Valil-Prolil-Prolina

Valyl-Prolyl-Proline (VPP): A Comprehensive Reference Article

1. Identity and Chemical Characterization

1.1 Nomenclature and Chemical Nature

Valyl-Prolyl-Proline (abbreviated VPP; systematic name L-valyl-L-prolyl-L-proline) is a naturally occurring bioactive tripeptide consisting of three amino acid residues in the sequence valine–proline–proline. The two tripeptides L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP) have been identified as possessing significant angiotensin-converting enzyme inhibitory activity and are therefore believed to be the source of normotensive effects observed with fermented milk. VPP belongs to the class of food-derived bioactive peptides collectively called lactotripeptides (LTPs). Milk-derived small peptides have held promise for reducing angiotensin II-mediated vasoconstriction, because mechanistically they imitate angiotensin-converting enzyme inhibitors (ACEIs). The best-characterised peptides are isoleucine–proline–proline (IPP) and valine–proline–proline (VPP), which are named lactotripeptides (LTPs).

Physical/chemical properties, molecular weights, chemical structures, normal consumption in the diet, manufacturing information, regulatory approval in Japan, and Japanese consumption of food containing enhanced levels of VPP plus IPP are established features of this peptide that have been formally characterized in published toxicological and regulatory documentation.

1.2 Natural Sources

Lactotripeptides are two naturally occurring milk peptides: isoleucine-proline-proline (IPP) and valine-proline-proline (VPP). These lactotripeptides are derived from casein, which is a milk protein also found in dairy products. Although most normal dairy products contain lactotripeptides, they are inactive within the original milk proteins. Dairy peptides can be effectively released through enzymatic predigestion — a process by which milk protein is enzymatically broken down into smaller pieces.

VPP and IPP are released from bovine milk proteins during in vitro oro-gastro-intestinal (OGI) digestion at final concentrations of 354.3 ± 29.8 and 973.8 ± 155.7 µg/L, respectively. In silico analysis of cleavage sites and mass spectrometry revealed that tetrapeptides VPPF, IPPL and IPPK are precursors of VPP and IPP.

VPP and IPP are well-known ACE inhibitory peptides, originally obtained by Nakamura et al. (1995) using Lactobacillus helveticus and Saccharomyces cerevisiae as fermenters to produce Calpis sour milk and isolated by a four-step HPLC method, with IC₅₀ values of 9 μM and 5 μM for their in vitro ACE inhibitory activities, respectively. Since then, researchers have isolated these two ACE inhibitory peptides using casein from buffalo, yak, goat, as well as sourdough made from rye, wheat, and malt.

1.3 Preparation Methods and Commercial Forms

The tripeptides isoleucine-prolyl-proline (IPP) and valyl-prolyl-proline (VPP) can be obtained through fermentation of milk by certain lactic acid bacteria, by enzymatic hydrolysis of casein, or by chemical synthesis. LTPs are obtained from either fermented or enzymatically treated milk (FLTPs or ELTPs).

VPP is commercially most prominently available as a proprietary ingredient. One well-characterized commercial product is produced through a patented fermentation and enzymatic process that releases two key tripeptides, Valyl-Prolyl-Proline (VPP) and Isoleucyl-Prolyl-Proline (IPP), made up of the three amino acid chains valine, proline and isoleucine, from casein. Such products provide VPP and IPP in concentrations not practical to achieve through the consumption of dairy products.

Milk fermented with Lactobacillus helveticus contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). Lactobacillus helveticus is one of the most efficient proteolytic Lactobacillus species compared to other lactic acid bacteria.

Two notable commercial fermented milk products incorporating VPP and IPP include the Japanese product Calpis and the Finnish product Evolus. Evolus, a fermented milk product by Valio Ltd. (Helsinki, Finland), and Calpis, a sour milk product by Calpis Co. Ltd. (Tokyo, Japan), both contain the bioactive peptides IPP and VPP, known for their antihypertensive benefits. Beyond fermented liquids, VPP and IPP have also been incorporated into liquid or tablet forms, administered for periods that ranged from four to 21 weeks in clinical research, generally with a run-in period of one to four weeks.

