**4. Bioavailability**

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

hypertensive patients

8 64 subjects with SBP 140-159 and DBP 90-99 mmHg

8 32 subjects with SBP 140 - 180 and DBP 90-105 mmHg

8 18 hypertensive and 26 normotensive subjects

8 30 subjects with SBP 140-180 and DBP 90-105 mmHg

21 39 subjects with SBP 133-176 and DBP 86-108 mmHg

10 94 hypertensive patients

> volunteers normal blood pressure (<130 mmHg SBP and <85 mmHg DPB).

8 135 Dutch subjects with untreated high-normal BP or mild hypertension

> subjects with prehypertension or

4 70 Caucasian

was carried out in crossover design.

stage 1 hypertension

1 20 healthy

60 Finnish subjects with SBP 140-180 and DBP 90-110 mmHg

(weeks) IPP

8 30 eldery

R, p-c, sbld, parallel

R, p-c, dbld, parallel

R, p-c, dbld, parallel

R, p-c, dbld, parallel

R, p-c, dbld, parallel

R, p-c, dbld, parallel 1)

R, p-c, dbld, parallel Crossover2)

R, p-c, dbld, parallel

R, p-c, dbld, parallel

R, p-c, dbld, parallel

R, p-c, dbld, crossover 10

7

Design Duration Study population Treatment BP changes

VPP mg/d

2.25 3.0 *Lb. helv* 

30 22.5 *Lb. helv* 

11.5 17.7 *Lb. helv* 

2.4-2.7 2.4- 2.7

Source of peptides

1.1 1.5 *Lb. helv* + *Str. cer* 1 x 95 ml

1.58 2.24 *Lb. helv* + *Str. cer.* 2 x 150 g

1·60 2·66 *Lb. helv* + *Str. cer.* 1 x 120 g

1.1 1.5 *Lb. helv* + *Str. cer.* 2 x 100 g

1.52 2.53 *Lb. helv* + *Str. cer.* 2 x 160 g

LBK-16H

*Lb. helv*  LBK-16H

LBK-16H

4.2 5.8 Fermentation 1 x 200 ml

endopeptidase

CM4

15 - Hydrolysis by

1) Results reported as changes in SBP and DBP after each month of treatment for all subjects (intention-to-treat analysis), and as mean changes over the total intervention period among subjects who had BP measurements for each month (per protocol analysis); 2) First part of the study was carried out in parallel design and second part of the study

**Table 3.** Hypotensive effects of fermented milks with bioactive peptides in humans

mg/d

mmHg








87


4.1 1.8 88

2.6 2 93





Formula SBP DBP

milk drink

milk drink

milk drink

milk drink

milk drink

2 x 150 ml milk drink

1 x 150 ml milk drink

2 x 150 ml milk drink

1 x 14 tablets

yoghurt drink

2 x 7.5 mg capsules

Ref.

Bioavilability of bioactive peptides is an important target to establish the relationship between *in vitro* and *in vivo* activities. The likelihood of any bioactive peptide released during fermentation mediating a physiological response is dependent on the ability of that peptide to reach an appropriate target site. Therefore, peptides may need to be resistant to further degradation by proteolytic and peptidolytic enzymes in digestive tract. Thereafter peptides should be absorbed and enter systemic circulation. Resistance to hydrolysis is one of the main factors influencing the bioavailability of bioactive peptides. The effects of digestive enzymes on bioactive peptides, in particular ACEI peptides derived from different food matrices, have been evaluated *in vitro* gastrointestinal simulated systems. The common purpose of these experiments was to assess the effects of the peptidases of the stomach and the pancreas on the preservation of the ACEI activity of different hydrolysates. Studies have shown that the ACEI is low after fermentation but increases during hydrolysis that simulates gastrointestinal digestion [35,103]. The ACEI peptides in rapeseed hydrolysate exhibited good stability in an *in vitro* digestion model using human gastric and duodenal fluids [104]. The digestion of some peptides have been reported. For example, Ile-Val-Tyr

