Title of Invention

"A METHOD FOR PRODUCING A FOOD HAVING ANTI-STRESS EFEECT"

Abstract

This invention is to provide an anti-stress agent and functional food which can be taken repeatedly and daily without any problems with safety, and which can mitigate and prevent mental and physical symptoms caused by stress. An anti-stress agent containing as an effective ingredient a tripeptide (particularly lie-Pro-Pro and/or Val-Pro-Pro) and/or a salt thereof having angiotensin converting enzyme inhibitory activity, and functional food containing the anti-stress agent.

Full Text

The present indention relates to a method for producing a food selected from the group consisting of a dairy product, a grain product and a soybean product, the food having anti-stress effect and containing a tripeptide and/or salt thereof having angiotensin converting enzyme inhibitory activity. In the modern society, people undergoes various kinds of stress caused by highly advanced and complicated scientific technology, or drastically changing social circumstances. Particularly, in the internationalized society, complex human relationships are formed, causing mental stress. It has been reported that a variety of symptoms are caused by mental stress. It is recognized that mental stress has a great influence on circulatory system and immune system. However, the scientific concept and definition of stress have not yet been well established, so that means of evaluation of stress still have many problems, combined with methodological difficulties. However, in the recent years, studies of stress have been made from the medical point of view. For example, it is reported that when one undergoes stress, angiotensin II increases, and intracorporeal sodium due to sodium reabsorbancy becomes excess, which causes rise in blood pressure (Osamu Mobara et al. : Taisha, 28 , 2, 323, 1991) . Based on such findings, studies have been made on the effect of enalapril and alacepril, which are angiotensin converting enzyme inhibitors and used as antihypertensive agents, on hypertension caused by stress (The American Journal of Cardiology; 68, 15, 1362(1991), Internal Medicine; 32, 9, 691(1993)). However, it is considered that suffering stress not only causes rise in blood pressure, but also influences various factors to cause stomach ulcer, ischemic heart diseases, cerebrovascular diseases, hyperlipemia, or the like. Therefore, though stress is regarded as one of the causes of hypertension, it is not believed that the anti-stress effect is achieved merely by suppressing the rise in blood pressure. As an agent for preventing and mitigating mental and physical symptoms caused by stress, chemically synthesized medicaments such as a tranquilizer, an antianxiety agent, and sleeping pills are presently used. However, these medicaments have habituation and side effect problems, so that it is not preferable to use them daily for the purpose of preventing mental and physical symptoms caused by stress. Accordingly, an anti-stress agent that can be taken repeatedly and daily without any problems with safety, and that can mitigate and prevent mental and physical symptoms caused by stress are desired and are under development. For example, there are proposals such as an anti-stress agent containing as an effective ingredient L-theanine contained in tea leaves (Japanese Laid-open Patent Application No. 6-100442), an anti-stress composition containing imidazole compounds such as anserine, valenine, n-methylhistidine, or r-methylhistidine (Japanese Laid-open Patent Application No. 9-20660) , and anti-stress food containing a composition of glutathione and antioxidant (Japanese Laid-open Patent Application No.8-275752). There is also a report on stress reducing effect of fragrance (Fragrance Journal: 1991-11, p44-49). However, there has not been reported that a tripeptide has the effect of mitigating and preventing mental and physical symptoms caused by stress. Disclosure of the Invention It is an object of the present invention to provide an anti-stress agent and functional food which can fulfill the social demand as mentioned above, which can be taken repeatedly and daily without any problems with safety, and which can mitigate and prevent mental and physical symptoms caused by stress. According to the present invention, there is provided an anti-stress agent comprising as an effective ingredient a tripeptide and/or a salt thereof having angiotensin converting enzyme inhibitory activity. According to the present invention, there is also provided the anti-stress agent wherein said tripeptide is lie-Pro-Pro and/or Val-Pro-Pro. According to the present invention, there is further provided use of the tripeptide and/or the salt thereof for the manufacture of an anti-stress agent. According to the present invention, there