2. Traditional and Historical Use

The consumption of fermented milk to maintain good health, including the maintenance of normal blood pressure, is an ancient tradition in a number of areas of the world (e.g., East Asia, France). VPP itself was not identified as an isolated bioactive compound in traditional practice; rather, it was consumed as an endogenous component of fermented dairy foods in these cultural traditions.

The formal scientific discovery and isolation of VPP as a specific antihypertensive component of such fermented milk dates to the mid-1990s. The Asahi Group's research on lactic acid bacteria and fermented milk can be traced back to Calpis, a company that produced and released Japan's first lactic acid bacteria beverage in 1919. Tests done on mice in the 1970s as part of a collaborative research project between Calpis and Riken found that longevity was promoted by a proprietary formulation of fermented milk made from lactic acid bacteria and yeast. This foundational animal research ultimately led to the identification of VPP and IPP as the specific bioactive constituents responsible for the cardiovascular effects observed in traditional fermented milk preparations.

Recent studies have suggested that fermented milk has a normotensive effect in hypertensive rats and humans, but no effect on blood pressure in normotensive rats and humans. This pattern is consistent with the observation that traditional populations consumed fermented milk broadly as part of general dietary health practices, rather than as a targeted pharmacological intervention.

3. Key Constituents, Active Compounds, and Mechanisms of Action

3.1 Angiotensin-Converting Enzyme (ACE) Inhibition

The primary and best-established mechanism of VPP is inhibition of angiotensin-converting enzyme (ACE). Human somatic angiotensin-I-converting enzyme (sACE) is composed of a catalytic N-domain (nACE) and C-domain (cACE) of similar size with different substrate specificities. It is involved in the regulation of blood pressure by converting angiotensin I to the vasoconstrictor angiotensin II and has been a major focus in the development of therapeutics for hypertension.

Structural research using X-ray crystallography and kinetic analysis has reported the structural basis for the role of Val-Pro-Pro and Ile-Pro-Pro in domain-specific inhibition of ACE. The lactotripeptides have preference for nACE due to altered polar interactions distal to the catalytic zinc ion. Elucidating the mechanism of binding and domain selectivity of these peptides also provides important insights into the functional roles of ACE.

BP reduction through lactotripeptides has been suggested to be due to inhibition of the renin-angiotensin system, which plays an important role in regulating arterial pressure. Renin converts angiotensinogen to the biologically inactive angiotensin I, which in turn undergoes proteolytic cleavage by ACE to the vasoconstrictor angiotensin II. Inhibition of ACE leads to an increase of the angiotensin I/angiotensin II ratio and a subsequent compensatory increase in renin activity.

3.2 Nitric Oxide Production and Endothelial Function

Beyond ACE inhibition, VPP has been demonstrated to exert direct effects on endothelial cells through stimulation of nitric oxide (NO) synthesis. For this purpose, researchers examined the effect of VPP and IPP on induction of nitric oxide (NO) production using cultured vascular endothelial cells and isolated arterial vessels. When both VPP and IPP were added to the medium of cultured endothelial cells at final concentrations of more than 100 nmol/l, the NO_x (NO₂ and NO₃) concentration in the medium was significantly higher than that of the control.

Moreover, both VPP and IPP induced endothelium-dependent relaxation of isolated aortic rings, and these effects were inhibited by NO synthase inhibitors, K channel inhibitors, and bradykinin B2 receptor antagonists.

VPP and IPP treatment reduced the level of reactive oxygen species and EV-induced expression of adhesion molecules, and restored the ability of tube formation by upregulating endothelial nitric oxide synthase expression.

3.3 Gene Expression Effects

DNA microarray studies in spontaneously hypertensive animal models have further clarified VPP's mechanisms at the genomic level. Significant and marked differences were detected for the endothelial nitric oxide synthase (eNOS) gene (1.89-fold, P<0.05) and the connexin 40 (gap junction 40) gene (2.81-fold, P<0.05). Administration of VPP and IPP led to a slight increase in the expression of the cyclooxygenase (COX-1) gene and a decrease in the expression of both the nuclear factor kappa B subunit (NF-κB) gene for vascular function and the peroxisome proliferator activator receptor gamma (PPARγ) gene. Taken together, these results suggest that VPP and IPP function as ACE inhibitors in the aorta, where they may have a preventive role in cardiovascular function.

