**2. Release and identification of peptides**

## **2.1. Peptides in cheese**

Proteolysis in cheese has been linked to its importance for texture, taste and flavour development during ripening. Changes of the cheese texture occur due to breakdown of the protein network. It contributes directly to taste and flavour by the formation of peptides and free amino acids as well as by liberation of substrates for further catabolic changes and thereby formation of volatile flavour compounds. Besides sensory quality aspects of proteolysis, formation of bioactive peptides as a result of proteolysis during cheese ripening has been reported. Cheese contains phosphopeptides as natural constituents [16, 17], and secondary proteolysis during cheese ripening leads to the formation of other bioactive peptides, such as those with ACEI activity. The findings by Meisel et al. [18] showed that inhibitory activity increased as proteolysis developed, however, the bioactivity decreased when proteolysis during ripening exceeded a certain level. Another link between potential antihypertensive peptides and proteolysis was found in Parmesan cheese [19]. A bioactive peptide derived from s1-casein was isolated from 6-month old cheese, but it was degraded further during maturation and was not detectable after 15 month of ripening. ACEI peptide fractions having different potencies have been isolated from various Italian cheeses, e.g. Crescenza (37% inhibition), mozzarella (59% inhibition), Gorgonzola (80% inhibition) and Italico (82% inhibition) [20]. ACEI peptides have also been found in enzyme-modified cheeses [21], in a low-fat cheese made in Finland [22] and Manchego cheeses manufactured with different starter cultures [23]. Mexican Fresco cheese manufactured with *Enterococcus faecium* or a *L. lactis* ssp. *lactis-Enterococcus faecium* mixture showed the largest number of fractions with ACEI activity among tested lactic acid strains [24]. Pripp et al. [25] investigated the relationship between proteolysis and ACE inhibition in Gamalost, Castello, Brie, Pultost, Norvegia, Port Salut and Kesam. The traditional Norwegian cheese Gamalost had per unit cheese weight higher ACE inhibition potential than Brie, Roquefort and Gouda-type cheese. However, ACE inhibition expressed as IC50 per unit peptide concentration from ethanol soluble fraction assessed by the OPA-assay was highest for Kesam, a Quark-type cheese with a low degree of proteolysis.

310 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

treatment of hypertension.

singlet oxygen quenching.

**2.1. Peptides in cheese** 

**2. Release and identification of peptides** 

estimates that by 2020, heart disease and stroke will have surpassed infectious diseases to become the leading cause of death and disability worldwide [10]. Consequently, there has been an increased focus on improving diet and lifestyle as a strategy for CVD risk reduction. Elevated blood pressure is one of the major independent risk factors for CVD [11]. Angiotensin I-converting enzyme (ACE) plays a crucial role in the regulation of blood pressure as it promotes the conversion of angiotensin I to the potent vasoconstrictor angiotensin II as well as inactivates the vasodilator bradykinin. By inhibiting these processes, synthetic ACE inhibitors (ACEI) have long been used as antihypertensive agents. In recent years, some food proteins have been identified as sources of ACEI peptides and are currently the best-known class of bioactive peptides [12, 13]. These nutritional peptides have received considerable attention for their effectiveness in both the prevention and the

Oxidant stress, the increased production of reactive oxygen species (ROS) in combination with outstripping endogenous antioxidant defense mechanisms, is another significant causative factor for the initiation or progression of several vascular diseases. ROS can cause extensive damage to biological macromolecules like DNA, proteins and lipids. Specifically, the oxidative modification of LDL results in the increased atherogenicity of oxidized LDL. Therefore, prolonged production of ROS is thought to contribute to the development of severe tissue injury [14]. Some peptides derived from hydrolyzed food proteins exert antioxidant activities against enzymatic (lipoxygenase-mediated) and nonenzymatic peroxidation of lipids and essential fatty acids [15]. The antioxidant properties of these peptides have been suggested to be due to metal ion chelation, free radical scavenging and

This review centers on liberation during fermentation, of bioactive peptides with properties relevant to cardiovascular health including the effects on blood pressure and oxidative stress. The focus is mainly to those peptides with in vivo blood pressure lowering effects. Moreover, bioavailability of peptides and aspects of necessary further information is given.

