**3.1. Animal studies**

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

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.

of fermented milk was attributed to peptides released from the casein fraction.

The search for *in vitro* ACEI is the most common strategy followed in the selection of potential antihypertensive peptides derived from food proteins. *In vitro* ACEI activity is generally measured by monitoring the conversion of an appropriate substrate by ACE in the presence and absence of inhibitors. The antihypertensive effects have been assessed by *in vivo* experiments using spontaneously hypertensive rats (SHR) as an animal model to study

**3. Antihypertensive effects in vivo** 

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

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 by other milk components.

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

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 decreased serum ACE activity [73]. In salt-loaded GK rats, fermented milk with lactotripeptides decreased serum ACE and aldosterone levels [68].

Lactic Fermentation and Bioactive Peptides 319

(VPP and IPP) from casein was reported to act as antihypertensive agents in SHR [81]. Recently, Nakahara et al. [82] used the Dahl salt-sensitive rats as a model of salt-sensitive hypertension to evaluate the antihypertensive effect of a peptide-enriched soy sauce-like seasoning. The results of these tests have highlighted an important lack of correlation between the *in vitro* ACEI activity and the *in vivo* action. This fact has provided doubts on the use of the *in vitro* ACEI activity as the exclusive criteria for potential antihypertensive substances, since physiological transformations may occur *in vivo*, and because other mechanisms of action than ACE inhibition might be responsible for the antihypertensive

Evidence of the beneficial effects of bioactive peptides has to be based on clinical data. Most research has been focused in lactotripeptides, VPP and IPP, and their antihypertensive properties. About twenty human studies have been published linking the consumption of products containing lactotripeptides with significant reductions in both SBP and DBP. Oral administration of these tri-peptides included in different formulas, fermented milk, dried product, fruit juice, etc., products. However, recent studies have provided some conflicting results. Most clinical trials have assessed BPlowering effects at multiple points over time. Most of the BP studies with lactotripeptides have been done in Japanese subjects, and several studies have been done in Finnish subjects [83-88]. Generally, maximum duration of treatment was 8 weeks at doses between 3 and 52 mg/day (Table 3). From these data, it becomes apparent that the largest part of the total BP reduction takes place in the first 1–2 weeks of treatment. Thereafter, a further gradual lowering is seen, but to a lesser extent than in the first period [84-86]. The first significant effects of lactotripeptides on BP in hypertensive subjects were observed after 1–2 weeks of treatment with dosages as low as 3.8 mg/d. Maximum BP-lowering effects of lactotripeptides approximate 13 mmHg SBP and 8 mmHg DBP active treatment v. placebo, and are likely reached after 8–12 weeks of treatment. Lactotripeptides exert a gradual effect on BP lowering after start of intake and return of BP after end of treatment as well [85, 86, 89]. The highest effective dosage of lactotripeptides was evaluated in a safety study, and consisted of 52.5 mg/d [88]. After 10 weeks of active treatment, mean SBP in subjects with hypertension decreased by 4.1 mmHg and DBP by 1.8 mmHg. The next highest dose of lactotripeptides that was tested amounted to 13.0 mg/d [89]. After 4 weeks of active treatment, SBP in subjects with mild hypertension decreased by 11.2 mmHg compared to placebo, and DBP tended to decrease by 6.5 mmHg. In none of the trials with normotensives were statistically significant BP changes found [90-92]. Even at the highest dosage of lactotripeptides used in normotensives, which included a total of 29.2 mg/d during a period of 7 d, no BP lowering effects by lactotripeptides were observed [93]. Thus lactotripeptides only seem to be active at elevated BP values. Evidence indicates that effectiveness is positively associated with BP level, which is in line

effect.

**3.2. Effects in clinical studies** 

with existing data for BP-lowering medication [94].

