JP4688457B2 - Immune enhancing composition - Google Patents

Description

  The present invention relates to an immune enhancing composition. More specifically, the present invention relates to an immune enhancing composition containing fermented milk obtained by co-culturing a plurality of types of lactic acid bacteria and yeast as active ingredients.

In recent years, immunity decline has become a problem due to nutritional reasons such as uneven eating habits and diet orientation, and due to decline in biological functions accompanying aging, and there is a substance that can correct this decline in immunity. Various researches and developments have been conducted. In particular, various researches and developments have been made on substances derived from natural products that can be taken for a long time without causing side effects. Strengthening of such biological immune function by administration of substances that enhance the immune force, various infectious diseases, as well as cancer only, arteriosclerosis, believed to be helpful in the prevention and treatment of diabetes and the like. However, development of an immunopotentiating substance that can sufficiently exhibit a desired immune enhancing effect and can be used as a pharmaceutical or a food has not yet been made.

  On the other hand, milk has the effect of increasing the natural healing power by controlling physiological systems such as the immune system, secretory system, nervous system, circulatory system, digestive system, growth promoting effect, cancer preventing effect, etc. Are known. More specifically, milk (milk), which is referred to as a complete diet, contains milk proteins such as casein and lactalbumin, and among the amino acid sequences of the milk proteins, sequences that are the same as or similar to peptides having various physiological activities Peptides that are these milk proteins or their degradation products (for example, degradation products by extracellular enzymes possessed by lactic acid bacteria) are thought to be capable of exhibiting various physiological activities useful for the human body. It is also known that lactic acid fermentation products such as yogurt have an intestinal effect. However, so far, there has been little research on substances having bioactive effects contained in milk and its fermented dairy products, and research on immune enhancing substances derived from these natural products has been sufficiently conducted. Not in the current situation.

The inventors of the present invention have conventionally conducted various studies on fermented milk. In the process, certain types of fermented milk obtained by co-culturing a plurality of types of lactic acid bacteria and yeast are excellent in flavor and the like. The present invention has been found to have an antibacterial action, an antimutagenic action, a blood pressure lowering action, etc., and has completed the invention relating to the method for producing this fermented milk (see Patent Document 1). This Patent Document 1 describes that the obtained fermented milk has an immunostimulatory action. This immunostimulatory action is an antibacterial activity demonstrated in each test example of Patent Document 1 (Test Example 1- 4) It is only used as a generic term for the antimutagenic activity (Test Example 5) and the blood pressure lowering action (Test Example 6) (action to maintain human health). It is described in Patent Document 1 that the fermented milk obtained by the method described in (1) directly has an immunostimulatory action or an immune enhancing action such as an IgM production promoting action and an IFN-γ production promoting action. Not. In addition, there has been no report that such fermented milk has an immunopotentiating action such as an IgM production promoting action and an IFN-γ production promoting action.
Patent No. 2791375 specification

  An object of the present invention is to provide a product derived from a natural product, particularly a fermented dairy product originating from cow milk, which has an excellent immunity enhancing action, particularly an IgM production promoting action and an IFN-γ production promoting action.

  Another object of the present invention is to provide a product having an immunopotentiating action derived from the above natural product, which is favorable in terms of its flavor and the like even when ingested as a food.

  As a result of intensive studies on the immunity enhancing action of dairy products, the present inventors have obtained a new finding that certain types of fermented milk have an IgM production promoting action and an IFN-γ production promoting action. In particular, the inventors of the present invention have excellent fermented milk obtained by a specific method (see Patent Document 1) previously developed by the inventors in addition to being excellent in flavor and the like. The present inventors have obtained new findings that they can exert IgM production promoting action and IFN-γ production promoting action.

  The present invention has been completed as a result of further research based on this knowledge. That is, the present invention is the discovery of the fact that fermented milk, particularly fermented milk obtained by co-culturing a plurality of types of lactic acid bacteria and yeast, contains a physiologically active peptide that exerts an IgM production promoting action and an IFN-γ production promoting action. It was completed based on. The present invention provides the following inventions.

  Item 1. An immune enhancement composition comprising fermented milk as an active ingredient.

  Item 2. The immune enhancing composition according to Item 1, wherein the fermented milk is obtained by co-culturing a plurality of types of lactic acid bacteria and yeast in raw milk.

  Item 3. The immune enhancing composition according to Item 1 or 2, which is an IgM production promoter.

  Item 4. The immune enhancing composition according to Item 1 or 2, which is an IFN-γ production promoter.

