identified in [23] was firstly demonstrated from *Brevibacterium* stain MSA13 (so-called *brevifactin*) and showed a different structure respect to that observed for other lipopeptides. Indeed, it was composed of a hydrophobic moiety of octadecanoic acid methyl ester and a peptide part contained a short sequence of four amino acids including pro-leu-gly-gly (**Figure 1**). Similarly, the possible production of novel BSs by *P. rettgeri*, *Psychrobacter* sp., and *B. anthracis* isolates was

In the marine context, the group of glycolipids has been widely studied because they are produced by a broad spectrum of bacteria isolated from various marine matrices, both animals (Annelida, sea pen *Pteroeides*, and fish gut) and contaminated soils (Arctic and Antarctic sediments) [14, 19, 20, 24, 26, 29, 42]. A glycolipopeptide BS produced by the coral-associated *Bacillus* sp. E34 was identified by

A list of various studied BS types with the respective microbial producers and

Finally, other studies drew their attention on a group of heterogeneous high molecular weight bioactive compounds not strictly defined as biosurfactants but endowed with interfacial properties, called exopolysaccharides (EPSs), often originated from marine both prokaryotes and eukaryotes (cyanobacteria and

*MS spectra of octadecanoic acid methyl ester (a), probable structure of octadecanoic acid methyl ester with peptide moiety (b), crystalline appearance of the recovered crystals of lipopeptide (MSA13) examined under a* 

**6**

**Figure 1.**

*light microscope at 40× [23].*

*Overview of various studied BS types with the respective marine producers and the source of isolation.*

#### **3. Natural roles and biotechnological applications**

Biosurfactants have various functions which are often unique for the physiology and ecology of their microbial producers. As stated above, one of the most interesting roles from the environmental point of view is represented by the different strategies adopted by microorganisms to enhance the bioavailability and the access to hydrophobic compounds as carbon source [44]. A mechanism proposed by which hydrocarbons became incorporated within the hydrophobic core of the BS micelles is shown in **Figure 2**. This process studied with alkanes as model substrates and referred to as "micelle solubilization" [45], favors their dispersion into the aqueous phase and their bioavailability for uptake by cells.

The potential application of BSs in hydrocarbon bioremediation has been investigated for marine microorganisms from different origins and allowed to obtain interesting results. In this case, the use of contaminated samples for isolation of potential BS producers is extremely encouraged. Strains affiliated to *Rhodococcus* spp. were reported as capable of reduce surface tension in the presence of oily substrates, and the extracted BSs have been proved optimal enhancer

#### **Figure 2.**

*Mechanism of hydrocarbon solubilization so called "micelle solubilization" within biosurfactant micelles: biosurfactants at the interface between the aqueous and hydrocarbon phases [45].*

for n-hexadecane biodegradation at 13°C and of tetradecane [11, 14]. The studies described in [26] evaluated the BS production in the presence of hydrocarbonic substrates to test the potential of *Brevibacterium* and *Vibrio* spp. strains in the field of bioremediation and reported the diesel oil as better utilized carbon source. The hydrocarbon remediation aspect was also deeply studied by investigating *Joostella* sp. A8, *Alcanivorax* sp. A53, and *Pseudomonas* sp. A6 for BS production and diesel oil degradation in pure culture and co-culture conditions [46]. Interestingly, the biodegradation rates and the efficiency increased in co-culture of 99.4 and 99.2%, respectively. Furthermore, in Ref. [27], a glycolipidic biosurfactant produced by the coral-associated *Bacillus* with a removing capacity of about 45% was reported as optimal candidate for oil removal. The bioremediation potential of bacterial BSs has been investigated also in terms of chelating activity toward heavy metals, despite in this context, the literature is scarce. In fact, several bacteria have been reported as able to produce BSs in the presence of heavy metals such as Cd, Cu, and Zn, as *Joostella* sp. A8 and *Alcanivorax* sp. A53 [47, 48]. Despite, the heavy metal removal is an interesting topic, it remains still largely unexplored [47–49] for marine bacteria and need to be improved especially for metal chelation in aqueous systems. BS-producing microorganisms have developed other important physiological functions as response to needs and life style, such as antimicrobial activity (mainly lipopeptide and glycolipid surfactants), biofilm formation or processes of motility and colonization of surfaces. Immunomodulation and enzyme inhibition, have been detected for several BSs from marine environments or not. The antimicrobial activity of BS has been studied *in vivo* and *in vitro* and a broad spectrum of this activity against Gram-positive, Gram-negative, fungi, viruses, algae, etc., so as different modes of action were detected [5]. Both lipopeptides and glycolipids of marine origin have been proved as active against several bacterial pathogens. Indeed, the lipopeptide produced by *N. alba* [22] exhibited antibacterial activity against *E. faecalis*, *B. subtilis*, and the pathogenic yeast *C. albicans*. Similarly, the sponge-associated *Brevibacterium aureum* MSA13 and *Brachybacterium paraconglomeratum* MSA21 were proved as BS producers with a wide antibacterial activity toward several pathogens such as *B. subtilis*, *C. albicans E. coli*, *E. faecalis*, *K. pneumonia, M. luteus*, *P. aeruginosa*, *P. mirabilis*, *Streptococcus* sp., *S. aureus,* and *S. epidermidis* [23, 24]. The antibacterial and antifungal activities were also found in the BSs produced by *Halobacterium salinarum* [50]. It is quite fascinating the mechanism by which BS producers act. Various studies observed that the formation of a film on an interface after the excretion of a BS determines the attachment of certain microorganisms to the interface while inhibiting others. Therefore, it can be stated that some microorganisms can use their BS to regulate the cell surface properties in order to attach or to detach from surfaces according to need. In this respect, some authors [51] reported about studies on the mechanism by which bioemulsifier producing microorganisms regulate biofilm formation. Interestingly, the biofilm formation inhibition of *P. aeruginosa* ATCC10145 is highlighted and this seems to be determined to the BSs produced by a coral mucus associated strains [28]. According to [49], the detection of microorganisms able to produce BSs with such activities is fundamental for a reduced utilization of synthetic surfactants, and it favors the increase of biodegradable compounds. Besides, the existence of a horizontal transfer of high molecular weight emulsifiers from the producing microorganisms to heterologous bacteria was highlighted. In this case, the first step of this process is to bind to the surface of a group of bacteria by changing their surface properties in order to transport the emulsifier into the recipient cells. This has significant ecological implications for building a network of microbial BS producing strains in natural microbial communities. Last but not least, a new role for rhamnolipids in stimulation of plant and animal defense responses has emerged [5].

**9**

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

more specific immune response is determined [5, 52].

sample, and simple and easily available equipment [56].

A second group of screening tests is differently based on the evaluation of emulsifying activity and is generally carried out with some modifications, with regards to volume of culture/supernatant, to the hydrocarbon used as test, and to vortexing time. The most used tests are the emulsifying activity test, based on a quick observation of emulsion occurrence [60] and the E24 index detection, based on the occurrence of emulsions stable over the time. It was applied in many screenings for marine BS producers [11–13, 19–25, 28, 30, 46–48, 50], and most authors reported some modifications: it was tested with cell broth instead of cell-free supernatant, whereas kerosene has been replaced in some cases by other hydrophobic compounds, for example, hexadecane, crude oil, vegetable oil, and diesel oil [13]. Surface activity and emulsification capacity do not always correlate [56]. Indeed, different studies [7, 14, 19] observed and explained this aspect considering that some BSs might stabilize (emulsifiers) or destabilize (de-emulsifiers) the emulsion so that the emulsification test alone fails to identify compounds with surfactant activity which destabilizes the emulsions. However, while the surface activity assay could give just an indication of BS production, the detection of stable emulsion

In particular, rhamnolipids have been demonstrated to have a direct biocide action on bacteria and fungi and to increase the susceptibility of certain Gram-positive and Gram-negative bacteria to specific antibiotics. Indeed, these biomolecules have been known as exotoxins produced by pathogens and are described as a new class of microbe-associated molecular patterns (MAMPs) that is, molecular signals which activate a large battery of defense-related genes of plants and animals by which a

The screening procedure is constantly based on the performance of a selection of standard tests, differently chosen by authors with the attempt to carry out a fast and economic selective procedure. The BS chemical diversity is very wide and also different in their properties; thus, the screening procedure has to probe all the multifaceted activities, from the interfacial to the emulsifying, from the chelating to the foaming stabilization functions, and so on. The interfacial actions are generally explored by direct measurements of surface tension, through the evaluation of the force required to detach a ring or loop of wire (Du-Noüy method), or a platinum plate (Wilhelmy plate method) from an interface or surface [53, 54]. These methods ensure the advantages of accuracy and easiness, despite they require specialized equipment, and the impossibility to perform the measurements on different samples simultaneously. Other direct surface tension measurements have been reported, but they are actually considered not recommendable for an efficient screening procedure [55, 56]. The surface tension evaluation by direct measurement is one of the most commonly test reported for marine isolates [11, 15, 19, 20, 25, 26, 28, 47]. Many screening methods are based on indirect measure of surface/interfacial tension, such as drop collapse method, titled glass slide test, oil spreading assay, penetration assay, and microplate assay. The main advantages are represented by the possibility to screen more samples in a quick way, although a low sensitivity due to the strong dependence on BS concentration was highlighted. The methods are indeed based on distortion visual effects caused by the BS presence and are generally performed on supernatants and some authors suggested to color them in order to evidence the visual effects [57–59]. Among them, the oil spreading and the drop collapse assays are the most reported for marine bacteria [11–13, 21–24, 26, 28, 30, 50] for its rapidity and easiness, in addition to the requirement of small volume of

**4. Methods and screening procedure for testing BS production**

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

*Metabolomics - New Insights into Biology and Medicine*

for n-hexadecane biodegradation at 13°C and of tetradecane [11, 14]. The studies described in [26] evaluated the BS production in the presence of hydrocarbonic substrates to test the potential of *Brevibacterium* and *Vibrio* spp. strains in the field of bioremediation and reported the diesel oil as better utilized carbon source. The hydrocarbon remediation aspect was also deeply studied by investigating *Joostella* sp. A8, *Alcanivorax* sp. A53, and *Pseudomonas* sp. A6 for BS production and diesel oil degradation in pure culture and co-culture conditions [46]. Interestingly, the biodegradation rates and the efficiency increased in co-culture of 99.4 and 99.2%, respectively. Furthermore, in Ref. [27], a glycolipidic biosurfactant produced by the coral-associated *Bacillus* with a removing capacity of about 45% was reported as optimal candidate for oil removal. The bioremediation potential of bacterial BSs has been investigated also in terms of chelating activity toward heavy metals, despite in this context, the literature is scarce. In fact, several bacteria have been reported as able to produce BSs in the presence of heavy metals such as Cd, Cu, and Zn, as *Joostella* sp. A8 and *Alcanivorax* sp. A53 [47, 48]. Despite, the heavy metal removal is an interesting topic, it remains still largely unexplored [47–49] for marine bacteria and need to be improved especially for metal chelation in aqueous systems. BS-producing microorganisms have developed other important physiological functions as response to needs and life style, such as antimicrobial activity (mainly lipopeptide and glycolipid surfactants), biofilm formation or processes of motility and colonization of surfaces. Immunomodulation and enzyme inhibition, have been detected for several BSs from marine environments or not. The antimicrobial activity of BS has been studied *in vivo* and *in vitro* and a broad spectrum of this activity against Gram-positive, Gram-negative, fungi, viruses, algae, etc., so as different modes of action were detected [5]. Both lipopeptides and glycolipids of marine origin have been proved as active against several bacterial pathogens. Indeed, the lipopeptide produced by *N. alba* [22] exhibited antibacterial activity against *E. faecalis*, *B. subtilis*, and the pathogenic yeast *C. albicans*. Similarly, the sponge-associated *Brevibacterium aureum* MSA13 and *Brachybacterium paraconglomeratum* MSA21 were proved as BS producers with a wide antibacterial activity toward several pathogens such as *B. subtilis*, *C. albicans E. coli*, *E. faecalis*, *K. pneumonia, M. luteus*, *P. aeruginosa*, *P. mirabilis*, *Streptococcus* sp., *S. aureus,* and *S. epidermidis* [23, 24]. The antibacterial and antifungal activities were also found in the BSs produced by *Halobacterium salinarum* [50]. It is quite fascinating the mechanism by which BS producers act. Various studies observed that the formation of a film on an interface after the excretion of a BS determines the attachment of certain microorganisms to the interface while inhibiting others. Therefore, it can be stated that some microorganisms can use their BS to regulate the cell surface properties in order to attach or to detach from surfaces according to need. In this respect, some authors [51] reported about studies on the mechanism by which bioemulsifier producing microorganisms regulate biofilm formation. Interestingly, the biofilm formation inhibition of *P. aeruginosa* ATCC10145 is highlighted and this seems to be determined to the BSs produced by a coral mucus associated strains [28]. According to [49], the detection of microorganisms able to produce BSs with such activities is fundamental for a reduced utilization of synthetic surfactants, and it favors the increase of biodegradable compounds. Besides, the existence of a horizontal transfer of high molecular weight emulsifiers from the producing microorganisms to heterologous bacteria was highlighted. In this case, the first step of this process is to bind to the surface of a group of bacteria by changing their surface properties in order to transport the emulsifier into the recipient cells. This has significant ecological implications for building a network of microbial BS producing strains in natural microbial communities. Last but not least, a new role for rhamnolipids in stimulation of plant and animal defense responses has emerged [5].

**8**

In particular, rhamnolipids have been demonstrated to have a direct biocide action on bacteria and fungi and to increase the susceptibility of certain Gram-positive and Gram-negative bacteria to specific antibiotics. Indeed, these biomolecules have been known as exotoxins produced by pathogens and are described as a new class of microbe-associated molecular patterns (MAMPs) that is, molecular signals which activate a large battery of defense-related genes of plants and animals by which a more specific immune response is determined [5, 52].

#### **4. Methods and screening procedure for testing BS production**

The screening procedure is constantly based on the performance of a selection of standard tests, differently chosen by authors with the attempt to carry out a fast and economic selective procedure. The BS chemical diversity is very wide and also different in their properties; thus, the screening procedure has to probe all the multifaceted activities, from the interfacial to the emulsifying, from the chelating to the foaming stabilization functions, and so on. The interfacial actions are generally explored by direct measurements of surface tension, through the evaluation of the force required to detach a ring or loop of wire (Du-Noüy method), or a platinum plate (Wilhelmy plate method) from an interface or surface [53, 54]. These methods ensure the advantages of accuracy and easiness, despite they require specialized equipment, and the impossibility to perform the measurements on different samples simultaneously. Other direct surface tension measurements have been reported, but they are actually considered not recommendable for an efficient screening procedure [55, 56]. The surface tension evaluation by direct measurement is one of the most commonly test reported for marine isolates [11, 15, 19, 20, 25, 26, 28, 47]. Many screening methods are based on indirect measure of surface/interfacial tension, such as drop collapse method, titled glass slide test, oil spreading assay, penetration assay, and microplate assay. The main advantages are represented by the possibility to screen more samples in a quick way, although a low sensitivity due to the strong dependence on BS concentration was highlighted. The methods are indeed based on distortion visual effects caused by the BS presence and are generally performed on supernatants and some authors suggested to color them in order to evidence the visual effects [57–59]. Among them, the oil spreading and the drop collapse assays are the most reported for marine bacteria [11–13, 21–24, 26, 28, 30, 50] for its rapidity and easiness, in addition to the requirement of small volume of sample, and simple and easily available equipment [56].

A second group of screening tests is differently based on the evaluation of emulsifying activity and is generally carried out with some modifications, with regards to volume of culture/supernatant, to the hydrocarbon used as test, and to vortexing time. The most used tests are the emulsifying activity test, based on a quick observation of emulsion occurrence [60] and the E24 index detection, based on the occurrence of emulsions stable over the time. It was applied in many screenings for marine BS producers [11–13, 19–25, 28, 30, 46–48, 50], and most authors reported some modifications: it was tested with cell broth instead of cell-free supernatant, whereas kerosene has been replaced in some cases by other hydrophobic compounds, for example, hexadecane, crude oil, vegetable oil, and diesel oil [13]. Surface activity and emulsification capacity do not always correlate [56]. Indeed, different studies [7, 14, 19] observed and explained this aspect considering that some BSs might stabilize (emulsifiers) or destabilize (de-emulsifiers) the emulsion so that the emulsification test alone fails to identify compounds with surfactant activity which destabilizes the emulsions. However, while the surface activity assay could give just an indication of BS production, the detection of stable emulsion

index correlates to the surfactant concentration. According to the Refs. [20, 25], the surface tension measurement and the emulsification activity assays could be complementary and represent the basic tests to include in a screening procedure, allowing to detect both low molecular mass BSs with efficiency in surface and interfacial tension reduction and high molecular mass BSs more effective as emulsion stabilizers. In addition to the above standard screening tests, several authors reported the use of specific assays, the cetyltrimethylammonium bromide (C-TAB) agar plate assay, and the blood agar assay, useful to detect anionic BSs, but not enough sensitive to detect BS producers. The first one is based on the interaction between anionic surfactants eventually present and the cationic surfactant cetyltrimethylammonium bromide contained in a methylene blue stained mineral salts agar plate; the consequent creation of a blue dark halo around the colonies detects the presence of the BS producers. The blood agar assay is differently based on the hemolytic actions of biosurfactants—α, β, and γ hemolysis—on solid medium containing defibrinated blood as greenish or clarification halos around the bacterial colonies. The use of these specific assays have been reported in most of the above mentioned references, but it has been reduced over the years, because some authors signaled the possible harmfulness toward bacterial growth, the low specificity, and the possible occurrence of false positive/negative. In [19, 20], the two assays are performed on 69 and 96 isolates of different marine origin, respectively, suggesting the use of such tests as integration of a more deep screening procedure. To the list of screening test, it is necessary to mention some other tests with important implications in the screening quality and for considerations about bioremediation purposes. In [46], the authors reported the BS-mediated hydrocarbon degradation by *Joostella* sp. A8 grown in pure culture and consortia, as result of a BS production monitoring in which the bacterial adhesion to hydrocarbons assay (BATH assay) was included. This is a simple photometrical assay described for the first time in Ref. [61] for indirect evaluation of BS production by measuring the hydrophobicity of bacteria. The use of such test in relation to BS production is still a debating issue in this field, because the authors are divided between those who believes that a correlation between cell hydrophobicity, biodegradation efficiency, and BS production exists [62, 63] and who instead claims that the changes in cellular surface properties could be affected by several parameters and may not be necessarily associated to biodegradation ability [64]. Finally, some other minor tests have been reported by several authors for BS production by marine bacteria, but their efficiency have to be improved: this is the case of penetration assay [26], the hydrocarbon overlay agar method [13, 30], tilted glass slide test (TGS test) [30], and lipase activity [21–24, 28]. A number of additional screening tests are well reviewed in [59].

#### **4.1 BS production conditions**

There are two primary pathways for bacterial BS biosynthesis: the way of hydrocarbons and the way of carbohydrates [9, 14], and their production is influenced by the availability, types of carbon sources, and the balance between carbon-nitrogen and other limiting nutrients [10]. The effects of several parameters on marine bacterial BS production have been explored by several authors, with particular focus on carbon source, temperature, pH, and NaCl concentration [20, 22–27]. The bacterial BS production has been generally observed at early stationary phase of growth [21, 22, 44, 65], or at exponential phase [25, 27]. In Ref. [22], the authors hypnotized that *N. alba* releases a cell bound biosurfactant into the culture broth which leads to an increase in extra cellular BSs. Moreover, the same authors evidenced a good BS production by the strains at all the tested conditions, even if they detected for *Nocardiopsis alba* the optimum conditions for BS production at pH 7, temperature

**11**

60 to 77%.