Proteolysis in cheese has been linked to its importance for texture, taste and flavour development during ripening. Changes of the cheese texture occur due to breakdown of the protein network. It contributes directly to taste and flavour by the formation of peptides and free amino acids as well as by liberation of substrates for further catabolic changes and thereby formation of volatile flavour compounds. Besides sensory quality aspects of proteolysis, formation of bioactive peptides as a result of proteolysis during cheese ripening has been reported. Cheese contains phosphopeptides as natural constituents [16, 17], and secondary proteolysis during cheese ripening leads to the formation of other bioactive peptides, such as those with ACEI activity. The findings by Meisel et al. [18] showed that inhibitory activity increased as proteolysis developed, however, the bioactivity decreased When -casomorphins were looked from commercial cheese products, no peptides were found or their concentration in the cheese extract was below 2 g/ml [26]. They further noted that the enzymatic degradation of -casomorphins was influenced by a combination of pH and salt concentration at the cheese ripening temperature. Therefore, if formed in cheese, -casomorphins may be degraded under conditions similar to Cheddar cheese ripening. Precursors of -casomorphins, on the other hand, have been identified in Parmesan cheese [19]. -Casomorphins were found at a higher level in the mould cheeses (166–648 mg/100 g), whereas the opioid peptides with antagonistic activity (casoxin-6) were identified at a higher level in the semi-hard cheeses (136–276 mg/100 g) and a low quantity of casomorphins (4–100 mg/100 g) [27]. Immunomodulating properties in water-soluble extracts from traditional French Alps cheeses, Abondance and Tomme de Savioe have been observed [28]. However, no correlation between peptide composition and *in vitro* immunomodulation of T-lymphocyte cells could be established.

A limited number of bioactive peptides have been isolated and identified in Gouda, Manchego, Festivo and Crescenza cheeses (Table 1). Several ACEI peptides have been identified from N-terminal of αs1-casein of Gouda, Festivo, Cheddar and Fresco cheeses [22, 24, 29, 30]. In addition, peptides from casein, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (β-cn, f(60–68)); and Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe (β-cn, f(109–119)) from Gouda [29] and Tyr-Gln-Glu-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val (-cn, f(193- 209)) from Cheddar [30] have been identified. Antihypertensive peptides Val-Pro-Pro (VPP) (β-cn, f(84–86)) and Ile-Pro-Pro (IPP) (β-cn, f(74–76) and κ-cn, f(108–110)), have also been identified and quantified in different cheese varieties [31-33]. In some varieties physiologically relevant amounts was observed, however, a large variation exists between samples of the same cheese variety, as well as between different varieties. The concentrations of VPP and IPP were in the range of 0-224 mg/kg and 0-95 mg/kg,

respectively, indicating that some cheese varieties contain similar concentrations of VPP and IPP to fermented milk products. Milk pretreatment, cultures, scalding conditions, and ripening time were identified as the key factors influencing the concentration of these two naturally occurring bioactive peptides in cheese. Thus, it is necessary to develop a reproducible cheese-making process with selected cultures to produce higher concentrations of these peptides that could be used for clinical trials.

Lactic Fermentation and Bioactive Peptides 313

SBP mmHg)

5 ml/kg -21.8 ±4.2 after 6 h

h

2 h

4 h

5 mg/ml SBP -22 after 8 weeks

10ml/kg -12 after 4-8 h


32.1 ±7.4 after 6


approx -12 after


ND 36

ND 43

Ref.

34

67

42

37

38

44

76

extracellular proteinase activity and the ability to release large amount of peptides in fermented milk. As a result, among various kinds of fermented milk, antihypertensive effect related to ACEI peptides were found in milk produced by *Lb. helveticus*. Two ACEI peptides

Sequence IC50 µM

Identified peptides Dose Response (Δ

27 ml/day

ml/kg

0.2 kg/kg

2 mg/kg 6 mg/kg

9 5

9 5

1.7 mg/ml 1.5 mg/ml

71 89

5.5 5.2

173.3 300.1 256.8

464.4 240.0

have been purified from sour milk and identified as VPP and IPP [34].