Research has reported that LTPs promote NO production through endothelial nitric oxide synthase (eNOS) activation and exert vasodilatory effects in humans. LTPs have been shown to inhibit vascular contraction through ACE inhibition, with antihypertensive effects confirmed in clinical trials. Improvements in endothelial function from LTP ingestion, via enhanced flow-mediated dilation (FMD) and pulse wave velocity, have also been observed in clinical studies.

4. Scientific Evidence by Area of Use

4.1 Blood Pressure Reduction

Meta-Analytic Evidence

The effects of milk protein-derived peptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) on blood pressure have been reported in more than 20 randomized, placebo-controlled clinical studies. Multiple independent meta-analyses have sought to pool this growing body of evidence, with varying conclusions depending on the populations studied and methodological criteria applied.

A meta-analysis covering nineteen randomized clinical intervention trials found that small daily doses (2.0–10.2 mg) of milk casein-derived tripeptides showed an overall lowering of systolic (4.0 mmHg) and diastolic (1.9 mmHg) blood pressure in mildly hypertensive subjects in a random effects meta-analysis. The results suggest that rather small daily doses of the lactotripeptides in different functional food products may offer a valuable option as a non-pharmacological treatment of prehypertension or mild hypertension as part of lifestyle advice.

A separate meta-analysis of 18 trials reported by Cicero et al. (2011) found that pooled effects of peptides were a reduction of −3.73 mmHg (95% CI: −6.70, −1.76) for systolic blood pressure (SBP) and −1.97 mmHg (95% CI: −3.85, −0.64) for diastolic blood pressure (DBP). The effect was more evident in Asian patients (SBP = −6.93 mmHg) than in Caucasian ones (SBP = −1.17 mmHg), and apparently not related to age, baseline BP values, dose of lactotripeptides assumed, or length of the treatment.

A DARE-reviewed meta-analysis (Xu et al., 2008) pooling twelve comparisons from randomized controlled trials found that IPP and VPP significantly reduced blood pressure compared to controls: systolic blood pressure reduced by −4.8 mmHg (95% CI −6.0 to −3.7) and diastolic by −2.2 mmHg (95% CI −3.1 to −1.3). There was no evidence of significant heterogeneity or publication bias. Subgroup analysis showed a more marked effect in the hypertensive group than in the pre-hypertensive group; the difference (2.4 mmHg) was statistically significant for systolic blood pressure.

A more recent and comprehensive meta-analysis of 24 studies with 28 trials involving 1,919 human subjects (Qin et al., 2013) found more modest pooled effects: in general analysis, there are small reductions in both systolic BP (SBP) and diastolic BP (DBP) with pooled mean effects of 1.66 (95% CI: −2.48 and −0.84) and 0.76 mmHg (−1.31 and −0.20) respectively. While the effect, though statistically significant, is small in magnitude — with pooled mean effects of only 1.66 and 0.76 mmHg reduction in SBP and DBP, respectively — the number of clinical trials continues to increase, but results have been inconsistent, especially in the last few years.

Japanese Population Evidence

A 2015 systematic review and meta-analysis in PLOS ONE, which is particularly notable for searching Japanese-language databases in addition to international ones, examined the SBP-lowering effect specifically in Japanese subjects. Eighteen studies including a total of 1,194 subjects were included in the meta-analysis. A random effect model using the restricted maximum likelihood (REML) estimator was used for the analysis. The analysis showed that consumption of IPP/VPP induced a significant reduction in SBP as compared with placebo in Japanese subjects, with an estimated effect of −5.63 mmHg (95% CI, −6.87 to −4.39, P<0.0001) and no evidence of publication bias.

