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.
Shah N 2007 Functional cultures and health benefitsInt. dairy j. 17 1262 1277
Takano T 2002 Anti-hypertensive activity of fermented dairy products containing biogenic peptides.Anton. leeuw. 82 333 340
Korhonen HJTPihlanto-Leppälä A ( 2004Milk-derived bioactive peptides : formation and prospects for health promotion. In: Edited by Colette Shortt and John O’Brien. Handbook of functional dairy productsFunctional foods and nutraceuticals series 6.0: 109 124
FitzGerald RJMurray BA ( 2006 Bioactive peptides and lactic fermentationsInt. j. dairy technology 59 118 125
Jäkälä P Vapaatalo H 2010 Antihypertensive peptides from milk proteins
Christensen J. E Dudley E. G Pederson J. A Steele J. L 1999 Peptidases and amino acid catabolism in lactic acid bacteria.Anton. leeuw. 76 217 246
Luoma S Peltoniemi K Joutsjoki V Rantanen T Tamminen M Heikkinen I Palva A 2001 Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactisAppl. environ. microb. 67 1232 1238
Foucaud C Juillard V 2000 Accumulation of casein-derived peptides during growth of proteinase-positive strains of Lactococcus lactis in milk: their contribution to subsequent bacterial growth is impaired by their internal transport.J dairy res. 67 233 240
Williams A. G Noble J Tammam J Lloyd D Banks J. M 2002 Factors affecting the activity of enzymes involved in peptide and amino acid catabolism in non starter lactic acid bacteria isolated from Cheddar cheeseInt. dairy j. 12 841 852
Lopez A. D Murray C. C 1998The global burden of disease, 1990-2020. Nat.med. 4 1241 1243
Harris T Cook E. F Kannel W Schatzkin A Goldman L 1985 Blood pressure experience and risk of cardiovascular disease in the elderly.
Pihlanto A Korhonen H 2003 Bioactive peptides and proteins.Adv. food res. 47 175 276
del Mar Contreras M, Recio I ( Hernández-ledesma B 2011 Antihypertensive peptides: production, bioavailability and incorporation into foodsAdv. colloid interface sci. 165 23 35
Van Gaal L. F Mertens I. L De Block C. E 2006 Mechanisms linking obesity with cardiovascular disease.
Pihlanto A 2006 Antioxidative peptides derived from milk proteinsInt. dairy j. 16 1306 1314
Roudot-algaron F Bars D. L Kerhoas L Einhorn J Gripon J. C 1994Phosptiopeptides from Comté Cheese: Nature and origin. J. food sci. 59 544 547
Singh T. K Fox P. F Healy A 1997 Isolation and identification of further peptides from diafiltration retentate of the water-soluble fraction of Cheddar cheese. Jdairy res. 64 433 443
Meisel H Goepfert A Günter S 1997 ACE-inhibitory activities in milk products
- 19. Addeo F, Chianes L, Salzano A, Sacchi R, Cappuccio U, Ferranti P, Malorni A (1992) Characterization of the 12% tricholoroacetic acid-insoluble oligopeptides of Parmigiano–Reggiano cheese. J. dairy res. 59: 401–411.