**5. Genetic regulation**

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

30°C, and 1% salinity with glucose and peptone supplementation as carbon and nitrogen sources, respectively. In [21], the authors confirmed that carbon source and its concentration are affecting parameters for BS production, and established that glycerol, peptone, ferrous sulfate, and incubation time exhibited significant effect, with optimum levels as pH 7, temperature at 37°C, and salt concentration 2% for *B. amyloliquefaciens.* In particular, the authors reported that glycerol used as a carbon source showed the highest BS production (up to 6.76 g/l). According to Ref. [24], the studies performed a more deep optimization analysis to investigate the BS production by the sponge-associated *Brachybacterium paraconglomeratum* and indicated a yeast extract nitrogen source as factor enhancing up to 60% biosynthesis activity. Positive effects were also exhibited by the supplementation with 2% of NaCl, a pH level of 7.0, and a 30°C temperature. Moreover, asparagine resulted highly effective for BS production followed by glycine, leucine, and valine, and a requirement of CuSO4 as a metal supplement was requested by the strain for optimum production of BS. In [20], the authors investigated the influence of salinity and temperature on the BS production by polychaete-associated isolates and showed that the NaCl concentration strongly influenced the surface tension reducing activity and emulsification rate in major level rather than temperature. Nevertheless, the authors reported that the strain *Marinobacter* sp. A1 exhibited the best performances at 15°C and in the absence of NaCl, by suggesting that limited conditions could act as stimulating factors. Interestingly, several researchers [19, 20, 25] also reported the BS production in the presence of hydrocarbons. The BS synthesis under solid state cultivation (SSC) was investigated for *Brevibacterium aureum* MSA13, which increased its production with pre-treated molasses, glucose, and acrylamide [23] as substrates. The report is interesting because it represents the first attempt in which acrylamide was used as nitrogen source, and the SSC conditions have been proven to be a preferred bioprocess for the BS production and optimization. In [27], the authors suggested a parallel relationship between bacterial growth and productivity and tested several carbon sources (sugar cane molasses, olive oil, corn oil, motor oil, and kerosene) among which molasses resulted the better one, as previously reported for non-marine BS producers [40]. This finding evidenced the possible importance of low cost substrate employment, which could solve the problems of high costs for BS production. As suggested by other researchers who revealed yeast extract and tryptone as significantly positive factors for BS production, nitrogen is an aspect to be carefully treated, as important constituent of the peptide part of lipopeptidic BSs [27]. Similarly, phosphate source has to be regulated to ensure a good bacterial growth, with positive influence on BS production. Finally, calcium source has also been reported as important positive factor and enhancer of emulsification activity. Due to this deep optimization approach, the authors achieved an increase of BS production with emulsification indexes from

The genetics of microbial surfactant synthesis is important because it represents a primary factor determining their productivity. It has been studied by the use of mutants naturally occurring or induced by transposition. However, the screening for such mutants is difficult because of the loss of ability to produce the surfactant that does not result in an easily selectable phenotype or may be lethal. The regulation at the molecular level of BS production has been mainly investigated for the rhamnolipid produced by *Pseudomonas aeruginosa* and for a few lipopeptides of Bacilli. The BS production has proved to be controlled by *quorum sensing*

#### *Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

*Metabolomics - New Insights into Biology and Medicine*

index correlates to the surfactant concentration. According to the Refs. [20, 25], the surface tension measurement and the emulsification activity assays could be complementary and represent the basic tests to include in a screening procedure, allowing to detect both low molecular mass BSs with efficiency in surface and interfacial tension reduction and high molecular mass BSs more effective as emulsion stabilizers. In addition to the above standard screening tests, several authors reported the use of specific assays, the cetyltrimethylammonium bromide (C-TAB) agar plate assay, and the blood agar assay, useful to detect anionic BSs, but not enough sensitive to detect BS producers. The first one is based on the interaction between anionic surfactants eventually present and the cationic surfactant cetyltrimethylammonium bromide contained in a methylene blue stained mineral salts agar plate; the consequent creation of a blue dark halo around the colonies detects the presence of the BS producers. The blood agar assay is differently based on the hemolytic actions of biosurfactants—α, β, and γ hemolysis—on solid medium containing defibrinated blood as greenish or clarification halos around the bacterial colonies. The use of these specific assays have been reported in most of the above mentioned references, but it has been reduced over the years, because some authors signaled the possible harmfulness toward bacterial growth, the low specificity, and the possible occurrence of false positive/negative. In [19, 20], the two assays are performed on 69 and 96 isolates of different marine origin, respectively, suggesting the use of such tests as integration of a more deep screening procedure. To the list of screening test, it is necessary to mention some other tests with important implications in the screening quality and for considerations about bioremediation purposes. In [46], the authors reported the BS-mediated hydrocarbon degradation by *Joostella* sp. A8 grown in pure culture and consortia, as result of a BS production monitoring in which the bacterial adhesion to hydrocarbons assay (BATH assay) was included. This is a simple photometrical assay described for the first time in Ref. [61] for indirect evaluation of BS production by measuring the hydrophobicity of bacteria. The use of such test in relation to BS production is still a debating issue in this field, because the authors are divided between those who believes that a correlation between cell hydrophobicity, biodegradation efficiency, and BS production exists [62, 63] and who instead claims that the changes in cellular surface properties could be affected by several parameters and may not be necessarily associated to biodegradation ability [64]. Finally, some other minor tests have been reported by several authors for BS production by marine bacteria, but their efficiency have to be improved: this is the case of penetration assay [26], the hydrocarbon overlay agar method [13, 30], tilted glass slide test (TGS test) [30], and lipase activity [21–24,

28]. A number of additional screening tests are well reviewed in [59].

There are two primary pathways for bacterial BS biosynthesis: the way of hydrocarbons and the way of carbohydrates [9, 14], and their production is influenced by the availability, types of carbon sources, and the balance between carbon-nitrogen and other limiting nutrients [10]. The effects of several parameters on marine bacterial BS production have been explored by several authors, with particular focus on carbon source, temperature, pH, and NaCl concentration [20, 22–27]. The bacterial BS production has been generally observed at early stationary phase of growth [21, 22, 44, 65], or at exponential phase [25, 27]. In Ref. [22], the authors hypnotized that *N. alba* releases a cell bound biosurfactant into the culture broth which leads to an increase in extra cellular BSs. Moreover, the same authors evidenced a good BS production by the strains at all the tested conditions, even if they detected for *Nocardiopsis alba* the optimum conditions for BS production at pH 7, temperature

**4.1 BS production conditions**

**10**

30°C, and 1% salinity with glucose and peptone supplementation as carbon and nitrogen sources, respectively. In [21], the authors confirmed that carbon source and its concentration are affecting parameters for BS production, and established that glycerol, peptone, ferrous sulfate, and incubation time exhibited significant effect, with optimum levels as pH 7, temperature at 37°C, and salt concentration 2% for *B. amyloliquefaciens.* In particular, the authors reported that glycerol used as a carbon source showed the highest BS production (up to 6.76 g/l). According to Ref. [24], the studies performed a more deep optimization analysis to investigate the BS production by the sponge-associated *Brachybacterium paraconglomeratum* and indicated a yeast extract nitrogen source as factor enhancing up to 60% biosynthesis activity. Positive effects were also exhibited by the supplementation with 2% of NaCl, a pH level of 7.0, and a 30°C temperature. Moreover, asparagine resulted highly effective for BS production followed by glycine, leucine, and valine, and a requirement of CuSO4 as a metal supplement was requested by the strain for optimum production of BS. In [20], the authors investigated the influence of salinity and temperature on the BS production by polychaete-associated isolates and showed that the NaCl concentration strongly influenced the surface tension reducing activity and emulsification rate in major level rather than temperature. Nevertheless, the authors reported that the strain *Marinobacter* sp. A1 exhibited the best performances at 15°C and in the absence of NaCl, by suggesting that limited conditions could act as stimulating factors. Interestingly, several researchers [19, 20, 25] also reported the BS production in the presence of hydrocarbons. The BS synthesis under solid state cultivation (SSC) was investigated for *Brevibacterium aureum* MSA13, which increased its production with pre-treated molasses, glucose, and acrylamide [23] as substrates. The report is interesting because it represents the first attempt in which acrylamide was used as nitrogen source, and the SSC conditions have been proven to be a preferred bioprocess for the BS production and optimization. In [27], the authors suggested a parallel relationship between bacterial growth and productivity and tested several carbon sources (sugar cane molasses, olive oil, corn oil, motor oil, and kerosene) among which molasses resulted the better one, as previously reported for non-marine BS producers [40]. This finding evidenced the possible importance of low cost substrate employment, which could solve the problems of high costs for BS production. As suggested by other researchers who revealed yeast extract and tryptone as significantly positive factors for BS production, nitrogen is an aspect to be carefully treated, as important constituent of the peptide part of lipopeptidic BSs [27]. Similarly, phosphate source has to be regulated to ensure a good bacterial growth, with positive influence on BS production. Finally, calcium source has also been reported as important positive factor and enhancer of emulsification activity. Due to this deep optimization approach, the authors achieved an increase of BS production with emulsification indexes from 60 to 77%.

#### **5. Genetic regulation**

The genetics of microbial surfactant synthesis is important because it represents a primary factor determining their productivity. It has been studied by the use of mutants naturally occurring or induced by transposition. However, the screening for such mutants is difficult because of the loss of ability to produce the surfactant that does not result in an easily selectable phenotype or may be lethal. The regulation at the molecular level of BS production has been mainly investigated for the rhamnolipid produced by *Pseudomonas aeruginosa* and for a few lipopeptides of Bacilli. The BS production has proved to be controlled by *quorum sensing*

mechanism, a cell density dependent gene regulation process allowing bacterial cells to express certain specific genes on attaining high cell density [66]. With this mechanism, at the base of the bacterial production of secondary metabolites, bacteria produce a small diffusible signal molecule which accumulates in the growth medium and determine the gene activation when the microorganisms are in high densities. According to [49], the genes responsible for lipopeptide BS biosynthesis from *Bacillus* and *Pseudomonas* species display a high degree of structural similarity among themselves. The lipopeptide surfactin is produced as a result of nonribosomal biosynthesis. The mechanism is quite complex, as peptide synthetase for the amino acid moiety of surfactin is encoded by four open reading frames (ORFs) in the *Srf*A operon. The gene controlling the peptide synthetase consists of *Srf*AA (SrfORF1), *Srf*AB (SrfORF2), and *Srf*AD which activate each other. As a matter of fact, a more complex mechanism drives the *srf*A operon expression by controlling the level of various molecular signals [67]. On the other hand, structural genes required for the synthesis of the lipopeptidic BS lichenysin have been isolated, and a high sequence homology with *srf*A of surfactin synthetase was observed. Indeed, the lichenysin biosynthesis operon (called *lic operon*) was cloned and sequenced by revealing a composition of three peptide synthetase genes (lic A, lic B, and lic C) [68]. With respect to the genes which regulate the synthesis of glycolipids as rhamnolipids, they had been isolated, characterized, and their introduction into other species allowed the production of rhamnolipids in heterologous hosts [66]. The genes involved in the rhamnolipid biosynthesis are plasmid encoded and act during the late exponential early stationary phase as a consequence of the higher cell density. The synthesis of rhamnolipids in *P. aeruginosa* is carried out by the *rhl*AB operon, and a few additional genes are required [51, 45]. In particular, the biosynthetic pathway involved the genes *rhl*A, B, C, R, and I. *rhl*AB operon and *rhl*C gene encode the two rhamnosyltransferases (proteins that resides in the periplasm) responsible for the synthesis, transport or the stabilization of the rhamnosyltransferase within and in the cytoplasmatic membrane. *Rhl*A, B genes are organized in one operon and are coexpressed from the same promoter [67]. *Rhl*R and *rhl*I genes act as regulators of the *rhl*A, B gene expressions, and in turn are regulated by other genes (*las*R and *las*I) of a second quorum sensing system found in a different region of the *Pseudomonas aeruginosa* chromosome [67, 69]. As a matter of fact, the circuit of rhamnolipid production is promoted by other regulatory factors triggered by environmental conditions such as C/N ratio and inhibited by higher iron concentration [70], while the transcription of *rhl*AB genes is overexpressed under nitrogenlimited medium. As regards to the genetic regulation of high molecular weight heteropolysaccharide bioemulsifiers, they are more complex than the low molecular weight lipopeptides or rhamnolipids because they require a larger number of genes, and the genetic organization is even more complicated for polysaccharides-protein complexes [49]. However, although several structural and regulatory genes have been identified for the BS production, this aspect has been mainly improved for bacteria of terrestrial origin, while it has been scarcely explored for marine microorganisms. To the best of our knowledge, the only reports in this regard are in Refs. [23, 71]. In particular, these studies were addressed to the polyketide synthases (PKSs), the nonribosomal peptide synthases (NRPS), and large multifunctional proteins, modular proteins involved in the production of bioactive molecules. The research group investigated the possible correlation between the PKS gene and the BS biosynthesis in sponge-associated BS actinobacterial producers. In Ref. [23], the authors detected the presence of *pks* II gene in *B. aureum* MSA13, by suggesting the possible biosynthesis of secondary metabolites with antibiotic and surfactant properties. Furthermore, the studies described in [71] provided interesting insights into the KS genes of *Brevibacterium* and *Brachybacterium*, by confirming through

**13**

provided the original work is properly cited.

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

ing approach to design novel biomolecules.

contributions and useful applications for humans.

\*, Carmen Rizzo2

Sciences, University of Messina, Messina, Italy

\*Address all correspondence to: rfloris@agrisricerca.it

**6. Conclusions**

**Author details**

Rosanna Floris1

Prodotti Ittici, Sassari, Italy

UOS Messina, Messina, Italy

molecular approaches that marine resources should be better explored for biodiscovery purposes. The molecular genetics of BS production is still in progress, and important genetic tools (plasmids, transposons, and gene libraries) are still to be developed, as well as further studies could allow to develop a biosynthetic engineer-

The research on BS is still undergoing evolution and requires many improvements in several aspects. The results obtained in the last decades from the marine resources are very encouraging, both in terms of BS chemical diversity and in terms of the BS effectiveness and microbial production capacity. Despite this, new tools and improvements are necessary for a better comprehension of the genetic regulation, in order to exploit these mechanisms for a potential large-scale production. The discovery of new BSs must represent the main goal, and should be accompanied by appropriate chemical analysis, and identification of the most active fractions. The screening methods must be standardized and refined, and above all the study should be planned on the basis of the application of interest. Marine bioprospecting and the blue biotechnology are research areas that deserve to be explored, which are worth focusing on, and which could allow significant scientific

© 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,

and Angelina Lo Giudice2,3

1 AGRIS-Sardegna-Agricultural Research Agency of Sardinia, Servizio Ricerca

2 Department of Chemical, Biological, Pharmaceutical and Environmental

3 Institute for the Coastal Marine Environment, National Research Council,

#### *Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

molecular approaches that marine resources should be better explored for biodiscovery purposes. The molecular genetics of BS production is still in progress, and important genetic tools (plasmids, transposons, and gene libraries) are still to be developed, as well as further studies could allow to develop a biosynthetic engineering approach to design novel biomolecules.

#### **6. Conclusions**

*Metabolomics - New Insights into Biology and Medicine*

mechanism, a cell density dependent gene regulation process allowing bacterial cells to express certain specific genes on attaining high cell density [66]. With this mechanism, at the base of the bacterial production of secondary metabolites, bacteria produce a small diffusible signal molecule which accumulates in the growth medium and determine the gene activation when the microorganisms are in high densities. According to [49], the genes responsible for lipopeptide BS biosynthesis from *Bacillus* and *Pseudomonas* species display a high degree of structural similarity among themselves. The lipopeptide surfactin is produced as a result of nonribosomal biosynthesis. The mechanism is quite complex, as peptide synthetase for the amino acid moiety of surfactin is encoded by four open reading frames (ORFs) in the *Srf*A operon. The gene controlling the peptide synthetase consists of *Srf*AA (SrfORF1), *Srf*AB (SrfORF2), and *Srf*AD which activate each other. As a matter of fact, a more complex mechanism drives the *srf*A operon expression by controlling the level of various molecular signals [67]. On the other hand, structural genes required for the synthesis of the lipopeptidic BS lichenysin have been isolated, and a high sequence homology with *srf*A of surfactin synthetase was observed. Indeed, the lichenysin biosynthesis operon (called *lic operon*) was cloned and sequenced by revealing a composition of three peptide synthetase genes (lic A, lic B, and lic C) [68]. With respect to the genes which regulate the synthesis of glycolipids as rhamnolipids, they had been isolated, characterized, and their introduction into other species allowed the production of rhamnolipids in heterologous hosts [66]. The genes involved in the rhamnolipid biosynthesis are plasmid encoded and act during the late exponential early stationary phase as a consequence of the higher cell density. The synthesis of rhamnolipids in *P. aeruginosa* is carried out by the *rhl*AB operon, and a few additional genes are required [51, 45]. In particular, the biosynthetic pathway involved the genes *rhl*A, B, C, R, and I. *rhl*AB operon and *rhl*C gene encode the two rhamnosyltransferases (proteins that resides in the periplasm) responsible for the synthesis, transport or the stabilization of the rhamnosyltransferase within and in the cytoplasmatic membrane. *Rhl*A, B genes are organized in one operon and are coexpressed from the same promoter [67]. *Rhl*R and *rhl*I genes act as regulators of the *rhl*A, B gene expressions, and in turn are regulated by other genes (*las*R and *las*I) of a second quorum sensing system found in a different region of the *Pseudomonas aeruginosa* chromosome [67, 69]. As a matter of fact, the circuit of rhamnolipid production is promoted by other regulatory factors triggered by environmental conditions such as C/N ratio and inhibited by higher iron concentration [70], while the transcription of *rhl*AB genes is overexpressed under nitrogenlimited medium. As regards to the genetic regulation of high molecular weight heteropolysaccharide bioemulsifiers, they are more complex than the low molecular weight lipopeptides or rhamnolipids because they require a larger number of genes, and the genetic organization is even more complicated for polysaccharides-protein complexes [49]. However, although several structural and regulatory genes have been identified for the BS production, this aspect has been mainly improved for bacteria of terrestrial origin, while it has been scarcely explored for marine microorganisms. To the best of our knowledge, the only reports in this regard are in Refs. [23, 71]. In particular, these studies were addressed to the polyketide synthases (PKSs), the nonribosomal peptide synthases (NRPS), and large multifunctional proteins, modular proteins involved in the production of bioactive molecules. The research group investigated the possible correlation between the PKS gene and the BS biosynthesis in sponge-associated BS actinobacterial producers. In Ref. [23], the authors detected the presence of *pks* II gene in *B. aureum* MSA13, by suggesting the possible biosynthesis of secondary metabolites with antibiotic and surfactant properties. Furthermore, the studies described in [71] provided interesting insights into the KS genes of *Brevibacterium* and *Brachybacterium*, by confirming through

**12**

The research on BS is still undergoing evolution and requires many improvements in several aspects. The results obtained in the last decades from the marine resources are very encouraging, both in terms of BS chemical diversity and in terms of the BS effectiveness and microbial production capacity. Despite this, new tools and improvements are necessary for a better comprehension of the genetic regulation, in order to exploit these mechanisms for a potential large-scale production. The discovery of new BSs must represent the main goal, and should be accompanied by appropriate chemical analysis, and identification of the most active fractions. The screening methods must be standardized and refined, and above all the study should be planned on the basis of the application of interest. Marine bioprospecting and the blue biotechnology are research areas that deserve to be explored, which are worth focusing on, and which could allow significant scientific contributions and useful applications for humans.