is also provided food having an anti-stress effect and comprising said anti-stress agent. According to the present invention, there is further provided use of the tripeptide and/or the salt thereof for the manufacture of food having an anti-stress effect. According to the present invention, there is further provided a method for producing the food having anti-stress effect comprising fermenting a medium containing a peptide and/or a protein including a sequence lie-Pro-Pro and/or Val-Pro-Pro with lactic acid bacteria under conditions for a tripeptide lie-Pro-Pro and/or Val-Pro-Pro to be produced in a resulting fermented medium. According to the present invention, there is also provided a method for reducing stress comprising orally administering an effective amount of a tripeptide and/or a salt thereof having angiotensin-converting enzyme inhibitory activity. Accordingly, the present invention relates to a method for the preparation of a food, particularly fermented milk product having anti-stress effect and containing lle-Pro-Pro (IPP) tripeptide and/or Val-Pro-Pro (VPP) tripeptide wherein the process comprises the step of fermentation of a medium containing a peptide and/or a protein including amino acid sequence lle-Pro-Pro and/or Val-Pro-Pro with lactic acid bacteria of the kind such as herein described wherein the step of fermentation is carried out by heat-sterilization of the medium followed by cooling and incubation with the lactic acid bacteria in the manner such as herein described to produced food path.cularly fermanted milk produce Preferred Erabodiments of the Invention The anti-stress agent of the present invention contains, as an effective ingredient, a tripeptide and/or a salt thereof having angiotensin converting enzyme inhibitory activity. In the present invention, "anti-stress" effect means an activity to cause approximation of the conditions of a subject to the conditions without stress, e.g., an activity to reduce systolic and diastolic blood pressures which have been increased due to stress, and an activity to inhibit or prevent lowering in immunological function caused by stress, such as lowering in spleen cell response. The tripeptide may be preferably selected from the group consisting of lie-Pro-Pro, Val-Pro-Pro (abbreviated hereinbelow as IPP and VPP, respectively) and mixtures thereof that have angiotensin converting enzyme inhibitory activity. The salt of the tripeptide may include pharmacologically acceptable salts, such as inorganic salts such as hydrochloride, sulf ate and phosphate, and organic salts such as citrate, maleate, fumarate, tartrate and lactate. The tripeptide may be produced by, e.g., fermentation with microorganism, enzyme hydrolysis, or chemical synthesis. The fermentation with microorganism can be performed by culturing lactic acid bacteria in a medium of a food material containing a peptide and/or a protein including an amino acid sequence corresponding to the tripeptide, such as sequences Ile-Pro-Pro and/or Val-Pro-Pro. The medium is preferably a food material containing a peptide and/or a protein including an amino acid sequence corresponding to the tripeptides. The food material may be milk, milk casein, corn, corn protein, wheat, wheat protein, soybean, de-fat soybean or soybean protein. The medium may further contain other ingredients such as yeast extract, vitamins and minerals, if necessary. As the lactic acid bacteria, lactic acid bacteria of the genus Lactobacillus may be employed. For example, Lactobacillus helveticus, Lactobacillus delbruekii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus fermentum or Lactobacillus casei subsp. caseimaybe employed. Specifically, Lactobacillus helveticus ATCC55796, Lactobacillus delbruekii subsp. bulgaricus ATCC11842, Lactobacillus acidophilus ATCC4356, Lactobacillus fermentum ATCC14931 or Lactobacillus casei subsp. casei ATCC393 may be employed. The fermentation may be performed by heat-sterilizing the medium, cooling the medium to a desired culturing temperature, and then inoculating the medium with a pre-cultured lactic acid bacteria starter. The inoculation amount of the lactic acid bacteria starter is preferably 105 to 107 cells of the lactic acid bacteria per Ig of the medium. The culture temperature may be 20 to 50° C, and preferably 30 to 45° C. The culturing time may be 3 to 48 hours, and preferably 6 to 24 hours. The culturing may be terminated when number of cells of the lactic acid bacteria reaches 108 cells/g or more and acidity of the lactic acid reaches I or more. The cultured medium itself containing live lactic acid bacteria may be used as the anti-stress agent of the present invention. Alternatively, the cultured medium may be used after heat-sterilizing, e.g., up to 80° C. Further, the cultured medium may be used after being