Smacchi E Gobbetti M 1998Peptides from several Italian cheeses inhibitory to proteolytic enzymes of lactic acid bacteria, Pseudomonas fluorescens ATCC 948 and to the angiotensin I-converting enzyme. Enzyme microb.tech. 22 687 694
Haileselassie S. S Lee B H Bibbs B. F 1999Purification and identification of potentially bioactive peptides from enzyme modified cheese. J. dairy sci. 82 1612 1617
Ryhänen E-L Pihlanto-leppälä A Pahkala E 2001A new type of ripened low-fat cheese with bioactive properties. Int. dairy j. 11 441 447
Gomez J. A Ramos M Recio I 2002Angiotensin-converting enzyme-inhibitory peptides in Manchego cheeses manufactured with different starter cultures. Int. dairy j. 12 697 706
Torres-llanez M. J González-córdova A. F Hernandez-mendoza A Garcia H. S Vallejo-cordoba B 2011Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese. J. dairy sci. 94 3794 3800
Pripp A. H Sorensen R and Stepaniak L Sorhaug T 2006Relationship between proteolysis and angiotensin I-converting enzyme inhibition in different cheeses LWT 39 677 683
Muehlenkamp M. R Warthesen J. J 1996casomorphins: Analysis in cheese and susceptibility to proteolytic enzymes from Lactococcus lactis ssp. cremoris. J. dairy sci. 79 20 26
Sienkiewicz- Szlapka E Jarmolowska B Krawczuk S Kostyra E Kostyra H Iwana M 2009Contents of agonistic and antagonistic opioid peptides in different cheese varieties. Int. dairy j. 19 258 263
Durrieu C Degraeve P Chappaz S Martial-gros A 2006Immunomodulating effects of water-soluble extracts of traditional French Alps cheeses on a human T-lymphocyte cell line. Int. dairy j. 16 1505 1514
Saito T Nakamura T Kitazawa H Kawai Y Itoh T 2000Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese J. dairy sci. 83 1434 1440
Ong L Shah N. P 2008Release and identification of angiotensin-converting enzyme-inhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses. LWT- Food sci. technol. 41 1555 1566
Bütikofer U Meyer J Sieber R Wechsler D 2007Quantification of the angiotensin-converting enzyme-inhibiting tripeptides Val-Pro-Pro and Ile-Pro-Pro in hard, semi-hard and soft cheeses. Int. dairy j. 17 968 975
Bütikofer U Meyer J Sieber R Walther B Wechsler D 2008Occurrence of the angiotensin-converting enzyme-inhibiting tripeptides Val-Pro-Pro and Ile-Pro-Pro in different cheese varieties of Swiss origin. J. dairy sci. 91 29 38
Meyer J Bütikofer U Walther B Wechsler D Sieber R 2009Hot topic: Changes in angiotensin-converting enzyme inhibition and concentrations of the tripeptides Val-Pro-Pro and Ile-Pro-Pro during ripening of different Swiss cheese varieties. J. dairy sci. 92 826 836
Nakamura Y Yamamoto N Sakai K Okubo A Yamazaki S Takano T 1995Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. J. dairy sci. 78 777 783
Pihlanto-leppälä A Rokka T Korhonen H 1998Angiotensin I converting enzyme inhibitory peptides derived from bovine milk proteins. Int. dairy j. 8 325 331
Gobbetti M Ferranti P Smacchi E Goffredi F Addeo F 2000Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4. Appl. environ. microb. 66 3898 3904
Fuglsang A Rattray F. P Nilsson D Nyborg C. B 2003Lactic acid bacteria: inhibition of angiotensin-converting enzyme in vitro and in vivo. Anton. leeuw. 83 27 34
Pihlanto A Virtanen T Korhonen H 2010Angiotensin I converting enzyme (ACE) inhibitory activity and antihypertensive effect of fermented milk. Int. dairy j. 20 3 10
Nielsen M. S Martinussen T Flambard B Sorensen K. I Otte J 2009Peptide profiles and angiotensin-I-converting enzyme inhibitory activity of fermented milk products: Effect of bacterial strain, fermentation pH, and storage time Int. dairy j. 19 155 165
Muguerza B Ramos M Sánchez E Manso M. A Miguel M Aleixander A Lopez-fandino R 2006Antihypertensive activity of milk fermented by Enterococcus faecalis strains isolated from raw milk. Int. dairy j. 16 61 69
Rodríguez-figueroa J. C Reyes-díaz R González-córdova A. F Troncoso-rojas R Vargas-arispuro I Vallejo-cordoba B 2010Angiotensin-converting enzyme inhibitory activity of milk fermented by wild and industrial Lactococcus lactis strains. J. dairy sci. 93 5032 5038
Yamamoto N Maeno M Takano T 1999Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by Lactobacillus helveticus CPN4. J. dairy sci. 82 1388 1393
Ashar M. N Chand R 2004Antihypertensive peptides purified form milks fermented with Lactobacillus belbrueckii ssp. bulgaricus. Milchwissenschaft 59 14 17
Quiros A Ramos M Muguerza B Delgado M. A Miguel M Aleixandre A Recio I 2007Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis. Int. dairy j. 17 33 41
Kunji E. R Mierau I Hagting A Poolman B Koning W. N 1996The proteolytic systems of lactic acid bacteria. Anton. leeuw. 70 187 221
Yamamoto N Ishida Y Kawakami N Yada H 1991Lactobacillus helveticus bacterium having high capability of producing tripeptide, fermented milk product, and process for preparing the same. EU Patent, 1016709A1.