### **Author details**

Rosanna Floris1 \*, Carmen Rizzo2 and Angelina Lo Giudice2,3

1 AGRIS-Sardegna-Agricultural Research Agency of Sardinia, Servizio Ricerca Prodotti Ittici, Sassari, Italy

2 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy

3 Institute for the Coastal Marine Environment, National Research Council, UOS Messina, Messina, Italy

\*Address all correspondence to: rfloris@agrisricerca.it

© 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] Baharum SN, Beng EK, Mokhtar MAA. Marine microorganisms: Potential application and challenges. Journal of Biological Sciences. 2010;**10**:555-564

[2] Dusane DH, Matkar P, Venugopalan VP, Kumar AR, Zinjarde SS. Cross-species induction of antimicrobial compounds, biosurfactants and quorum-sensing inhibitors in tropical marine epibiotic bacteria by pathogens and biofouling microorganisms. Current Microbiology. 2011;**62**:974-980

[3] Paniagua-Michel J, Olmos-Soto J, Morales-Guerrero ER. Algal and microbial exopolysaccharides: New insights as biosurfactants and bioemulsifiers. Advances in Food and Nutrition Research. 2014;**73**:221-257. (chapter eleven)

[4] Rodrigues L, Banat IM, Teixeira J, Oliveira R. Biosurfactants: Potential applications in medicine. Journal of Antimicrobial Chemotherapy. 2006;**57**:609-618

[5] Vatsa P, Sanchez L, Clement C, Baillieul F, Dorey S. Rhamnolipid biosurfactants as new players in animal and plant defense against microbes. International Journal of Molecular Sciences. 2010;**11**(12):5095-5108

[6] Rizzo C, Lo GA. Marine invertebrates: Underexplored sources of bacteria producing biologically active molecules. Diversity. 2018;**10**:52

[7] Chen C-Y, Baker SC, Darton RC. The application of high throughput analysis method for the screening of potential biosurfactants from natural sources. Journal of Microbiology Methods. 2007;**70**:503-510

[8] Batista SB, Mounteer AH, Amorim FR, Tòtola MR. Isolarion and characterization of biosurfactants/ bioemulsifier-producing bacteria from petroleum contaminated sites. Bioresource Technology. 2006;**97**:868-875

[9] Desai JD, Banat IM. Microbial production of surfactants and their commercial potential. Microbiology and Molecular Biology Reviews. 1997;**61**:47-64

[10] Lotfabad TB, Shourian M, Roostaazad R, Najafabadi AR, Adelzadeh MR, Noghabi KA. An efficient biosurfactant-producing bacterium *Pseudomonas aeruginosa* MR01, isolated from oil excavation areas in south of Iran. Colloids and Surfaces B: Biointerfaces. 2009;**69**:183-193

[11] Dang NP, Landfald B, Willassen NP. Biological surface-active compounds from marine bacteria. Environmental Technology. 2016;**37**(9):1151-1158

[12] Dhail S, Jasuja ND. Isolation of biosurfactant-producing marine bacteria. African Journal of Environmental Science and Technology. 2012;**6**(6):263-266

[13] Shoeb E, Ahmed N, Akhter J, Badar U, Siddiqui K, Ansari FA, Waqar M, Imtiaz S, Akhtar N, Shaikh QA, Baig R, Butt S, Khan S, Khan S, Hussain S, Ahmed B, Ansari MA. Screening and characterization of biosurfactantproducing bacteria isolated from the Arabian Sea coast of Karachi. Turkish Journal of Biology. 2015;**39**:210-216

[14] Malavenda R, Rizzo C, Michaud L, Gerçe B, Bruni V, Syldatk C, Hausmann R, Lo Giudice A. Biosurfactant production by Arctic and Antarctic bacteria growing on hydrocarbons. Polar Biology. 2015;**38**:1565-1574

[15] Thavasi R, Sharma S, Jayalakshmi S. Evaluation of screening methods for

**15**

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

the isolation of biosurfactant producing marine bacteria. Journal of Petroleum & Environmental Biotechnology.

marine actinomycetes *Nocardiopsis alba* MSA10. Bioprocess and Biosystems Engineering. 2009;**32**:825-835

[23] Kiran GS, Anto TT, Selvin J, Sabarathnam B. Optimization and characterization of a new lipopeptide biosurfactant produced by marine *Brevibacterium aureum* MSA13 in solid state culture. Bioresource Technology.

[24] Kiran GS, Sabarathnam B, Thajuddin N, Selvin J. Production of glycolipid biosurfactant from spongeassociated marine actinobacterium *Brachybacterium paraconglomeratum* MSA21. Journal of Surfactants and Detergents. 2014;**17**:531-542

[25] Rizzo C, Syldatk C, Hausmann R,

Conte A, De Domenico E, Michaud L, Lo Giudice A. The demosponge *Halichondria* (Halichondria) *panicea* (Pallas, 1766) as a novel source of biosurfactant-producing bacteria. Journal of Basic Microbiology. 2018:1-11. https://doi.org/10.1002/

[26] Graziano M, Rizzo C, Michaud L, Porporato EMD, De Domenico E, Spanò N, Lo Giudice A. Biosurfactant production by hydrocarbon-degrading *Brevibacterium* and *Vibrio* isolates from the sea pen *Pteroeides spinosum* (Ellis, 1764). Journal of Basic Microbiology.

[27] Mabrouk MEM, Youssif EM, Sabry SA. Biosurfactant production by a newly isolated soft coral-associated marine *Bacillus* sp. E34: Statistical optimization and characterization. Life

[28] Padmavathi AR, Pandian SK. Antibiofilm activity of biosurfactant producing coral associated bacteria isolated from Gulf of Mannar. Indian Journal of Microbiology.

Science Journal. 2014;**11**:10

Gerçe B, Longo C, Papale M,

jobm.201700669

2016;**56**:963-974

2014;**54**:376-382

2010;**101**:2389-2396

[16] Pini F, Grossi C, Nereo S, Michaud L, Lo Giudice A, Bruni V, Baldi F, Fani R. Molecular and physiological characterisation of psychrotrophic hydrocarbon-degrading bacteria isolated from Terra Nova Bay (Antarctica). European Journal of Soil

Biology. 2007;**43**:368-379

2012;**28**:401-419

[17] Chrzanowski L, Ławniczak L, Czaczyk K. Why do microorganisms produce rhamnolipids? World Journal of Microbiology and Biotechnology.

[18] Ławniczak L, Marecik R, Chrzanowski L. Contributions of biosurfactants to natural or induced bioremediation. Applied Microbiology and Biotechnology. 2013;**97**:2327-2339

Bulletin. 2013;**70**:125-133

2014;**21**:2988-3004

2015;**5**:443-454

[19] Rizzo C, Michaud L, Hörmann B, Gerçe B, Syldatk C, Hausmann R, De Domenico E, Lo Giudice A. Bacteria associated with Sabellids (Polychaeta: Annelida) as a novel source of surface active compounds. Marine Pollution

[20] Rizzo C, Michaud L, Syldatk C, Hausmann R, De Domenico E, Lo Giudice A. Influence of salinity and temperature on the activity of biosurfactants by polychaeteassociated isolates. Environmental Science and Pollutution Research.

[21] Dhasayan A, Selvin J, Kiran S. Biosurfactant production from marine bacteria associated with sponge *Callyspongia diffusa*. Biotechnology.

[22] Gandhimathi R, Kiran SG, Hema TA, Selvin J. Production and characterization of lipopeptide biosurfactant by a sponge associated

2011;**S-1**(1):1-6

#### *Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

the isolation of biosurfactant producing marine bacteria. Journal of Petroleum & Environmental Biotechnology. 2011;**S-1**(1):1-6

[16] Pini F, Grossi C, Nereo S, Michaud L, Lo Giudice A, Bruni V, Baldi F, Fani R. Molecular and physiological characterisation of psychrotrophic hydrocarbon-degrading bacteria isolated from Terra Nova Bay (Antarctica). European Journal of Soil Biology. 2007;**43**:368-379

[17] Chrzanowski L, Ławniczak L, Czaczyk K. Why do microorganisms produce rhamnolipids? World Journal of Microbiology and Biotechnology. 2012;**28**:401-419

[18] Ławniczak L, Marecik R, Chrzanowski L. Contributions of biosurfactants to natural or induced bioremediation. Applied Microbiology and Biotechnology. 2013;**97**:2327-2339

[19] Rizzo C, Michaud L, Hörmann B, Gerçe B, Syldatk C, Hausmann R, De Domenico E, Lo Giudice A. Bacteria associated with Sabellids (Polychaeta: Annelida) as a novel source of surface active compounds. Marine Pollution Bulletin. 2013;**70**:125-133

[20] Rizzo C, Michaud L, Syldatk C, Hausmann R, De Domenico E, Lo Giudice A. Influence of salinity and temperature on the activity of biosurfactants by polychaeteassociated isolates. Environmental Science and Pollutution Research. 2014;**21**:2988-3004

[21] Dhasayan A, Selvin J, Kiran S. Biosurfactant production from marine bacteria associated with sponge *Callyspongia diffusa*. Biotechnology. 2015;**5**:443-454

[22] Gandhimathi R, Kiran SG, Hema TA, Selvin J. Production and characterization of lipopeptide biosurfactant by a sponge associated marine actinomycetes *Nocardiopsis alba* MSA10. Bioprocess and Biosystems Engineering. 2009;**32**:825-835

[23] Kiran GS, Anto TT, Selvin J, Sabarathnam B. Optimization and characterization of a new lipopeptide biosurfactant produced by marine *Brevibacterium aureum* MSA13 in solid state culture. Bioresource Technology. 2010;**101**:2389-2396

[24] Kiran GS, Sabarathnam B, Thajuddin N, Selvin J. Production of glycolipid biosurfactant from spongeassociated marine actinobacterium *Brachybacterium paraconglomeratum* MSA21. Journal of Surfactants and Detergents. 2014;**17**:531-542

[25] Rizzo C, Syldatk C, Hausmann R, Gerçe B, Longo C, Papale M, Conte A, De Domenico E, Michaud L, Lo Giudice A. The demosponge *Halichondria* (Halichondria) *panicea* (Pallas, 1766) as a novel source of biosurfactant-producing bacteria. Journal of Basic Microbiology. 2018:1-11. https://doi.org/10.1002/ jobm.201700669

[26] Graziano M, Rizzo C, Michaud L, Porporato EMD, De Domenico E, Spanò N, Lo Giudice A. Biosurfactant production by hydrocarbon-degrading *Brevibacterium* and *Vibrio* isolates from the sea pen *Pteroeides spinosum* (Ellis, 1764). Journal of Basic Microbiology. 2016;**56**:963-974

[27] Mabrouk MEM, Youssif EM, Sabry SA. Biosurfactant production by a newly isolated soft coral-associated marine *Bacillus* sp. E34: Statistical optimization and characterization. Life Science Journal. 2014;**11**:10

[28] Padmavathi AR, Pandian SK. Antibiofilm activity of biosurfactant producing coral associated bacteria isolated from Gulf of Mannar. Indian Journal of Microbiology. 2014;**54**:376-382

**14**

2007;**70**:503-510

[8] Batista SB, Mounteer AH,

Amorim FR, Tòtola MR. Isolarion and

*Metabolomics - New Insights into Biology and Medicine*

characterization of biosurfactants/ bioemulsifier-producing bacteria from petroleum contaminated sites. Bioresource Technology.

[9] Desai JD, Banat IM. Microbial production of surfactants and their commercial potential. Microbiology and Molecular Biology Reviews.

[10] Lotfabad TB, Shourian M, Roostaazad R, Najafabadi AR, Adelzadeh MR, Noghabi KA. An efficient biosurfactant-producing bacterium *Pseudomonas aeruginosa* MR01, isolated from oil excavation areas in south of Iran. Colloids and Surfaces B: Biointerfaces. 2009;**69**:183-193

[11] Dang NP, Landfald B, Willassen NP. Biological surface-active compounds from marine bacteria. Environmental Technology. 2016;**37**(9):1151-1158

[12] Dhail S, Jasuja ND. Isolation of biosurfactant-producing marine bacteria. African Journal of

2012;**6**(6):263-266

Environmental Science and Technology.

[13] Shoeb E, Ahmed N, Akhter J, Badar U,

[14] Malavenda R, Rizzo C, Michaud L, Gerçe B, Bruni V, Syldatk C, Hausmann R, Lo Giudice A. Biosurfactant production

growing on hydrocarbons. Polar Biology.

[15] Thavasi R, Sharma S, Jayalakshmi S. Evaluation of screening methods for

by Arctic and Antarctic bacteria

2015;**38**:1565-1574

Siddiqui K, Ansari FA, Waqar M, Imtiaz S, Akhtar N, Shaikh QA, Baig R, Butt S, Khan S, Khan S, Hussain S, Ahmed B, Ansari MA. Screening and characterization of biosurfactantproducing bacteria isolated from the Arabian Sea coast of Karachi. Turkish Journal of Biology. 2015;**39**:210-216

2006;**97**:868-875

1997;**61**:47-64

[1] Baharum SN, Beng EK, Mokhtar MAA. Marine microorganisms: Potential application and challenges. Journal of Biological Sciences.

Zinjarde SS. Cross-species induction of antimicrobial compounds, biosurfactants and quorum-sensing inhibitors in tropical marine epibiotic bacteria by pathogens and biofouling microorganisms. Current Microbiology.

[3] Paniagua-Michel J, Olmos-Soto J, Morales-Guerrero ER. Algal and microbial exopolysaccharides: New insights as biosurfactants and bioemulsifiers. Advances in Food and Nutrition Research. 2014;**73**:221-257.

[4] Rodrigues L, Banat IM, Teixeira J, Oliveira R. Biosurfactants: Potential applications in medicine. Journal of Antimicrobial Chemotherapy.

[5] Vatsa P, Sanchez L, Clement C, Baillieul F, Dorey S. Rhamnolipid biosurfactants as new players in animal and plant defense against microbes. International Journal of Molecular Sciences. 2010;**11**(12):5095-5108

[6] Rizzo C, Lo GA. Marine

invertebrates: Underexplored sources of bacteria producing biologically active molecules. Diversity. 2018;**10**:52

[7] Chen C-Y, Baker SC, Darton RC. The application of high throughput analysis method for the screening of potential biosurfactants from natural sources. Journal of Microbiology Methods.

2010;**10**:555-564

**References**

2011;**62**:974-980

(chapter eleven)

2006;**57**:609-618

[2] Dusane DH, Matkar P, Venugopalan VP, Kumar AR, [29] Floris R, Scanu G, Fois N, Rizzo C, Malavenda R, Spanò N, Lo Giudice A. Intestinal bacterial flora of Mediterranean gilthead seabream (*Sparus aurata*, L.) as a novel source of natural surface active compounds. Aquaculture Research. 2018;**49**:1262- 1273. DOI: 10111/are.13580

[30] Satpute SK, Banat IM, Dhakephalkar PK, Banpurkar AG, Chopade BA. Biosurfactants, bioemulsifiers and exopolysaccharides from marine microorganisms. Biotechnology Advances. 2010;**28**:436-450

[31] Mulligan CN. Environmental applications for biosurfactants. Environmental Pollution. 2005;**133**:183-198

[32] Rosenberg E, Ron EZ. High and low molecular mass microbial surfactants. Applied Microbiology and Biotechnology. 1999;**52**:154-162

[33] Pattanatu KSM, Gakpe E. Production, characterisation and applications of biosurfactants-Review. Biotechnology. 2008;**7**(2):360-370

[34] Rahman KSM, Street G, Lord R, Kane G, Rahman TJ, Marchant R, Banat IM. Bioremediation of petroleum sludege using bacterial consortium with biosurfactant. In: Singh SN, Tripathi RD, editors. Environmental Bioremediation Technologies. Springer Publication; 2006. pp. 391-408. DOI:10.1007/978-3-540-34793-4-17

[35] Reis CBLD, Morandini LMB, Bevilacqua CB, Bublitz F, Ugalde G, Mazutti MA, Jacques RJS. First report of the production of a potent biosurfactant with ß-trehalose by *Fusarium fujikuroi* under optimized conditions of submerged fermentation. Brazilian Journal of Microbiology. 2018;**S1517-8382**(17):30993-0

[36] Rosenberg E, Ron EZ. Surface active polymers from the genus *Acinetobacter*.

In: Kaplan DL, editor. Biopolymers from Renewable Resources. New York: Springer, Berlin Heidelberg; 1998. pp. 281-291

[37] Desjardine K, Pereira A, Wright H, Matainaho T, Kelly M, Andersen RJ. Tauramamide, a lipopeptide antibiotic produced in culture by *Brevibacillus laterosporus* isolated from a marine habitat. Journal of Natural Products. 2007;**70**:1850-1853

[38] Kalinovvskaya NI, Kuznetsova TA, Rashkes YV, Milgrom YM, Milgrom EG, Willis RH. Surfactin-like structures of five cyclic depsipeptides from the marine isolate of *Bacillus pumillus*. Russian Chemical Bulletin. 1995;**44**(5):951-955

[39] Gerard J, Lloyd R, Barsby T, Haden P, Kelly MT, Andersen RJ. Massetolides A-H, antimycobacterial cyclic depsipeptides produced by two pseudomonads isolated from marine habitats. Journal of Natural Products. 1997;**60**(3):223-229

[40] Joshi SJ, Suthar H, Yadav AK, Hinguaro K, Nerurkar A. Occurrence of biosurfactants producing *Bacillus* spp. in diverse habitats. 2013 International Scholarly Research Network ISRN Biotechnology. Article ID 652340. p. 6. DOI: 10.5402/2013/652340

[41] Yakimov MM, Timmis KN, Wray V, Fredrickson HL. Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface *Bacillus licheniformis* Bas50. Applied and Environmental Microbiology. 1995;**61**:1706-1713

[42] Gutierrez T, Shimmied T, Haidon C, Black K, Green DH. Emulsifying and metal ion binding activity of a glycoprotein exopolymer produced by *Pseudoalteromonas* sp. Strain TG12. Applied and Environmental Microbiology. 2008, 2008;**74**:4867-4876

**17**

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

[43] Kumar AK, Mody K, Jha B. Bacterial exopolysaccharides—A perception. Journal of Basic Microbiology.

[50] Sumaiya M, Anchana Devi C, Leela K. A study on biosurfactant production from marine bacteria. International Journal of Scientific and Research Publications. 2017;**7**:12

[51] Ron EZ, Rosenberg E. Natural roles of biosurfactants. Environmental Microbiology. 2001;**3**(4):229-236

[52] Mackey D, McFall AJ. MAMPs and MIMPs: Proposed classifications for inducers of innate immunity. Molecular Microbiology. 2006;**61**:1365-1371

[53] Tadros T. Adsorption of surfactants at the air/liquid and liquid/liquid interfaces. In: Tadros TF, editor. Applied Surfactants: Principles and Applications. Weinheim: Wiley VCH;

[54] Tuleva B, Christova N, Jordanov B, Jordanov B, Nikolova-Damyanova B, Petrov P. Naphthalene degradation and biosurfactant activity by *Bacillus cereus* 28BN. Zeitschrift für Naturforschung.

