isoleucil-prolil-prolina

Isoleucyl-Prolyl-Proline (IPP): A Comprehensive Reference

1. Identity, Chemical Characterization, and Natural Sources

1.1 Chemical Identity and Nomenclature

L-isoleucyl-L-prolyl-L-proline (IPP) is a tripeptide composed of the three amino acid residues isoleucine, proline, and proline. Its molecular formula is C₁₆H₂₇N₃O₄, and its SMILES representation encodes the specific stereochemistry of the three residues in the all-L configuration. The compound is registered under CAS number 26001-32-1 and is assigned PubChem Compound ID (CID) 9949212. Its InChIKey is FQYQMFCIJNWDQZ-CYDGBPFRSA-N. The full IUPAC name recognized by chemical databases is L-isoleucyl-L-prolyl-L-proline, sometimes abbreviated in the literature as Ile-Pro-Pro or simply IPP. IPP is most commonly studied alongside its structural analog valine-proline-proline (VPP); together the two are designated lactotripeptides (LTPs).

IPP is a peptide composed of the amino acids proline and isoleucine, and is specifically characterized as L-Proline, L-isoleucyl-L-prolyl-. It was originally identified as an antihypertensive tripeptide isolated from fermented milk. IPP inhibited angiotensin I-converting enzyme (ACE) with an IC₅₀ of 5 µM in vitro.

1.2 Natural Sources and Location Within Milk Proteins

Lactotripeptides are naturally derived milk peptides and part of our daily diet. The tripeptides isoleucyl-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.

The VPP sequence is embedded in the β-casein fraction at positions 84–86, while IPP appears in β-casein at positions 74–76 and in κ-casein at positions 108–110; no direct embedding of these specific tripeptides has been identified in αs1-casein, though the fraction contributes to overall peptide diversity through hydrolysis. Caseins are characterized by high proline content that protects embedded tripeptide sequences from premature degradation.

The exact IPP and VPP sequences occur in ruminant milks such as bovine, goat, and sheep due to similar casein structures. 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 were 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 from casein derived from buffalo, yak, and goat milks, as well as from sourdough made from rye, wheat, and malt.

1.3 Common Preparations and Dosage Forms

IPP can be obtained through fermentation of milk by certain lactic acid bacteria, by enzymatic hydrolysis of casein, or by chemical synthesis, and all clinical studies presented for regulatory review have been conducted with either directly fermented milk, with powdered fermented milk, or with tripeptides obtained from enzymatically hydrolyzed casein.

Bioactive peptides and protein hydrolysate products for treatment of hypertension have been marketed in Japan under Food for Specified Health Use (FOSHU) designation for a number of years. These include a proprietary standardized LTP product (known as AmealPeptide) derived from enzymatic hydrolysis of casein, which contains levels of VPP and IPP higher than what is found in common dairy products. A fermented milk product (FMP) containing these peptides is commercially available in Japan under the trade name Ameel S®, approved as a FOSHU product.

In clinical research settings, IPP has been delivered in several formats: study treatments have been offered to volunteers in capsules, to exclude any pre-ingestion interference with food matrices; treatments consisted of consumption for 4 weeks, with 500 mg hydrolyzed protein per capsule used for each of the milk protein hydrolysate (MPH) treatments. IPP and VPP have also been studied in tablet form, in fermented milk beverages, in yogurt drinks, and incorporated into food spreads alongside plant sterols.

2. Traditional and Historical Use

2.1 Fermented Milk in East Asia and Europe

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, including East Asia and France. 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.

The specific identification of IPP (and VPP) as the active peptides responsible for antihypertensive properties is a product of modern analytical science, not ancient knowledge. Traditional fermented milk preparations across East Asia — including Japanese-style sour milk — were consumed for general health maintenance and digestive wellbeing without any precise awareness of these peptides. Nakamura et al. (1995b) demonstrated that the peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) represented most of the ACE-inhibitory activity of Calpis sour milk (Calpis Co., Ltd., Tokyo, Japan), which is prepared by fermenting skim milk with a starter containing L. helveticus and Saccharomyces cerevisiae. This 1995 work was pivotal in scientifically defining the active components within a long-standing traditional food.