inhibition

ND VPP

*Lb.helveticus* CPN4 ND YP 720 10

IPP

IPP

 SKVYPFPGPI SKVYP

QN

QNIPP

ND NIPPLTQTPV

GVW

0.053 LHLPLP

0.24 GTW

LVYPFPGPIH

LNVPGEIVE DKIHPF

LVYPFPGPIPNSLP

**Table 2.** ACE-inhibitory and antihypertensive activity in spontaneously hypertensive rats of peptides

mg/ml

0.16 0.26

*Lb. jensenii* 0.52 LVYPFPGPIHNSLP

Organisms ACE-

IC50

*Lb. helveticus* VPP

*Lb. helveticus and Str.* 

*Lb. helveticus* CHCC637 *Lb. helveticus* CHCC641

*Lact. delbrueckii* ssp.

*Enterococcus faecalis* CECT 5727

*Lb. delbrueckii* subsp. *bulgaricus* SS1

*L. lactis* subsp. *cremoris*

One letter amino acid codes used

produced by fermentation of milk

1) Pure synthetic peptides were used in the study

Mixed lactic acid bacteria (*Lb. casei, acidophilus, bulcaricus, Str. themophilus, Bifidobacterium*) and

FT4

protease

ND Not described

*bulgaricus Str. salivarius* ssp *thermophilus* and *L.lactis* biovar *diacetylactis*

*thermophilus*


ND: Not described

IC50: Peptide concentration that shows 50% inhibition of ACE activity One letter amino acid codes used

**Table 1.** Examples of identified bioactive peptides in different cheese varieties

### **2.2. Fermented milk**

During fermentation process, lactic acid bacteria hydrolyze milk proteins, mainly caseins, into peptides and amino acids which are used as nitrogen sources necessary for their growth. Hence, bioactive peptides can be generated by starter and non-starter bacteria used in the manufacture of fermented dairy products (Table 2). Proteolytic system of *Lb. helveticus, Lb. delbrueckii* ssp *bulgaricus, L. lactis* ssp. *diacetylactis, L. lactis* ssp. *cremoris,* and *Streptococcus (Str.) salivarius* ssp. *thermophilu*s strains have demonstrated to hydrolyze milk proteins and release ACEI peptides. Among lactic acid bacteria, *Lb. helveticus* has high extracellular proteinase activity and the ability to release large amount of peptides in fermented milk. As a result, among various kinds of fermented milk, antihypertensive effect related to ACEI peptides were found in milk produced by *Lb. helveticus*. Two ACEI peptides have been purified from sour milk and identified as VPP and IPP [34].


One letter amino acid codes used

ND Not described

312 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

of these peptides that could be used for clinical trials.

ovine s1-cn f(205-208)

s1-cn f(1-13) -cn f(68-66) β-cn f(109–119)

s1-cn f(1-9) s1-cn f(1-7) s1-cn f(1-6) s1-cn f(24-32) -cn f(193-209)

β-cn, f(84–86) β-cn, f(74–76) and κ-cn, f(108–110)

s1-cn f(1-22) s1-cn f(14-23) s1-cn f(24-34) -cn f(193-205) -cn f(193-207) -cn f(193-209)

IC50: Peptide concentration that shows 50% inhibition of ACE activity

**Table 1.** Examples of identified bioactive peptides in different cheese varieties

Manchego ovine s1-cn f(102-109)

Gouda s1-cn f( 1-9)

Fresco cheese s1-cn f(1-15)

Cheddar (with probiotics)

Swiss cheese varieties

ND: Not described

One letter amino acid codes used

**2.2. Fermented milk** 

respectively, indicating that some cheese varieties contain similar concentrations of VPP and IPP to fermented milk products. Milk pretreatment, cultures, scalding conditions, and ripening time were identified as the key factors influencing the concentration of these two naturally occurring bioactive peptides in cheese. Thus, it is necessary to develop a reproducible cheese-making process with selected cultures to produce higher concentrations