  Item 5. Fermented milk individually inoculates raw milk with multiple types of lactic acid bacteria, and each inoculated product is cultured individually for an appropriate period of time under the optimal temperature environment for each microorganism. Collect appropriate amount of liquid individually, inoculate milk together with yeast and incubate for appropriate time under different temperature environment, then add each starter obtained at each temperature to milk at a predetermined ratio Item 2. The immune enhancing composition according to Item 1, which is a fermentation broth extracted from the obtained fermentation card after culturing for a certain period of time in a constant temperature environment.

  Item 6.The fermented milk is Lactococcus lactis subsp. Streptococcus thermophilus, Leuconostoc cremoris, Lactobacillus delbreckii subspice bulgaricus, Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus casei, Lactobacillus helveticus Item 1. The immune enhancement according to item 1, which is a centrifugal supernatant of a culture product obtained by culturing skim milk for 2 weeks using lactic acid bacteria and yeast consisting of Bifidobacterium longum and Saccharomyces cerevisiae. Composition.

  Item 7. The immune enhancing composition according to Item 1, wherein the fermented milk contains a 19 kDa protein that is a degradation product of milk protein.

  Item 8. The composition according to any one of Items 1-7, which is in a food form.

  Item 9. The composition according to any one of Items 1-7, which is in a pharmaceutical form.

  As described above, the immune enhancing composition of the present invention exhibits excellent IgM production promoting action and IFN-γ production promoting action based on the use of specific fermented milk as an active ingredient. These excellent physiologically active effects found in the composition of the present invention will be described in detail in Examples below.

  Hereinafter, fermented milk as an active ingredient of the immune enhancing composition of the present invention and the composition of the present invention containing the same will be described in detail.

(1) Preparation of fermented milk Fermented milk as an active ingredient of the composition of the present invention is obtained by causing lactic acid bacteria or the like to act on raw milk and decomposing the milk protein, and promotes IgM production. And a physiologically active peptide capable of promoting IFN-γ production. The production method is not particularly limited as long as the obtained fermented milk contains such a physiologically active peptide.

  A preferable fermented milk is obtained by co-culturing a plurality of types of lactic acid bacteria and yeast in raw milk. This co-cultivation can be carried out, for example, according to the method previously established by the present inventors (the method described in Patent Document 1). More specifically, it can be carried out as follows. That is, a plurality of types of lactic acid bacteria (including bifidobacteria) are individually inoculated into raw milk, and each inoculated product is cultured individually for an appropriate time under the optimum growth temperature environment for each bacteria. Collect the appropriate amount of the fungus individually, inoculate the milk together with the yeast and incubate it under different temperature conditions for an appropriate time, and then add each starter obtained at each temperature to the milk at a predetermined ratio. Add and incubate for a certain period of time in a constant temperature environment. Thus, fermented milk in the form of a fermented curd (including casein) can be obtained.

  The fermented milk may be in the form of a fermented liquid obtained by further extracting the fermented card thus obtained. Furthermore, fermented milk is in the form of powder and tablet obtained by pulverizing (freeze-drying) the above fermentation card and fermentation broth according to conventional methods such as vacuum freeze-drying means, or tableting them. Also good.

As a preferred combination of the above-mentioned multiple types of lactic acid bacteria and yeast, for example, the following combinations of bacteria can be mentioned.
・ Lactococcus lactis subsp. Lactis,
・ Lactococcus lactis subsp. Cremoris,
・ Streptococcus lactis subsp. Diacetylactis,
-Streptococcus thermophillus (Streptococcus thermophillus),
・ Leuconostoc cremoris,
・ Lactobacillus delbrueckii subsp.bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus),
Lactobacillus delbrueckii subsp. Lactis,
・ Lactobacillus acidophilus,
・ Lactobacillus casei,
・ Lactobacillus helveticus (Lactobacillus helveticus),
・ Bifidobacterium longum,
-Saccharomyces cerevisiae.

  The method for producing fermented milk of the present invention according to the above method is roughly divided into three steps: preculture, main culture, and extraction of lactic acid bacteria fermentation broth. As the pre-culture, a plurality of types of lactic acid bacteria (including bifidobacteria) are individually inoculated into milk as single bacteria, and the inoculated ones are individually cultured under the optimum temperature environment for each bacteria. On the other hand, yeast is inoculated into milk and cultured in an optimum temperature environment for an appropriate time. This yeast may be added to a nutrient solution containing vitamins, amino acids and the like without being cultured in milk. Next, each culture solution obtained by the pre-culture is individually collected and main culture is performed in which the inoculated milk is cultured in a different temperature environment for an appropriate time. The starter obtained for each temperature by the main culture is added to milk at a predetermined ratio, and further cultured in a constant temperature environment for an appropriate time. By this cultivation, a desired fermentation card can be obtained.