[55] Dilmohamud B, Seeneevassen J, Rughooputh S, Ramasami P. Surface tension and related thermodynamic parameters of alcohols using the Traube stalagmometer. European Journal of Physics. 2005;**26**(6):1079-1084

[56] Plaza G, Zjawiony I, Banat I. Use of different methods for detection of thermophilic biosurfactant-

producing bacteria from hydrocarboncontaminated bioremediated soils. Journal of Petroleum Science and Engineering. 2006;**50**(1):71-77

[57] Chen C, Baker S, Darton R. The application of a high throughput analysis method for the screening of potential biosurfactants from natural sources. Journal of Microbiological

[58] Tugrul T, Cansunar E. Detecting surfactant-producing microorganisms

Methods. 2007;**70**:503-510

Section C. 2005;**60**:577-582

2015. pp. 81-82

[44] Zhang Y, Miller RM. Enhanced

rhamnolipid surfactant (biosurfactant).

octadecane dispersion and biodegradation by a *Pseudomonas*

Applied and Environmental Microbiology. 1992;**58**:3276-3282

[45] Perfumo A, Smyth TJP,

2010. pp. 1502-1510. DOI: 10.1007/978-3-540-77587-4\_103

degradation and biosurfactant production by *Joostella* sp. A8 when grown in pure culture and consortia. Journal of Environmental Sciences.

Lo Giudice A. Biosurfactant activity, heavy metal tolerance and characterization of *Joostella* strain A8 from the Mediterranean polychaete *Megalomma claparedei* (Gravier, 1906). Ecotoxicology. 2015;**24**:1294-1304

[48] Rizzo C, Lo Giudice A. Heavy metal tolerance and chelating activity of bacteria associated with Mediterranean polychaetes. SF Journal of Environmental and Earth Science.

[49] Das P, Mukherjee S, Sen R. Genetic regulations of the biosynthesis of microbial surfactants: An overview. Biotechnology & Genetic Engineering

Reviews. 2008;**25**(1):165-186

2018;**67**:115-126

2018;**1**(2):1015

Marchant R, Banat IM. Production and roles of biosurfactants and

bioemulsifiers in accessing hydrophobic substrates. In: Timmis KN, editor. Handbook of Hydrocarbon and Lipid Microbiology. Chichester: Springer-Verlag Berlin Heidelberg;

[46] Rizzo C, Rappazzo AC, Michaud L, De Domenico E, Rochera C, Camacho A, Lo Giudice A. Efficiency in hydrocarbon

[47] Rizzo C, Michaud L, Graziano M, De Domenico E, Syldatk C, Hausmann R,

2007;**47**:103-117

*Biosurfactants from Marine Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.80493*

*Metabolomics - New Insights into Biology and Medicine*

In: Kaplan DL, editor. Biopolymers from Renewable Resources. New York: Springer, Berlin Heidelberg; 1998.

[37] Desjardine K, Pereira A, Wright H, Matainaho T, Kelly M, Andersen RJ. Tauramamide, a lipopeptide antibiotic produced in culture by *Brevibacillus laterosporus* isolated from a marine habitat. Journal of Natural Products.

[38] Kalinovvskaya NI, Kuznetsova TA, Rashkes YV, Milgrom YM, Milgrom EG, Willis RH. Surfactin-like structures of five cyclic depsipeptides from the marine isolate of *Bacillus* 

*pumillus*. Russian Chemical Bulletin.

[39] Gerard J, Lloyd R, Barsby T, Haden P, Kelly MT, Andersen RJ. Massetolides A-H, antimycobacterial cyclic depsipeptides produced by two pseudomonads isolated from marine habitats. Journal of Natural Products.

[40] Joshi SJ, Suthar H, Yadav AK, Hinguaro K, Nerurkar A. Occurrence of biosurfactants producing *Bacillus* spp. in diverse habitats. 2013 International Scholarly Research Network ISRN Biotechnology. Article ID 652340. p. 6. DOI:

[41] Yakimov MM, Timmis KN, Wray V, Fredrickson HL. Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface *Bacillus licheniformis* Bas50. Applied and Environmental Microbiology. 1995;**61**:1706-1713

[42] Gutierrez T, Shimmied T, Haidon C,

Black K, Green DH. Emulsifying and metal ion binding activity of a glycoprotein exopolymer produced by *Pseudoalteromonas* sp. Strain TG12. Applied and Environmental Microbiology. 2008, 2008;**74**:4867-4876

pp. 281-291

2007;**70**:1850-1853

1995;**44**(5):951-955

1997;**60**(3):223-229

10.5402/2013/652340

[29] Floris R, Scanu G, Fois N, Rizzo C, Malavenda R, Spanò N, Lo Giudice A. Intestinal bacterial flora of Mediterranean gilthead seabream (*Sparus aurata*, L.) as a novel source of natural surface active compounds. Aquaculture Research. 2018;**49**:1262-

1273. DOI: 10111/are.13580

[30] Satpute SK, Banat IM, Dhakephalkar

PK, Banpurkar AG, Chopade BA. Biosurfactants, bioemulsifiers and exopolysaccharides from marine microorganisms. Biotechnology Advances. 2010;**28**:436-450

[31] Mulligan CN. Environmental applications for biosurfactants. Environmental Pollution.

[32] Rosenberg E, Ron EZ. High and low molecular mass microbial surfactants. Applied Microbiology and

Biotechnology. 1999;**52**:154-162

[33] Pattanatu KSM, Gakpe E. Production, characterisation and applications of biosurfactants-Review. Biotechnology. 2008;**7**(2):360-370

[34] Rahman KSM, Street G, Lord R, Kane G, Rahman TJ, Marchant R, Banat IM. Bioremediation of petroleum sludege using bacterial consortium with biosurfactant. In: Singh SN, Tripathi RD, editors. Environmental Bioremediation Technologies. Springer Publication; 2006. pp. 391-408. DOI:10.1007/978-3-540-34793-4-17

[35] Reis CBLD, Morandini LMB, Bevilacqua CB, Bublitz F, Ugalde G, Mazutti MA, Jacques RJS. First report of the production of a potent biosurfactant with ß-trehalose by *Fusarium fujikuroi* under optimized conditions of submerged fermentation. Brazilian Journal of Microbiology. 2018;**S1517-8382**(17):30993-0

[36] Rosenberg E, Ron EZ. Surface active polymers from the genus *Acinetobacter*.

2005;**133**:183-198

**16**

[43] Kumar AK, Mody K, Jha B. Bacterial exopolysaccharides—A perception. Journal of Basic Microbiology. 2007;**47**:103-117

[44] Zhang Y, Miller RM. Enhanced octadecane dispersion and biodegradation by a *Pseudomonas* rhamnolipid surfactant (biosurfactant). Applied and Environmental Microbiology. 1992;**58**:3276-3282

[45] Perfumo A, Smyth TJP, Marchant R, Banat IM. Production and roles of biosurfactants and bioemulsifiers in accessing hydrophobic substrates. In: Timmis KN, editor. Handbook of Hydrocarbon and Lipid Microbiology. Chichester: Springer-Verlag Berlin Heidelberg; 2010. pp. 1502-1510. DOI: 10.1007/978-3-540-77587-4\_103

[46] Rizzo C, Rappazzo AC, Michaud L, De Domenico E, Rochera C, Camacho A, Lo Giudice A. Efficiency in hydrocarbon degradation and biosurfactant production by *Joostella* sp. A8 when grown in pure culture and consortia. Journal of Environmental Sciences. 2018;**67**:115-126

[47] Rizzo C, Michaud L, Graziano M, De Domenico E, Syldatk C, Hausmann R, Lo Giudice A. Biosurfactant activity, heavy metal tolerance and characterization of *Joostella* strain A8 from the Mediterranean polychaete *Megalomma claparedei* (Gravier, 1906). Ecotoxicology. 2015;**24**:1294-1304

[48] Rizzo C, Lo Giudice A. Heavy metal tolerance and chelating activity of bacteria associated with Mediterranean polychaetes. SF Journal of Environmental and Earth Science. 2018;**1**(2):1015

[49] Das P, Mukherjee S, Sen R. Genetic regulations of the biosynthesis of microbial surfactants: An overview. Biotechnology & Genetic Engineering Reviews. 2008;**25**(1):165-186

[50] Sumaiya M, Anchana Devi C, Leela K. A study on biosurfactant production from marine bacteria. International Journal of Scientific and Research Publications. 2017;**7**:12

[51] Ron EZ, Rosenberg E. Natural roles of biosurfactants. Environmental Microbiology. 2001;**3**(4):229-236

[52] Mackey D, McFall AJ. MAMPs and MIMPs: Proposed classifications for inducers of innate immunity. Molecular Microbiology. 2006;**61**:1365-1371

[53] Tadros T. Adsorption of surfactants at the air/liquid and liquid/liquid interfaces. In: Tadros TF, editor. Applied Surfactants: Principles and Applications. Weinheim: Wiley VCH; 2015. pp. 81-82

[54] Tuleva B, Christova N, Jordanov B, Jordanov B, Nikolova-Damyanova B, Petrov P. Naphthalene degradation and biosurfactant activity by *Bacillus cereus* 28BN. Zeitschrift für Naturforschung. Section C. 2005;**60**:577-582

[55] Dilmohamud B, Seeneevassen J, Rughooputh S, Ramasami P. Surface tension and related thermodynamic parameters of alcohols using the Traube stalagmometer. European Journal of Physics. 2005;**26**(6):1079-1084

[56] Plaza G, Zjawiony I, Banat I. Use of different methods for detection of thermophilic biosurfactantproducing bacteria from hydrocarboncontaminated bioremediated soils. Journal of Petroleum Science and Engineering. 2006;**50**(1):71-77

[57] Chen C, Baker S, Darton R. The application of a high throughput analysis method for the screening of potential biosurfactants from natural sources. Journal of Microbiological Methods. 2007;**70**:503-510

[58] Tugrul T, Cansunar E. Detecting surfactant-producing microorganisms by the drop-collapse test. World Journal of Microbiology and Biotechnology. 2005;**21**:851-853

[59] Walter V, Syldatk C, Hausmann R. Screening concepts for the isolation of biosurfactant producing microorganisms. Advances in Experimental Medicine and Biology. 2010;**672**:1-13

[60] Christova N, Tuleva B, Lalchev Z, Jordanovac A, Jordanov B. Rhamnolipid biosurfactants produced by *Renibacterium salmoninarum* 27BN during growth on n-hexadecane. Zeitschrift für Naturforschung. Section C. 2004;**59**(1-2):70-74

[61] Rosenberg M, Gutnick D, Rosenberg E. Adherence of bacteria to hydrocarbons—A simple method for measuring cell-surface hydrophobicity. FEMS Microbiology Letters. 1980;**9**(1):29-33

[62] Franzetti A, Caredda P, Colla PL, Pintus M, Tamburini E, Papacchini M, Bestetti G. Cultural factors affecting biosurfactant production by *Gordonia* sp. BS29. International Biodeterioration and Biodegradation. 2009;**63**:943-947

[63] Obuekwe CO, Al-Jadi ZK, Al-Saleh ES. Insight into heterogeneity in cell-surface hydrophobocity and ability to degrade hydrocarbons among cells of two hydrocarbon-degrading bacterial populations. Canadian Journal of Microbiology. 2007;**53**:252-260

[64] Chakraborty S, Mukherji S, Mukherji S. Surface hydrophobicity of petroleum hydrocarbon degrading *Burkholderia* strains and their interactions with NAPLs and surfaces. Colloids and Surfaces. B, Biointerfaces. 2010;**78**:101-108

[65] Antoniou E, Fodelianakis S, Korkakaki E, Kalogerakis N. Biosurfactant production from marine hydrocarbon-degrading consortia

and pure bacterial strains using crude oil as carbon source. Frontiers in Microbiology. 2015;**6**:274

[66] Daniels R, Vanderleyden J, Michiels J. Quorum sensing and swarming migration in bacteria. FEMS Microbiology. 2004;**28**:261-289

[67] Sullivan ER. Molecular genetics of biosurfactants production. Current Opinion in Biotechnology. 1998;**9**(3):263-269

[68] Yakimov MM, Kroeger A, Slepak TN, Giuliano L, Timmis KN, Golyshin PN. A putative lichenysis A Synthetase operon in *Bacillus licheniformis*: Initial characterization. Biochimica et Biophysica Acta. 1998;**1399**:141-153

[69] Ochsner UA, Fiechter A, Reiser J, Witholt B. Production of *Pseudomonas aeruginosa* rhamnolipid biosurfactant in heterologous hosts. Applied and Environmental Microbiology. 1995;**61**:3503-3506

[70] Guerra-Santos LH, Kappel O, Fiechter A. Dependence of *Pseudomonas aeruginosa* biosurfactant production in continuous culture biosurfactant production on nutritional and environmental factors. Applied Microbiology and Biotechnology. 1986;**24**:443-448

[71] Selvin J, Sathiyanarayanan G, Lipton AN, Al-Dhabi NA, Arasu MV, Kiran GS. Ketide synthase (KS) domain prediction and analysis of iterative type II PKS gene in marine sponge-associated actinobacteria producing biosurfactants and antimicrobial agents. Frontiers in Microbiology. 2016;**7**:63

**19**

**Chapter 2**

Mice

**Abstract**

and associated metabolic syndrome.

**1. Introduction**

overweight, fecal metabolites, biomarkers

Fecal Metabolomics Insights of

*Alicia Huazano-García, Horacio Claudio Morales-Torres,* 

Targeted and non-targeted metabolite profiling can identify biomarkers after a dietary treatment leading to a better understanding of interactions between diet and health. This study was conducted to establish enriched or depleted metabolites in the feces of overweight mice after a diet shift plus agavins or inulins supplementation, and their possible association with beneficial effects on host health. Thirty-eight male C57BL/6 mice were fed with a high-fat diet for 5 weeks followed by a diet shift to a standard diet supplemented with agavins (HF-ST + A) or inulins (HF-ST + I) for five more weeks. Feces were collected before and after prebiotic supplementation for metabolomics analyses. HF-ST + I group increased the fecal excretion of two methyl esters: linoleic and oleic acid, while HF-ST + A mice showed a substantial augment of 2-decenal, fructose, cyclohexanol, and the acids: 10-undecenoic, 3-phenyllactic, nicotinic, 5-hydroxyvaleric, and lactic. From the metabolites identified in HF-ST + A, only lactic acid has been reported previously and associated with beneficial effects on host health. However, the identification of new metabolites, coming from the microbial fermentation of agavins, opens opportunities to transform this information into practical solutions to tackle overweight

**Keywords:** agavins, branched neo-fructans, metabolomics, postbiotics, prebiotics,

In the last decade, an increasing number of studies have been strongly associated with a high-fat consumption altering the gut microbiota composition and/or its functionality [1, 2]. It has also been related to overweight and obesity as well as the metabolic syndrome [3–5]. Overweight and obesity not only affects the wellbeing of an individual but also places an unwanted economic burden on society [6]. Therefore, it is necessary and urgent to find an effective way to prevent and/or treat these worldwide pathologies. In this sense, prebiotics might be a good nutritional alternative in the management of overweight and obesity and its associated metabolic syndrome, since their supplementation or consumption can modulate the

*Juan Vázquez-Martínez and Mercedes G. López*

Agavins Intake in Overweight

#### **Chapter 2**

*Metabolomics - New Insights into Biology and Medicine*

and pure bacterial strains using crude oil as carbon source. Frontiers in

Microbiology. 2015;**6**:274

1998;**9**(3):263-269

1998;**1399**:141-153

1995;**61**:3503-3506

1986;**24**:443-448

Microbiology. 2016;**7**:63

[66] Daniels R, Vanderleyden J, Michiels J. Quorum sensing and swarming migration in bacteria. FEMS

Microbiology. 2004;**28**:261-289

[68] Yakimov MM, Kroeger A, Slepak TN, Giuliano L, Timmis KN, Golyshin PN. A putative lichenysis A Synthetase operon in *Bacillus licheniformis*: Initial characterization. Biochimica et Biophysica Acta.

[67] Sullivan ER. Molecular genetics of biosurfactants production. Current Opinion in Biotechnology.

[69] Ochsner UA, Fiechter A, Reiser J, Witholt B. Production of *Pseudomonas aeruginosa* rhamnolipid biosurfactant in heterologous hosts. Applied and Environmental Microbiology.

[70] Guerra-Santos LH, Kappel O, Fiechter A. Dependence of *Pseudomonas aeruginosa* biosurfactant production in continuous culture biosurfactant production on nutritional and environmental factors. Applied Microbiology and Biotechnology.

[71] Selvin J, Sathiyanarayanan G, Lipton AN, Al-Dhabi NA, Arasu MV, Kiran GS. Ketide synthase (KS) domain prediction and analysis of iterative type II PKS gene in marine sponge-associated actinobacteria producing biosurfactants and antimicrobial agents. Frontiers in

by the drop-collapse test. World Journal of Microbiology and Biotechnology.

[59] Walter V, Syldatk C, Hausmann R. Screening concepts for the isolation

[60] Christova N, Tuleva B, Lalchev Z, Jordanovac A, Jordanov B. Rhamnolipid

of biosurfactant producing microorganisms. Advances in Experimental Medicine and Biology.

biosurfactants produced by *Renibacterium salmoninarum* 27BN during growth on n-hexadecane. Zeitschrift für Naturforschung. Section

[61] Rosenberg M, Gutnick D,

FEMS Microbiology Letters.

[63] Obuekwe CO, Al-Jadi ZK,

[64] Chakraborty S, Mukherji S, Mukherji S. Surface hydrophobicity of petroleum hydrocarbon degrading

*Burkholderia* strains and their

[65] Antoniou E, Fodelianakis S, Korkakaki E, Kalogerakis N.

2010;**78**:101-108

interactions with NAPLs and surfaces. Colloids and Surfaces. B, Biointerfaces.

Biosurfactant production from marine hydrocarbon-degrading consortia

Al-Saleh ES. Insight into heterogeneity in cell-surface hydrophobocity and ability to degrade hydrocarbons among cells of two hydrocarbon-degrading bacterial populations. Canadian Journal of Microbiology. 2007;**53**:252-260

1980;**9**(1):29-33

Rosenberg E. Adherence of bacteria to hydrocarbons—A simple method for measuring cell-surface hydrophobicity.

[62] Franzetti A, Caredda P, Colla PL, Pintus M, Tamburini E, Papacchini M, Bestetti G. Cultural factors affecting biosurfactant production by *Gordonia* sp. BS29. International Biodeterioration and Biodegradation. 2009;**63**:943-947

C. 2004;**59**(1-2):70-74

2005;**21**:851-853

2010;**672**:1-13

**18**

## Fecal Metabolomics Insights of Agavins Intake in Overweight Mice

*Alicia Huazano-García, Horacio Claudio Morales-Torres, Juan Vázquez-Martínez and Mercedes G. López*

#### **Abstract**

Targeted and non-targeted metabolite profiling can identify biomarkers after a dietary treatment leading to a better understanding of interactions between diet and health. This study was conducted to establish enriched or depleted metabolites in the feces of overweight mice after a diet shift plus agavins or inulins supplementation, and their possible association with beneficial effects on host health. Thirty-eight male C57BL/6 mice were fed with a high-fat diet for 5 weeks followed by a diet shift to a standard diet supplemented with agavins (HF-ST + A) or inulins (HF-ST + I) for five more weeks. Feces were collected before and after prebiotic supplementation for metabolomics analyses. HF-ST + I group increased the fecal excretion of two methyl esters: linoleic and oleic acid, while HF-ST + A mice showed a substantial augment of 2-decenal, fructose, cyclohexanol, and the acids: 10-undecenoic, 3-phenyllactic, nicotinic, 5-hydroxyvaleric, and lactic. From the metabolites identified in HF-ST + A, only lactic acid has been reported previously and associated with beneficial effects on host health. However, the identification of new metabolites, coming from the microbial fermentation of agavins, opens opportunities to transform this information into practical solutions to tackle overweight and associated metabolic syndrome.