The scientific investigation of IPP thus bridges traditional fermented dairy culture — particularly in Japan and Finland, where L. helveticus-fermented milks have longstanding popularity — and modern functional food science. The peptides themselves are not isolated folk remedies; rather, they are bioactive constituents of foods with deep traditional roots whose activity was only characterized in the late twentieth century.

3. Key Constituents, Structure, and Mechanisms of Action

3.1 Structural Features Relevant to Bioactivity

The stability of lactotripeptides in the gastrointestinal tract is attributed to structural features such as C-terminal proline residues and branched-chain N-terminal amino acids, which allow them to reach target tissues intact and exert physiological effects. Proline- and hydroxyproline-containing peptides are relatively resistant to degradation by digestive enzymes. The double proline at the C-terminus of IPP is particularly critical, as proline-containing peptide bonds are poor substrates for most mammalian peptidases and carboxypeptidases.

These inhibitors have an Ile-Pro-Pro (IPP) motif at their C terminus, and this structural motif was used to guide the structure-based design of the widely utilized synthetic ACE inhibitor captopril.

3.2 Primary Mechanism: ACE Inhibition

ACE is involved in the regulation of blood pressure by converting angiotensin I to the vasoconstrictor angiotensin II. ACE functions by activating angiotensin I to the potent vasoconstrictor angiotensin II, and through the metabolic inactivation of the vasodilatory peptide bradykinin.

The lactotripeptides Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP), isolated from fermented milk and casein hydrolysates, provide vasodilatory effects through the inhibition of somatic ACE (sACE), with IC₅₀ values for sACE of 5 and 9 µM respectively. The structural basis for the role of IPP in domain-specific inhibition of ACE has been elucidated using X-ray crystallography and kinetic analysis; the lactotripeptides show preference for the N-domain of ACE (nACE) due to altered polar interactions distal to the catalytic zinc ion.

3.3 Nitric Oxide Induction and Endothelial Mechanisms

IPP and VPP 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. Additionally, casein hydrolysate containing both VPP and IPP has been reported to improve vascular endothelial function in subjects with stage I hypertension.

Research examining the effect of VPP and IPP on nitric oxide (NO) production using cultured vascular endothelial cells and isolated arterial vessels found that 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. IPP and VPP have been reported to promote NO production through endothelial nitric oxide synthase (eNOS) activation and exert vasodilatory effects in humans.

3.4 Gene Expression and Additional Mechanisms

In spontaneously hypertensive rat (SHR) studies using DNA microarray analysis, significant and marked differences in gene expression 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 also 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. These results together suggest that VPP and IPP function as ACE inhibitors in the aorta, where they may have a preventive role in cardiovascular function.

Although most ACE-inhibitory peptides were characterized based on in vitro ACE inhibitory activity, a relationship between ACE inhibition and physiological antihypertensive effect is not fully apparent, indicating the involvement of other mechanisms of action. LTPs have been shown to inhibit vascular contraction through ACE inhibition, with antihypertensive effects confirmed in clinical trials, and improvements in endothelial function from LTP ingestion — via enhanced flow-mediated dilation (FMD) and pulse wave velocity — have also been observed in clinical studies.

Lactotripeptides may exert blood pressure-lowering effects either via ACE inhibition or via non-ACE-dependent pathways, but only limited in vivo evidence is currently available for the physiological basis of their antihypertensive action. Other mechanisms that might be involved include production of vasodilative substances or an effect on sympathetic nervous activity.

4. Scientific Evidence by Area of Use

4.1 Blood Pressure Reduction: Overview of the Clinical Evidence Base

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. Between 1996 and 2012, more than 30 human clinical trials evaluated blood pressure responses after consuming fermented milks, yogurt drinks, or casein hydrolysates containing known amounts of IPP and VPP. Most were randomized controlled trials with hypertensive or prehypertensive populations. Nine systematic reviews and/or meta-analyses and a Cochrane review and meta-analysis, as well as a European Food Safety Authority (EFSA) review, have been published.