A significant heterogeneity between series was evident, which could be explained by a significant influence of the baseline blood pressure status of subjects; the effect of IPP/VPP on SBP was stronger in hypertensive subjects (−8.35 mmHg, P<0.0001) than in non-hypertensive subjects (−3.42 mmHg, P<0.0001). Furthermore, the effect of IPP/VPP on SBP remained significant when limiting the analysis to series that tested the usual doses of IPP/VPP consumed daily (below 5 mg/d), with estimated effects of −6.01 mmHg in the overall population and −3.32 mmHg in non-hypertensive subjects.

European Population Evidence

A meta-analysis specifically examining European subjects found the decrease in SBP with IPP/VPP was 1.28 mmHg (95% CI, −2.09 to −0.48, P = 0.0017) and the decrease in diastolic BP (DBP) was 0.59 mmHg (95% CI, −1.18 to −0.01, P = 0.047). The peptides IPP and VPP are effective in moderately reducing SBP in European subjects, as is known for Asian populations. These two peptides could therefore have a role in controlling blood pressure, a prospect that merits their further study.

Evidence Limitations and Inconsistencies

Data derived from clinical trials have reached conflicting conclusions. Some clinical studies have suggested that these lactotripeptides help promote healthy blood pressure levels as part of a healthy diet and lifestyle. However, other clinical trials have seen no effects from these compounds.

In studies of lactotripeptides, VPP and IPP appear more effective in reducing BP of subjects with a higher starting BP. Indeed, in none of the trials with normotensives were any statistically significant BP changes found. This echoes the pattern observed in early animal studies: research suggests that in adult normotensive volunteers, consumption of up to 7.92 mg of VPP and 4.52 mg IPP daily for 2 weeks causes neither clinical signs nor biologically meaningful effects on systolic or diastolic blood pressure, pulse rate, or clinical pathology (serum chemistry or hematology). However, when a similar study was performed using mildly and moderately hypertensive adults as subjects and they consumed 2.52 mg of VPP and 1.64 mg of IPP per day, a significant drop in systolic blood pressure was detected for a prolonged time interval.

4.2 Arterial Stiffness and Central Blood Pressure

Beyond peripheral blood pressure, evidence has accumulated for VPP and IPP effects on arterial stiffness and central aortic blood pressure. A randomized, double-blind, placebo-controlled trial by Nakamura et al. (2011), published in Atherosclerosis, demonstrated that casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro improves central blood pressure and arterial stiffness in hypertensive subjects.

Eight weeks of LTP ingestion has been reported to improve arterial stiffness, endothelial function, and cerebral blood flow velocity in middle-aged and older adults. A clinical trial published in the Journal of Medicinal Food also examined the effect of casein hydrolysate containing the antihypertensive tripeptides VPP and IPP on vascular endothelial function independent of blood pressure-lowering effects, contributing to inhibitory action of angiotensin-converting enzyme (Hypertension Research, 2007).

4.3 Endothelial Function and Vascular Health

The hydrolysate of milk casein containing both VPP and IPP improved the vascular endothelial function of subjects with stage I hypertension, providing impetus to elucidate the mechanism of the improvement of endothelial dysfunction by these peptides.

The milk-derived peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) inhibit angiotensin-converting enzyme, dilate blood vessels ex vivo, and stimulate nitric oxide (NO) production in cells. Both endothelial dysfunction and arterial stiffness are surrogate markers of atherosclerosis and thus cardiovascular (CV) events.

A study in postmenopausal women (Yoshizawa et al., 2010) found additive beneficial effects of lactotripeptide intake with regular exercise on endothelium-dependent dilatation in postmenopausal women (American Journal of Hypertension, 2010).

4.4 Cognitive Function and Cerebral Blood Flow

A smaller but emerging body of clinical research has examined VPP's effects on cerebrovascular function. An 8-week intake of LTP increased cerebral oxygenation in the prefrontal cortex region in middle-aged and older adults, with and without exercise. Age-related declines in cognitive function and cerebral perfusion increase the risk of dementia. Although nutrition and exercise may be effective in reducing cognitive decline, the effect of lactotripeptide (LTP) on cerebral oxygenation and hemodynamics remains under investigation.