powdered by freeze drying, spray drying or drum dryer drying. The cultured medium may be concentrated to purify the tripeptide before use as the anti-stress agent of the present invention. The concentration and purification may be carried out by subjecting the cultured medium to centrifugation and taking supernatant of the centrifuged liquid. The supernatant may further be subjected to electrodialysis, ion exchange resin treatment, hollow fiber membrane dialysis, reverse osmosis treatment, hydrophobic column chromatography, or combinations thereof, to obtain a further concentrated and purified tripeptide. The enzyme hydrolysis for producing the tripeptide may include treating a food material containing a peptide and/or a protein including an amino acid sequence corresponding to the tripeptides such as lie-Pro-Pro and Val-Pro-Pro, successively with proteinase and carboxypeptidase. The proteinase may be proteinase derived from microorganisms, from plants, or from animals, and may be prepared by a publicly known method. The carboxypeptidase may be carboxypeptidase derived from microorganisms, fromplants, or from animals, and may be prepared by a publicly known method. The chemical synthesis for producing the tripeptide may include publicly known organic synthesis. For example, the tripeptide may be obtained by preparing amino acids that are components of the objective tripeptide, protecting the amino group of each amino acid with a fluorenylmethoxycarbonyl group, successively reacting the amino acids protected with the fluorenylmethoxycarbonyl group in accordance with the amino acid sequence of the objective tripeptide by any conventional method, to obtain a tripeptide bound to a resin, and then removing the resin by any conventional method and purifying the tripeptide. The content of the tripeptide in the anti-stress agent of the present invention is not particularly limited as long as it enables administration of the effective amount of the tripeptide to be discussed later. The content of the tripeptide may usually be 0.001 to 1 wt% of the agent. The anti-stress agent of the present invention may contain, in addition to the tripeptide and/or the salt thereof, other additives such as sugars, proteins, lipids, vitamins, minerals, flavoring agents and coloring agents. The anti-stress agent of the present invention may be administered to humans or animals. The administration may be performed orally or intravenously, with the oral administration being preferable. Effective administration amount of the anti-stress agent of the present invention may usually be, for example, in a range of 0.1 to 40mg/kg body weight'day in terms of the tripeptide for oral administration, in order to achieve the effect of the present invention such as mitigation and prevention of stress in human. However, the anti-stress agent may be administered in an amount exceeding this range. When the anti-stress agent of the present invention is administered to a subject, changes in various physiological indices in response to stress, such as rise in blood pressure, lowering in immunological functions, becomes smaller than that in a case in which the present agent is not administered, and approximates to that in a case in which stress is not loaded. The functional food of the present invention contains the anti-stress agent. Taking this functional food prevents or mitigates the stress. The content of the anti-stress agent in the functional food of the present invention is not particularly limited as long as it is in a range to enable the tripeptide and/or the salt thereof in the functional food achieves the anti-stress effect. The functional food may usually contain the anti-stress agent in an amount to make the content of the tripeptide and/or the salt thereof in the functional food be 0.001 to 0.1 wt%. By taking the functional food of the present invention in an amount ranging from 0.1 to 40 mg in terms of the tripeptide per kg body weight-day, preferable effects of the present invention may be obtained. However, the functional food may be taken in an amount exceeding this range. The functional food of the present invention may include yogurt, milk-containing acidified beverages, cheese, processed foods and beverages containing fermented sour milk or health foods, and may be in the form of solid such as powders, granules and tablets, or of fluid such as paste, gel and liquid. The tripeptide, the effective ingredient of the anti-stress agent and the functional food of the present invention may be obtained by fermenting a food material with lactic acid bacteria, and thus is quite safe. Since the anti-stress agent and the functional food of the present invention contain the tripeptide, they are quite safe, and repeated and daily intake thereof can mitigate and prevent mental and