4.2 Meta-Analyses and Systematic Reviews

Nineteen randomized clinical intervention trials with small daily doses (2.0–10.2 mg) of milk casein-derived tripeptides showed an overall lowering of systolic blood pressure (4.0 mmHg) and diastolic blood pressure (1.9 mmHg) in mildly hypertensive subjects in a random effects meta-analysis.

In a general analysis of 24 studies with 28 trials on 1919 human subjects, there were small reductions in both systolic BP (SBP) and diastolic BP (DBP) with the pooled mean effects of 1.66 mmHg (95% CI: −2.48 and −0.84) and 0.76 mmHg (−1.31 and −0.20), respectively.

In a meta-analysis of 18 clinical trials, the pooled effect of these peptides was a reduction of −3.73 mmHg (95% CI: −6.70, −1.76) for SBP and −1.97 mmHg (95% CI: −3.85, −0.64) for DBP. The effect was more evident in Asian patients (SBP = −6.93 mmHg; DBP = −3.98 mmHg) than in Caucasian subjects (SBP = −1.17 mmHg; DBP = −0.52 mmHg), and was apparently not related to age, baseline BP values, dose of lactotripeptides assumed, or length of treatment.

4.3 Ethnicity-Stratified Evidence: Japanese/Asian Populations

A systematic review and meta-analysis of 18 studies totaling 1,194 Japanese subjects used a random effects model with restricted maximum likelihood (REML) estimation. The analysis showed that consumption of IPP/VPP induced a significant reduction in SBP 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 studies was evident, which could be explained by a significant influence of baseline blood pressure status: 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 remained significant when limiting the analysis to series that tested the usual doses consumed daily (below 5 mg/day), with estimated effects of −6.01 mmHg in the overall population and −3.32 mmHg in non-hypertensive subjects.

4.4 European Populations

The milk-derived peptides IPP and VPP have been shown to reduce systolic blood pressure; this decrease is convincingly shown in subjects of Asian origin, but less consistent results have been obtained in European populations. A PRISMA-compliant meta-analysis 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 was 0.59 mmHg (95% CI: −1.18 to −0.01, P=0.047) in European subjects. There was no evidence of publication bias or heterogeneity (P=0.13). A significant effect was seen for age, with each additional year of age reducing the effect on SBP by 0.09 mmHg. The authors concluded that the peptides IPP and VPP are effective in moderately reducing SBP in European subjects, as is known for Asian populations.

4.5 Key Individual Randomized Controlled Trials

One Dutch randomized, placebo-controlled, double-blind crossover trial enrolled subjects with prehypertension and stage 1 hypertension. The study included 70 Caucasian subjects with prehypertension or stage 1 hypertension. Study treatments consisted of daily consumption of two capsules of MPH1 (each containing 7.5 mg Isoleucine-Proline-Proline; IPP), MPH2 (each containing 6.6 mg Methionine-Alanine-Proline, 2.3 mg Leucine-Proline-Proline, and 1.8 mg IPP), or placebo (containing cellulose) for 4 weeks. In subjects with prehypertension, daily intake of both MPH1 and MPH2 did not affect blood pressure. However, among subjects with stage 1 hypertension a reduction was observed with the IPP-rich hydrolysate.

A Finnish spread study tested the combination of IPP/VPP with plant sterols. Inclusion criteria were age 30–55 years, systolic blood pressure ≥140 mmHg, and diastolic pressure 85–99 mmHg, with elevated LDL cholesterol; subjects with antihypertensive or lipid-lowering medication, coronary artery disease, diabetes, malignant disease, alcohol abuse, or milk allergy were excluded. A 5 mg daily dose of IPP/VPP peptides decreased systolic blood pressure on average 2–10 mmHg and diastolic blood pressure by 2–7 mmHg. Little further benefit was obtained with a 50 mg daily dose of peptides.

A Japanese crossover trial studied the effect of a standardized lactotripeptide product on endothelial function. Results suggested that VPP and IPP might have beneficial effects on arterial health; in this placebo-controlled, double-blind crossover trial, 24 male subjects received either a placebo or 1.25 g of standardized LTP product (3.42 mg of VPP and 3.87 mg of IPP) per day for 1 week.