was hydrolysed by pepsin, trypsin and chymotrypsin alone or in combination and IC50 value did not change significantly during digestion [105]. Proline- and hydroxyprolinecontaining peptides are usually resistant to degradation by digestive enzymes. Tripeptides containing C-terminal proline-proline are generally resistant to proline-specific peptidases [106]. In some cases, pancreatic digestion is needed to produce active peptide. For instance, the active form of peptide Lys-Val-Leu-Pro-Val-Pro-Glu is generated by hydrolysis of the glutamine residue at the C-terminal during pancreatic digestion [107]. The results are not completely predictive of the resistance of the bioactive peptides because they do not mimic all the physiological factors affecting food digestion, as pH variations, the relative amounts of the enzymes, the interactions with other molecules, and the ratio peptidase/tested compound. These variations may affect the rate of enzymatic degradation of the bioactive peptides under study, therefore affecting the estimated bioavailability of these bioactive peptides. Moreover, commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities [108]. This could lead to conflicting results when comparing human *in vivo* protein digestion with digestion using purified enzymes of non-human species.

Lactic Fermentation and Bioactive Peptides 323

Jauhiainen et al. [115] used radiolabelled tripeptide and showed that it absorbed partly intact from the gastrointestinal tract after a single oral dose to rats. Considerable amounts of radioactivity were found from several tissues, e.g., liver, kidney and aorta. The excretion of IPP was slow; even after 48 hours the radiolabelled peptide had not been completely excreted. IPP did not bind to albumin or other plasma proteins *in vitro*. Considering this and the long-lasting retention of the radioactivity in the tissues, accumulation of IPP may occur in sufficient concentrations to cause blood pressure lowering effects e.g., by ACE-inhibition in the vascular wall. In another study the absolute bioavailability of the tripeptides in pigs was below 0.1%, with an extremely short elimination half-life ranging from 5 to 20 min [116]. In humans, maximal plasma concentration did not exceed picomolar concentration

The improvement of limited absorption and stability of peptides has been a goal when evaluating their effectiveness. For example, some carriers interact with the peptide molecule to create an insoluble entity at low pH which later dissolves and facilitates intestinal uptake, by enhancing peptide transport over the non-polar biological membrane [118]. Bioavailability of bioactive tripeptides (VPP, IPP, LPP) was improved by administering them with a meal containing fiber, as compared to a meal containing no fiber. High methylated citrus pectin was used as a fiber [119]. Ko et al. [120] applied emulsification, microencapsulation and lipophilization to enhance the antihypertensive activity of a hydrolysate of tuna cooking juice. Among these treatments, lipophilization was the most effective, followed by microencapsulation and lecithin emulsification, getting for each of them a stronger effect than the obtained with the double untreated dosage. Antihypertensive effect of ovokinin (Phe-Arg-Ala-Asp-Pro-Phe-Leu) increased four-times compared to the untreated dosage after administration with egg yolk [121]. In this case, phospholipids were identified as responsible for enhancing the antihypertensive effect, particularly phosphatidylcholine, that could improve intestinal absorption or by protecting ovokinin of peptidases. Among drug delivery systems, emulsions have been used to enhance oral bioavailability or promoting absorption through mucosal surfaces of peptides and proteins [118]. Individually, various components of emulsions have been considered as

The interest on foods possessing health-promoting or disease-preventing properties has been increasing. An increasing number of foods sold in developed countries bears nutrition and health claims. Fermented milk with putative antihypertensive effect in humans could be an easy applicable lifestyle intervention against hypertension. In fact, much work has been done with dietary antihypertensive peptides and evidence of their effect in animal and clinical studies. Moreover, there are numerous available patents of products containing antihypertensive bioactive peptides. However, certain aspects, such as identification of the active form in the organism and the different mechanisms of action that contribute in the antihypertensive effect still need to be further investigated. Recent advances on specific

candidates for improving bioavailability of peptides.

**5. General conclusions** 

[117].