Yamamoto N Akino A Takano T 1993Purification and specificity of a cell-wall associated proteinase from Lactobacillus helveticus CP790. J. biochem, 114 740 745
Ueno K Mizuno S Yamamoto N 2004Purification and characterization of an endopeptidase has an important role in the carboxyl terminal processing of antihypertensive peptides in Lactobacillus helveticus CM4. Lett. appl. microbiol. 39 313 318
Kilpi E Kahala M Steele J. M Pihlanto A Joutsjoki V 2007Angiotensin I-converting enzyme inhibitory activity in milk fermented by wild-type and peptidase-deletion derivatives of Lactobacillus helveticus CNRZ32. Int. dairy j. 17 976 984
Kwon D. Y Daily J. W Kim H. J Park S 2010Antidiabetic effects of fermented soybean products on type 2 diabetes. Nutr. res. 30 1 13
Shin Z. I Yu R Park S. A Chung D. K Ahn C. W Nam H. S Kim K. S Lee H. J 2001His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo. J. agric. food chem. 49 3004 3009
Hu Y Stromeck A Loponen J Lopes-lutz D Schieber A Gänzle M. G 2011LC-MS/MS quantification of bioactive angiotensin I-converting enzyme inhibitory peptides in rye malt sourdoughs. J. agric. food chem. 59 11983 11989
Pihlanto A Johansson T Mäkinen S 2012Inhibition of angiotensin I-converting enzyme and lipid peroxidation by fermented rapeseed and flaxseed meal. Eng. life sci. 12 DOI:elsc.201100137
Lin M. Y Yen C. L 1999Antioxidative ability of lactic acid bacteria J. agric. food chem. 47 1460 1466
Ou C. C Lu T. M Tsai J. J Yen J. H Chen H. W Lin M. Y 2009Antioxidative effect of lactic acid bacteria: Intact cells vs. intracellular extracts. J. food drug anal. 17 209 216
Kudoh Y Matsuda S Igoshi K Oki T 2001Antioxidative peptide from milk fermented with Lactobacillus delbrueckii subsp. bulgaricus IFO13953. Nippon Shokuhin Kagaku Kaishi 48 44 55
Hernández-ledesma B Miralles B Amigo L Ramos M Recio I 2005Identification of antioxidant and ACE-inhibitory peptides in fermented milk. J. sci. food agric. 85 1041 1048
Virtanen T Pihlanto A Akkanen S Korhonen H 2007Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. J. appl. microbial. 102 106 115
Libby P 2006Inflammation and cardiovascular disease mechanisms. Am. j. clin. nutr. 83: 456S- 460S.
LeBlanc AMMatar C, Valdéz JC, LeBlanc N, Perdigón G ( 2002Immunomodulatory effects of peptidic fractions issued from milk fermented with Lactobacillus helveticus. J. dairy sci. 85 2733 2742
Matar C Nadathur S. S Bakalinsky A. T Goulet J 1997Antimutagenic effects of milk fermented by Lactobacillus helveticus L89 and a protease-deficient derivative. J. dairy sci. 80 1965 1970
Laffineur E Genetet N Leonil J 1996Immunomodulatory activity of β-casein permeate medium fermented by lactic acid bacteria. J. dairy sci. 79 2112 2120
Tompa G Laine A Pihlanto A Korhonen H Rogel I Marnila P 2011Chemiluminescence of non-differentiated THP-1 promonocytes: developing an assay for screening anti-inflammatory milk proteins and peptides. Luminescence 26: 251-258,
Matar C Valdez J. C Medina M Rachid M Perdigon G 2001Immunomodulating effects of milks fermented by Lactobacillus helveticus and its non-proteolytic variant. J. dairy res. 68 601 609
Yamamoto N Akino A Takano T 1994Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from Lactobacillus helveticus CP790. J. dairy sci. 77 917 922
Nakamura Y Yamamoto N Sakai K Takano T 1995Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. J. dairy sci. 78 1253 1257
Sipola M Finckenberg P Korpela R Vapaatalo H Nurminen M. L 2002Effect of long-term intake of milk products on blood pressure in hypertensive rats. J. dairy res. 69 103 111
Jäkälä P Hakala A Turpeinen A Korpela R Vapaatalo H 2009Casein-derived bioactive tripeptides Ile-Pro-Pro and Val-Pro-Pro attenuate the development of hypertension and improve endothelial function in salt-loaded Goto-Kakizaki rats. J. funct. foods 1 366 374
Jauhiainen T Pilvi T Cheng Z. J Kautiainen H Müller D. N Vapaatalo H Korpela R Mervaala E 2010Milk products containing bioactive tripeptides have an antihypertensive effect in double transgenic rats (dTGR) harbouring human renin and human angiotensinogen genes. J. nutr. metab. doi:10.1155/2010/287030.