Researchers have hypothesized that a lactotripeptide with ACE inhibitory activity and capacity to increase the production of a vasodilator could reduce cognitive decline and cerebral atrophy by improving the speed of blood flow to the brain in middle-aged and older adults. A randomized, placebo-controlled, double-blind design was used to assess the health benefits of daily supplementation, for 8 weeks, of a casein hydrolysate containing VPP and IPP peptides, in healthy middle-aged and older adults. This area of evidence remains preliminary and requires larger, adequately powered trials.

4.5 Fatigue

LTP ingestion may be effective in mitigating fatigue, as an observational study reported that endothelial function is associated with fatigue status. One study reported that a single dose of LTP temporarily alleviated fatigue in middle-aged and older men. However, the effects of long-term LTP ingestion on fatigue have not yet been formally determined. This area of research is at a very early stage and should be regarded as preliminary.

5. Body Systems and Health Areas

• Cardiovascular system: The primary area of VPP research. Milk casein-derived tripeptides VPP and IPP inhibit angiotensin-converting enzyme (ACE), and both fermented milk and proteolytic hydrolysates of milk casein containing these peptides exert blood pressure-lowering effects in animals and humans.
• Vascular endothelium: Improvements in endothelial function from LTP ingestion, via enhanced flow-mediated dilation (FMD) and pulse wave velocity, have been observed in clinical studies.
• Central nervous system / cerebrovascular function: Preliminary human trial evidence suggests effects on cerebral oxygenation and blood flow velocity, though this evidence base is nascent.
• Oxidative stress and inflammation: In vitro evidence indicates that VPP and IPP treatment reduced the level of reactive oxygen species and EV-induced expression of adhesion molecules.

6. Dosage Forms and Dosages Reported in Studies

VPP is administered in clinical studies both as a fermented milk beverage and in tablet or powder form. The lowest and highest effective dosages of these two antihypertensive tripeptides administered to humans were reported to be 3 mg/d (2 mg VPP plus 1 mg IPP) and 52.5 mg/day (30 mg VPP plus 22.5 mg IPP), respectively.

Effective dosages have been reported to range from 3.07 mg/d (1.60 mg IPP and 1.47 mg VPP) to 52.5 mg/d (30 mg IPP and 22.5 mg VPP).

In one specific randomized, placebo-controlled study on fatigue in middle-aged and older adults, three LTP tablets contained 1.4 mg VPP and 2.0 mg IPP. The administered dose was defined based on a previous study, which confirmed that a single dose of 1.4 mg VPP and 2.0 mg IPP reduced fatigue in middle-aged and older men.

Nineteen randomized clinical intervention trials used small daily doses of 2.0–10.2 mg of milk casein-derived tripeptides (VPP plus IPP combined). IPP and VPP were administered as liquid or tablet forms for periods that ranged from four to 21 weeks, generally with a run-in period of one to four weeks.

The Japanese regulatory and research context has also characterized acceptable intake. Products containing IPP and VPP peptides in effective doses (>3.0 mg/day) can be considered a safe and effective non-pharmacological intervention in patients with mild hypertension.

7. Safety Considerations

7.1 General Safety Profile

Toxicity studies in animals and safety trials in humans substantiate the safety of lactotripeptides. A comprehensive series of toxicological studies was organized under the auspices of US regulatory evaluation. Single-dose (oral administration of 2,000 mg/kg pasteurized casein hydrolysate, 4,000 mg/kg powdered Lactobacillus helveticus-fermented milk, 400 mg/kg synthesized VPP), repeated-dose (16 mg/kg/day VPP for continuously 8 weeks or 40 mg IPP/kg body weight/day for 90 days), and multiple-dose (greater than 1,000 mg/kg body weight/day powdered casein hydrolysate) were found to show no systemic/local toxicity in male and female rats.

A NOAEL was formally established in a 90-day subchronic study. The NOAEL is equivalent to an overall mean intake of 2 g Tensguard™/kg body weight/day and corresponds to 40 mg IPP/kg body weight/day. This is 141-fold higher than the maximal anticipated intake. In conclusion, the product is safe under the conditions of intended use.