Besides the most extensively studied lactotripeptides, also other fermented milk products and peptides have been found. Different strains of lactic acid bacteria, such as *Lb. helveticus* CPN4, *Lb. bulgaricus, Lb. jensenii* and *Str. thermophilus*, have been also shown to provoke liberation of peptides with antihypertensive activity in SHR [36, 37, 41]. Two peptides, corresponding to -casein fragments Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro and Leu-His-Leu-Pro-Leu-Pro, have been isolated in fermented milk with *Enterococcus faecalis* and their antihypertensive effect in SHR, after acute and long-term administration has been proved. The administration of 2 mg/kg of peptide Leu-His-Leu-Pro-Leu-Pro resulted in a significant decrease of the SBP in SHR 4 h post-administration [74,75]. Fermentation of milk with one or more lactic acid bacteria strains followed by hydrolysis using food-grade enzymes liberated tripeptides (Gly-Thr-Trp and Gly-Val-Trp). Oral administration of this fermented whey lowered significantly SBP in SHR from 9 to 15 weeks of age. Bioactive substances, tripeptides and -aminobutyric acid (GABA), contributed to lowering blood pressure of SHR [76].

Some of ACE-inhibitory peptide fractions from cheese have shown *in vivo* activities. A water-soluble peptide preparation isolated from Gouda ripened for 8 months was found to have the most potent antihypertensive activity (maximum decrease in SBP = 24.7 (± 0.3) mmHg (P ≤ 0.01) after 6 h) when administered to SHR by gastric intubation at doses between 6.1 and 7.5 mg/kg body weight. Three peptide fractions were isolated from watersoluble extract by hydrophobic chromatography using different concentrations of acetonitrile. The fractions eluting between 15% and 30%, 30–45% and 60–75% acetonitrile decreased SBP in SHR by 15.0, 29.3 and 18.8 mmHg (P ≤ 0.01), respectively, 6 h after gastric intubation. The peptide fraction eluting between 30% and 45% acetonitrile was shown to contain the sequences (αs1-cn f(1–9)) Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln and (β-cn f(60– 68)) Tyr-Pro-Phe-Gly-Pro-Ile-Pro-Asn (Table 1), which, respectively, decreased SBP in SHR by 9.3 (± 4.8) and 7.0 (± 3.8) mmHg 6 h after gastric intubation [29].

Several sequences have been proposed as responsible for the antihypertensive activity of soy protein hydrolysates and fermented products, but only the peptide His-His-Leu derived from fermented soy paste was assayed in pure form in SHR, where a decrease of 32 mm Hg of SBP was reached at a dose of 100 mg/kg. Moreover, the synthetic tripeptide His-His-Leu resulted in a significant decrease of ACE activity in the aorta [77]. Soybean-derived products contain isoflavones, which are thought to possess a favourable effect in reducing cardiovascular risk factors as well as vascular function [78]. However, on the basis of *in vitro* results and literature review, Wu and Muir [79] have indicated that the contribution of isoflavones to a blood-pressure-lowering effect in soybean ACEI peptides may be negligible. Similarly, it has been reported that the reduction of hypertension of a fermented product from soy milk was contributed mainly by peptides of 800–900 Da but it could be also attributable to GABA [80]. Moreover, fermented soy product, miso, with added tripeptides (VPP and IPP) from casein was reported to act as antihypertensive agents in SHR [81]. Recently, Nakahara et al. [82] used the Dahl salt-sensitive rats as a model of salt-sensitive hypertension to evaluate the antihypertensive effect of a peptide-enriched soy sauce-like seasoning. The results of these tests have highlighted an important lack of correlation between the *in vitro* ACEI activity and the *in vivo* action. This fact has provided doubts on the use of the *in vitro* ACEI activity as the exclusive criteria for potential antihypertensive substances, since physiological transformations may occur *in vivo*, and because other mechanisms of action than ACE inhibition might be responsible for the antihypertensive effect.

### **3.2. Effects in clinical studies**

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

lactotripeptides decreased serum ACE and aldosterone levels [68].

(GABA), contributed to lowering blood pressure of SHR [76].

by 9.3 (± 4.8) and 7.0 (± 3.8) mmHg 6 h after gastric intubation [29].

decreased serum ACE activity [73]. In salt-loaded GK rats, fermented milk with

Besides the most extensively studied lactotripeptides, also other fermented milk products and peptides have been found. Different strains of lactic acid bacteria, such as *Lb. helveticus* CPN4, *Lb. bulgaricus, Lb. jensenii* and *Str. thermophilus*, have been also shown to provoke liberation of peptides with antihypertensive activity in SHR [36, 37, 41]. Two peptides, corresponding to -casein fragments Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro and Leu-His-Leu-Pro-Leu-Pro, have been isolated in fermented milk with *Enterococcus faecalis* and their antihypertensive effect in SHR, after acute and long-term administration has been proved. The administration of 2 mg/kg of peptide Leu-His-Leu-Pro-Leu-Pro resulted in a significant decrease of the SBP in SHR 4 h post-administration [74,75]. Fermentation of milk with one or more lactic acid bacteria strains followed by hydrolysis using food-grade enzymes liberated tripeptides (Gly-Thr-Trp and Gly-Val-Trp). Oral administration of this fermented whey lowered significantly SBP in SHR from 9 to 15 weeks of age. Bioactive substances, tripeptides and -aminobutyric acid