Jauhiainen T Collin M Narva M Cheng Z. J Poussa T Vapaatalo H Korpela R 2005Effect of long-term intake of milk peptides and minerals on blood pressure and arterial function in spontaneously hypertensive rats. Milchwissenschaft 60 358 362
Jäkälä P Jauhiainen T Korpela R Vapaatalo H 2009Milk protein-derived bioactive tripeptides Ile-Pro-Pro and Val-Pro-Pro protect endothelial function in vitro in hypertensive rats. J. funct. foods 1 266 273
Masuda O Nakamura Y Takano T 1996Antihypertensive peptides are present in aorta after oral administration of sour milk containing these peptides to spontaneously hypertensive rats. J. nutr. 126 3063 3068
Jäkälä P Pere E Lehtinen R Turpeinen A Korpela R Vapaatalo H 2009Cardiovascular activity of milk casein-derived tripeptides and plant sterols in spontaneously hypertensive rats. J. physiol. pharmacol. 60 11 20
Miguel M Recio I Ramos M Delgado M. A Aleixandre A 2006Antihypertensive effect of peptides obtained from Enterococcus faecalis-fermented milk in rats J. dairy sci. 89 3352 3359
Quiros A Ramos M Muguerza B Delgado M. A Migue M Aleixandre A Recio I 2007Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis Int. dairy j. 17 33 41
Chen G. W Tsai J. S Pan B. S 2007Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation. Int. dairy j. 17 641 647
Shin Z. I Yu R Park S. A Chung D. K Ahn C. W Nam H. S Kim K. S Lee H. J 2001His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo. J. agric. food chem. 49 3004 3009
Sacks F. S Lichtenstein A Van Horn L Harris W Kris-etherton P Winston M 2006Soy protein, isoflavones, and cardiovascular health. Circulation 113 1034 1044
Wu J Muir A. D 2008Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats J. agric. food chem. 56 9899 9904
Tsai J. S Lin Y. S Pan B. S Chen T. J 2006Antihypertensive peptides and γaminobutyric acid from prozyme 6 facilitated lactic acid bacteria fermentation of soymilk. Process biochem. 41 1282 1288
Inoue K Gotou T Kitajima H Mizuno S Nakazawa T Yamamoto N 2009Release of antihypertensive peptides in miso paste during its fermentation, by the addition of casein. J. biosci. bioeng. 108 111 115
Nakahara T Sano A Yamaguchi H Sugimoto K Chikata H Kinoshita E Uchida R 2010Antihypertensive effect of peptide-enriched soy sauce-like seasoning and identification of its angiotensin I-converting enzyme inhibitory substances, J. agric. food chem. 58 821 827
study of the effect of sour milk on blood pressure in hypertensive subjects. Am. j. clin. nutr. Hata Y Yamamoto M Ohni M Nakajima K Nakamura Y Takano T 1996 A Placebo-controlled 64 767 771
Kajimoto O Kurosaki T Mizutani J Ikeda N Kaneko K Yabune M Nakamura Y 2002Antihypertensive effects of liquid yogurts containing ‘lactotripeptides (VPP, IPP)’ in mild hypertensive subjects. J. nutr. food 5 55 66
Hirata H Nakamura Y Yada H Moriguchi S Kajimoto O Takahashi T 2002Clinical effects of new sour milk drink on mild or moderate hypertensive subjects. J. new. rem. clin 51 61 69
Seppo L Jauhiainen T Poussa T Korpela R 2003A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. Am. j. clin. nutr. 77 326 330