physical symptoms caused by various kinds of stress. Examples of the Invention The present invention will be explained more in detail referring to the Examples, but the present invention is not limited thereto. Example 1 9g of skim milk powders were dissolved in lOOg of water. The resulting solution was sterilized at 115° C for 20 minutes, cooled to room temperature, inoculated with 1 platinum loop of Lactobacillus helveticus ATCC-8205, and cultured at 37° C for 24 hours, to prepare a primary starter (5xl08 cells/ml, pH 3.5) . Subsequently, 2kg of skim milk (containing 9wt% solids) sterilized by heating up to 90° C was inoculated with 80g of the primary starter, and cultured at 37° C for 24 hours, to prepare a secondary starter. Then, 4.5kg of skim milk powders were dissolved in 50kg of water. The resulting solution was sterilized by heating up to 90° C, cooled to room temperature, inoculated with 2 kg of the secondary starter, and cultured at 37° C for 24 hours, to obtain 56kg of fermented milk. The content of IPP and VPP in the fermented milk thus obtained was 5.4mg and 9.5mg per litter, respectively. Example 2 6 kg of the fermented milk prepared in Example 1 was adjusted to pH 7.3 with 10N aqueous solution of sodium hydroxide, admixed with one liter of an ion exchange resin (trade name "Amberlite XAD-2" manufactured by ORGANO CORP.), and further admixed with distilled water so that the total weight of the resulting mixture was 20kg. The mixture was stirred with a stirrer for 90 minutes, and filtrated with a suction filtrationdevice to separate the resin. The resin on the filter was washed with 20kg of distilled water, and then the washed resin was collected. The collected resin was admixed with 0.8kg of methanol, and stirred for 30 minutes with a stirrer. The resulting mixture was filtrated through nylon wool (200 mesh) and then through a hard filter paper by suction. The obtained filtrate was concentrated under a reduced pressure at 55° C with an evaporator, to obtain 200 g of concentrated liquid. The liquid was admixed with 250 ml of an ion exchange resin (trade name "Amberlite IRA-400 (OH type)", manufactured by ORGANO CORP.), stirred for 10 minutes, and filtrated through a filter paper by suction. The filtrate was adjusted to pH 7 with IN hydrochloric acid solution, and freeze dried. The product thus purified and dried was uniformly dissolved in 5ml of distilled water, applied to a column (trade name "Sephadex G-25" manufactured by PHARMACIA CO.) , and eluted with distilled water, to collect a tripeptide fraction. The fraction was freeze dried to obtain 50 mg of powders of the purified tripeptide fraction. 50mg of the powders of the purified tripeptide fraction contained 0.6mg of IPP and 1. Omg of VPP. Example 3 IPP and VPP were synthesized by the following organic chemistry synthesis. The synthesis was performed in accordance with solid phase method using automatic peptide synthesis apparatus (Type PSSM-8) manufactured by SHIMADZU CO. As a solid phase carrier, 20 µ mol of a polystyrene resin of benzyloxybenzyl alcohol type to which proline having an amino group protected with a fluorenylmethoxycarbonyl group (referred to hereinbelow as Fmoc) was bound was employed. In accordance with the amino acid sequence, Fmoc-Ile, Fmoc-Pro and Fmoc-Val each having an amino group protected with the Fmoc group were successively reacted by l00umol each by a conventional method, to obtain a peptide-bound resin. The peptide-bound resin was suspended in 1 ml of a reaction liquid (containing lwt% ethanedithiol, 5wt% anisole and 94wt% trifluoroacetic acid) and reacted for 2 hours at room temperature to separate the peptides from the resin and to remove the side chain protecting group from the peptides . The reaction mixture was filtrated through a glass filter, admixed with 10ml of anhydrous ether to precipitate the purified peptide, and centrifuged at 3000rpm for 5 minutes to separate the precipitate. The precipitate was washed with anhydrous ether several times, and dried by blowing nitrogen gas. All the crude synthesized peptides thus obtained was dissolved in 2 ml of 0. IN hydrochloric acid aqueous solution, and then purified by HPLC on a reverse phase column of C18 in accordance with the following conditions: Pump: Type L6200 intelligent pump (HITACHI, LTD.) Detector: Type L4000 UV detector (HITACHI, LTD.) for detecting UV absorbance at 215 nm Column: MICROBONDASPHERE 5 /z C18 (manufactured by WATERS INC.) Eluent: liquid A; 0.1 wt% TFA aqueous solution liquid B; acetonitrile containing 0.1 wt% TFA (B/A+B)xl00(%): 0 → 40% (60 minutes) Flow rate: 1ml/min. Eluate fraction at the maximum absorbance were collected and freeze dried to obtain the objective synthesized peptides, i.e., 2 . Img of IPP and 0. 