Some of ACE-inhibitory peptide fractions from cheese have shown *in vivo* activities. A water-soluble peptide preparation isolated from Gouda ripened for 8 months was found to have the most potent antihypertensive activity (maximum decrease in SBP = 24.7 (± 0.3) mmHg (P ≤ 0.01) after 6 h) when administered to SHR by gastric intubation at doses between 6.1 and 7.5 mg/kg body weight. Three peptide fractions were isolated from watersoluble extract by hydrophobic chromatography using different concentrations of acetonitrile. The fractions eluting between 15% and 30%, 30–45% and 60–75% acetonitrile decreased SBP in SHR by 15.0, 29.3 and 18.8 mmHg (P ≤ 0.01), respectively, 6 h after gastric intubation. The peptide fraction eluting between 30% and 45% acetonitrile was shown to contain the sequences (αs1-cn f(1–9)) Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln and (β-cn f(60– 68)) Tyr-Pro-Phe-Gly-Pro-Ile-Pro-Asn (Table 1), which, respectively, decreased SBP in SHR

Several sequences have been proposed as responsible for the antihypertensive activity of soy protein hydrolysates and fermented products, but only the peptide His-His-Leu derived from fermented soy paste was assayed in pure form in SHR, where a decrease of 32 mm Hg of SBP was reached at a dose of 100 mg/kg. Moreover, the synthetic tripeptide His-His-Leu resulted in a significant decrease of ACE activity in the aorta [77]. Soybean-derived products contain isoflavones, which are thought to possess a favourable effect in reducing cardiovascular risk factors as well as vascular function [78]. However, on the basis of *in vitro* results and literature review, Wu and Muir [79] have indicated that the contribution of isoflavones to a blood-pressure-lowering effect in soybean ACEI peptides may be negligible. Similarly, it has been reported that the reduction of hypertension of a fermented product from soy milk was contributed mainly by peptides of 800–900 Da but it could be also attributable to GABA [80]. Moreover, fermented soy product, miso, with added tripeptides Evidence of the beneficial effects of bioactive peptides has to be based on clinical data. Most research has been focused in lactotripeptides, VPP and IPP, and their antihypertensive properties. About twenty human studies have been published linking the consumption of products containing lactotripeptides with significant reductions in both SBP and DBP. Oral administration of these tri-peptides included in different formulas, fermented milk, dried product, fruit juice, etc., products. However, recent studies have provided some conflicting results. Most clinical trials have assessed BPlowering effects at multiple points over time. Most of the BP studies with lactotripeptides have been done in Japanese subjects, and several studies have been done in Finnish subjects [83-88]. Generally, maximum duration of treatment was 8 weeks at doses between 3 and 52 mg/day (Table 3). From these data, it becomes apparent that the largest part of the total BP reduction takes place in the first 1–2 weeks of treatment. Thereafter, a further gradual lowering is seen, but to a lesser extent than in the first period [84-86]. The first significant effects of lactotripeptides on BP in hypertensive subjects were observed after 1–2 weeks of treatment with dosages as low as 3.8 mg/d. Maximum BP-lowering effects of lactotripeptides approximate 13 mmHg SBP and 8 mmHg DBP active treatment v. placebo, and are likely reached after 8–12 weeks of treatment. Lactotripeptides exert a gradual effect on BP lowering after start of intake and return of BP after end of treatment as well [85, 86, 89]. The highest effective dosage of lactotripeptides was evaluated in a safety study, and consisted of 52.5 mg/d [88]. After 10 weeks of active treatment, mean SBP in subjects with hypertension decreased by 4.1 mmHg and DBP by 1.8 mmHg. The next highest dose of lactotripeptides that was tested amounted to 13.0 mg/d [89]. After 4 weeks of active treatment, SBP in subjects with mild hypertension decreased by 11.2 mmHg compared to placebo, and DBP tended to decrease by 6.5 mmHg. In none of the trials with normotensives were statistically significant BP changes found [90-92]. Even at the highest dosage of lactotripeptides used in normotensives, which included a total of 29.2 mg/d during a period of 7 d, no BP lowering effects by lactotripeptides were observed [93]. Thus lactotripeptides only seem to be active at elevated BP values. Evidence indicates that effectiveness is positively associated with BP level, which is in line with existing data for BP-lowering medication [94].