IC50 M

13.4 ND 14.8 ND

77.2 24.1

9 5

ND 30

ND 24

Ref

29

23

31- 34

Cheese variety Milk protein fragment Peptide sequence ACE-inhibition

RPKHPIKHQ RPKHPIKHQGLPQ YPFPGPIPN MPFPKYPVQPF

KKYNVPQL VRYL

RPKHPIKHQ RPKHPIK RPKHPI FVAPFPEVFGK YQEPVLGPVRGPFPIIV

RPKHPIKHQGLPQEV RPKHPIKHQGLPQEVLNEN

EVLNENLLRF FVAPFPEVFGK YQEPVLGPVRGPF YQEPVLGPVRGPFPI YQEPVLGPVRGPFPIIV

During fermentation process, lactic acid bacteria hydrolyze milk proteins, mainly caseins, into peptides and amino acids which are used as nitrogen sources necessary for their growth. Hence, bioactive peptides can be generated by starter and non-starter bacteria used in the manufacture of fermented dairy products (Table 2). Proteolytic system of *Lb. helveticus, Lb. delbrueckii* ssp *bulgaricus, L. lactis* ssp. *diacetylactis, L. lactis* ssp. *cremoris,* and *Streptococcus (Str.) salivarius* ssp. *thermophilu*s strains have demonstrated to hydrolyze milk proteins and release ACEI peptides. Among lactic acid bacteria, *Lb. helveticus* has high

VPP IPP

LLR

1) Pure synthetic peptides were used in the study

**Table 2.** ACE-inhibitory and antihypertensive activity in spontaneously hypertensive rats of peptides produced by fermentation of milk

Pihlanto-Leppälä et al. [35] studied the potential formation of ACEI peptides from cheese whey and caseins during fermentation with various commercial dairy starters used in the manufacture of yogurt, ropy milk and sour milk. No ACEI activity was observed in these hydrolysates. Further digestion of the above samples with pepsin and trypsin resulted in the release of several strong ACEI peptides derived primarily from αs1-casein and β-casein. The formation of ACEI peptides was demonstrated in two dairy strains, *Lb. delbrueckii* ssp. *bulgaricus* and *L. lactis* ssp. *cremoris,* after fermentation of milk separately with each strain for 72 hours [36]. The most inhibitory fractions of the fermented milk mainly contained casein-derived peptides with inhibitory concentration (IC50) values ranging from 8.0 to 11.2 g/ml. Fuglsang et al. [37] tested a total of 26 strains of wild-type lactic acid bacteria, mainly belonging to *L. lactis* and *Lb. helveticus*, for their ability to produce a milk fermentate with ACEI activity. All tested strains produced ACEI substances in varying amounts, and two of the strains exhibited high ACE inhibition and a high OPA index, which correlates well with peptide formation. In another study 25 lactic acid strains of *Lactobacillus, Lactococccus* and *Leuconsotoc* were used [38]. The strains were tested alone or in combination and the highest activities were observed in *Lb. jensenii, Lb. acidophilus* and *Leuc. mesenteroides* strains and all strains showed correlation between ACE inhibition and degree of proteolysis. In a recent study, milk was fermented to defined pH values with 13 strains of lactic acid bacteria. The highest ACEI activity was obtained with two highly proteolytic strains of *Lb. helveticus* and with the *Lactococcus* strains. Fermentation from pH 4.6 to 4.3 with these strains slightly increased the ACEI activity, whilst fermentation to pH 3.5 with *Lb. helveticus* reduced the ACEI activity [39]. Moreover, four different *Enterococcus faecalis* strains, isolated from raw milk, produced fermented milk with potent ACEI activity [40]. In a recent research it was found that *L. lactis* strains isolated from artisanal dairy starters or commercial starter cultures are potential for the production of fermented dairy products with ACEI properties. Especially, a strain isolated from artisanal cheese presented the lowest IC50 (13µg/ml) [41].