Tuomilehto J Lindstrom J Hyyrynen J Korpela R Karhunen M. L Mikkola L Jauhiainen T Seppo L Nissinen A 2004Effect of ingesting sour milk fermented using Lactobacillus helveticus bacteria producing tripeptides on blood pressure in subjects with mild hypertension. J. hum. hypertens. 18 795 802
Jauhiainen T Vapaatalo H Poussa T Kyrönpalo S Rasmussen M Korpela R 2005Lactobacillus helveticus fermented milk lowers blood pressure in hypertensive subjects in 24-h ambulatory blood pressure measurement. Am. j. hypertens. 18 1600 1605
Arihara K Kajimoto O Hirata H Takahashi R Nakamura Y 2005Effect of powdered fermented milk with Lactobacillus helveticus on subjects with high-normal blood pressure or mild hypertension. J. am. coll. nutr. 24 257 265
Kajimoto O Aihara K Hirata H Takahashi R Nakamura Y 2001Hypotensive effects of the tablets containing lactotripeptides (VPP, IPP). J. nutr. food 4 51 61
Itakura H Ikemoto S Terada S Kondo K 2001The effect of sour milk on blood pressure in untreated hypertensive and normotensive subjects. J. jap. soc. clin. nutr. 23 26 31
Kajimoto O Nakamura Y Yada H Moriguchi S Hirata H Takahashi T 2001Hypotensive effects of sour milk in subjects with mild or moderate hypertension. J. jpn. soc. nutr. food sci. 54 347 354
Yasuda K Aihara K Komazaki K Mochii M Nakamura Y 2001Effect of large high intake of tablets containing ‘lactotripeptides (VPP, IPP)’ on blood pressure, pulse rate and clinical parameters in healthy volunteers. J. nutr. food 4 63 72
Law M. R Wald N. J Morris J. K Jordan J. E 2003Value of low dose combination treatment with blood pressure lowering drugs: analysis of 354 randomised trials. Br. med. j. 326 1427 1431
Pripp A. H 2008Effect of peptides derived from food proteins on blood pressure: a meta-analysis of randomized controlled trials. Food nutr. res. 5 1 9
Xu J. Y Qin L. Q Wang P. Y Li W Chang C 2008Effect of milk tripeptides on blood pressure: a meta-analysis of randomized controlled trials. Nutrition 24 933 940
Engberink M. F Schouten E. G Kok F. J Van Mierlo L. A Brouwer I. A Geleijnse J. M 2009Lactotripeptides show no effect on human blood pressure: results from a double-blind randomized controlled trial. Hypertension 51 399 405
Usinger L Jensen L. T Flambard B Linneberg A Ibsen H 2010The antihypertensive effect of fermented milk in individuals with prehypertension or borderline hypertension. J.hum. hypertens. 24 678 683
Cicero AFGGerocarni B, Laghi L, Borghi C ( 2011Blood pressure lowering effect of lactotripeptides assumed as functional foods: a meta-analysis of current available clinical trials. J. hum. hypertens. 25 425 436
Vaidyanathan S Jermany J Yeh C Bizot M. N Camisasca R 2006Aliskiren, a novel orally effective renin inhibitor, exhibits similarpharmacokinetics and pharmacodynamics in Japanese and Caucasian subjects. Br. j. clin. pharmacol. 62 690 698
Lanthier L Cabana J, Guillemette G, Lavigne P, Leduc R, Escher E ( Arsenault J Lehoux J 2010A single-nucleotide polymorphism of alanine to threonine at position 163 of the human angiotensin II type 1 receptor impairs losartan affinity. Pharmacogenet Genomics 20 377 388
Boelsma E Kloek J 2010IPP-rich milk protein hydrolysate lowers blood pressure in subjects with stage 1 hypertension, a randomized controlled trial. J. nutr. 9:52 doi:10.1186/1475-2891-9-52.