Lactic Fermentation and Bioactive Peptides 321

The results have been included in two meta-analysis [95, 96], which described decreases around 5 mmHg for SBP and 2.3 mmHg for DBP. In general, the effects described in Japanese studies on lactotripeptides are larger than those reported in Finnish studies. However, it is unlikely that genetic differences can account for these differential effects. Moreover, clinical trials in Dutch and Danish subjects have described controversial results since no effect on blood pressure was found [97, 98]. In a recent meta-analysis with a total of 18 trials, it was found a reduction of 3.73 mm Hg for SBP and 1.97 mm Hg for DBP but it was highlighted that the effect was more evident in Asian subjects that in Caucasian ones [99]. The relevance of these findings in genetics or dietary patterns should be further investigated. Comparative studies on antihypertensive medication in different races/ethnic groups have demonstrated that pharmacokinetic parameters and haemodynamic effects are essentially the same in

Hypertension is a complex multifactor disorder that is thought to result from an interaction between environmental factors and genetic background. Subject characteristics such as age and race/ethnicity can affect BP, including the BP response to specific antihypertensive medication. For certain antihypertensive drugs, it has been reported that a polymorphism found in humans can affect the clinical effectiveness, and similarly, these differences could be also affecting clinical trials of functional ingredients [101]. Although ACE inhibition has been postulated as the underlying mechanism of these lactotripeptides, results about the inhibition of this enzyme are not conclusive in humans. Several studies have shown that rennin or ACE activity was not affected by the oral administration of the tripeptides [95, 102]. Therefore, other mechanisms could be implicated in the observed blood pressure reduction. It has been found that the intake of fermented milk containing these peptides may decrease sympathetic activity, leading to a diminished heart rate variability, heart rate and total peripheral resistance,

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

Chinese and Japanese subjects compared with Caucasian subjects [100].

although differences did not reach statistical significance [98].

**4. Bioavailability** 

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 was carried out in crossover design.

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

The results have been included in two meta-analysis [95, 96], which described decreases around 5 mmHg for SBP and 2.3 mmHg for DBP. In general, the effects described in Japanese studies on lactotripeptides are larger than those reported in Finnish studies. However, it is unlikely that genetic differences can account for these differential effects. Moreover, clinical trials in Dutch and Danish subjects have described controversial results since no effect on blood pressure was found [97, 98]. In a recent meta-analysis with a total of 18 trials, it was found a reduction of 3.73 mm Hg for SBP and 1.97 mm Hg for DBP but it was highlighted that the effect was more evident in Asian subjects that in Caucasian ones [99]. The relevance of these findings in genetics or dietary patterns should be further investigated. Comparative studies on antihypertensive medication in different races/ethnic groups have demonstrated that pharmacokinetic parameters and haemodynamic effects are essentially the same in Chinese and Japanese subjects compared with Caucasian subjects [100].

Hypertension is a complex multifactor disorder that is thought to result from an interaction between environmental factors and genetic background. Subject characteristics such as age and race/ethnicity can affect BP, including the BP response to specific antihypertensive medication. For certain antihypertensive drugs, it has been reported that a polymorphism found in humans can affect the clinical effectiveness, and similarly, these differences could be also affecting clinical trials of functional ingredients [101]. Although ACE inhibition has been postulated as the underlying mechanism of these lactotripeptides, results about the inhibition of this enzyme are not conclusive in humans. Several studies have shown that rennin or ACE activity was not affected by the oral administration of the tripeptides [95, 102]. Therefore, other mechanisms could be implicated in the observed blood pressure reduction. It has been found that the intake of fermented milk containing these peptides may decrease sympathetic activity, leading to a diminished heart rate variability, heart rate and total peripheral resistance, although differences did not reach statistical significance [98].
