Examples of identified bioactive peptides in different cheese varieties
Fermented milk products have naturally high nutritional value, and as an extra benefit many health-promoting effects, such as improvement of lactose metabolism, reduction of serum cholesterol and reduction of cancer risk . The beneficial health effects associated with some fermented dairy products may, in part, be attributed to the release of bioactive peptide sequences during the fermentation process. Numerous peptides and peptide fractions, having bioactive properties have been isolated from fermented dairy products. These activities include immunomodulatory, cytomodulatory, hypocholesterolemic, antioxidative, antimicrobial, mineral binding, opioid and bone formation activities. Many recent articles and book chapters have reviewed the release of various bioactive peptides from milk proteins through microbial proteolysis [2-5].
Many industrially utilized dairy starter cultures are highly proteolytic. The use of bioactive peptides producers microbial cultures (starter and non-starter) may allow the development new fermented dairy products. The proteolytic system of lactic acid bacteria e.g.
Cardiovascular disease (CVD) is the single leading cause of death for both males and females in technologically advanced countries in the world. In lesser-developed countries it generally ranks among the top five causes of death. The World Health Organization estimates that by 2020, heart disease and stroke will have surpassed infectious diseases to become the leading cause of death and disability worldwide . 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 . 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 treatment of hypertension.
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 . Some peptides derived from hydrolyzed food proteins exert antioxidant activities against enzymatic (lipoxygenase-mediated) and nonenzymatic peroxidation of lipids and essential fatty acids . The antioxidant properties of these peptides have been suggested to be due to metal ion chelation, free radical scavenging and singlet oxygen quenching.
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.
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.  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 . A bioactive peptide derived from αs1-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) . ACEI peptides have also been found in enzyme-modified cheeses , in a low-fat cheese made in Finland  and Manchego cheeses manufactured with different starter cultures . Mexican Fresco cheese manufactured with
When β-casomorphins were looked from commercial cheese products, no peptides were found or their concentration in the cheese extract was below 2 μg/ml . They further noted that the enzymatic degradation of β-casomorphins was influenced by a combination of pH and salt concentration at the cheese ripening temperature. Therefore, if formed in cheese, β-casomorphins may be degraded under conditions similar to Cheddar cheese ripening. Precursors of β-casomorphins, on the other hand, have been identified in Parmesan cheese . β-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) . Immunomodulating properties in water-soluble extracts from traditional French Alps cheeses, Abondance and Tomme de Savioe have been observed . However, no correlation between peptide composition and
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 β−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  and Tyr-Gln-Glu-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val (β-cn, f(193-209)) from Cheddar  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.
|Cheese variety||Milk protein fragment||Peptide sequence||ACE-inhibition|
|Gouda||αs1-cn f( 1-9)|
|Manchego||ovine αs1-cn f(102-109)|
ovine αs1-cn f(205-208)
|Cheddar (with probiotics)||αs1-cn f(1-9)|
|Swiss cheese varieties||β-cn, f(84–86)|
β-cn, f(74–76) and
|Fresco cheese||αs1-cn f(1-15)|
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
|Organisms||ACE-inhibition||Identified peptides||Dose||Response (Δ SBP mmHg)||Ref.|
|IC50 mg/ml||Sequence||IC50 µM|
|5 ml/kg||-21.8 ±4.2 after 6 h||34|
|27 ml/day||-21 after 4 weeks||67|
|ND||YP||720||10 ml/kg||32.1 ±7.4 after 6 h||42|
|10ml/kg||-12 after 4-8 h|
-11 after 4-8 h
Str. salivarius ssp thermophilus and L.lactis biovar diacetylactis
|0.2 kg/kg||approx -12 after 2 h||38|
|-21.87 ±4.51 after 4h1)|
approx -15 after 4 h
|Mixed lactic acid bacteria (||0.24||GTW|
|5 mg/ml||SBP -22 after 8 weeks||76|
Pihlanto-Leppälä et al.  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,
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
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 . 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 . The enzymatic process generating the antihypertensive peptides VPP and IPP in
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
2.3. 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
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 . 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
3. Antihypertensive effects in vivo
The search for
3.1. Animal studies
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
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. , 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 . Protection of endothelial function after incubation with tripeptides IPP and VPP for 24 h was found in a study with isolated SHR mesenteric arteries .
Evidence from ACE inhibition was gained by Masuda et al. , 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 . In addition, treatment with fermented milk containing lactotripeptides and plant sterols decreased serum ACE activity . In salt-loaded GK rats, fermented milk with lactotripeptides decreased serum ACE and aldosterone levels .
Besides the most extensively studied lactotripeptides, also other fermented milk products and peptides have been found. Different strains of lactic acid bacteria, such as
Some of ACE-inhibitory peptide fractions from cheese have shown
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 . Soybean-derived products contain isoflavones, which are thought to possess a favourable effect in reducing cardiovascular risk factors as well as vascular function . However, on the basis of
3.2. Effects in clinical studies
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 BP-lowering 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 . 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 . 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 . 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 .
|Design||Duration||Study population||Treatment||BP changes mmHg||Ref.|
|(weeks)||IPP mg/d||VPP mg/d||Source of peptides||Formula||SBP||DBP|
|R, p-c, s-bld,|
|8||30 eldery hypertensive patients||1.1||1.5||1 x 95 ml|
|R, p-c, d-bld,|
|8||64 subjects with SBP 140-159 and DBP 90-99 mmHg||1.58||2.24||2 x 150 g|
|R, p-c, d-bld,|
|8||32 subjects with SBP 140 - 180 and DBP 90-105 mmHg||1·60||2·66||1 x 120 g|
|R, p-c, d-bld,|
|8||18 hypertensive and 26 normotensive subjects||1.1||1.5||2 x 100 g|
|R, p-c, d-bld, parallel||8||30 subjects with SBP 140-180 and DBP 90-105 mmHg||1.52||2.53||2 x 160 g|
|R, p-c, d-bld, parallel 1)||21||39 subjects with SBP 133-176 and DBP 86-108 mmHg||2.25||3.0||Lb. helv|
|2 x 150 ml|
|R, p-c, d-bld,|
|60 Finnish subjects with SBP 140-180 and DBP 90-110 mmHg||2.4-2.7||2.4-2.7||Lb. helv|
|1 x 150 ml|
|R, p-c, d-bld,|
|10||94 hypertensive patients||30||22.5||Lb. helv|
|2 x 150 ml|
|R, p-c, d-bld,|
|1||20 healthy volunteers|
normal blood pressure (<130 mmHg SBP and <85 mmHg DPB).
|1 x 14 tablets||2.6||2||93|
|R, p-c, d-bld, parallel||8||135 Dutch subjects with untreated high-normal BP or mild hypertension||4.2||5.8||Fermentation||1 x 200 ml|
|R, p-c, d-bld, crossover||4||70 Caucasian subjects with prehypertension or stage 1 hypertension||15||-||Hydrolysis by endopeptidase||2 x 7.5 mg capsules||-3.8||-2.3||102|
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 . 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 .
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 . 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 .
Bioavilability of bioactive peptides is an important target to establish the relationship between
Peptides have been reported to have poor permeation across biological barriers (e.g. intestinal mucosa) . 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 . The intestinal absorption of peptides have been performed using
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 . 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
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 . 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 . Ko et al.  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 . 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 . Individually, various components of emulsions have been considered as candidates for improving bioavailability of peptides.
5. General conclusions
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.
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 non-pharmacological, nutritional treatment of elevated blood pressure for some groups of people.