Hernández-ledesma B Amigo L Ramos M Recio I 2004Application of high-performance liquid chromatography-tandem mass spectrometry to the identification of biologically active peptides produced by milk fermentation and simulated gastrointestinal digestion. J. chromatogr. A 1049 107 114
Mäkinen S Johansson T Vegarud G Pihlava J. M Pihlanto A 2012Angiotensin I-converting enzyme inhibitory and antioxidant properties of rapeseed hydrolysates. J. funct. foods (in press)
Matsui T Li C. H Osajima Y 1999Preparation and characterization of novel bioactive peptides responsible for angiotensin I-converting enzyme inhibition from wheat germ. J. pept. sci. 5 289 297
Vermeirssen V Van Camp J Verstraete W 2004Bioavailability of angiotensin I converting enzyme inhibitory peptides. Br. j. nutr. 92 357 366
Maeno M Yamamoto N Takano T 1996Identification of an antihypertensive peptide from casein hydrolysate produced by a proteinase from Lactobacillus helveticus CP790. J. dairy sci. 79 1316 1321
Eriksen E. K Holm H Jensen E Aaboe R Devold T. G Jacobsen M Vegarud G. E 2010Different digestion of caprine whey proteins by human and porcine gastrointestinal enzymes. Br. j. nutr. 104 374 381
Pauletti G. M Gangwar S Knipp G. T Nerurkar M. M Okumu F. W Tamura T Siahaan T. J 1996Structural requirements for intestinal absorption of peptide drugs. J. control release. 41 3 17
Daniel H 2004Molecular and integrative physiology of intestinal peptide transport. Annu. rev. physiol. 66 361 384
Duchateau GSMJE, Augustijns Foltz M Cerstiaens A Van Meensel A Mols R Van Der Pijl P. C 2008The angiotensin converting enzyme inhibitory tripeptides Ile-Pro-Pro and Val-Pro-Pro show increasing permeabilities with increasing physiological relevance of absorption models. Peptides 29: 1312-1320.
Satake M Enjoh M Nakamura Y Takano T Kawamura Y Arai S Shimizu M 2002Transepithelial transport of the bioactive tripeptide Val-Pro-Pro, in human intestinal Caco-2 cell monolayers. Biosci. biotech. bioch. 66 378 384
Quiros A Davalos A Lasuncion M. A Ramos M Recio I 2008Bioavailability of the antihypertensive peptide LHLPLP: Transepithelial flux of HLPLP Int. dairy j. 18 279 286
Deacon C. F Nauck M. A Toft-nielsen M Pridal L Willms B Holst J. J 1995Both subcutaneously and intravenously administered glucagon-like peptide I are rapidly degraded from the NH2-terminus in type II diabetic patients and in healthy subjects. Diabetes. 44 1126 1131
Jauhiainen T Wuolle K Vapaatalo H Kerojoki O Nurmela K Lowrie C Korpela R 2007Oral absorption, tissue distribution and excretion of a radiolabelled analog of a milk-derived antihypertensive peptide, Ile-Pro-Pro, in rats. Int. dairy j. 17 1216 1223
Ten Have GA, Duchateau GS, Deutz NE ( Van Der Pijl P. C Kies A. K 2008Pharmacokinetics of proline-rich tripeptides in the pig. Peptides. 29 2196 2202
Koning TMMG, Kloek J ( Foltz M Meynen E. E Bianco V Van Platerink C 2007Angiotensin converting enzyme inhibitory peptides from a lactotripeptide-enriched milk beverage are absorbed intact into the circulation. J. nutr. 137 953 958
Shaji J Patole V 2008Protein and peptide drug delivery: Oral approaches. J. pharm. sci. 70 269 277
Kies A. K Van Der Pijl P 2012Peptide availability USA Patent Application 20120040895.
Ko W. C Cheng M. L Hsu K. C Hwang Y. S 2006Absorption-enhancing treatments for antihypertensive activity of oligopeptides from tuna cooking juice: In vivo evaluation in spontaneously hypertensive rats. J. food sci. 71 13 17
Fujita H Sasaki R Kurahashi K Yoshikawa M 1995Potentiation of the antihypertensive activity of orally administered ovokinin, a vasorelaxing peptide derived from ovalbumin, by emulsification in egg phosphatidyl-choline. Biosci. biotech. bioch. 59 2344 2345