  The milk used as a raw material is usually preferably milk or skim milk, but is not particularly limited to this, and may be other animal milk.

(2) Composition of the present invention It is important that the composition of the present invention contains the above fermented milk (fermented card, fermented liquor, powder and tablet thereof) as an essential component. The fermented milk itself is derived from milk used as a raw material for fermented milk and lactic acid bacteria that act on the fermented milk, together with a physiologically active substance having an IgM production promoting action and an IFN-γ production promoting action as an active ingredient of the composition of the present invention. It contains proteins, lipids, components that constitute the cells themselves, and metabolites that result from fermentation. This metabolite includes, for example, aromatic components such as diacetyl, acetoin and citric acid, antibacterial components such as niacin, and organic acids such as amino acids and lactic acid. Therefore, fermented milk alone is useful as the immune enhancing composition of the present invention. In particular, since this product has excellent flavor, taste and the like, it can be advantageously applied to patients who are required to have the above physiological activity or therapeutic effects on various diseases based on the above in the form of food and drink.

  The composition of the present invention purifies a physiologically active substance exhibiting an IgM production promoting action and an IFN-γ production promoting action from the fermented milk (including powders and tablets), and uses the resulting purified product as an active ingredient. Depending on the composition, it may be a composition in the form of various foods and beverages or pharmaceuticals, which is contained together with an edible carrier, a pharmaceutical preparation carrier and the like.

  Here, the physiologically active substance is, for example, a supernatant obtained by dissolving fermented milk (powder) in water or alkaline water and then centrifuging, or a precipitate obtained by centrifuging after dissolving the powder in water. Is a water-soluble fraction such as a supernatant obtained by further dissolving in water or a substance contained therein. Among these, the particularly excellent effect of the present invention can be achieved by dissolving the fermented milk powder in water and then centrifuging the supernatant (pH about 4) further with a 10 mM sodium phosphate buffer. And water-soluble fractions obtained by dialysis with s.

  In addition, as an edible carrier and a pharmaceutical preparation carrier, various nutrient sources conventionally used in the food and pharmaceutical fields, such as carbohydrates, lipids, protein materials, etc., and pharmaceutically acceptable fillers, Examples include fillers and diluents such as extenders, binders, wetting agents, disintegrants, surfactants, lubricants and the like.

  Representative examples of food forms prepared using these include beverages such as soft drinks, carbonated drinks, fruit drinks, whey drinks, coffee drinks, vegetable juices, nutritional drinks, soy milk, tea drinks, sports drinks, Examples include oolong tea drinks and green tea drinks. Examples of other food forms include dairy products, lactic acid bacteria beverages, fermented milk, milk beverages, jellies, fresh confectionery, confectionery, health foods, and the like. Examples of the pharmaceutical form include ordinary tablets, pills, powders, liquids (suspensions, emulsions, injections, etc.), granules, capsules, suppositories, ointments and the like. Preparation into each of these forms can be carried out according to a conventional method using a commonly used carrier, and the amount of the carrier used at that time can be the same as the general amount used. . Further, the composition of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals as necessary.

  The various forms of the composition of the present invention described above are applied by an appropriate intake or administration method depending on the form. The composition of the present invention in a food form can be taken orally as it is or with other foods. The pharmaceutical composition of the present invention is administered orally, for example, tablets, pills, granules, capsules and the like, and injections are administered intravenously, intramuscularly, intradermally, subcutaneously or intraperitoneally according to the formulation form. And suppositories can be administered rectally.

  The intake or dose of the composition of the present invention can be appropriately selected depending on its usage, patient age, sex and other conditions, degree of disease, desired desired effect, and the like.

  The composition of the present invention can be ingested or administered in a pharmaceutical form or a food form to exhibit an excellent IgM production promoting action and IFN-γ production promoting action.