Lactic Fermentation and Bioactive Peptides 315

Leu-Val-Try-Pro-Phe-Pro-Gly-Pro-Ile-His [38]. Quirós et al. [44] identified two peptides in fermented milk with *Enterococcus faecalis* that corresponded to -casein fragments Lys-His-Leu-Pro-Leu-Pro and Lys-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-ASn-Ser-Leu-Pro-Gln-Asn-

Many kinds of proteolytic enzymes have been reported from lactic acid bacteria, and have been reviewed extensively [6, 45]. The components of the proteolytic systems of lactic acid bacteria are divided into three groups, including the extracellular proteinase that catalyzes casein breakdown to peptides, peptidases that hydrolyze peptides to amino acids and a peptide transport system. The extracellular proteinase activity was almost correlated with ACEI activity in the fermented milk, suggesting that the proteolysis of casein by the extracellular proteinase is the most important parameter in the processing of active components [46]. The importance of the proteinase was also supported by the fact that a proteinase negative mutant was not able to generate antihypertensive peptides in the fermented milk, whereas the wild-type strain had the ability to release strong antihypertensive peptides in the fermented milk [47]. The enzymatic process generating the antihypertensive peptides VPP and IPP in *Lb. helveticus* has been elucidated. By the proteolytic action of the extracellular proteinase long peptide with amino acid residue including VPP and IPP sequences were generated. Next the long peptide would be hydrolyzed to shorter peptides by intracellular peptidases. A key enzyme that can catalyze C-terminal processing of Val-Pro-Pro-Phe-Leu and Ile-Pro-Pro-Leu-Thr to VPP and IPP has been purified from *Lb. helveticus* CM4. The endopeptidase has sequence homology in amino terminal sequence to a previously reported pepO-gene product [48]. Kilpi et al. [49] found out higher ACE inhibition in milk fermentation using peptidase-deletion mutants compared to the wild-type of *Lb. helveticus* strain. Unlike with the wild type strain, ACEI remained constant during the course of fermentation with the proline-specific peptidase mutant. The

mutant strains had also different peptide profiles than the wild-type strain.

Various types of fermented soybean foods are consumed in Asian countries such as Korea, China, Japan, Indonesia and Vietnam. Soybeans are traditionally fermented primarily by *Bacilli* species during the early stage of fermentation followed by *Aspergillus* species, which predominate during the remaining fermentation period [50]. ACEI peptides have been found in many traditional Asian fermented soy foods, such as soybean paste, soy sauce, natto and tempeh. ACEI peptide His-His-Leu was isolated from Korean fermented soybean paste [51]. Rye gluten sourdoughs fermented with *Lb. reuteri* and added protease were found to contain the lactoripeptides VPP, IPP [52]. Moreover, our recent studies showed that fermentation of rapeseed or flaxseed meals with *Bacillus subtils* or *Lb. helveticus* strains

It is reasonable to expect that lactic acid bacteria produce scavengers for hydroxyl radical, which can be metabolic compounds produced by bacteria or degradation products of milk

Ile-Pro-Pro, with potent ACEI activity.

**2.3. Other** 

produced ACEI activity [53].

**2.4. Other activities** 

Bioactive peptides isolated from skim milk and whey fermented using a range of organisms are summarized in Table 2**.** The majority of identified peptides are casein-derived ACEI peptides having IC50 values ranging from 5 to 500 µM. The best characterized ACEI and antihypertensive peptides liberated with *Lb. helveticus* alone or in combination with *Saccharomyces cerevisiae* are the tripeptides IPP, and VPP. Yamamoto et al. [42] identified an ACEI dipeptide (Tyr-Pro) from a yogurt-like product fermented with *Lb. helveticus* CPN4 strain. This peptide sequence is present in all major casein fractions, and its concentration was found to increase during fermentation, reaching a maximum concentration of 8.1 g/ml in the product. Ashar and Chand [43] identified an ACEI peptide from milk fermented with *Lb. delbrueckii* ssp. *bulgaricus.* The peptide showed the sequence Ser-Lys-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile from casein with an IC50 value of 1.7 mg/ml. In combination with *Str. salivarius* ssp. *thermophilus* and *L. lactis* biovar. *diacetylactis*, a peptide structure with a sequence of Ser-Lys-Val-Tyr-Pro was obtained from β-casein with an IC50 value of 1.4 mg/ml. Both peptides were markedly stable to digestive enzymes, acidic and alkaline pH, as well as during storage at 5 and 10 ºC for four days. Two -casein-derived peptides were identified from water soluble fraction of milk fermented with *Lb. jensenii.* The identified peptides were Leu-Val-Try-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser-Leu-Pro-Gln-Asn, and Leu-Val-Try-Pro-Phe-Pro-Gly-Pro-Ile-His [38]. Quirós et al. [44] identified two peptides in fermented milk with *Enterococcus faecalis* that corresponded to -casein fragments Lys-His-Leu-Pro-Leu-Pro and Lys-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-ASn-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro, with potent ACEI activity.