**Keywords:** agavins, branched neo-fructans, metabolomics, postbiotics, prebiotics, overweight, fecal metabolites, biomarkers

#### **1. Introduction**

In the last decade, an increasing number of studies have been strongly associated with a high-fat consumption altering the gut microbiota composition and/or its functionality [1, 2]. It has also been related to overweight and obesity as well as the metabolic syndrome [3–5]. Overweight and obesity not only affects the wellbeing of an individual but also places an unwanted economic burden on society [6]. Therefore, it is necessary and urgent to find an effective way to prevent and/or treat these worldwide pathologies. In this sense, prebiotics might be a good nutritional alternative in the management of overweight and obesity and its associated metabolic syndrome, since their supplementation or consumption can modulate the

gut microbiota producing a wide range of metabolites (postbiotics), consequently generating positive effects on host health [6, 7].

Agavins are relatively new prebiotics that in pre-clinical studies have shown several beneficial effects on the health of individuals [8–10]. Agavins are neo-fructans composed of complex and highly branched molecules with β(2–1) and β(2–6) linkages as well as an internal glucose unit [11, 12].

Our research group has evidenced that agavins can decrease glucose, triglycerides, and cholesterol concentrations as well as increase the anorexigenic peptide glucagon-like peptide1 (GLP-1; appetite-suppressing peptide) secretion on mice fed with a normal diet [8, 9]. Moreover, recently, we reported that agavins intake led to the reversion of metabolic disorders in overweight mice (induced by highfat diet consumption) and also a substantial decrement of orexigenic peptide ghrelin (appetite-stimulating) and adipokines (leptin and insulin) levels in the portal vein, in such a way that all mice showed an integral improvement on their health [10].

On the other hand, due to the structural complexity of agavins, they cannot be degraded by endogenous gastrointestinal enzymes during their passage through the stomach and the small intestine; so, they reach the colon structurally unchanged, where they are fermented by the gut microbiota present in this organ [13, 14]. Fermentation of complex carbohydrates, such as agavins, might involve the collaboration of a highly diverse selection of gut microbes, which produce a myriad of different metabolites (postbiotics) that are suggested as key links in the communication between bacterial communities of the gut and the host [15, 16]. Nonetheless, only short-chain fatty acids (SCFA) such as acetate, propionate, butyrate, lactate, and succinate are among the metabolites reported up to now, derived from the agavins fermentation in *in vitro* and/or *in vivo* studies [9, 10, 17, 18]; the generation of these acids has been associated with different beneficial effects in the context of obesity, since SCFA reduce body weight gain, through G-protein-coupled receptors (GPRs), influencing the secretion of hormones involved in appetite control [19–21]. Moreover, SCFA are used as energy sources and may contribute to several metabolic pathways, including gluconeogenesis [22] and lipogenesis [23], thus contributing to whole-body energy homeostasis.

In spite of the above, other secondary metabolic products from the microbiota such as amino acids, nucleotides, bile acids, phenolic acids, fatty acids, and sterols, to mention some, can come from the agavins-microbiota interactions that have yet to be established. In the last decades, developed metabolomic tools have allowed researchers to study and characterize a wide range of metabolites in a non-invasive manner and also on biological systems, obtaining a large set of metabolites (metabolomics) that derive from gut microbes, enriched or depleted, after a dietary intervention [24]. This area of studies has been increased on the last decade, since this opens an opportunity to propose new biomarkers with new therapeutic approaches, through selective alterations of microbial production molecules to promote host health and prevent diseases [25].

In the present work, we established general and unique metabolites in overweight mice after agavins intake. We have previously showed that agavins consumption by overweight mice led to a gut microbiota modulation (these changes differed from those originated by inulins intake [14]); then, we hypothesized that agavins structure and the changes in the composition of gut microbiota in relation to inulins could lead to changes in colonic metabolic activity. The identification of microbial metabolites derived exclusively from agavins consumption may help to propose new biomarkers with huge potential and applicability on the prevention and/or treatment of overweight and their comorbidities.

**21**

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice*

Thirty-eight male C57BL/6 mice (12 weeks old at the beginning of the experiment were obtained from Universidad Autonoma Metropolitana, Mexico city, Mexico) and housed in a temperature and humidity controlled room with a 12-h light–dark cycles. Mice were maintained in individual cages and subject to two experimental phases, to gain and loss weight, respectively. In the first phase, mice were fed with a high-fat diet (n = 30; 58Y1 Test Diet, St. Louis, MO, USA) for 5 weeks to induce overweight in the animals. In the second phase, overweight mice (HF) were shifted to the standard diet (5053 Lab Diet, St. Louis, MO, USA) alone (HF-ST; n = 8) or supplemented with agavins (HF-ST + A; n = 8) or inulins (HF-ST + I; n = 8) for five more weeks. Moreover, we had a healthy control group of mice (ST; n = 8), which were fed with the standard diet (5053 Lab Diet, St. Louis,

The high-fat diet (58Y1 Test Diet) had 20.3% calories from carbohydrates (16.15% maltodextrin, 8.85% sucrose, and 6.46% powdered cellulose), 18.1% from proteins, and 61.6% from fat (31.7% lard and 3.2% soybean oil), whereas the standard diet (5053 Lab Diet) contained 62.4% calories from carbohydrates (28.6% starch, 3.24% sucrose, 1.34% lactose, 0.24% fructose, and 0.19% glucose), 24.5%

Food and water were provided *at libitum* along the experiment. Mice experiments were conducted according to the Mexican Norm NOM-062-ZOO-1999 and approved by the Institutional Care and Use of Laboratory Animals Committee from

Agavins from 4-year-old *Agave tequilana* Weber blue variety plants were extracted and purified in our laboratory and presented an average degree polymerization (DP) of 8, whereas inulins (oligofructose) was bought from Megafarma® (Mexico city, Mexico) and possess an average DP of 5. Agavins and inulins were

**2.3 Feces collection and preparation for untargeted and organic acids metabolic** 

Feces were collected from each mouse at the end of the first and second experimental phases, before and after prebiotic supplementation, respectively. The feces of mice were pooled by treatment, lyophilized, triturated, and homogenized to generate fecal metabolites profiles. Untargeted metabolic analysis was carried out following a method adjusted from Eneroth et al. [26] and Gao et al. [27] as follows: 100 mg of feces were extracted tree times with chloroform/methanol (2:1), 1 mL each time. After that, the extracts were combined and solvent freed. The residue was resuspended in 1 mL of chloroform/methanol (2:1) and an aliquot of 50 μL was transferred to a vial. The aliquot was solvent freed under nitrogen flux and then was derivatized using BSTFA with 1% TCMS (80 μL) and pyridine (20 μL) at 80°C for 25 min. Once the system was at room temperature, isooctane was added to a final volume of 200 μL. Heptadecanoic acid, at final concentration of 3 mg/mL, was used

On the other hand, extraction of organic acids was performed according to García-Villalba et al. [28]. Briefly, 100 mg of feces were suspended in 1 mL of

*DOI: http://dx.doi.org/10.5772/intechopen.89844*

MO, USA) throughout the experiment.

from proteins, and 13.1% from fat.

**2.2 Agavins and inulins**

**analyses**

as internal standard.

Cinvestav-Mexico (CICUAL; protocol number 0091-14).

added in the water at a concentration of 0.38 g/mouse/day [10].

**2. Materials and methods**

**2.1 Animals and diets**

### **2. Materials and methods**

#### **2.1 Animals and diets**

*Metabolomics - New Insights into Biology and Medicine*

generating positive effects on host health [6, 7].

linkages as well as an internal glucose unit [11, 12].

health [10].

whole-body energy homeostasis.

health and prevent diseases [25].

gut microbiota producing a wide range of metabolites (postbiotics), consequently

Agavins are relatively new prebiotics that in pre-clinical studies have shown several beneficial effects on the health of individuals [8–10]. Agavins are neo-fructans composed of complex and highly branched molecules with β(2–1) and β(2–6)

Our research group has evidenced that agavins can decrease glucose, triglycerides, and cholesterol concentrations as well as increase the anorexigenic peptide glucagon-like peptide1 (GLP-1; appetite-suppressing peptide) secretion on mice fed with a normal diet [8, 9]. Moreover, recently, we reported that agavins intake led to the reversion of metabolic disorders in overweight mice (induced by highfat diet consumption) and also a substantial decrement of orexigenic peptide ghrelin (appetite-stimulating) and adipokines (leptin and insulin) levels in the portal vein, in such a way that all mice showed an integral improvement on their

On the other hand, due to the structural complexity of agavins, they cannot be degraded by endogenous gastrointestinal enzymes during their passage through the stomach and the small intestine; so, they reach the colon structurally unchanged, where they are fermented by the gut microbiota present in this organ [13, 14]. Fermentation of complex carbohydrates, such as agavins, might involve the collaboration of a highly diverse selection of gut microbes, which produce a myriad of different metabolites (postbiotics) that are suggested as key links in the communication between bacterial communities of the gut and the host [15, 16]. Nonetheless, only short-chain fatty acids (SCFA) such as acetate, propionate, butyrate, lactate, and succinate are among the metabolites reported up to now, derived from the agavins fermentation in *in vitro* and/or *in vivo* studies [9, 10, 17, 18]; the generation of these acids has been associated with different beneficial effects in the context of obesity, since SCFA reduce body weight gain, through G-protein-coupled receptors (GPRs), influencing the secretion of hormones involved in appetite control [19–21]. Moreover, SCFA are used as energy sources and may contribute to several metabolic pathways, including gluconeogenesis [22] and lipogenesis [23], thus contributing to

In spite of the above, other secondary metabolic products from the microbiota such as amino acids, nucleotides, bile acids, phenolic acids, fatty acids, and sterols, to mention some, can come from the agavins-microbiota interactions that have yet to be established. In the last decades, developed metabolomic tools have allowed researchers to study and characterize a wide range of metabolites in a non-invasive manner and also on biological systems, obtaining a large set of metabolites (metabolomics) that derive from gut microbes, enriched or depleted, after a dietary intervention [24]. This area of studies has been increased on the last decade, since this opens an opportunity to propose new biomarkers with new therapeutic approaches, through selective alterations of microbial production molecules to promote host

In the present work, we established general and unique metabolites in overweight mice after agavins intake. We have previously showed that agavins consumption by overweight mice led to a gut microbiota modulation (these changes differed from those originated by inulins intake [14]); then, we hypothesized that agavins structure and the changes in the composition of gut microbiota in relation to inulins could lead to changes in colonic metabolic activity. The identification of microbial metabolites derived exclusively from agavins consumption may help to propose new biomarkers with huge potential and applicability on the prevention and/or treatment of overweight and their

**20**

comorbidities.

Thirty-eight male C57BL/6 mice (12 weeks old at the beginning of the experiment were obtained from Universidad Autonoma Metropolitana, Mexico city, Mexico) and housed in a temperature and humidity controlled room with a 12-h light–dark cycles. Mice were maintained in individual cages and subject to two experimental phases, to gain and loss weight, respectively. In the first phase, mice were fed with a high-fat diet (n = 30; 58Y1 Test Diet, St. Louis, MO, USA) for 5 weeks to induce overweight in the animals. In the second phase, overweight mice (HF) were shifted to the standard diet (5053 Lab Diet, St. Louis, MO, USA) alone (HF-ST; n = 8) or supplemented with agavins (HF-ST + A; n = 8) or inulins (HF-ST + I; n = 8) for five more weeks. Moreover, we had a healthy control group of mice (ST; n = 8), which were fed with the standard diet (5053 Lab Diet, St. Louis, MO, USA) throughout the experiment.

The high-fat diet (58Y1 Test Diet) had 20.3% calories from carbohydrates (16.15% maltodextrin, 8.85% sucrose, and 6.46% powdered cellulose), 18.1% from proteins, and 61.6% from fat (31.7% lard and 3.2% soybean oil), whereas the standard diet (5053 Lab Diet) contained 62.4% calories from carbohydrates (28.6% starch, 3.24% sucrose, 1.34% lactose, 0.24% fructose, and 0.19% glucose), 24.5% from proteins, and 13.1% from fat.

Food and water were provided *at libitum* along the experiment. Mice experiments were conducted according to the Mexican Norm NOM-062-ZOO-1999 and approved by the Institutional Care and Use of Laboratory Animals Committee from Cinvestav-Mexico (CICUAL; protocol number 0091-14).

#### **2.2 Agavins and inulins**

Agavins from 4-year-old *Agave tequilana* Weber blue variety plants were extracted and purified in our laboratory and presented an average degree polymerization (DP) of 8, whereas inulins (oligofructose) was bought from Megafarma® (Mexico city, Mexico) and possess an average DP of 5. Agavins and inulins were added in the water at a concentration of 0.38 g/mouse/day [10].

#### **2.3 Feces collection and preparation for untargeted and organic acids metabolic analyses**

Feces were collected from each mouse at the end of the first and second experimental phases, before and after prebiotic supplementation, respectively. The feces of mice were pooled by treatment, lyophilized, triturated, and homogenized to generate fecal metabolites profiles. Untargeted metabolic analysis was carried out following a method adjusted from Eneroth et al. [26] and Gao et al. [27] as follows: 100 mg of feces were extracted tree times with chloroform/methanol (2:1), 1 mL each time. After that, the extracts were combined and solvent freed. The residue was resuspended in 1 mL of chloroform/methanol (2:1) and an aliquot of 50 μL was transferred to a vial. The aliquot was solvent freed under nitrogen flux and then was derivatized using BSTFA with 1% TCMS (80 μL) and pyridine (20 μL) at 80°C for 25 min. Once the system was at room temperature, isooctane was added to a final volume of 200 μL. Heptadecanoic acid, at final concentration of 3 mg/mL, was used as internal standard.

On the other hand, extraction of organic acids was performed according to García-Villalba et al. [28]. Briefly, 100 mg of feces were suspended in 1 mL of

aqueous 0.5% phosphoric acid solution and mixed in vortex for 2 min. After that, samples were centrifuged for 10 min at 10,000 g. Then, the supernatant was transferred to a vial and was extracted with an equal volume of ethyl acetate. 2-Methyl valeric acid was used as internal standard at final concentration of 2 mM. This system was centrifuged for 10 min at 10,000 g, and then 200 μL of the ethyl acetate phase were transferred to a vial, dried under nitrogen flux, and derivatized using 80 μL of BSFTA with 1% TMCS and 20 μL of pyridine. The mix was allowed to react at 80°C for 25 min. After the mix was at room temperature, isooctane was added to a final volume of 200 μL.

#### **2.4 Gas chromatography/mass spectrometry analysis**

For GC/MS analysis, 1 μL of the organic phase was injected in the pulsedsplitless mode. Injector temperature was set to 260°C. A HP-5-MS capillary column (30 m × 25 μm × 0.25 μm) was used with helium as carrier gas at constant flow rate of 1 mL/min. Oven program began at 40°C (held 5 min), then increased at rate of 6°C/min until 170°C, then a second temperature ramp of 12°C/min until 290°C was applied. Transfer line temperature was set at 260°C. Mass spectrometer operated at 70 eV of electron energy, quadrupole and ion-source temperatures were 150 and 230°C, respectively. Data were obtained scanning from 40 to 550 m/z, while MassHunter Workstation software version B.0.0.6 (Agilent Technologies, Inc.) was used to collect the data. Components mass spectra and retention times were obtained using the AMDIS (automated mass spectral deconvolution and identification system, http://www.amdis.net/) software. Compounds identification was achieved by comparing their respective extracted mass spectrum with the mass spectra of the standards and/or with the mass spectra data of the NIST library and software (National Institute of Standards and Technology, USA).

#### **2.5 Statistics and data analysis**

Results are present as mean ± standard deviation. Differences between the diets were determined using one-way ANOVA followed by a Tukey post hoc test or a Dunett T3 post hoc test. Differences were considered significant when p < 0.05. Statistical analyses were performed using the IBM SPSS Statistics software version 22. Principal component analysis and heatmap were conducted using a language and environment for statistical computing R and the ade4 and gplots packages.

#### **3. Results and discussion**

Previous studies carried out by our research group evidenced that agavins intake led to improvement on health and wellness of the host, which has been associated with gut microbiota modulation and their metabolic products such as SCFA [10, 14]; nonetheless, other bioactive chemical compounds coming from agavins fermentation that could also contribute with the beneficial agavins consumption effects are unknown yet. In the present work, we performed a metabolomics analysis to establish and propose general and unique metabolites (postbiotics) in the feces of overweight mice after agavins (prebiotic) intake. Since it has been recommended the use of combination of methodologies to extend the metabolic coverage of microbiota [29], we performed an untargeted metabolic as well as organic acids profile analyses to carry out this task.

**23**

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice*

Untargeted metabolic analysis showed a total of 300 metabolites, from which only 109 were identified completely. Those 109 compounds mainly included fatty acids and their esters, carbohydrates, sterols, alcohols, alkanes, SCFA, aldehydes,

PCA was applied to the data to investigate metabolomics changes derived from agavins consumption. The variance explained by each principal component (PC) is displayed on the X and Y axes. PC1 and PC2 account for 62.5 and 20.5% of the variance, respectively. Very clear and separated clusters were observed among overweight mice (HF) and the other mice groups, suggesting differences on fecal metabolomics profiling (**Figure 1A**). In addition, mice that received agavins supplement are clearly separate, into a distinct cluster, from rodents fed with inulins supplementation. Interestingly, HF-ST + A group appear very close to standard diets (HF-ST and ST), evidencing a large similarity on metabolites among them

Moreover, the loading plot illustrates the variables (metabolites) that are responsible for the discrimination (clustering of the samples) observed in the PCA plot. Then, according to the loading plot, HF and HF-ST + I groups shared the PC2 due to high content of oleic acid, cholesterol, and stigmasterol in the feces (**Figure 1B**).

**ID RT Compound Family** 12.62 Cyclohexanol Alcohol 13.028 Carbonic acid Others 13.183 β-Hydroxybutyric acid βOH-SCFA 13.234 Heptanoic acid FA 13.281 α-Hydroxyvaleric acid αOH-SCFA 14.008 Benzaldehyde, 2,5-dimethyl- Aldehyde 14.049 L-Valine Amino acid 14.483 Urea Others 14.623 2-Decenal, (E)- Aldehyde 14.94 Glycerol Polyalcohol 15.074 2,5-dihydroxy hexane, 2,5-dimethyl- Polyalcohol 15.406 Succinic acid DiCAc 15.778 Uracil Nucleobase 16.153 Butane, 1,2,4, triol Polyalcohol 16.646 Thymine Nucleobase 16.774 Hydrocinnamic acid PhePr

17 17.096 Bicyclo[2.2.1]heptane-1-carboxylic acid,

7-Hydroxy, methyl ester

18 17.258 Decanoic acid FA 19 17.55 Decane, 2,3,5,8-tetramethyl- Alkane

Ester

Of these 109 compounds (**Table 1**), 32 presented a significantly differential abundance between the different evaluated diets (Tukey's test, p < 0.05; **Table 2**). These 32 metabolites were grouped mostly in fatty acids and sterols and subsequently used for the principal component analysis (PCA) and heat map

amino acids, nucleobases, bile salts, and phenylpropanoids (**Table 1**).

*DOI: http://dx.doi.org/10.5772/intechopen.89844*

compared to HF-ST + I group (**Figure 1A**).

**3.1 Untargeted metabolic profiles**

construction.

#### **3.1 Untargeted metabolic profiles**

*Metabolomics - New Insights into Biology and Medicine*

**2.4 Gas chromatography/mass spectrometry analysis**

a final volume of 200 μL.