Hereinafter, in order to explain the present invention in more detail, a fermented milk production example used in the present invention (performed in the same manner as in Example 1 of Patent No. 2791375) is given as a reference example, and then the present invention example. Give up. In each example,% indicates w / w% unless otherwise specified. In addition, the protein such as test fermented milk in each Example was quantified by the Lowry method (Lowry method, Lowry, .OH, et al., Protein measurement with the folin phenol reagent, J. Biol. Chem., 1951; 193 : 265-275).
Reference example 1
Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Cremoris, Streptococcus lactis subsp. Diacetylactis and Str. Each of Leu. Cremoris (Leu. Cremoris) was individually inoculated into milk (fat milk) having a concentration of about 25% or less, and cultured under a constant temperature environment at an optimum temperature of each bacterium for 48 hours. Each fungus individually grew in milk, and the produced lactic acid produced fermented milk with an acidity of about 0.9%.

  Streptococcus salivarius subsp. Thermophilus (Lb. delbrueckii subsp. Bulgaricus), Lactobacillus delbrikkii subsp. ) And Lactobacillus helveticus (Lb. helveticus) were individually inoculated into milk (fat milk) having a concentration of 25% or less, and cultured under a constant temperature environment at an optimum temperature of each bacterium for 48 hours. Each fungus grew in milk, producing fermented milk with an acidity of about 1.0%.

  Lactobacillus acidophilus (Lb. acidophilus), Lactobacillus casei (Lb. casei) and bifidobacterium (Bifidobacterium. Longum), each containing about 0.5% soybean peptide (milk) Milk) was inoculated and cultured for 48 hours in a constant temperature environment that is the optimum temperature for each bacterium. According to this culture, activation of each bacterium can be enhanced.

  Saccharomyces cerevisiae was inoculated into milk (fat milk) containing yeast extract and glucose and cultured at about 30 to 32 ° C. for about 48 hours. Saccharomyces cerevisiae may be a yeast solution added to an aqueous solution containing vitamins and amino acids without being cultured in milk. The yeast is not limited to Saccharomyces cerevisiae, and may be Saccharomyces delbrueckii, Torulopsis kefir, dry yeast, and the like.

  Each culture solution obtained by each of the above precultures was inoculated to the same amount of milk (fat milk) having a concentration of about 25% or less. The amount of inoculation is about 0.01 to 4.00 v / v%, preferably 2.00 v / v% or less, with respect to milk. After inoculation, high temperature, medium temperature and low temperature were selected within the range of 22 to 40 ° C., and the cells were cultured for about 24 to 60 hours in each constant temperature environment. A starter 1 is a culture in a high temperature environment, a starter 2 is a culture in an intermediate temperature environment, and a starter 3 is a culture in a low temperature environment.

  As a result of microscopic observation of starter 1, starter 2 and starter 3 at 400 magnifications, starter 1 has one yeast per field, lactic acid bacteria exist in a ratio of gonococci and cocci 8: 2, and as a whole, Many were observed. In Starter 2, it was found that three yeasts were present in one field of view, and gonococci and cocci were present in the same proportion in lactic acid bacteria. did. In Starter 3, it was found that yeast was one in 10 fields, and lactic acid bacteria were abundant in a ratio of gonococci and cocci in a ratio of 3 to 7, with a large number of cocci in general, and in particular, there were many diococci.

  Starter 1, starter 2 and starter 3 were added to milk (fat milk) according to the composition shown in Table 1 below, and cultured at about 22 to 40 ° C. for each time to obtain a fermentation card. In this test, in order to obtain a metabolite rich in physiologically active substances by the symbiosis of lactic acid bacteria and enzyme, a long incubation time is selected.

  The results of examining the pH, the number of lactic acid bacteria and the number of yeasts of the obtained fermentation card were as shown in Table 2 below. The number of lactic acid bacteria and yeast were measured according to the following method.

(1) Measurement of the number of lactic acid bacteria The lactic acid bacteria in this test are acid-producing bacteria that grow aerobically at a medium temperature on a BCP-added plate count agar medium.

  1 Select a dilution that will yield 30-300 colonies on a flat plate, prepare two or more sterile Petri dishes for the same-stage diluted sample, and accurately take 1 mL of each dilution with a sterile pipette. Add about 15 mL of BCP-added plate count agar medium that has been heated and dissolved in advance and kept at about 50 ° C., mix gently, and cool and solidify. Incubate at 35-37 ° C for 72 ± 3 hours. After culturing, the yellowing out of the developed colonies is counted as a colony of lactic acid bacteria.