Peptides have been reported to have poor permeation across biological barriers (e.g. intestinal mucosa) [109]. Peptides can be transported by active transcellular transport or by passive processes. Although substantial amino acid absorption occurs in the form of di- and tripeptides at the apical side of enterocytes, efflux of intact peptides via the basolateral membrane into the general circulation seems to be negligible [110]. The intestinal absorption of peptides have been performed using *in vitro* tests with monolayer of intestinal cell lines, simulating intestinal epithelium, as well as analysis of peptides and derivatives in blood samples after animal and clinical studies. Foltz et al. [111] investigated the transport of IPP and VPP by using three different absorption models and demonstrated that these tripeptides are transported in small amounts intact across the barrier of the intestinal epithelium. The major transport mechanisms of IPP and VPP were demonstrated to be paracellular transport and passive diffusion [112]. Another ACEI peptide, Leu-His-Leu-Pro-Leu-Pro resisted gastrointestinal simulation but was degraded to His-Leu-Pro-Leu-Pro by cellular peptidases before crossing Caco-2 cell monolayer. The pentapeptide was rapidly transported through Caco-2 cell monolayers through paracellular route [113].

Vascular endothelial tissue peptidases and soluble plasma peptidases further contribute to peptide hydrolysis. As a consequence, for most peptides, the plasma half-life is limited to minutes as shown for endogenous peptides such as angiotensin II and glucagon-like peptide 1 [114]. In order to exert antihypertensive effect ACEI peptides need to resist different peptidases such as ACE. In this regard ACEI peptides can be classified into three groups: the inhibitor type, of which the IC50-value is not affected by preincubation with ACE; the substrate type, peptides that are hydrolysed by ACE to give peptides with a weaker activity; the pro-drug type inhibitor, peptides that are converted to true inhibitors by ACE or other proteases/peptidases. Only peptides belonging to pro-drug or inhibitor type exert antihypertensive properties after oral administration. There are some examples showing that peptides are absorbed and can exert *in vivo* activities. As regard to casein-derived IPP, Jauhiainen et al. [115] used radiolabelled tripeptide and showed that it absorbed partly intact from the gastrointestinal tract after a single oral dose to rats. Considerable amounts of radioactivity were found from several tissues, e.g., liver, kidney and aorta. The excretion of IPP was slow; even after 48 hours the radiolabelled peptide had not been completely excreted. IPP did not bind to albumin or other plasma proteins *in vitro*. Considering this and the long-lasting retention of the radioactivity in the tissues, accumulation of IPP may occur in sufficient concentrations to cause blood pressure lowering effects e.g., by ACE-inhibition in the vascular wall. In another study the absolute bioavailability of the tripeptides in pigs was below 0.1%, with an extremely short elimination half-life ranging from 5 to 20 min [116]. In humans, maximal plasma concentration did not exceed picomolar concentration [117].

The improvement of limited absorption and stability of peptides has been a goal when evaluating their effectiveness. For example, some carriers interact with the peptide molecule to create an insoluble entity at low pH which later dissolves and facilitates intestinal uptake, by enhancing peptide transport over the non-polar biological membrane [118]. Bioavailability of bioactive tripeptides (VPP, IPP, LPP) was improved by administering them with a meal containing fiber, as compared to a meal containing no fiber. High methylated citrus pectin was used as a fiber [119]. Ko et al. [120] applied emulsification, microencapsulation and lipophilization to enhance the antihypertensive activity of a hydrolysate of tuna cooking juice. Among these treatments, lipophilization was the most effective, followed by microencapsulation and lecithin emulsification, getting for each of them a stronger effect than the obtained with the double untreated dosage. Antihypertensive effect of ovokinin (Phe-Arg-Ala-Asp-Pro-Phe-Leu) increased four-times compared to the untreated dosage after administration with egg yolk [121]. In this case, phospholipids were identified as responsible for enhancing the antihypertensive effect, particularly phosphatidylcholine, that could improve intestinal absorption or by protecting ovokinin of peptidases. Among drug delivery systems, emulsions have been used to enhance oral bioavailability or promoting absorption through mucosal surfaces of peptides and proteins [118]. Individually, various components of emulsions have been considered as candidates for improving bioavailability of peptides.