Technology, USA).

**2.5 Statistics and data analysis**

**3. Results and discussion**

profile analyses to carry out this task.

aqueous 0.5% phosphoric acid solution and mixed in vortex for 2 min. After that, samples were centrifuged for 10 min at 10,000 g. Then, the supernatant was transferred to a vial and was extracted with an equal volume of ethyl acetate. 2-Methyl valeric acid was used as internal standard at final concentration of 2 mM. This system was centrifuged for 10 min at 10,000 g, and then 200 μL of the ethyl acetate phase were transferred to a vial, dried under nitrogen flux, and derivatized using 80 μL of BSFTA with 1% TMCS and 20 μL of pyridine. The mix was allowed to react at 80°C for 25 min. After the mix was at room temperature, isooctane was added to

For GC/MS analysis, 1 μL of the organic phase was injected in the pulsedsplitless mode. Injector temperature was set to 260°C. A HP-5-MS capillary column (30 m × 25 μm × 0.25 μm) was used with helium as carrier gas at constant flow rate of 1 mL/min. Oven program began at 40°C (held 5 min), then increased at rate of 6°C/min until 170°C, then a second temperature ramp of 12°C/min until 290°C was applied. Transfer line temperature was set at 260°C. Mass spectrometer operated at 70 eV of electron energy, quadrupole and ion-source temperatures were 150 and 230°C, respectively. Data were obtained scanning from 40 to 550 m/z, while MassHunter Workstation software version B.0.0.6 (Agilent Technologies, Inc.) was used to collect the data. Components mass spectra and retention times were obtained using the AMDIS (automated mass spectral deconvolution and identification system, http://www.amdis.net/) software. Compounds identification was achieved by comparing their respective extracted mass spectrum with the mass spectra of the standards and/or with the mass spectra data of the NIST library and software (National Institute of Standards and

Results are present as mean ± standard deviation. Differences between the diets were determined using one-way ANOVA followed by a Tukey post hoc test or a Dunett T3 post hoc test. Differences were considered significant when p < 0.05. Statistical analyses were performed using the IBM SPSS Statistics software version 22. Principal component analysis and heatmap were conducted using a language and environment

Previous studies carried out by our research group evidenced that agavins intake

led to improvement on health and wellness of the host, which has been associated with gut microbiota modulation and their metabolic products such as SCFA [10, 14]; nonetheless, other bioactive chemical compounds coming from agavins fermentation that could also contribute with the beneficial agavins consumption effects are unknown yet. In the present work, we performed a metabolomics analysis to establish and propose general and unique metabolites (postbiotics) in the feces of overweight mice after agavins (prebiotic) intake. Since it has been recommended the use of combination of methodologies to extend the metabolic coverage of microbiota [29], we performed an untargeted metabolic as well as organic acids

for statistical computing R and the ade4 and gplots packages.

**22**

Untargeted metabolic analysis showed a total of 300 metabolites, from which only 109 were identified completely. Those 109 compounds mainly included fatty acids and their esters, carbohydrates, sterols, alcohols, alkanes, SCFA, aldehydes, amino acids, nucleobases, bile salts, and phenylpropanoids (**Table 1**).

Of these 109 compounds (**Table 1**), 32 presented a significantly differential abundance between the different evaluated diets (Tukey's test, p < 0.05; **Table 2**). These 32 metabolites were grouped mostly in fatty acids and sterols and subsequently used for the principal component analysis (PCA) and heat map construction.

PCA was applied to the data to investigate metabolomics changes derived from agavins consumption. The variance explained by each principal component (PC) is displayed on the X and Y axes. PC1 and PC2 account for 62.5 and 20.5% of the variance, respectively. Very clear and separated clusters were observed among overweight mice (HF) and the other mice groups, suggesting differences on fecal metabolomics profiling (**Figure 1A**). In addition, mice that received agavins supplement are clearly separate, into a distinct cluster, from rodents fed with inulins supplementation. Interestingly, HF-ST + A group appear very close to standard diets (HF-ST and ST), evidencing a large similarity on metabolites among them compared to HF-ST + I group (**Figure 1A**).

Moreover, the loading plot illustrates the variables (metabolites) that are responsible for the discrimination (clustering of the samples) observed in the PCA plot. Then, according to the loading plot, HF and HF-ST + I groups shared the PC2 due to high content of oleic acid, cholesterol, and stigmasterol in the feces (**Figure 1B**).



28.125, 28.661,

29.646, 29.932

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice*

**ID RT Compound Family** 25.198 Linoleic acid FA 25.25 Oleic acid FA 25.298 *trans*-11-Octadecenoic acid FA 25.315 *cis*-11-Octadecenoic acid FA 25.481 Stearic acid FA 26.262 2-O-glycerol-α-D-galactose CHO 26.288 Nonadecanoic acid FA 26.546 Arachidonic acid FA 26.648 tert-Hexadecanethiol Thiol 26.716 Tetratriacontane Alkane 26.743 Octadecane, 3-ethyl-5-(2-ethylbutyl)- Alkane 26.774 Succinylacetone Others 26.838 Hentriacontane Alkane 26.9 Oleamide Amide 27.006 Sebacic acid DiCAc 27.095 Eicosanoic acid FA 27.509 1-O-hexadecylglycerol Glycerol-ether 27.584 Heneicosanoic acid FA 27.618 Propyl myristate Ester 27.716 2-Octadecenoic acid FA 27.866 Heneicosanoic acid FA 28.498 4-n-octadecylcyclohexane, 1,3,5-trimethyl- Alkane 28.62 Docosanoic acid FA 28.76 α-Hydroxy sebacic acid αOH-DiCAc 28.967 1-O-Octadecylglycerol Glycerol-ether 29.01 *cis*-4-Tetradecene, 2-methyl- Alkene 29.339 Tricosanoic acid FA 29.578 1-Monooleoylglycerol Monoglyceride 29.709 1-Docosanol Alcohol 29.895 *cis*-15-Tetracosenoic acid FA

Disaccharides (including sucrose) CHO

 30.026 Octadecane, 3-ethyl-5-(2-ethylbutyl)- Alkane 30.221 Enterolactone Lignin 30.329 1-O-hexadecylglycerol Glycerol-ether 30.383 Tricosanol Alcohol 30.428 Cholesta-2,4-diene Sterol 30.673 Cholesta-3,5-diene Sterol 31.31 β-Tocopherol Vitamin 31.454 Hexacosanoic acid FA 31.891 Coprostan-3-ol Sterol

*DOI: http://dx.doi.org/10.5772/intechopen.89844*


#### *Fecal Metabolomics Insights of Agavins Intake in Overweight Mice DOI: http://dx.doi.org/10.5772/intechopen.89844*

*Metabolomics - New Insights into Biology and Medicine*

**ID RT Compound Family** 17.666 Dodecane, 4,6-dimethyl- Alkane 17.741 Dodecane, 2,6,11-trimethyl- Alkane 17.95 2,4-Ditert-butylphenol Phenolic 18.011 1-Butene, 1-phenyl-3-(hydroxy)-, E Ar 18.168 Pyroglutamic acid Amino acid 18.422 2,6-Ditert-butylphenol Phenolic 18.642 Pentadecane Alkane 18.637 Undecanoic acid FA 18.724 Heptadecane, 2,6,10,15-tetramethyl- Alkane 19.173 *m*-Hydroxyphenylacetic acid OH-Ar-SCFA 19.228 Cyanuric acid Triazine 19.428 D-Arabinose CHO 19.52 *p*-Hydroxyphenylacetic acid OH-Ar-SCFA 19.599 *n*-Dodecanoic acid FA 20.172 Hexadecane, 2,6,11,15-tetramethyl- Alkane 20.204 Xylulose CHO 19.153, 20.341 D-Mannose CHO 20.438 10-Undecenoic acid FA 20.51 Benzenepropanoic acid PhePr 21.051 Glycerol phosphate Others 21.159 Tetradecanoic acid, 12-methyl-, methyl ester FAME 21.225 Azelaic acid DiCAc 19.549, 21.389 D-Galactose CHO 21.583, 21.652 D-Fructose CHO 21.345 Tetradecanoic acid FA 21.878 Inositol Polyalcohol 22.039 Adenine Nucleobase

22.352, 22.436 Methyl-tetradecanoic acid isomers (C15 fatty acid

22.716 Pentadecanoic acid FA

 23.428 *cis*-9-Hexadecenoic acid FA 23.481 *trans*-9-Hexadecenoic acid FA 23.691 Hexadecanoic acid FA 24.128 Linoleic acid, methyl ester FAME 24.177 Oleic acid, methyl ester FAME 24.37 *cis*-10-Heptadecenoic acid FA 24.399 Stearic acid, methyl ester FAME 24.404 α-D-glucose, 2-(acetylamino)-2-deoxy CHO 25.102 5-Hydroxyindoleacetic acid IndolAc

isomers)

D-Glucose CHO

FA

21.966, 22.483,

23.336


*Some metabolites have more than one retention time (RT) due to the presence of isomers. SCFA, short-chain fatty acid; FA, fatty acid; FAME, fatty acid methyl ester; CHO, carbohydrate; αOH, alfa-hydroxy; βOH, beta-hydroxy; DiCAc, dicarboxylic acid; PyrCAc, pyridine carboxylic acid; ωOH, omega-hydroxy; Cy, cyclic; Ar, aromatic; PhePr, phenylpropanoid; TCA, tricarboxylic acid; IndolAc, indolic acid.*

#### **Table 1.**

*Metabolites identified in the feces of mice.*


**27**

biomarker for HF diet.

**Table 2.**

*supplementation.*

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice*

**ID RT Compound Family Fold change**

54 24.177 Oleic acid, methyl ester FAME −1.00 −0.07 −0.15 1.90

59 25.198 Linoleic acid FA −0.71 −0.12 −0.41 0.34 60 25.25 Oleic acid FA 0.34 −0.09 −0.35 0.18 81 28.62 Docosanoic acid FA −0.55 −0.01 −0.03 0.25 98 31.891 Coprostan-3-ol Sterol −0.08 0.21 0.54 1.53 100 32.768 Cholesterol Sterol 0.52 −0.09 −0.35 0.27 103 34.207 Stigmasterol Sterol −0.13 −0.17 −0.47 0.04 106 34.918 β-Sitosterol Sterol −0.75 −0.14 −0.44 −0.04 107 35.064 24-Ethylcoprostanol Sterol −0.86 −0.12 −0.29 −0.09 *Fold change value was calculated by comparison with the healthy mice fed with a standard diet (ST). HF, overweight mice; HF-ST, overweight mice that were switched to a standard diet; HF-ST + A, overweight mice changed to standard diet plus agavins; HF-ST + I, overweight mice changed to standard diet plus inulins. All the metabolites listed here have significant difference at least in one treatment p < 0.5. ID numbers correspond with those of Table 1. SCFA, short-chain fatty acid; FA, fatty acid; FAME, fatty acid methyl ester; CHO, carbohydrate; αOH, alfa-hydroxy; βOH, beta-hydroxy; DiCAc, dicarboxylic acid; Ar, aromatic; PhePr, phenylpropanoid; IndolAc, indolic acid.*

**HF HF-ST HF-ST+ A HF-ST+ I**

FAME −1.00 0.02 0.01 2.94

FAME −0.60 −0.15 0.02 1.15

IndolAc −1.00 0.57 0.86 1.38

D-Glucose CHO −0.98 −0.40 0.02 0.47

Besides, hierarchical clustering analysis (**Figure 2**) revealed that HF group had a very low content of most identified metabolites, with exception of cholesterol and oleic acid. In addition, the bile salt chenodeoxycholic acid was detected exclusively in the HF treatment, and although it was not included in the hierarchical analysis since it was not detected in any other treatment, this metabolite could be used a

*Fold-change of differential metabolites detected in the feces of overweight mice after a diet switch and prebiotic* 

Interestingly, only HF-ST + I mice presented a high content of cholesterol and oleic acid in its feces; therefore, in the heatmap, this group appears very close to HF (**Figure 2**). Moreover, HF-ST treatment is closely linked to ST group due to similar content of all evaluated compounds. While, HF-ST + A group is located as the link between HF-ST + I and the cluster of standard diets (HF-ST and ST **Figure 2**).

In contrast to HF-ST group, HF-ST + A and HF-ST + I exhibited an enrichment of the following acids: succinic, β-hydroxybutyric (BHB), α-hydroxyvaleric, and pyroglutamic; as well as 12-methyl-tetradecanoic acid methyl ester and adenine,

On the other hand, HF-ST + A mice showed the highest content of 2-decenal, 10-undecenoic acid (UDA), cyclohexanol, and fructose as well as the lowest levels of oleic acid and cholesterol compared to HF, HF-ST, and HF-ST + I groups; hence, these metabolites might be used as biomarkers for agavins prebiotic. While, HF-ST + I mice had an increment of three methyl esters: linoleic, oleic, and stearic

which could be used as biomarkers for mice groups with prebiotics.

*DOI: http://dx.doi.org/10.5772/intechopen.89844*

53 24.128 Linoleic acid, methyl

56 24.399 Stearic acid, methyl

58 25.102 5-Hydroxyindoleacetic

ester

ester

acid

49 21.966 22.483 23.336


#### *Fecal Metabolomics Insights of Agavins Intake in Overweight Mice DOI: http://dx.doi.org/10.5772/intechopen.89844*

*Fold change value was calculated by comparison with the healthy mice fed with a standard diet (ST). HF, overweight mice; HF-ST, overweight mice that were switched to a standard diet; HF-ST + A, overweight mice changed to standard diet plus agavins; HF-ST + I, overweight mice changed to standard diet plus inulins. All the metabolites listed here have significant difference at least in one treatment p < 0.5. ID numbers correspond with those of Table 1. SCFA, short-chain fatty acid; FA, fatty acid; FAME, fatty acid methyl ester; CHO, carbohydrate; αOH, alfa-hydroxy; βOH, beta-hydroxy; DiCAc, dicarboxylic acid; Ar, aromatic; PhePr, phenylpropanoid; IndolAc, indolic acid.*

#### **Table 2.**

*Metabolomics - New Insights into Biology and Medicine*

*phenylpropanoid; TCA, tricarboxylic acid; IndolAc, indolic acid.*

*Metabolites identified in the feces of mice.*

**ID RT Compound Family Fold change**

 12.62 Cyclohexanol Alcohol −1.00 0.10 0.57 −0.13 13.183 β-Hydroxybutyric acid βOH-SCFA −0.62 −0.48 0.15 0.34 13.234 Heptanoic acid FA −0.88 −0.39 −0.13 −0.13 13.281 α-Hydroxyvaleric acid αOH-SCFA −0.30 −0.18 0.56 1.10 14.623 2-Decenal, (E)- Aldehyde −1.00 0.18 1.79 −0.90 14.94 Glycerol Polyalcohol −0.90 −0.15 −0.16 0.01 15.406 Succinic acid DiCAc −1.00 −0.27 1.07 2.25 16.774 Hydrocinnamic acid PhePr −1.00 −0.15 −0.30 0.05 17.95 2,4-Ditert-butylphenol Phenolic −0.40 0.39 0.52 −0.16 18.168 Pyroglutamic acid Amino acid −0.78 0.00 0.17 1.29

**ID RT Compound Family** 32.551 α-Tocopherol Vitamin 32.768 Cholesterol Sterol 33.336 Lanosterol Sterol 33.852 Campesterol Sterol 34.207 Stigmasterol Sterol 34.346 Chenodeoxycholic acid Bile salt 34.563 Xi-Ergost-7-ene, 3β- Sterol 34.918 β-Sitosterol Sterol 35.064 24-Ethylcoprostanol Sterol 35.268 *trans*-Dehydroandrosterone Steroid 38.518 Urs-12-en-28-al, 3-(acetyloxy)-, (3β)- Sterol *Some metabolites have more than one retention time (RT) due to the presence of isomers. SCFA, short-chain fatty acid; FA, fatty acid; FAME, fatty acid methyl ester; CHO, carbohydrate; αOH, alfa-hydroxy; βOH, beta-hydroxy; DiCAc, dicarboxylic acid; PyrCAc, pyridine carboxylic acid; ωOH, omega-hydroxy; Cy, cyclic; Ar, aromatic; PhePr,* 

37 20.438 10-Undecenoic acid FA −0.75 0.01 1.03 −0.68 38 20.51 Benzenepropanoic acid PhePr −0.95 −0.28 −0.32 −0.24 39 21.051 Glycerol phosphate Others −0.98 −0.07 −0.16 0.24

44 21.345 Tetradecanoic acid FA −0.57 0.12 0.01 0.18 46 22.039 Adenine Nucleobase −0.92 −0.34 0.08 0.36 48 22.716 Pentadecanoic acid FA −0.68 −0.12 −0.04 0.48

**HF HF-ST HF-ST+ A HF-ST+ I**

Ar-acid −0.75 −0.27 −0.36 0.19

FAME −0.31 0.02 0.58 2.27

D-Fructose CHO −0.93 −0.88 0.36 −0.57

**26**

32 19.52 *p*-Hydroxyphenylacetic

40 21.159 Tetradecanoic acid,

43 21.583 21.652

**Table 1.**

acid

12-methyl, methyl ester

*Fold-change of differential metabolites detected in the feces of overweight mice after a diet switch and prebiotic supplementation.*

Besides, hierarchical clustering analysis (**Figure 2**) revealed that HF group had a very low content of most identified metabolites, with exception of cholesterol and oleic acid. In addition, the bile salt chenodeoxycholic acid was detected exclusively in the HF treatment, and although it was not included in the hierarchical analysis since it was not detected in any other treatment, this metabolite could be used a biomarker for HF diet.

Interestingly, only HF-ST + I mice presented a high content of cholesterol and oleic acid in its feces; therefore, in the heatmap, this group appears very close to HF (**Figure 2**). Moreover, HF-ST treatment is closely linked to ST group due to similar content of all evaluated compounds. While, HF-ST + A group is located as the link between HF-ST + I and the cluster of standard diets (HF-ST and ST **Figure 2**).

In contrast to HF-ST group, HF-ST + A and HF-ST + I exhibited an enrichment of the following acids: succinic, β-hydroxybutyric (BHB), α-hydroxyvaleric, and pyroglutamic; as well as 12-methyl-tetradecanoic acid methyl ester and adenine, which could be used as biomarkers for mice groups with prebiotics.

On the other hand, HF-ST + A mice showed the highest content of 2-decenal, 10-undecenoic acid (UDA), cyclohexanol, and fructose as well as the lowest levels of oleic acid and cholesterol compared to HF, HF-ST, and HF-ST + I groups; hence, these metabolites might be used as biomarkers for agavins prebiotic. While, HF-ST + I mice had an increment of three methyl esters: linoleic, oleic, and stearic

#### **Figure 1.**

*Metabolites enriched or depleted in the feces of overweight mice after a diet shift and prebiotic supplementation. (A) PCA and (B) loading plot of the two first PCs. Overweight mice (HF) after a diet change (HF-ST) and agavins (HF-ST + A) or inulins (HF-ST + I) supplementation. ST was a healthy mice group.*

#### **Figure 2.**

*Heatmap of differential metabolites found in the feces of overweight mice after a diet shift and prebiotic supplementation. HF, overweight mice; HF-ST, overweight mice after a diet shift; HF-ST + A, overweight mice that were switched to standard diet plus agavins supplement; HF-ST + I, overweight mice that were switched to standard diet plus inulins supplement. ST was a healthy mice group.*

**29**

supplementation.

with inulins intake.