(2) Method for measuring the number of yeast
1 Select a dilution that will yield 30-300 colonies on a flat plate, prepare two or more sterile Petri dishes for the same-stage diluted sample, and accurately take 1 mL of each dilution with a sterile pipette. Add about 15 mL of potato dextrose agar medium (adjusted to pH 3.5 with 10% tartaric acid sterilized before use) previously heated and dissolved at about 50 ° C, mix gently, and solidify by cooling. Incubate at 25 ° C for 5-7 days. After culturing, the generated colonies are measured, and the average colony number is multiplied by the dilution factor to obtain the number of yeasts.

  The resulting fermentation curd was stirred individually while heating to about 70 ° C. By this stirring heat treatment, the fermentation curd was separated into a solid and a liquid. After the stirring is completed, the mixture is left to cool to room temperature, and then the solid is filtered through Melita filter paper (trade name of Melita Japan Co., Ltd.). An emulsion was obtained.

  The obtained fermented milky milk had a transparent yellowish brown color. Analytical values (moisture content, lactic acid concentration, pH, protein content, lipid content, presence of coliforms) of fermented milk obtained by culturing for 14 days (Table 1, fermented milk 4) are shown in Table 3 below. It was as shown. In Table 3, the analytical values of the freeze-dried product of the fermented milk are also shown. Moreover, the measurement of each said analysis item of fermented milk was based on the following method.

Water content: atmospheric drying method,
Lactic acid acidity: neutralization titration method,
pH: Glass electrode method,
Protein: Semi-micro Kjeldahl method,
Lipid: Rese-Gottlieb method,
Coliform group: desoxycholate agar medium method.

IgM production promotion test
(1) Preparation of test fermented milk The fermented milk of fermented milk 4 prepared in Reference Example 1 was centrifuged at 100,000 × g for 30 minutes at 4 ° C., and the supernatant was collected. The pH of this was about 4. Next, the obtained supernatant was dialyzed against 10 mM sodium phosphate buffer (NaPB) (pH = 7.4) to prepare test solution 1.
(2) Test antibody-producing cells and their culture Human peripheral blood lymphocytes (PBL) were prepared from the peripheral blood of healthy donors as reported by Murakami (Murakami, H., et al., Cytotechnology, 24, 177-182 (1997)). In this preparation, donor peripheral blood was diluted 1: 1 with physiological saline phosphate buffer (PBS), then centrifuged at room temperature for 30 minutes in lymphocyte separation medium (Nycomed, Pharma, Oslo, Norway), and 3% in ERDF medium. This was performed by washing twice. The obtained PBL was pre-cultured with ERDF supplemented with 10% fetal calf serum (FCS) for 1 day in order to remove adherent cells such as monocytes and fibroblasts. After pre-culture, PBL was inoculated into ITES-ERDF medium.
(3) Measurement of produced IgM Inoculate 1 × 10 ⁶ cells / mL of PBL prepared in (2) above into the ITES-ERDF medium supplemented with various concentrations of the test solution prepared in (1) above for 4 days. Culture (37 ° C, wet, 5% carbon dioxide incubator) and measure the amount of IgM secreted and accumulated in each culture medium according to the ELISA method using anti-human IgM antibody (Biosource International) reported by Sugahara et al. (Sugahara, T., et al., Bioscience, Biotechnology and Biochemistry, 58, 2212-2214 (1994)). Each measurement was performed by repeating the same test three times, and the measured values were expressed as means ± SD.

Moreover, the time course (time course) effect in the IgM production was examined as follows. That is, human PBL was inoculated at 1 × 10 ⁶ cells / mL in the ITES-ERDF medium supplemented with 530 μg / mL of each test solution, and cultured for 5 days in the same manner as above, in the medium after 1, 3 and 5 days. The amount of IgM was measured in the same manner.
(4) Results
The results of examining the amount of IgM produced after 4 days of culture are shown in Fig. 1 (horizontal axis: protein concentration (μg / mL), vertical axis: IgM production amount (ng / mL) (white circles). As a control, the measured value of IgM in the medium when PBL cells were cultured in the same manner as described above using the ITES-ERDF medium without addition of the test solution is indicated by a black circle.

  From FIG. 1, it is clear that the use of the test solution 1 shows an increase in the production amount of IgM as compared with the case where it is not added.

  Next, the results of examining the time course of IgM production are shown in FIG. 2 (horizontal axis: culture time (days), vertical axis: IgM production (ng / mL)) (white circles). In FIG. 2, the results (IgM production amount) of the control (the same test using the ITES-ERDF medium without addition of the test solution) are indicated by black circles.