Already in 1976, the Life Science Research Office of the Federation of American Societies for Experimental Biology (FASEB) concluded that there are no suspicious hazards related to protein hydrolysates as food ingredients for public health aspects. The FDA lists protein hydrolysates as 'generally recognized as safe' (GRAS) under 21 CFR 102.22.

In randomized controlled trials, most reported minor adverse events in both VPP/IPP and placebo groups, none of which were considered to be treatment related.

7.2 Effect in Normotensive Individuals

An important safety feature is the absence of hypotensive effects in people with normal blood pressure. Studies have suggested that fermented milk has a normotensive effect in hypertensive rats and humans, but no effect on blood pressure in normotensive rats and humans. Research suggests that in adult normotensive volunteers, consumption of up to 7.92 mg of VPP and 4.52 mg IPP daily for 2 weeks causes neither clinical signs nor biologically meaningful effects on systolic or diastolic blood pressure, pulse rate, or clinical pathology (serum chemistry or hematology).

7.3 Allergen Consideration

VPP is derived from bovine milk casein. Individuals with confirmed cow's milk protein allergy should exercise caution, as VPP-containing products originate from a major allergenic source. This is a factual source-identity consideration rather than a documented clinical adverse event from VPP per se; the peptide is not itself a recognized allergen in formal regulatory assessments, but its bovine casein origin is relevant for individuals with established dairy protein allergies.

7.4 EFSA Health Claim Assessment and Regulatory Status

The regulatory status of VPP and IPP health claims in Europe reflects the complexity and inconsistency of the clinical evidence base. The EFSA Panel on Dietetic Products, Nutrition and Allergies concluded that a cause and effect relationship has not been established between the consumption of IPP and VPP and maintenance of normal blood pressure in its 2011 Article 13(5) opinion submitted under Regulation (EC) No 1924/2006. The interpretation of the results from seven out of eight studies which reported an effect of IPP and VPP on office SBP was hampered by major methodological limitations. The animal studies did not provide additional information on the effect of IPP and VPP on BP in humans, and no convincing evidence for a mechanism by which IPP and VPP could exert their effects was presented.

Following an application that was assessed, EFSA concluded that on the basis of the data presented, a cause and effect relationship had not been established between the consumption of IPP and VPP and the claimed effect, resulting in the European Commission refusing to authorise the health claim under EU Regulation No 1017/2013.

This regulatory outcome underscores that while the totality of the evidence — including numerous RCTs and multiple meta-analyses — shows consistent, albeit modest, blood pressure reductions in hypertensive individuals (particularly Asian populations), European regulatory bodies required a higher and more methodologically rigorous standard of proof than was available at the time of review, particularly with respect to adequately powered trials in European populations.

In Japan, by contrast, VPP and IPP have received approval and have been incorporated into Foods for Specified Health Uses (FOSHU). Regulatory approval in Japan and Japanese consumption of food containing enhanced levels of VPP plus IPP are formally documented in published regulatory and toxicological summaries.

8. Summary of Evidence Strength

The overall scientific evidence for VPP's blood pressure effects in hypertensive individuals is moderate, supported by a large number of randomized controlled trials and multiple independent meta-analyses, with consistent but modest effects (approximately −2 to −6 mmHg SBP reduction depending on the population). Evidence is strongest in Japanese/Asian hypertensive populations; effects in Caucasian/European normotensive individuals are weak to absent based on adequately powered trials. VPP and IPP lactotripeptides assumed as functional foods may significantly reduce SBP particularly in Asian subjects. The relevance of these findings in other ethnicities or associated with different dietary patterns should be further investigated.

Evidence for vascular endothelial function and arterial stiffness is preliminary but mechanistically coherent, supported by in vitro, animal, and a small number of human trials. Evidence for cognitive function, cerebrovascular effects, and fatigue is very early stage, confined to small single trials, and cannot be considered established. The EFSA's 2011 and 2012 scientific opinions concluded insufficient evidence for a health claim under European law, despite the available data.

References

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