4.6 Negative and Null Trials

Several IPP and VPP intervention studies were published that found no significant blood pressure-lowering effect. Thirteen of the RCTs reviewed by EFSA, four of which were adequately powered to detect small between-group differences in systolic BP, did not observe an effect of IPP and VPP on SBP or diastolic BP. The interpretation of the results from seven out of the eight studies that reported an effect of IPP and VPP on office SBP was hampered by major methodological limitations. While the number of clinical trials continues to increase, the results have been inconsistent, especially in more recent studies.

4.7 Normotensive Individuals

Fermented milk has been reported to have a normotensive effect in hypertensive rats and humans, but no effect on blood pressure in normotensive rats and humans. In clinical studies, fermented milk containing biologically active peptides significantly decreased systolic and diastolic blood pressure in hypertensive subjects; in contrast, no significant effect on blood pressure was observed in normotensives.

4.8 Vascular Endothelial Function and Arterial Stiffness

Lactotripeptides have additionally been shown to exert beneficial effects other than lowering systemic BP, including improvement of vascular endothelial function in subjects with mild hypertension. One study reported no change in systemic BP, suggesting that the improvement of vascular endothelial function attributable to VPP and IPP is independent of hemodynamic changes.

Apart from ACE-inhibitory activity, the lactotripeptides VPP and IPP have been shown to exert a beneficial effect on arterial stiffness of hypertensive rats and mildly hypertensive human subjects. In a rat model of L-NAME-induced hypertension, L-NAME treatment gradually increased pulse wave velocity (PWV) over time from 460.0 cm/s after 1 week to 572.9 cm/s after 4 weeks of treatment. In contrast, 4 weeks of treatment with VPP or IPP significantly attenuated this increase in PWV. This result is in agreement with a previous 8-week human study that evaluated the intake of food containing VPP and IPP, which improved PWV in hypertensive subjects.

A randomized, double-blind, placebo-controlled trial found that casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro improves central blood pressure and arterial stiffness in hypertensive subjects.

4.9 Cholesterol and Lipid Effects

Antihypertensive and cholesterol-lowering effects have been reported for a spread containing bioactive peptides IPP and VPP together with plant sterols. However, these results were from studies combining IPP/VPP with plant sterols, making it difficult to attribute the lipid-lowering effect to the lactotripeptides alone. This area requires further study with IPP/VPP as isolated variables.

4.10 Atherosclerosis: Preliminary Animal Data

Milk-derived peptides Val-Pro-Pro and Ile-Pro-Pro have been reported to attenuate atherosclerosis development in apolipoprotein E-deficient mice in a preliminary study. These findings are pre-clinical and cannot yet be extrapolated to humans.

5. Regulatory Status and EFSA Assessment

Following an application from Valio Ltd submitted pursuant to Article 13(5) of Regulation (EC) No 1924/2006, the EFSA Panel on Dietetic Products, Nutrition and Allergies was asked to deliver an opinion on the scientific substantiation of a health claim related to isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP) and maintenance of normal blood pressure.

EFSA determined that the tripeptides IPP and VPP are sufficiently characterized, and that maintenance of normal blood pressure is a beneficial physiological effect. Nevertheless, the EFSA Panel concluded that a cause and effect relationship had not been established between the consumption of IPP and VPP and maintenance of normal blood pressure.

On the basis of all data presented, the Panel reiterated that a cause and effect relationship has not been established between the consumption of IPP and VPP and maintenance of normal blood pressure. Antihypertensive lactotripeptide-containing milk products do not have approved health claims in the EU. EFSA considered the evidence on the antihypertensive effect of lactotripeptides to be insufficient. Although a number of clinical studies showing positive results have been published, there are a few studies showing no effect on blood pressure.

In Japan, by contrast, bioactive peptides and protein hydrolysate products for treatment of hypertension have been marketed under Japan's Food for Specified Health Use (FOSHU) designation for a number of years.