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice*

acid in relation to the other mice groups. These results evidence a clear difference in the fecal metabolites profiles between agavins and inulins, which was associated to their structural differences, such as the presence of fructose observed exclusively in HF-ST + A group. Agavins structure presents at least four terminal fructose units, so that some gut bacteria might start breaking down this prebiotic releasing fructose and agavins of smaller degree of polymerization [30]. In addition, 2-decenal and UDA were the metabolites mostly enriched with agavins intake. 2-decenal has a broad antimicrobial spectrum against pathogenic bacteria [31], while UDA is a neuroprotectant compound [32, 33] used as a nutritional supplement for maintaining a healthy balance of gut microbiota [34]. Besides, UDA also might be acting through GPR84 (a newly described medium chain fatty acid receptor) associated to immunological responses [35], and this mechanism could also contribute to improve the host health. Whereas in comparison to agavins, inulins led to a significant increase of methyl esters and sterols, coinciding with previous studies which proposed this

event as a mechanism to improve the lipid metabolism of host [36, 37].

A total of 21 organic acids were identified, including SCFA, hydroxy-SCFA, dicarboxylic acids, and aromatic carboxylic acids (**Table 3**). PCA analysis of organic acids showed the HF group in a separate cluster, while standard diets (HF-ST and ST) displayed an overlap due to presence of similar metabolites in both groups (**Figure 3A**). In addition, PCA and the loading plot evidenced that HF-ST + A and HF-ST + I groups had a more similar organic acids profiles among them in relation

Moreover, hierarchical clustering analysis evidenced that HF group had the lowest content of all organic acids, while HF-ST + A mice exhibited the highest content of the majority of them; therefore, this group is located at one end of the heatmap (**Figure 4**). Noticeably, HF-ST + I group showed various metabolites with a similar content as HF group; for instance the following acids: 2 methyl butanoic, lactic, and hexanoic (**Table 3**); hence, HF-ST + I appears very close to HF group in the heatmap (**Figure 4**). Once again, HF-ST and ST are the closes groups because they

Interestingly, and despite that the hierarchical analyses locate fructans diets very

On the other hand, only agavins supplementation led to a notably enrichment of four acids: nicotinic, 3-phenyllactic, 5-hydroxyvaleric, and lactic; therefore, these metabolites could be used as specifics biomarkers for this prebiotic. It is known that nicotinic acid also stimulate the HCA2, thereby decreasing plasma lipids and protecting against atherosclerotic disorders [41], and this event could be contributing to improve lipid metabolism of overweight mice that we previously reported [10]. Whereas 3-phenyllactic (the metabolite with the highest increment after agavins consumption) is synthesized by *Lactobacillus* strains and exerts direct antipatho-

Surprisingly, we did not find any differential abundance of any organic acid

distant from each other (in addition to the great structural differences between agavins and inulins), HF-ST + A and HF-ST + I groups showed an increment of the following acids: succinic, BHB, α-hydroxyisovaleric, and α-hydroxyglutaric. Stunningly, succinic acid is involved in glucose homeostasis [38], while BHB has a neuroprotective effect in mice [39] as well as may inhibit the release of fatty acids from adipose tissue by the hydroxy-carboxylic acid receptor 2 (HCA2) [40]. Then, these metabolites might be employed in general as biomarkers of prebiotic

*DOI: http://dx.doi.org/10.5772/intechopen.89844*

**3.2 Organic acid profiles**

to the other diets (**Figure 3B**).

presented similar levels of most analyzed organic acids.

genic activities against bacteria, viruses, and fungi [42].

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice DOI: http://dx.doi.org/10.5772/intechopen.89844*

acid in relation to the other mice groups. These results evidence a clear difference in the fecal metabolites profiles between agavins and inulins, which was associated to their structural differences, such as the presence of fructose observed exclusively in HF-ST + A group. Agavins structure presents at least four terminal fructose units, so that some gut bacteria might start breaking down this prebiotic releasing fructose and agavins of smaller degree of polymerization [30]. In addition, 2-decenal and UDA were the metabolites mostly enriched with agavins intake. 2-decenal has a broad antimicrobial spectrum against pathogenic bacteria [31], while UDA is a neuroprotectant compound [32, 33] used as a nutritional supplement for maintaining a healthy balance of gut microbiota [34]. Besides, UDA also might be acting through GPR84 (a newly described medium chain fatty acid receptor) associated to immunological responses [35], and this mechanism could also contribute to improve the host health. Whereas in comparison to agavins, inulins led to a significant increase of methyl esters and sterols, coinciding with previous studies which proposed this event as a mechanism to improve the lipid metabolism of host [36, 37].

#### **3.2 Organic acid profiles**

*Metabolomics - New Insights into Biology and Medicine*

*Metabolites enriched or depleted in the feces of overweight mice after a diet shift and prebiotic supplementation. (A) PCA and (B) loading plot of the two first PCs. Overweight mice (HF) after a diet change (HF-ST) and* 

*Heatmap of differential metabolites found in the feces of overweight mice after a diet shift and prebiotic supplementation. HF, overweight mice; HF-ST, overweight mice after a diet shift; HF-ST + A, overweight mice that were switched to standard diet plus agavins supplement; HF-ST + I, overweight mice that were switched to* 

*standard diet plus inulins supplement. ST was a healthy mice group.*

*agavins (HF-ST + A) or inulins (HF-ST + I) supplementation. ST was a healthy mice group.*

**28**

**Figure 2.**

**Figure 1.**

A total of 21 organic acids were identified, including SCFA, hydroxy-SCFA, dicarboxylic acids, and aromatic carboxylic acids (**Table 3**). PCA analysis of organic acids showed the HF group in a separate cluster, while standard diets (HF-ST and ST) displayed an overlap due to presence of similar metabolites in both groups (**Figure 3A**). In addition, PCA and the loading plot evidenced that HF-ST + A and HF-ST + I groups had a more similar organic acids profiles among them in relation to the other diets (**Figure 3B**).

Moreover, hierarchical clustering analysis evidenced that HF group had the lowest content of all organic acids, while HF-ST + A mice exhibited the highest content of the majority of them; therefore, this group is located at one end of the heatmap (**Figure 4**). Noticeably, HF-ST + I group showed various metabolites with a similar content as HF group; for instance the following acids: 2 methyl butanoic, lactic, and hexanoic (**Table 3**); hence, HF-ST + I appears very close to HF group in the heatmap (**Figure 4**). Once again, HF-ST and ST are the closes groups because they presented similar levels of most analyzed organic acids.

Interestingly, and despite that the hierarchical analyses locate fructans diets very distant from each other (in addition to the great structural differences between agavins and inulins), HF-ST + A and HF-ST + I groups showed an increment of the following acids: succinic, BHB, α-hydroxyisovaleric, and α-hydroxyglutaric. Stunningly, succinic acid is involved in glucose homeostasis [38], while BHB has a neuroprotective effect in mice [39] as well as may inhibit the release of fatty acids from adipose tissue by the hydroxy-carboxylic acid receptor 2 (HCA2) [40]. Then, these metabolites might be employed in general as biomarkers of prebiotic supplementation.

On the other hand, only agavins supplementation led to a notably enrichment of four acids: nicotinic, 3-phenyllactic, 5-hydroxyvaleric, and lactic; therefore, these metabolites could be used as specifics biomarkers for this prebiotic. It is known that nicotinic acid also stimulate the HCA2, thereby decreasing plasma lipids and protecting against atherosclerotic disorders [41], and this event could be contributing to improve lipid metabolism of overweight mice that we previously reported [10]. Whereas 3-phenyllactic (the metabolite with the highest increment after agavins consumption) is synthesized by *Lactobacillus* strains and exerts direct antipathogenic activities against bacteria, viruses, and fungi [42].

Surprisingly, we did not find any differential abundance of any organic acid with inulins intake.

#### **Figure 3.**

*Organic acids detected in the feces of overweight mice after a diet shift and prebiotic supplementation. (A) PCA and (B) loading plot of the two first PCs. Overweight mice (HF) after a diet change (HF-ST) and agavins (HF-ST + A) or inulins (HF-ST + I) supplementation. ST was a healthy mice group.*

#### **Figure 4.**

*Hierarchically clustered heatmap of organic acids found in the feces of overweight mice after a diet shift and prebiotic supplementation. HF, overweight mice; HF-ST, overweight mice after a diet shift; HF-ST + A, overweight mice that were switched to standard diet plus agavins supplement; HF-ST + I, overweight mice that were switched to standard diet plus inulins supplement. ST was a healthy mice group.*

**31**

**4. Conclusions**

*prebiotic supplementation.*

**Table 3.**

Microbial metabolites found in agavins group exhibited greater similarity to healthy mice, plus enrichment of specific metabolites (biomarkers) such as 2-decenal, UDA, cyclohexanol, fructose as well as some organic acids that undoubtedly are

*Fold-change of differential organic acids detected in the feces of overweight mice after a diet switch and* 

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice*

**ID RT Compound Family tFold change**

110 11.015 Butanoic acid, 2-methyl SCFA −0.46 −0.15 −0.38 −0.32 111 12.382 *n*-Pentanoic acid SCFA −0.71 0.07 −0.37 −0.23

SCFA

SCFA

SCFA

SCFA

SCFA

SCFA

Ar-SCFA

OH-Cy-SCFA

SCFA

124 29.137 *cis*-Aconitic acid TCA −1.00 0.06 1.62 3.48

41 29.634 Azelaic acid DiCAc −0.69 0.20 0.25 −0.41 58 29.2929 5-Hydroxyindoleacetic acid IndolAc −0.90 −0.40 −0.03 −0.22

*Fold change value was calculated by comparison with the healthy mice fed with a standard diet (ST). HF, overweight mice; HF-ST, overweight mice that were switched to a standard diet; HF-ST + A, overweight mice changed to standard diet plus agavins; HF-ST + I, overweight mice changed to standard diet plus inulins. All the metabolites listed here have significant difference at least in one treatment p < 0.5. ID numbers correspond with those of Table 1. SCFA, short-chain fatty acid; αOH, alfa-hydroxy; βOH, beta-hydroxy; DiCAc, dicarboxylic acid; PyrCAc, pyridine carboxylic acid; ωOH, omega-hydroxy; Cy, cyclic; Ar, aromatic; PhePr, phenylpropanoid; TCA,* 

119 21.483 Fumaric acid DiCAc −1.00 0.29 1.05 0.38

116 18.248 Malonic acid DiCAc −0.53 0.08 0.29 0.18 117 20.133 Nicotinic acid PyrCAc −0.80 −0.18 0.56 −0.06 12 20.773 Succinic acid DiCAc −0.88 0.01 2.01 0.96

113 14.826 *n*-Hexanoic acid SCFA −0.66 0.12 −0.27 −0.54

**HF HF-ST HF-ST+ A HF-ST+ I**

−1.00 −0.41 0.05 −0.89

−0.89 −0.08 0.00 −0.39

−0.74 −0.98 0.62 0.23

−0.86 −0.44 2.26 0.11

−1.00 −0.18 0.51 −0.40

−1.00 −0.24 0.88 0.39

−0.80 −0.32 0.66 −0.32

−0.80 −0.15 −0.07 −0.53

−0.98 −0.02 −0.17 −0.55

PhePr −0.95 −0.02 −0.10 −0.51

PhePr −0.62 0.04 0.18 −0.09

*DOI: http://dx.doi.org/10.5772/intechopen.89844*

112 14.666 Lactic acid αOH-

114 15.028 Hydroxyacetic acid αOH-

3 17.177 β-Hydroxybutyric acid βOH-

115 17.312 α-Hydroxyisovaleric acid αOH-

118 21.186 5-Hydroxyvaleric acid ωOH-

120 26.404 α-Hydroxyglutaric acid αOH-

121 26.483 3-Phenyllactic acid αOH-

32 27.406 *p*-Hydroxyphenylacetic acid OH-Ar-

acid

acid

acid

122 26.993 *p*-Hydroxycyclohexaneacetic

123 28.745 *m*-Hydroxyphenylpropanoic

125 29.186 *p*-Hydroxyhydrocinnamic

*tricarboxylic acid; IndolAc, indolic acid.*


#### *Fecal Metabolomics Insights of Agavins Intake in Overweight Mice DOI: http://dx.doi.org/10.5772/intechopen.89844*

*Metabolomics - New Insights into Biology and Medicine*

*Organic acids detected in the feces of overweight mice after a diet shift and prebiotic supplementation. (A) PCA and (B) loading plot of the two first PCs. Overweight mice (HF) after a diet change (HF-ST) and* 

*Hierarchically clustered heatmap of organic acids found in the feces of overweight mice after a diet shift and prebiotic supplementation. HF, overweight mice; HF-ST, overweight mice after a diet shift; HF-ST + A, overweight mice that were switched to standard diet plus agavins supplement; HF-ST + I, overweight mice that* 

*were switched to standard diet plus inulins supplement. ST was a healthy mice group.*

*agavins (HF-ST + A) or inulins (HF-ST + I) supplementation. ST was a healthy mice group.*

**30**

**Figure 4.**

**Figure 3.**

*Fold change value was calculated by comparison with the healthy mice fed with a standard diet (ST). HF, overweight mice; HF-ST, overweight mice that were switched to a standard diet; HF-ST + A, overweight mice changed to standard diet plus agavins; HF-ST + I, overweight mice changed to standard diet plus inulins. All the metabolites listed here have significant difference at least in one treatment p < 0.5. ID numbers correspond with those of Table 1. SCFA, short-chain fatty acid; αOH, alfa-hydroxy; βOH, beta-hydroxy; DiCAc, dicarboxylic acid; PyrCAc, pyridine carboxylic acid; ωOH, omega-hydroxy; Cy, cyclic; Ar, aromatic; PhePr, phenylpropanoid; TCA, tricarboxylic acid; IndolAc, indolic acid.*

#### **Table 3.**

*Fold-change of differential organic acids detected in the feces of overweight mice after a diet switch and prebiotic supplementation.*

#### **4. Conclusions**

Microbial metabolites found in agavins group exhibited greater similarity to healthy mice, plus enrichment of specific metabolites (biomarkers) such as 2-decenal, UDA, cyclohexanol, fructose as well as some organic acids that undoubtedly are

playing a very important role on overweight mice health. For instance, 2-decenal possess antimicrobial properties; UDA is a neuroprotectant compound; nicotinic acid can decrease plasma lipids levels; while 3-phenyllatic acid shown antipathogenic activities versus bacteria, viruses and fungi. Nevertheless, further studies are needed to clarify the underlying mechanisms by which metabolites derived from agavins fermentation induce a beneficial effect on health of host. Finally, these findings open new and exciting opportunities to explore new biomarkers with applicability on prevention, therapy, or treatment of overweight people.

### **Acknowledgements**

We deeply appreciate Inulina y Miel de Agave, S.A. de C.V. for its constant support.

### **Conflict of interest**

All authors report no financial interests or potential conflicts of interest.

#### **Nomenclature**


### **Author details**

Alicia Huazano-García, Horacio Claudio Morales-Torres, Juan Vázquez-Martínez and Mercedes G. López\* Department of Biotechnology and Biochemistry, Center of Research and Advanced Studies of the National Polytechnic Institute, Irapuato, Mexico

\*Address all correspondence to: mercedes.lopez@cinvestav.mx

© 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.

**33**

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice*

of dietary fructans extracted from *Agave tequilana* Gto. And *Dasylirion* spp. The British Journal of Nutrition. 2008;**99**:254-261. DOI: 10.1017/

[9] Santiago-García PA, López MG. Agavins from *Agave angustifolia* and *Agave potatorum* affect food intake, body weight gain and satiety-related hormones (GLP-1 and ghrelin) in mice. Food and Function. 2014;**5**:3311-3319.

[10] Huazano-García A, López MG. Agavins reverse the metabolic disorders

[11] López MG, Mancilla-Margalli NA, Mendoza-Diaz G. Molecular structures of fructans from *Agave tequilana* Weber var. Azul. Journal of Agricultural and Food Chemistry. 2003;**51**:7835-7840.

[12] Mancilla-Margalli NA, López MG. Water-soluble carbohydrates and fructan structure patterns from agave and Dasylirion species. Journal of Agricultural and Food Chemistry. 2006;**54**:7832-7839. DOI: 10.1021/

Rastall RA, Tuohy KM, Hotchkiss A, Dubert-Ferrandon A, et al. Dietary prebiotics: Current status and new definition. Food Science and Technology Bulletin. 2010;**7**:1-19. DOI:

in overweight mice through the increment of short chain fatty acids and hormones. Food and Function. 2015;**6**:3720-3727. DOI: 10.1039/

S0007114507795338

DOI: 10.1039/c4fo00561a

DOI: 10.1021/jf030383v

[13] Gibson GR, Scott KP,

10.1616/1476-2137.15880

DOI: 10.3390/nu9090821

[14] Huazano-García A, Shin H, López MG. Modulation of gut microbiota of overweight mice by agavins and their association with body weight loss. Nutrients. 2017;**9**:E821.

c5fo00830a

jf060354v

*DOI: http://dx.doi.org/10.5772/intechopen.89844*

[1] Flint HJ, Duncan SH, Scott KP, Louis P. Links between diet, gut microbiota composition and gut metabolism. The Proceedings of the Nutrition Society. 2015;**74**:13-22. DOI:

[2] Turnbaugh PJ. Microbes and dietinduced obesity: Fast, cheap, and out of control. Cell Host and Microbe. 2017;**21**:278-281. DOI: 10.1016/j.

[3] Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: Human gut microbes associated with obesity. Nature. 2006;**444**:1022-1023. DOI:

[4] Turnbaugh PJ, Bäckhed F, Fulton L, Gordon JI. Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome. Cell Host and Microbe. 2008;**3**:213-223. DOI: 10.1016/j.

[5] Mazidi M, Rezaie P, Kengne AP, Mobarhan MG, Ferns GA. Gut

microbiome and metabolic syndrome. Diabetes and Metabolic Syndrome. 2016;**10**:S150-S157. DOI: 10.1016/j.

[6] Dahiya DK, Puniya M, Shandilya UK, Dhewa T, Kumar N, Kumar S, et al. Gut microbiota modulation and its relationship with obesity using prebiotic

fibers and probiotics: A review. Frontiers in Microbiology. 2017;**8**:563. DOI: 10.3389/fmicb.2017.00563

[7] He M, Shi B. Gut microbiota as a potential target of metabolic syndrome: The role of probiotics and prebiotics. Cell and Bioscience. 2017;**7**:54. DOI:

10.1186/s13578-017-0183-1

[8] Urias-Silvas JE, Cani PD, Delmée E, Neyrinck A, López MG, Delzenne NM. Physiological effects

10.1017/s0029665114001463

chom.2017.02.021

10.1038/nature4441022a

chom.2008.02.015

dsx.2016.01.024

**References**

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice DOI: http://dx.doi.org/10.5772/intechopen.89844*

#### **References**

*Metabolomics - New Insights into Biology and Medicine*

therapy, or treatment of overweight people.

**Acknowledgements**

**Conflict of interest**

**Nomenclature**

HF overweight mice

ST healthy mice

SCFA short-chain fatty acids

BHB β-hydroxybutyric acid UDA 10-undecenoic acid

provided the original work is properly cited.

PCA principal component analysis

GC/MS gas chromatography/mass spectrometry

support.

playing a very important role on overweight mice health. For instance, 2-decenal possess antimicrobial properties; UDA is a neuroprotectant compound; nicotinic acid can decrease plasma lipids levels; while 3-phenyllatic acid shown antipathogenic activities versus bacteria, viruses and fungi. Nevertheless, further studies are needed to clarify the underlying mechanisms by which metabolites derived from agavins fermentation induce a beneficial effect on health of host. Finally, these findings open new and exciting opportunities to explore new biomarkers with applicability on prevention,

We deeply appreciate Inulina y Miel de Agave, S.A. de C.V. for its constant

All authors report no financial interests or potential conflicts of interest.