9mg of VPP. The purified peptides were analyzed from their N-terminus with a full automatic protein primary structure analyzer (Type PPSQ-10 manufactured by SHIMADZU CO.), and with an amino acid analyzer (Type 800 series, manufactured by NIHON BUNKOU KOUGYO CO.). As a result, these peptides were found to be as designed. Example 4 24 male Wister rats (body weight about 300g) were preliminarily fed for one week. During the preliminary feeding period and the experimental period, the rats were restrictively fed with commercial diet (trade name "CE-2" manufactured by NIHON CREA CO.), and allowed to take water ad libitum. After the preliminary feeding period, the rats were divided into three groups (eight rats each), namely, (1) the group not exposed, to stress and given saline, (2) the group exposed to stress and given saline, and (3) the group exposed to stress, and given VPP and IPP. The animals of groups (2) and (3) were exposed to stress for 9 days by putting them in a cold room (4° C) for 4 hours per one day. On the day 10, rats of groups (1) and (2) were given 1ml of saline, and rats of group (3) were given 1ml of saline containing 3 mg/kg body weight each of IPP and VPP synthesized in Example 3, by forcible administration into stomach via an oral sonde. After the administration, the rats of groups (2) and (3) were exposed to cold stress for 4 hours. Two hours after finishing the stress loading, blood pressure of the rats in each group was measured by tail-cuff method using a non-warming, non-invasive rat sphygmomanometer (Type PE-300, manufactured by NARUCO BIOSYSTEM). The results are shown in Table 1. The results of blood pressure are shown in Table 1. As shown in Table 1, both systolic and diastolic blood pressures were higher in group (2) which were exposed to stress than in group (1) which were not exposed to stress. On the contrary, both systolic and diastolic blood pressures of group (3) rats which received VPP and IPP were lower than those of group (2) rats which received saline, and approximated to those of group (1) rats which were not exposed to stress. Table 1(Table Removed)*significantly different from group (1) with significance level of 5% Changes in blood pressure before and after the cold stress are shown in Table 2. As shown in Table 2, both systolic and diastolic blood pressures were increased significantly in group (2) which were exposed to stress, compared to group (1) which were not exposed to stress. Increase in both systolic and diastolic blood pressures were suppressed in group (3) which received VPP and IPP, compared to group (2) which received saline. Particularly, suppression of increase in systolic blood pressure was significant. From these results, it was confirmed that administration of IPP and VPP results in suppression of increase in blood pressure induced by stress. Table 2 (Table Removed) *significantly different from group (1) with significance level of 5% #significantly different from group (2) with significance level of 5% Example 5 24 male Wister rats (body weight about 300g) were preliminarily fed for one week. During the preliminary feeding period and the experimental period, the rats were restrictively fed with commercial diet (trade name "CE-2" manufactured by NIHON CREA CO.), and allowed to take water ad libitum. After the preliminary feeding period, the rats were divided into three groups (eight rats each) , namely, (1) the group given saline, (2) the group given VPP, and (3) the group given IPP. The rats of groups (1) to (3) were exposed to stress for 9 days by, as in Example 4, putting them in a cold room (4° C) for 4 hours per one day. On the day 10, rats of group (1) were given 1ml of saline, and rats of group (2) and (3) were given 1ml of saline containing 3 mg/kg body weight of IPP or 3mg/kg body weight of VPP, respectively, synthesized in Example 3, by forcible administration into stomach via an oral sonde. After the administration, the rats of groups (1) to (3) were exposed to cold stress for 4 hours, and blood pressure was measured as in Example 4. The results are shown in Table 3. The results of blood pressure are shown in Table 3. As shown in Table 3, both systolic and diastolic blood pressures were significantly lower in groups (2) and (3) which received tripeptides than in group (1) which received saline. It was recognized that administration of the tripeptides results in suppression of increase in blood pressure induced by stress. Table 3(Table Removed) *significantly different from group (1) with significance level of 5% Example 6 The test was carried out in the same way as in Example 5 except that the group (1) rats were given 2ml of saline, and the rats in group (2) were given 5ml/kg body weight of the fermented milk obtained in Example 1, and the rats in group (3) were given 2ml of saline containing 150mg/kg of the purified tripeptide fraction