6. Bioavailability and Pharmacokinetics

To exert physiological effects after oral ingestion, it is of crucial importance that lactotripeptides remain active during gastrointestinal digestion and absorption and reach the cardiovascular system. IPP, VPP, and Leucine-Proline-Proline (LPP) are named together as XPP as models for C-terminal proline-containing food-derived peptides in general, because they are relatively resistant against breakdown in the gastrointestinal tract and therefore expected to be similar in their kinetics.

Using liquid chromatography/MS, research found the absolute bioavailability of proline-containing tripeptides including IPP to be approximately 0.1%. In human subjects, following an oral dose of lactotripeptide in enriched yoghurt (250 ml containing approximately 20 mg of both IPP and VPP), the maximal plasma concentration of IPP was determined to be less than 1 pmol/ml, concluded to be far below its effective concentration for ACE inhibition determined in vitro.

Data from a study assessing bioavailability of IPP and VPP suggest that IPP may have better bioavailability than VPP. Lactotripeptides (VPP and IPP) from milk were reported to be able to cross the intestinal barrier and, post-absorption, inhibit the production of angiotensin II in the bloodstream.

The disconnect between the very low measured plasma concentrations and the clinically observed blood pressure effects is a recognized puzzle in the field. Preclinical and in vitro studies suggest that a portion of the orally ingested dose of these peptides can be absorbed in the intact form from the gastrointestinal tract, can inhibit the tissue renin-angiotensin system, and can produce significant reductions in blood pressure. Local inhibition of ACE within the gut wall or in peripheral vascular tissue, without the requirement for high systemic concentrations, has been proposed as an explanation, but has not been definitively established.

7. Biosynthesis and Manufacturing

7.1 Microbial Fermentation

Among lactic acid bacteria, L. helveticus can grow rapidly in milk because of its high proteolytic activity and resistance to acid stress, and can therefore release a large amount of peptides, including bioactive peptides, in fermented milk by means of proteolysis of milk proteins. The contribution of L. helveticus cell-wall proteases to the activation of antihypertensive sequences, namely Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP) tripeptides, from the hydrolysis of casein has been demonstrated.

The biosynthetic mechanism consists of the hydrolysis of β-casein by cell-wall proteinase; then, the generated peptides are transferred into the cell by oligopeptide transporter, where endopeptidases such as PepO2, PepC2, and PepX can act on the C- and N-terminal sequences to produce VPP and IPP.

7.2 Enzymatic Hydrolysis

Milk protein hydrolysates rich in IPP can be produced through hydrolysis of glycomacropeptide and casein, respectively, using a proline-specific endoprotease. The pentapeptides QNIPP and VVVPP, incorporating IPP and VPP respectively, are generated from β-casein; aminopeptidase activity is required to release the final tripeptides, and this can theoretically be provided by lysed lactobacilli generated during the fermentation process.

8. Dosages Used in Clinical Studies

Nineteen randomized clinical intervention trials tested small daily doses of 2.0–10.2 mg of milk casein-derived tripeptides (IPP + VPP combined).

In one crossover trial, daily consumption consisted of two capsules of MPH1, each containing 7.5 mg IPP, for 4 weeks.

In one Japanese endothelial function trial, 24 male subjects received 1.25 g of standardized LTP product (3.42 mg of VPP and 3.87 mg of IPP) per day for 1 week.

A 5 mg daily dose of IPP/VPP peptides was used in Finnish clinical trials, decreasing systolic blood pressure on average 2–10 mmHg and diastolic blood pressure by 2–7 mmHg. Little further benefit was obtained with a 50 mg daily dose of peptides.

The effect of IPP/VPP on SBP remained significant when limiting the analysis to studies testing the usual doses consumed daily (below 5 mg/day).

Maximum blood pressure reductions approximating 13 mmHg of SBP and 8 mmHg of DBP, respectively, after active treatment compared with placebo, are likely reached after 8–12 weeks of treatment.