### **5. General conclusions**

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

digestion using purified enzymes of non-human species.

was hydrolysed by pepsin, trypsin and chymotrypsin alone or in combination and IC50 value did not change significantly during digestion [105]. Proline- and hydroxyprolinecontaining peptides are usually resistant to degradation by digestive enzymes. Tripeptides containing C-terminal proline-proline are generally resistant to proline-specific peptidases [106]. In some cases, pancreatic digestion is needed to produce active peptide. For instance, the active form of peptide Lys-Val-Leu-Pro-Val-Pro-Glu is generated by hydrolysis of the glutamine residue at the C-terminal during pancreatic digestion [107]. The results are not completely predictive of the resistance of the bioactive peptides because they do not mimic all the physiological factors affecting food digestion, as pH variations, the relative amounts of the enzymes, the interactions with other molecules, and the ratio peptidase/tested compound. These variations may affect the rate of enzymatic degradation of the bioactive peptides under study, therefore affecting the estimated bioavailability of these bioactive peptides. Moreover, commercial enzymes appear to digest whey proteins more efficiently compared with human digestive juices when used at similar enzyme activities [108]. This could lead to conflicting results when comparing human *in vivo* protein digestion with

Peptides have been reported to have poor permeation across biological barriers (e.g. intestinal mucosa) [109]. Peptides can be transported by active transcellular transport or by passive processes. Although substantial amino acid absorption occurs in the form of di- and tripeptides at the apical side of enterocytes, efflux of intact peptides via the basolateral membrane into the general circulation seems to be negligible [110]. The intestinal absorption of peptides have been performed using *in vitro* tests with monolayer of intestinal cell lines, simulating intestinal epithelium, as well as analysis of peptides and derivatives in blood samples after animal and clinical studies. Foltz et al. [111] investigated the transport of IPP and VPP by using three different absorption models and demonstrated that these tripeptides are transported in small amounts intact across the barrier of the intestinal epithelium. The major transport mechanisms of IPP and VPP were demonstrated to be paracellular transport and passive diffusion [112]. Another ACEI peptide, Leu-His-Leu-Pro-Leu-Pro resisted gastrointestinal simulation but was degraded to His-Leu-Pro-Leu-Pro by cellular peptidases before crossing Caco-2 cell monolayer. The pentapeptide was rapidly

transported through Caco-2 cell monolayers through paracellular route [113].

Vascular endothelial tissue peptidases and soluble plasma peptidases further contribute to peptide hydrolysis. As a consequence, for most peptides, the plasma half-life is limited to minutes as shown for endogenous peptides such as angiotensin II and glucagon-like peptide 1 [114]. In order to exert antihypertensive effect ACEI peptides need to resist different peptidases such as ACE. In this regard ACEI peptides can be classified into three groups: the inhibitor type, of which the IC50-value is not affected by preincubation with ACE; the substrate type, peptides that are hydrolysed by ACE to give peptides with a weaker activity; the pro-drug type inhibitor, peptides that are converted to true inhibitors by ACE or other proteases/peptidases. Only peptides belonging to pro-drug or inhibitor type exert antihypertensive properties after oral administration. There are some examples showing that peptides are absorbed and can exert *in vivo* activities. As regard to casein-derived IPP,

The interest on foods possessing health-promoting or disease-preventing properties has been increasing. An increasing number of foods sold in developed countries bears nutrition and health claims. Fermented milk with putative antihypertensive effect in humans could be an easy applicable lifestyle intervention against hypertension. In fact, much work has been done with dietary antihypertensive peptides and evidence of their effect in animal and clinical studies. Moreover, there are numerous available patents of products containing antihypertensive bioactive peptides. However, certain aspects, such as identification of the active form in the organism and the different mechanisms of action that contribute in the antihypertensive effect still need to be further investigated. Recent advances on specific

analytical techniques able to follow small amounts of the peptides or derivatives from them in complex matrices and biological fluids will allow performing these kinetic studies in model animals and humans. Similarly, advances in new disciplines such as nutrigenomic and nutrigenetic will open new ways to follow bioactivity in the organism by identifying novel and more complex biomarkers of exposure and/or of activity. There is still poor knowledge on the resistance of peptides to gastric degradation, and low bioavailability of peptides has been observed. This reinforces the need of various strategies to improve the oral bioavailability of peptides.

Lactic Fermentation and Bioactive Peptides 325

Handbook of functional dairy products. Functional foods and nutraceuticals series 6.0:

[4] FitzGerald RJ, Murray BA (2006) Bioactive peptides and lactic fermentations. Int. j.

[5] Jäkälä P, Vapaatalo H (2010) Antihypertensive peptides from milk proteins.