powders obtained in Example 2, respectively. After the administration of the samples and exposure to stress, the blood pressure of each rat was measured. The results of blood pressure are shown in Table 4 . As shown in Table 4, comparing group (2) which received the fermented milk and group (3) which received the purified tripeptide fraction powders with group (1) which received saline, both systolic and diastolic blood pressures were lower in groups (2) and (3) than in group (1) . It was thus recognized that administration of the fermented milk and the purified tripeptide fraction results in suppression of increase in blood pressure induced by stress. Table 4 (Table Removed) *significantly different from group (1) with significance level of 5% Example 7 24 male Wister rats (body weight about 300g) were preliminarily fed for one week. During the preliminary feeding period, the rats were restrictively fed with commercial diet (trade name "CE-2" manufactured by NIHON CREA CO.) , and allowed to take water ad libitum. After the preliminary feeding period, the rats were divided into three groups (eight rats each), namely, (1) the group not exposed to stress and given saline, (2) the group exposed to stress and given saline, and (3) the group exposed to stress, and given VPP and IPP. The animals of groups (2) and (3) were exposed to water immersion restraint stress for 6 hours per one day for consecutive 5 days by putting the animals in a wire cage for restraint and immersing them in a water bath at 25°C with their heads above the water surface for breathing. During the stress loading period, the rats were allowed to take commercial diet (trade name "CE-2" manufactured by NIHON CREA CO.) and water ad libitum. The rats were given samples throughout the consecutive 5 days of stress. As the samples, 1 ml of saline was given to groups (1) and (2), and 1 ml of saline containing 3mg/kg body weight each of IPP and VPP synthesized in Example 3 to group (3), by forcible administration into stomach via an oral sonde. From the second day to the third day of stress loading, urine was collected using a metabolic cage, and analyzed with HPLC to measure the levels of catecholamine and indoleamine. A silica reverse phase column (trade name "Catecholpack") manufactured by NIHON BUNKOU KOUGYO CO. and an electrochemical detector (trade name "Coulochem") manufactured by esa CO. were employed in the analysis. After the exposure to stress was finished on the final day of loading stress, the rats were sacrificed by decapitation, to collect blood and take out thymus and spleen. Amino acid composition of the serum was measured by amino acid analyzer (Type 800 series, manufactured by NIHON BUNKOU KOGYO CO.), to calculate Fischer ratio (molar ratio of branched chain amino acid /aromatic amino acid). The weight of thymus and lever was measured. Spleen cells were prepared from spleen and measured for interleukin 2 productivity and mitogen reactivity in accordance with the following procedure. (Preparation of Spleen Cells) Spleen was finely ground in a homogenizer, subjected to hypotonic treatment to remove hemocyte, washed with MEM containing 2% fetal calf serum (FCS) , and suspended in RPMI 1640 medium containing 10% FCS, to prepare a free cell suspension containing 1xl07 cells/ml. (Measurement of Interleukin 2 Productivity) RPMI 1640 medium containing 2.5xl06 cells/ml of the spleen cells prepared above, 5 µ g/ml of concanavalin A (ConA) and 10% FCS was prepared, and cultured for 24 hours. The amount of interleukin 2 in the supernatant of the cultured medium was measured by bioassay using as an index proliferation of an interleukin 2 reactive cell strain. (Mitogen Reactivity) RPMI 1640 medium containing 5 µ g/ml of either ConA or a pokeweed mitogen (PWM) as a mitogen, 5xl06 cells/ml of the spleen cells prepared above and 10% FCS was prepared, and cultured for 24 hours. The number of the cells after 24 hours was measured by absorbance, using intake of MTT (3-(4,5-dimethylthiazoil-2-yl)-2,5-diphenyltetrazolium bromide) as an index, and represented as the ratio with respect to the number of cells without mitogen. The amount of noradrenaline and dopamine excreted in urine from the second day to the third day of stress loading is shown in Table 5. As shown in Table 5, excretion of noradrenaline and dopamine in urine was significantly smaller in group (2) which was exposed to stress than in group (1) which were not exposed to stress. In group (3) which received the tripeptides, the decrease in excretion of noradrenaline and dopamine tended to be suppressed, compared to group (2) which received saline. It was thus recognized that IPP and VPP have a suppressive effect in stress induced decrease in excretion of noradrenaline and dopamine in urine. Table 5(Table Removed) *significantly