Many kinds of proteolytic enzymes have been reported from lactic acid bacteria, and have been reviewed extensively [6, 45]. The components of the proteolytic systems of lactic acid bacteria are divided into three groups, including the extracellular proteinase that catalyzes casein breakdown to peptides, peptidases that hydrolyze peptides to amino acids and a peptide transport system. The extracellular proteinase activity was almost correlated with ACEI activity in the fermented milk, suggesting that the proteolysis of casein by the extracellular proteinase is the most important parameter in the processing of active components [46]. The importance of the proteinase was also supported by the fact that a proteinase negative mutant was not able to generate antihypertensive peptides in the fermented milk, whereas the wild-type strain had the ability to release strong antihypertensive peptides in the fermented milk [47]. The enzymatic process generating the antihypertensive peptides VPP and IPP in *Lb. helveticus* has been elucidated. By the proteolytic action of the extracellular proteinase long peptide with amino acid residue including VPP and IPP sequences were generated. Next the long peptide would be hydrolyzed to shorter peptides by intracellular peptidases. A key enzyme that can catalyze C-terminal processing of Val-Pro-Pro-Phe-Leu and Ile-Pro-Pro-Leu-Thr to VPP and IPP has been purified from *Lb. helveticus* CM4. The endopeptidase has sequence homology in amino terminal sequence to a previously reported pepO-gene product [48]. Kilpi et al. [49] found out higher ACE inhibition in milk fermentation using peptidase-deletion mutants compared to the wild-type of *Lb. helveticus* strain. Unlike with the wild type strain, ACEI remained constant during the course of fermentation with the proline-specific peptidase mutant. The mutant strains had also different peptide profiles than the wild-type strain.

### **2.3. Other**

314 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

Pihlanto-Leppälä et al. [35] studied the potential formation of ACEI peptides from cheese whey and caseins during fermentation with various commercial dairy starters used in the manufacture of yogurt, ropy milk and sour milk. No ACEI activity was observed in these hydrolysates. Further digestion of the above samples with pepsin and trypsin resulted in the release of several strong ACEI peptides derived primarily from αs1-casein and β-casein. The formation of ACEI peptides was demonstrated in two dairy strains, *Lb. delbrueckii* ssp. *bulgaricus* and *L. lactis* ssp. *cremoris,* after fermentation of milk separately with each strain for 72 hours [36]. The most inhibitory fractions of the fermented milk mainly contained casein-derived peptides with inhibitory concentration (IC50) values ranging from 8.0 to 11.2 g/ml. Fuglsang et al. [37] tested a total of 26 strains of wild-type lactic acid bacteria, mainly belonging to *L. lactis* and *Lb. helveticus*, for their ability to produce a milk fermentate with ACEI activity. All tested strains produced ACEI substances in varying amounts, and two of the strains exhibited high ACE inhibition and a high OPA index, which correlates well with peptide formation. In another study 25 lactic acid strains of *Lactobacillus, Lactococccus* and *Leuconsotoc* were used [38]. The strains were tested alone or in combination and the highest activities were observed in *Lb. jensenii, Lb. acidophilus* and *Leuc. mesenteroides* strains and all strains showed correlation between ACE inhibition and degree of proteolysis. In a recent study, milk was fermented to defined pH values with 13 strains of lactic acid bacteria. The highest ACEI activity was obtained with two highly proteolytic strains of *Lb. helveticus* and with the *Lactococcus* strains. Fermentation from pH 4.6 to 4.3 with these strains slightly increased the ACEI activity, whilst fermentation to pH 3.5 with *Lb. helveticus* reduced the ACEI activity [39]. Moreover, four different *Enterococcus faecalis* strains, isolated from raw milk, produced fermented milk with potent ACEI activity [40]. In a recent research it was found that *L. lactis* strains isolated from artisanal dairy starters or commercial starter cultures are potential for the production of fermented dairy products with ACEI properties. Especially, a strain isolated from artisanal cheese presented the lowest IC50 (13µg/ml) [41].