HF-ST overweight mice that were switched to a standard diet HF-ST + A overweight mice changed to standard diet plus agavins HF-ST + I overweight mice changed to standard diet plus inulins

**32**

**Author details**

and Mercedes G. López\*

Alicia Huazano-García, Horacio Claudio Morales-Torres, Juan Vázquez-Martínez

Studies of the National Polytechnic Institute, Irapuato, Mexico

\*Address all correspondence to: mercedes.lopez@cinvestav.mx

Department of Biotechnology and Biochemistry, Center of Research and Advanced

© 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,

[1] Flint HJ, Duncan SH, Scott KP, Louis P. Links between diet, gut microbiota composition and gut metabolism. The Proceedings of the Nutrition Society. 2015;**74**:13-22. DOI: 10.1017/s0029665114001463

[2] Turnbaugh PJ. Microbes and dietinduced obesity: Fast, cheap, and out of control. Cell Host and Microbe. 2017;**21**:278-281. DOI: 10.1016/j. chom.2017.02.021

[3] Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: Human gut microbes associated with obesity. Nature. 2006;**444**:1022-1023. DOI: 10.1038/nature4441022a

[4] Turnbaugh PJ, Bäckhed F, Fulton L, Gordon JI. Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome. Cell Host and Microbe. 2008;**3**:213-223. DOI: 10.1016/j. chom.2008.02.015

[5] Mazidi M, Rezaie P, Kengne AP, Mobarhan MG, Ferns GA. Gut microbiome and metabolic syndrome. Diabetes and Metabolic Syndrome. 2016;**10**:S150-S157. DOI: 10.1016/j. dsx.2016.01.024

[6] Dahiya DK, Puniya M, Shandilya UK, Dhewa T, Kumar N, Kumar S, et al. Gut microbiota modulation and its relationship with obesity using prebiotic fibers and probiotics: A review. Frontiers in Microbiology. 2017;**8**:563. DOI: 10.3389/fmicb.2017.00563

[7] He M, Shi B. Gut microbiota as a potential target of metabolic syndrome: The role of probiotics and prebiotics. Cell and Bioscience. 2017;**7**:54. DOI: 10.1186/s13578-017-0183-1

[8] Urias-Silvas JE, Cani PD, Delmée E, Neyrinck A, López MG, Delzenne NM. Physiological effects of dietary fructans extracted from *Agave tequilana* Gto. And *Dasylirion* spp. The British Journal of Nutrition. 2008;**99**:254-261. DOI: 10.1017/ S0007114507795338

[9] Santiago-García PA, López MG. Agavins from *Agave angustifolia* and *Agave potatorum* affect food intake, body weight gain and satiety-related hormones (GLP-1 and ghrelin) in mice. Food and Function. 2014;**5**:3311-3319. DOI: 10.1039/c4fo00561a

[10] Huazano-García A, López MG. Agavins reverse the metabolic disorders in overweight mice through the increment of short chain fatty acids and hormones. Food and Function. 2015;**6**:3720-3727. DOI: 10.1039/ c5fo00830a

[11] López MG, Mancilla-Margalli NA, Mendoza-Diaz G. Molecular structures of fructans from *Agave tequilana* Weber var. Azul. Journal of Agricultural and Food Chemistry. 2003;**51**:7835-7840. DOI: 10.1021/jf030383v

[12] Mancilla-Margalli NA, López MG. Water-soluble carbohydrates and fructan structure patterns from agave and Dasylirion species. Journal of Agricultural and Food Chemistry. 2006;**54**:7832-7839. DOI: 10.1021/ jf060354v

[13] Gibson GR, Scott KP, Rastall RA, Tuohy KM, Hotchkiss A, Dubert-Ferrandon A, et al. Dietary prebiotics: Current status and new definition. Food Science and Technology Bulletin. 2010;**7**:1-19. DOI: 10.1616/1476-2137.15880

[14] Huazano-García A, Shin H, López MG. Modulation of gut microbiota of overweight mice by agavins and their association with body weight loss. Nutrients. 2017;**9**:E821. DOI: 10.3390/nu9090821

[15] Flint HJ, Scott KP, Duncan SH, Louis P, Forano E. Microbial degradation of complex carbohydrates in the gut. Gut Microbes. 2012;**3**:289-306. DOI: 10.4161/gmic.19897

[16] Cani PD, Van Hul M, Lefort C, Depommier C, Rastelli M, Everard A. Microbial regulation of organismal energy homeostasis. Nature Metabolism. 2019;**1**:34-46. DOI: 10.1038/s42255-018-0017-4

[17] García-Curbelo Y, Bocourt R, Savón LL, García-Vieyra MI, López MG. Prebiotic effect of *Agave fourcroydes* fructans: An animal model. Food and Function. 2015;**6**:3177-3182. DOI: 10.1039/C5FO00653H

[18] Ramnani P, Costabile A, Bustillo AGR, Gibson GR. A randomised, double-blind, cross-over study investigating the prebiotic effect of agave fructans in healthy humans subject. Journal of Nutritional Science. 2015;**4**:e10. DOI: 10.1017/JNS.2014.68

[19] Psichas A, Sleeth ML, Murphy KG, Brooks L, Bewick GA, Hanyaloglu AC, et al. The short chain fatty acid propionate stimulates GLP-1 and PYY secretion via free fatty acid receptor 2 in rodents. International Journal of Obesity. 2015;**39**:424-429. DOI: 10.1038/ijo.2014.153

[20] Tolhurst G, Heffron H, Lam YS, Parker HE, Habib AM, Diakogiannaki E, et al. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2. Diabetes. 2012;**61**:364-371. DOI: 10.2337/db11-1019

[21] Lin HV, Frasseto A, Kowalik EJ Jr, Nawrocki AR, Lu MM, Kosinski JR, et al. Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acids receptor 3-independent mechanisms. PLoS One. 2012;**7**:e35240. DOI: 10.1371/journal.pone.0035240

[22] De Vadder F, Kovatcheva-Datchary P, Goncalves D, Vinera J, Zitoun C, Duchampt A, et al. Microbiota-generated metabolites promote metabolic benefits via gutbrain neural circuits. Cell. 2014;**156**:84- 96. DOI: 10.1016/j.cell.2013.12.016

[23] Singh V, Chassaing B, Zhang L, San Yeoh B, Xiao X, Baker MT, et al. Microbiota-dependent hepatic lipogenesis mediated by stearoyl CoA desaturase 1 (SCD1) promotes metabolic syndrome in TLR5-deficient mice. Cell Metabolism. 2015;**22**:983-996. DOI: 10.1016/j.cmet.2015.09.028

[24] Wishart DS. Emerging application of metabolomics in drug discovery and precision medicine. Nature Reviews. Drug Discovery. 2016;**15**:473-484. DOI: 10.1038/nrd.2016.32

[25] Hamer HM, De Preter V, Windey K, Verbeke K. Functional analysis of colonic bacterial metabolism: Relevant to health? American Journal of Physiology. Gastrointestinal and Liver Physiology. 2012;**302**:G1-G9. DOI: 10.1152/ ajpgi.00048.2011

[26] Eneroth P, Hellstroem K, Ryhage R. Identification and quantification of neutral fecal steroids by gasliquid chromatography and mass spectrometry: Studies of human excretion during two dietary regimens. Journal of Lipid Research. 1964;**5**:245-262

[27] Gao X, Pujos-Guillot E, Sébédio JL. Development of a quantitative metabolomic approach to study clinical human fecal water metabolome based on trimethylsilylation derivatization and GC/MS analysis. Analytical Chemistry. 2010;**82**:6447-6456. DOI: 10.1021/ ac1006552

[28] García-Villalba R, Giménez-Bastida JA, García-Conesa MT, Tomás-Barberán FA, Carlos Espín J, Larrosa M.

**35**

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice*

10-undecylenic acid, a medium chain fatty acid derivative. The AAPS Journal. 2014;**16**:1064-1076. DOI: 10.1208/

[35] Alvarez-Curto E, Milligan G. Metabolism meets immunity: The role of free fatty acid receptors in the immune system. Biochemical Pharmacology. 2016;**114**:3-13. DOI:

[36] Yang J, Zhang S, Henning SM, Lee R, Hsu M, Grojean E, et al.

[37] Catry E, Bindels LB, Tailleus A, Lestavel S, Neyrinck AM, Goossens JF, et al. Targeting the gut microbiota with inulin-type fructans: Preclinical demonstration of a novel approach in the management of endothelial dysfunction. Gut. 2018;**67**:271-283. DOI:

[38] Hoque M, Ali S, Hoda M. Current status of G-protein coupled receptors as potential targets against type 2 diabetes mellitus. International Journal of Biological Macromolecules. 2018;**118**:2237-2244. DOI: 10.1016/j.

[39] Rahman M, Muhammad S, Khan MA, Chen H, Ridder DA, Müller-Fielitz H, et al. The β-hydroxybutyrate receptor HCA2 activates a neuroprotective subset of macrophages. Nature Communications. 2014;**5**:3944. DOI:

[40] Taggart AK, Kero J, Gan X, Cai TQ, Cheng K, Ippolito M, et al. (D)-betahydroxybutyrate inhibits adipocyte lipolysis via the nicotinic acid receptor PUMA-G. The Journal of Biological Chemistry. 2005;**280**:26649-26652. DOI:

10.1136/gutjnl-2016-313316

ijbiomac.2018.07.091

10.1038/ncomms4944

10.1074/jbc.c500213200

Cholesterol-lowering effects on dietary pomegranate extract and inulin in mice fed an obesogenic diet. The Journal of Nutritional Biochemistry. 2018;**52**:62-69. DOI: 10.1016/j.

10.1016/j.bcp.2016.03.017

jnutbio.2017.10.003

s12248-014-9634-3

*DOI: http://dx.doi.org/10.5772/intechopen.89844*

chromatography-mass spectrometry analysis of short-chain fatty acids in fecal samples. Journal of Separation Science. 2012;**35**:1906-1913. DOI:

[29] Matysik S, Le Roy CI, Liebisch G, Claus SP. Metabolomics of fecal samples: A practical consideration. Trends in Food Science and Technology. 2016;**57**:244-255. DOI: 10.1016/j.

[30] Huazano-García A, López MG.

fructooligosaccharides (a-FOS). Applied Biochemistry and Biotechnology. 2018;**184**:25-34. DOI: 10.1007/

Enzymatic hydrolysis of agavins to generate branched

[31] Bisignano G, Laganà MG, Trombetta D, Arena S, Nostro A, Uccella N, et al. *In vitro* antibacterial activity of some aliphatic aldehydes from *Olea europaea* L. FEMS Microbiology Letters. 2001;**198**:9- 13. DOI: 10.1111/j.1574-6968.2001.

[32] Jantas D, Piotrowski M,

Lason W. An involvement of PI3-K/ Akt activation and inhibition of AIF translocation in neuroprotective effects of undecylenic acid (UDA) against pro-apoptotic factors-induced cell death in human neuroblastoma SH-SY5Y cells. Journal of Cellular Biochemistry. 2015;**116**:2882-2895. DOI: 10.1002/

[33] Lee E, Eom JE, Kim HL, Kang DH, Jun KY, Jung DS, et al. Neuroprotective effect of undecylenic acid extrated from *Ricinus communis* L. through inhibition of μ-calpain. European Journal of Pharmaceutical Sciences. 2012;**46**:17-25.

DOI: 10.1016/j.ejps.2012.01.015

[34] Brayden DJ, Walsh E. Efficacious intestinal permeation enhancement induced by the sodium salt of

s12010-017-2526-0

tb10611.x

jcb.25236

Alternative method for gas

10.1002/jssc.201101121

tifs.2016.05.011

*Fecal Metabolomics Insights of Agavins Intake in Overweight Mice DOI: http://dx.doi.org/10.5772/intechopen.89844*

Alternative method for gas chromatography-mass spectrometry analysis of short-chain fatty acids in fecal samples. Journal of Separation Science. 2012;**35**:1906-1913. DOI: 10.1002/jssc.201101121

*Metabolomics - New Insights into Biology and Medicine*

[22] De Vadder F, Kovatcheva-Datchary

[23] Singh V, Chassaing B, Zhang L, San Yeoh B, Xiao X, Baker MT, et al. Microbiota-dependent hepatic lipogenesis mediated by stearoyl CoA desaturase 1 (SCD1) promotes metabolic syndrome in TLR5-deficient mice. Cell Metabolism. 2015;**22**:983-996.

DOI: 10.1016/j.cmet.2015.09.028

10.1038/nrd.2016.32

ajpgi.00048.2011

1964;**5**:245-262

ac1006552

[24] Wishart DS. Emerging application of metabolomics in drug discovery and precision medicine. Nature Reviews. Drug Discovery. 2016;**15**:473-484. DOI:

[25] Hamer HM, De Preter V, Windey K, Verbeke K. Functional analysis of colonic bacterial metabolism: Relevant to health? American Journal of Physiology. Gastrointestinal and Liver Physiology.

[26] Eneroth P, Hellstroem K, Ryhage R. Identification and quantification of neutral fecal steroids by gasliquid chromatography and mass spectrometry: Studies of human excretion during two dietary

regimens. Journal of Lipid Research.

[28] García-Villalba R, Giménez-Bastida JA, García-Conesa MT, Tomás-Barberán

FA, Carlos Espín J, Larrosa M.

[27] Gao X, Pujos-Guillot E, Sébédio JL. Development of a quantitative metabolomic approach to study clinical human fecal water metabolome based on trimethylsilylation derivatization and GC/MS analysis. Analytical Chemistry. 2010;**82**:6447-6456. DOI: 10.1021/

2012;**302**:G1-G9. DOI: 10.1152/

P, Goncalves D, Vinera J, Zitoun C, Duchampt A, et al. Microbiota-generated metabolites promote metabolic benefits via gutbrain neural circuits. Cell. 2014;**156**:84- 96. DOI: 10.1016/j.cell.2013.12.016

[15] Flint HJ, Scott KP, Duncan SH, Louis P, Forano E. Microbial degradation of complex carbohydrates in the gut. Gut Microbes. 2012;**3**:289-306. DOI:

[16] Cani PD, Van Hul M, Lefort C, Depommier C, Rastelli M, Everard A. Microbial regulation of organismal energy homeostasis. Nature Metabolism. 2019;**1**:34-46. DOI: 10.1038/s42255-018-0017-4

[17] García-Curbelo Y, Bocourt R, Savón LL, García-Vieyra MI, López MG. Prebiotic effect of *Agave fourcroydes* fructans: An animal model. Food and Function. 2015;**6**:3177-3182. DOI:

10.1039/C5FO00653H

[18] Ramnani P, Costabile A, Bustillo AGR, Gibson GR. A

[19] Psichas A, Sleeth ML,

DOI: 10.1038/ijo.2014.153

10.2337/db11-1019

randomised, double-blind, cross-over study investigating the prebiotic effect of agave fructans in healthy humans subject. Journal of Nutritional Science. 2015;**4**:e10. DOI: 10.1017/JNS.2014.68

Murphy KG, Brooks L, Bewick GA, Hanyaloglu AC, et al. The short chain fatty acid propionate stimulates GLP-1 and PYY secretion via free fatty acid receptor 2 in rodents. International Journal of Obesity. 2015;**39**:424-429.

[20] Tolhurst G, Heffron H, Lam YS, Parker HE, Habib AM, Diakogiannaki E, et al. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2. Diabetes. 2012;**61**:364-371. DOI:

[21] Lin HV, Frasseto A, Kowalik EJ Jr, Nawrocki AR, Lu MM, Kosinski JR, et al. Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acids receptor 3-independent mechanisms. PLoS One. 2012;**7**:e35240. DOI: 10.1371/journal.pone.0035240

10.4161/gmic.19897

**34**

[29] Matysik S, Le Roy CI, Liebisch G, Claus SP. Metabolomics of fecal samples: A practical consideration. Trends in Food Science and Technology. 2016;**57**:244-255. DOI: 10.1016/j. tifs.2016.05.011

[30] Huazano-García A, López MG. Enzymatic hydrolysis of agavins to generate branched fructooligosaccharides (a-FOS). Applied Biochemistry and Biotechnology. 2018;**184**:25-34. DOI: 10.1007/ s12010-017-2526-0

[31] Bisignano G, Laganà MG, Trombetta D, Arena S, Nostro A, Uccella N, et al. *In vitro* antibacterial activity of some aliphatic aldehydes from *Olea europaea* L. FEMS Microbiology Letters. 2001;**198**:9- 13. DOI: 10.1111/j.1574-6968.2001. tb10611.x

[32] Jantas D, Piotrowski M, Lason W. An involvement of PI3-K/ Akt activation and inhibition of AIF translocation in neuroprotective effects of undecylenic acid (UDA) against pro-apoptotic factors-induced cell death in human neuroblastoma SH-SY5Y cells. Journal of Cellular Biochemistry. 2015;**116**:2882-2895. DOI: 10.1002/ jcb.25236

[33] Lee E, Eom JE, Kim HL, Kang DH, Jun KY, Jung DS, et al. Neuroprotective effect of undecylenic acid extrated from *Ricinus communis* L. through inhibition of μ-calpain. European Journal of Pharmaceutical Sciences. 2012;**46**:17-25. DOI: 10.1016/j.ejps.2012.01.015

[34] Brayden DJ, Walsh E. Efficacious intestinal permeation enhancement induced by the sodium salt of

10-undecylenic acid, a medium chain fatty acid derivative. The AAPS Journal. 2014;**16**:1064-1076. DOI: 10.1208/ s12248-014-9634-3

[35] Alvarez-Curto E, Milligan G. Metabolism meets immunity: The role of free fatty acid receptors in the immune system. Biochemical Pharmacology. 2016;**114**:3-13. DOI: 10.1016/j.bcp.2016.03.017

[36] Yang J, Zhang S, Henning SM, Lee R, Hsu M, Grojean E, et al. Cholesterol-lowering effects on dietary pomegranate extract and inulin in mice fed an obesogenic diet. The Journal of Nutritional Biochemistry. 2018;**52**:62-69. DOI: 10.1016/j. jnutbio.2017.10.003

[37] Catry E, Bindels LB, Tailleus A, Lestavel S, Neyrinck AM, Goossens JF, et al. Targeting the gut microbiota with inulin-type fructans: Preclinical demonstration of a novel approach in the management of endothelial dysfunction. Gut. 2018;**67**:271-283. DOI: 10.1136/gutjnl-2016-313316

[38] Hoque M, Ali S, Hoda M. Current status of G-protein coupled receptors as potential targets against type 2 diabetes mellitus. International Journal of Biological Macromolecules. 2018;**118**:2237-2244. DOI: 10.1016/j. ijbiomac.2018.07.091

[39] Rahman M, Muhammad S, Khan MA, Chen H, Ridder DA, Müller-Fielitz H, et al. The β-hydroxybutyrate receptor HCA2 activates a neuroprotective subset of macrophages. Nature Communications. 2014;**5**:3944. DOI: 10.1038/ncomms4944

[40] Taggart AK, Kero J, Gan X, Cai TQ, Cheng K, Ippolito M, et al. (D)-betahydroxybutyrate inhibits adipocyte lipolysis via the nicotinic acid receptor PUMA-G. The Journal of Biological Chemistry. 2005;**280**:26649-26652. DOI: 10.1074/jbc.c500213200

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Section 2

Metabolomics: New

Insights into Medicine

Section 2