9. Body Systems and Health Areas of Association

9.1 Cardiovascular System

IPP is principally associated with cardiovascular health, specifically blood pressure regulation. IPP and VPP 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. LTPs have been shown to inhibit vascular contraction through ACE inhibition, and improvements in endothelial function from LTP ingestion via enhanced flow-mediated dilation (FMD) and pulse wave velocity have been observed in clinical studies.

9.2 Renin-Angiotensin-Aldosterone System (RAAS)

Milk-derived small peptides have held promise for reducing angiotensin II-mediated vasoconstriction, because mechanistically they imitate angiotensin-converting enzyme inhibitors (ACEIs). A clinical study showed significant decrease of the angiotensin II/angiotensin I ratio in circulating blood by consumption of fermented milk containing VPP and IPP.

9.3 Endothelial and Vascular Function

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 events.

10. Safety Considerations and Interactions

10.1 Toxicological Profile

Toxicity studies in animals and safety trials in humans substantiate the safety of lactotripeptides. The safety-in-use of commercial lactotripeptide-containing products has been confirmed in several in vitro and in vivo toxicity studies and in studies with humans. In genotoxicity testing including bacterial reverse mutation tests, mammalian cell gene mutation tests, and mammalian chromosomal aberration tests, IPP-containing milk protein hydrolysate (Tensguard™) was found not to be mutagenic or clastogenic.

In sub-chronic toxicity testing in rats exposed to the casein hydrolysate product, the 'no observable adverse effect level' (NOAEL) resulted in >1000 mg casein hydrolysate/kg body weight per day (corresponding to 3 mg IPP + 3 mg VPP/kg body weight per day). Many published human studies and a battery of toxicity studies with several lactotripeptide-containing products have been performed and results showed no safety concerns. There has been no evidence of target organ toxicity associated with administration of Val-Pro-Pro and Ile-Pro-Pro.

10.2 No Effect in Normotensives

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 selective activity in hypertensive individuals, without significant pressure reduction in normotensive individuals, is considered a favorable safety characteristic distinguishing IPP from pharmaceutical ACE inhibitors.

10.3 Interaction with ACE Inhibitor Drugs

A study investigated interactions between enalapril (a pharmaceutical ACE inhibitor) and a fermented milk product (FMP) containing the ACE-inhibitory peptides VPP and IPP. Single-dose and long-term (6-week) in vivo studies were used. Single-dose oral administration of concomitant enalapril and FMP (VPP, IPP: 3.5 mg/kg) produced a lower antihypertensive effect than enalapril monotherapy. However, this effect was not observed in animals administered a lower dose of FMP (VPP, IPP: 1.75 mg/kg) along with enalapril. At present, a fermented milk product (FMP) containing these peptides is commercially available in Japan (trade name, Ameel S®) and this FOSHU product is readily available to people, irrespective of whether they are taking other antihypertensive medications. There are few published studies on the effects of combined intake of FMP and a prescribed ACE inhibitor. These animal data raise a question of potential pharmacodynamic interaction at higher doses; the clinical significance in humans at typical dietary doses has not been established.

10.4 Milk Allergy Considerations

In IPP/VPP clinical trials, subjects with milk allergy were excluded from participation. Since IPP is derived from bovine milk casein, individuals with cow's milk protein allergy require specific consideration, though the extensive hydrolysis involved in manufacturing IPP-enriched products substantially alters the allergenic protein structures relative to intact casein.

10.5 Evidence Gaps

Future research is expected to look into the effects of bioaccessibility, bioavailability, stability, and reactivity of the peptides with food and gut matrices, as well as the gut microbiota, on blood pressure reduction. The striking ethnic difference in clinical response between Asian and Caucasian subjects — statistically significant in multiple meta-analyses — remains an unresolved question, and differences in diet, gut microbiota, renin-angiotensin system activity, or genetic polymorphisms in ACE have been proposed as explanations but not confirmed.