[6] Christensen JE, Dudley EG, Pederson JA, Steele JL (1999) Peptidases and amino acid

[7] Luoma S, Peltoniemi K, Joutsjoki V, Rantanen T, Tamminen M, Heikkinen I, Palva A (2001) Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis.

[8] Foucaud C, Juillard V (2000) Accumulation of casein-derived peptides during growth of proteinase-positive strains of Lactococcus lactis in milk: their contribution to subsequent bacterial growth is impaired by their internal transport*.* J dairy res. 67: 233-

[9] Williams AG, Noble J, Tammam J, Lloyd D, Banks JM (2002) Factors affecting the activity of enzymes involved in peptide and amino acid catabolism in non starter lactic

[10] Lopez AD, Murray CC (1998) The global burden of disease, 1990–2020. Nat. med.

[11] Harris T, Cook EF, Kannel W, Schatzkin A, Goldman L (1985) Blood pressure experience and risk of cardiovascular disease in the elderly. Hypertension 7:118–24. [12] Pihlanto A, Korhonen H (2003) Bioactive peptides and proteins. Adv. food res. 47: 175–

[13] Hernández-Ledesma B, del Mar Contreras M, Recio I (2011) Antihypertensive peptides: production, bioavailability and incorporation into foods. Adv. colloid interface sci.

[14] Van Gaal LF, Mertens IL, De Block CE (2006) Mechanisms linking obesity with

[15] Pihlanto A (2006) Antioxidative peptides derived from milk proteins. Int. dairy j. 16:

[16] Roudot-Algaron F, Bars DL, Kerhoas L, Einhorn J, Gripon JC (1994) Phosptiopeptides

[17] Singh TK, Fox PF, Healy A (1997) Isolation and identification of further peptides from diafiltration retentate of the water-soluble fraction of Cheddar cheese. J. dairy res.

[18] Meisel H, Goepfert A, Günter S (1997) ACE-inhibitory activities in milk products.

[19] Addeo F, Chianes L, Salzano A, Sacchi R, Cappuccio U, Ferranti P, Malorni A (1992) Characterization of the 12% tricholoroacetic acid-insoluble oligopeptides of

from Comté Cheese: Nature and origin. J. food sci. 59: 544–547.

Parmigiano–Reggiano cheese. J. dairy res. 59: 401–411.

acid bacteria isolated from Cheddar cheese. Int. dairy j. 12: 841–852.

catabolism in lactic acid bacteria. Anton. leeuw. 76: 217-246.

p. 109-124.

240.

276.

165:23-35

1306–1314.

64:433-443.

Milchwissenschaft 52: 307–311.

4:1241–1243.

dairy technology 59: 118-125.

Pharmaceuticals 3: 251-272.

Appl. environ. microb. 67: 1232–1238.

cardiovascular disease. Nature 444: 876-880.

More emphasis has been put on the legal regulation of the health claims attached to the products. Authorities around the world have developed systematic approaches for review and assessment of scientific data. Evidence on the beneficial effects of a functional food product should be enough detailed, extensive and conclusive for the use of a health claim in the product labeling and marketing. Besides being based on generally accepted scientific evidence, the claims should be well understood by the average consumer. First, it is necessary to identify and quantify the active sequences. Antihypertensive peptides are only minor constituents in highly complex food matrices and, therefore, a monitoring of the large-scale production by hydrolytic or fermentative industrial process is mandatory. Second, extensive investigations to prove the antihypertensive effect in humans as well as the minimal dose to show this effect are necessary to fulfill the requirements of the legislation concerning functional foods. Japan was the pioneer with the Foods for Special Health Use (FOSHU) legislation in 1991. Europe adopted a joint Regulation on Nutrition and Health Claims made on Foods in 2006 being the European Food Safety Authority (EFSA). At present, EFSA have concludes that the evidence is insufficient to establish a cause and effect relationship between the consumption of the tripeptides VPP and IPP and the maintenance of normal blood pressure. Bearing in mind that 'essential hypertension' consists of disparate mechanisms that ultimately lead to elevations in systemic BP, it is most probably that that products containing lactotripeptides offer a valuable option as a nonpharmacological, nutritional treatment of elevated blood pressure for some groups of people.