Bioactive peptides isolated from skim milk and whey fermented using a range of organisms are summarized in Table 2**.** The majority of identified peptides are casein-derived ACEI peptides having IC50 values ranging from 5 to 500 µM. The best characterized ACEI and antihypertensive peptides liberated with *Lb. helveticus* alone or in combination with *Saccharomyces cerevisiae* are the tripeptides IPP, and VPP. Yamamoto et al. [42] identified an ACEI dipeptide (Tyr-Pro) from a yogurt-like product fermented with *Lb. helveticus* CPN4 strain. This peptide sequence is present in all major casein fractions, and its concentration was found to increase during fermentation, reaching a maximum concentration of 8.1 g/ml in the product. Ashar and Chand [43] identified an ACEI peptide from milk fermented with *Lb. delbrueckii* ssp. *bulgaricus.* The peptide showed the sequence Ser-Lys-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile from casein with an IC50 value of 1.7 mg/ml. In combination with *Str. salivarius* ssp. *thermophilus* and *L. lactis* biovar. *diacetylactis*, a peptide structure with a sequence of Ser-Lys-Val-Tyr-Pro was obtained from β-casein with an IC50 value of 1.4 mg/ml. Both peptides were markedly stable to digestive enzymes, acidic and alkaline pH, as well as during storage at 5 and 10 ºC for four days. Two -casein-derived peptides were identified from water soluble fraction of milk fermented with *Lb. jensenii.* The identified peptides were Leu-Val-Try-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser-Leu-Pro-Gln-Asn, and

Various types of fermented soybean foods are consumed in Asian countries such as Korea, China, Japan, Indonesia and Vietnam. Soybeans are traditionally fermented primarily by *Bacilli* species during the early stage of fermentation followed by *Aspergillus* species, which predominate during the remaining fermentation period [50]. ACEI peptides have been found in many traditional Asian fermented soy foods, such as soybean paste, soy sauce, natto and tempeh. ACEI peptide His-His-Leu was isolated from Korean fermented soybean paste [51]. Rye gluten sourdoughs fermented with *Lb. reuteri* and added protease were found to contain the lactoripeptides VPP, IPP [52]. Moreover, our recent studies showed that fermentation of rapeseed or flaxseed meals with *Bacillus subtils* or *Lb. helveticus* strains produced ACEI activity [53].

### **2.4. Other activities**

It is reasonable to expect that lactic acid bacteria produce scavengers for hydroxyl radical, which can be metabolic compounds produced by bacteria or degradation products of milk

proteins. The results have demonstrated that the antioxidant production is commonly higher within the group of obligately homofermentative lactobacilli, than within the facultatively or obligately heterofermentative strain groups. Also heterofermentative *Lactobacillus* sp. have been reported to exhibit antioxidative activity. *Lb. acidophilus*, *Lb. bulgaricus*, *Str. thermophilus* and *Bifidobacterium longum* exhibited antioxidative activity by various mechanisms, like metal ion chelating capacity, scavenging of reactive oxygen species (ROS), reducing activity and superoxide dismutase activity [54, 55]. Peptides liberated during fermentation can be partially responsible for the reported antioxidative properties. An antioxidative peptide derived from -casein was detected in milk after fermentation with *Lb. delbrueckii* subs. *bulgaricus* [56]. Moreover, Hernández-Ledesma et al. [57] found a moderate ABTS radical scavenging capacity in commercial fermented milk from Europe. Further studies of this radical scavenging activity in different HPLC fractions showed low TEAC values. Virtanen et al. [58] found that fermentation with *Leuc. mesenteroides* ssp. *cremoris*, *Lb. jensenii* and *Lb. acidophilus* strains produced compounds that showed both radical scavenging activity and inhibition of lipid peroxidation.