References

• PubChem CID 9949212: Isoleucyl-prolyl-proline — National Library of Medicine
• ChemSpider CSID 8124823: L-Isoleucyl-L-prolyl-L-proline — Royal Society of Chemistry
• Bernard BK et al. (2005). Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): II. Introduction. PubMed PMID 16419575
• EFSA Panel on Dietetic Products, Nutrition and Allergies (2011). Scientific Opinion on the substantiation of a health claim related to IPP and VPP and maintenance of normal blood pressure pursuant to Article 13(5) of Regulation (EC) No 1924/2006. EFSA Journal 9(6):2380
• EFSA Panel on Dietetic Products, Nutrition and Allergies (2012). Scientific Opinion on the substantiation of health claims related to IPP and VPP and maintenance of normal blood pressure (further assessment). EFSA Journal 10(6):2715
• Turpeinen AM et al. (2013). Antihypertensive effects of bioactive tripeptides — a random effects meta-analysis. Annals of Medicine 45(1):51–56
• Qin LQ et al. (2013). Lactotripeptides intake and blood pressure management: A meta-analysis of randomised controlled clinical trials. Nutrition, Metabolism and Cardiovascular Diseases
• Cicero AFG et al. (2011). Blood pressure lowering effect of lactotripeptides assumed as functional foods: a meta-analysis of current available clinical trials. Journal of Human Hypertension 25:425–436
• Fekete AA et al. (2015). Influence of the Lactotripeptides IPP and VPP on Systolic Blood Pressure in Japanese Subjects: A Systematic Review and Meta-Analysis. PLOS ONE. PMC4633157
• Engberink MF et al. (2013). Do the Lactotripeptides IPP and VPP Reduce Systolic Blood Pressure in European Subjects? A Meta-Analysis of Randomized Controlled Trials. American Journal of Hypertension 26(3):442–452
• Boelsma E & Kloek J (2010). IPP-rich milk protein hydrolysate lowers blood pressure in subjects with stage 1 hypertension, a randomized controlled trial. Nutrition Journal 9:52. PMC2989300
• Hirota T et al. (2011). Milk casein-derived tripeptides, VPP and IPP induced NO production in cultured endothelial cells and endothelium-dependent relaxation of isolated aortic rings. PubMed PMID 21221598
• Yamaguchi N et al. (2009). Study of the mechanism of antihypertensive peptides VPP and IPP in spontaneously hypertensive rats by DNA microarray analysis. European Journal of Pharmacology. PubMed PMID 19686729
• Gregory M et al. (2024). Structural insights into the inhibitory mechanism of angiotensin-I-converting enzyme by the lactotripeptides IPP and VPP. FEBS Letters
• Nonaka A et al. (2014). The milk-derived peptides Val-Pro-Pro and Ile-Pro-Pro attenuate arterial dysfunction in L-NAME-treated rats. Hypertension Research
• Boelsma E & Kloek J (2009). Lactotripeptides and antihypertensive effects: a critical review. British Journal of Nutrition 101(6):776–786
• Van der Zander K et al. (2008). Safety evaluation of an IPP tripeptide-containing milk protein hydrolysate. Food and Chemical Toxicology
• Sasaki K et al. (2015). The influence of dietary peptide inhibitors of angiotensin-converting enzyme on the hypotensive effects of enalapril. Journal of Pharmaceutical Health Care and Sciences. PMC4729115
• Turpeinen AM et al. (2009). Antihypertensive and cholesterol-lowering effects of a spread containing bioactive peptides IPP and VPP and plant sterols. Journal of Functional Foods 1(3):260–265
• Broadbent JR et al. (2012). Genome sequence and analysis of Lactobacillus helveticus. Frontiers in Microbiology. PMC3542869
• Fan M et al. (2023). Bioactive peptides derived from fermented foods: Preparation and biological activities. Journal of Functional Foods
• van der Pijl PC et al. (2015). Enhanced Lacto-Tri-Peptide Bio-Availability by Co-Ingestion of Macronutrients. PLOS ONE
• Miner-Williams WM et al. (2014). Are intact peptides absorbed from the healthy gut in the adult human? Nutrition Research Reviews
• McGregor RA & Poppitt SD (2021). Dairy bioactive proteins and peptides: a narrative review. Nutrition Reviews
• Sipola M et al. (2010). Antihypertensive Peptides from Milk Proteins. Pharmaceuticals 3(1):251–272