different from group (1) with significance level of 5% #significantly different from group (2) with significance level of 10% Fischer ratio of serum amino acids after sacrifice is shown in Table 6. As shown in Table 6, Fischer ratio was significantly lower in group (2) which were exposed to stress than in group (1) which were not exposed to stress . On the other hand, Fischer ratio was significantly higher in group (3) which received the tripeptide than in group (2) which received saline. Table 6(Table Removed) *significantly different from group (1) with significance level of 5% ftsignificantly different from group (2) with significance level of 5% Weight of thymus and spleen after sacrifice is shown in Table 7 . The weight of thymus and spleen was lowered more in group (2) which were exposed to stress than in group (1) which were not exposed to stress. Weight of thymus and spleen tended to be slightly greater in group (3) which received the tripeptides, than in group (2) which received saline. Table 7(Table Removed) *significantly different from group (1) with significance level of 5% Mitogen reactivity of the spleen cells is shown in Table 8, and interleukin 2 productivity of the spleen cells is shown in Table 9. Decrease in mitogen reactivity and tendency toward decrease in interleukin 2 productivity were recognized in group (2) which were exposed to stress, compared to group (1) which were not exposed to stress. On the other hand, increase in mitogen reactivity and tendency toward increase in interleukin 2 productivity were recognized in group (3) which received the tripeptides, compared to group (2) which received saline. Table 8(Table Removed) *significantly different from group (1) with significance level of 5% #significantly different from group (2) with significance level of 5% Table 9(Table Removed) #significantly different from group (2) with significance level of 10%From these results, it is thus recognized that administration of IPP and VPP can suppress decrease in indices of immunological function caused by stress such as change in balance of blood amino acid (Fischer ratio), atrophy of thymus and spleen, and decrease in spleen cell reactivity. WE CLAIM: A process for the preparation of a food, particularly fermented milk product having anti-stress effect and containing lie-Pro-Pro (IPP) tripeptide and/or Val-Pro-Pro (VPP) tripeptide characterized in that said process comprises the step of fermentation of a medium containing a peptide and/or a protein including amino acid sequence lie-Pro-Pro and/or Val-Pro-Pro with lactic acid bacteria, wherein said step of fermentation is carried out by heat-sterilization of said medium followed by cooling and incubation with said lactic acid bacteria in the manner such as hereindescribed to produce food, particularly said fermented milk product. A process as claimed in claim 1, wherein said medium containing a peptide and/or a protein including amino acid sequence lie-Pro-Pro and/or Val-Pro-Pro is food material. A process as claimed in claim 2, wherein said food material is preferably selected from a group consisting of milk, milk casein, corn, corn protein, wheat, wheat protein, soybean, de-fat soyabean or soyabean protein. A process as claimed in claim 3, wherein said food material preferably comprises yeast extract, vitamins and minerals. A process as claimed in claim 1, wherein said lactic acid bacteria is genus of Lactobacillus. A process as claimed in claim 5, wherein said lactic acid bacteria is preferably Lactobacillus helveticus, Lactobacillus delbruekii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus fermentum or Lactobacillus casei subsp. casei. 7 A process as claimed in claim 6, wherein said lactic acid bacteria is more preferably Lactobacillus helveticus. 8 A process as claimed in any of the preceding claims, wherein said step of heat-sterilization is carried out upto a temperature of about 115°C or less. 9 A process as claimed in any of the preceding claims, wherein said step of incubation is carried out at a temperature of about 50°C or less. 10 A process as claimed in claim 9, wherein said step of incubation is preferably carried out at a temperature varying from 20°C to 50°C, more preferably from 30°C to 45°C. 11 A process for the preparation of a food, particularly fermented milk product substantially as herein described with the help of accompanying examples.

Documents:

2884-del-1998-abstract.pdf

2884-del-1998-claims.pdf

2884-del-1998-correspondence-others.pdf

2884-del-1998-correspondence-po.pdf

2884-del-1998-description (complete).pdf

2884-del-1998-form-1.pdf

2884-del-1998-form-13.pdf

2884-del-1998-form-2.pdf

2884-del-1998-form-3.pdf

2884-del-1998-form-4.pdf

2884-del-1998-form-6.pdf

2884-del-1998-form-9.pdf

2884-del-1998-gpa.pdf

2884-del-1998-pct-210.pdf