Lactic Fermentation and Bioactive Peptides 317

human essential hypertension [7]. Following a positive response in animal studies human

A great number of studies have addressed the effects of both short-term and long-term administration of potential antihypertensive peptides using this animal model. Fermented milks with different IC50-values ranging from from 0.08 to 1.88 mg/ml have been shown to

The first antihypertensive effect of milk casein-derived peptides was first demonstrated by casein hydrolysate formed by purified proteinase from *Lb. helveticus* CP790 and milk fermented with the same bacteria [65]. The authors concluded that peptides deliberated from casein by extracellular proteinases were responsible for the antihypertensive effect. The active substances were liberated during fermentation of milk with *Lb. helveticus* and *Saccharomyces cerevisiae* and were identified to be IPP and VPP. Oral administration of fermented milk or pure tripeptides were shown to produce strong antihypertensive effect in SHR after single-dose [34, 66]. Thereafter, several animal studies have been conducted to characterize the long-term effects of lactotripeptides or fermented milk containing them. These studies were mainly conducted with SHR but also Goto-Kakizaki (GK) rats and double transgenic rats (dTGR) with malignant hypertension have been used. The development of hypertension was attenuated significantly in rats receiving fermented milk product containing lactotripeptides, attenuation in systolic blood pressure was 12-21 mmHg in SHR, 10 mmHg in high salt-fed GK rats and 19 mmHg in dTGR in comparison to control group [67-69]. Pure tripeptides did not produce as strong antihypertensive effect as the milk products containing them. In addition, minerals alone did not attenuate the development of blood pressure as much as the fermented milk products [68]. These studies indicate that the bioavailability of peptides may be better from milk in comparison of water or is improved

After the blood pressure monitoring has been completed the effect of long-term intake of lactotripeptides on vascular function has been assessed [68,70,71]. Jauhiainen et al. [70], showed improved endothelium-dependent relaxation in mesenteric arteries and aortas of rats that had received minerals and lactotripeptide. Endothelial function of mesenteric arteries was strongly impaired in all groups of salt-loaded GK rats, and significantly improved endothelium-dependent relaxations were observed after treatment with different fermented milk products [68]. Protection of endothelial function after incubation with tripeptides IPP and

Evidence from ACE inhibition was gained by Masuda et al. [72], who found that after receiving a single-dose of Calpis™ sour milk, ACE activity was decreased in SHR aorta. The lactotripeptides were detected in solubilized fraction from the abdominal aorta of SHR but not from WKY given the sour milk. Moreover, in SHR, plasma rennin activity increased after long-term treatment of fermented milk product containing the lactotripeptides [67]. In addition, treatment with fermented milk containing lactotripeptides and plant sterols

VPP for 24 h was found in a study with isolated SHR mesenteric arteries [71].

studies may be carried out to ascertain the ACEI potential

decrease blood pressure in SHR from 10 to 32 mmHg (Table 2).

**3.1. Animal studies** 

by other milk components.

Inflammation plays a key role in the development of cardiovascular disease. It often begins with inflammatory changes in the endothelium, which begins to express the adhesion molecule VCAM-1. VCAM-1 attracts monocytes, which then migrate through the endothelial layer under the influence of various proinflammatory chemoattractants [59]. Accordingly, fermentation by lactic acid may be able to release components that possess immunomodulatory properties. Most of the studies have been done with synthetic peptides derived from enzymatic treatment of milk proteins using different *in vitro* models. Leblanc et al. [60] investigated the effect of peptides released during the fermentation of milk by *Lb. helveticus* on the humoral immune system and on the growth of fibrosacromas. The study showed that bioactive components were released during fermentation that contributed to the immunoenhancing and antitumor properties. Antimutagenic compounds were produced during fermentation by *Lb. helveticus*, and release of peptides is one possible explanation [61]. The permeate fraction obtained from milk fermented by *Lb. helveticus* was able to modulate the *in vitro* proliferation of lymphocytes by acting on the production of cytokines [62]. Tompa et al. [63] found that peptide fractions form *Lb. helveticus* BGRA43 fermented milk have anti-inflammatory potential. Matar et al. [64] fed milk fermented with a *Lb. helveticus* strain to mice for three days and detected significantly higher numbers of IgA secreting cells in their intestinal mucosa, compared with control mice fed with similar milk incubated with a non-proteolytic variant of the same strain. The immunostimulatory effect of fermented milk was attributed to peptides released from the casein fraction.