  From the results shown in FIG. 2, it was found that the IgM producing action of PBL continued to increase clearly after 1 day by adding the test solution, and increased to about 9 times after 1 day after 5 days. In contrast, it was found that such a daily increase in IgM production was not observed in the control.

   From the results of this test, it was revealed that fermented milk (test solution) has an IgM production promoting effect.

  In addition, as a result of the same test by an ELISA method using an IgG antibody, the present inventors have confirmed that the test solution exhibits not only IgM but also an IgG production promoting effect.

  FIGS. 3 and 4 show the IgG production promoting effect in the same manner as FIGS. 1 and 2 described above.

IFN-γ production promoting effect test
(1) Measurement of IFN-γ Using human PBL used in Example 1, it was examined how the addition of fermented milk had an effect on IFN-γ production of the cells. This test was performed in the same manner as in Example 1 using a medium in which 5.0 μg / mL of lipopolysaccharide (LPS, Sigma) was further added to the ITES-ERDF medium supplemented with test solution 1 having various concentrations. Then, 1 × 10 ⁶ cells / mL of human PBL is cultured for 5 days, and the amount of IFN-γ secreted and accumulated in the medium is measured using a cytokine measurement kit based on ELISA (Biosouurce International). It was measured. For comparison, the same operation was repeated using a predetermined concentration (200 μg / mL or 400 μg / mL) of α-lactalbumin (α-LA, manufactured by Sigma) instead of the above test solution 1. Each measurement was performed three times for the same sample, and the measured value was determined by means ± SD.

Further, the time course (time-dependent effect) in the production of IFN-γ was examined as follows. That is, human PBL was inoculated at 1 × 10 ⁶ cells / mL in ITES-ERDF medium supplemented with 50 μg / mL of Test Solution 1 (stimulated by adding LPS 5.0 μg / mL), and the same as above. After culturing for 5 days, the amount of IFN-γ in the medium after 1, 3 and 5 days was measured in the same manner. In this test, as a control, the amount of IFN-γ produced by PBL cells when cultured in a medium to which the test solution 1 was not added was examined in the same manner.
(2) Results The obtained results (results of IFN-γ production measurement test) are shown in FIG. 5 (horizontal axis: control, α-lactalbumin and test solution 1, α-lactalbumin and test solution 1) Each protein concentration (μg / mL) is shown), and the vertical axis: IFN-γ production (pg / mL)). In FIG. 5, the asterisk indicates a significant difference from the control.

  The results of examining the time course are shown in FIG. 6 (horizontal axis: culture time (days), vertical axis: IFN-γ production (pg / mL)). In FIG. 6, white circles are the results when test solution 1 was used, and black circles are the results of control.

  As is clear from the results shown in each figure, it can be seen that test solution 1 (fermented milk) can exert an effect of promoting IFN-γ production on human PBL. The effect is significant even in comparison with α-lactalbumin (see FIG. 5), and it is clear from this fact that the fermented milk according to the present invention has an effect of producing a significant amount of IFN-γ. It was.

SDS-PAGE
(1) Gel filtrationFor the fermented milk described in (1) of Example 1 (test solution 1), gel filtration was performed using a Superdex 75 gel filtration column, and the absorbance at 280 nm was determined for each fraction, and each fraction was determined for each fraction. The amount of IgM produced was determined in the same manner as in Example 1 above, and the fraction in which IgM production promoting activity was observed (Figure 7 showing the results of gel filtration (vertical axis: absorbance at 280 nm, horizontal axis: eluate volume ( mL)), the fractional portion enclosed with diagonal lines, and its IgM production promoting activity is 3.2 ± 0.3 ng / mL, which is approximately twice that of control 1.6 ± 0.6 ng / mL). As described above, SDS-PAGE was performed.

The fractions were collected for each peak of the 280 nm signal that had been eluted by gel filtration, and the measured IgM was measured using 10 mM sodium phosphate buffer (1 fraction approx. 2-5 mL). After dialysis against pH = 7.4), the procedure was as described in Example 1. The gel filtration conditions are as follows.
<Gel filtration conditions>
Equipment used: Pharmacia FPLC system Column: Pharmacia Superdex75 HR 10/30 Column Developing solvent: PBS
Flow rate: 1mL / min
conditions
(2) SDS-PAGE
The collected fractions were subjected to SDS-PAGE using a 15% polyacrylamide slab gel according to the method of Lamelli et al. (Laemmli, UK, et al., Nature, 227, 680 (1970)). The protein was stained with Coomassie Brilliant Blue R-250 (ICN Biomedicals). As molecular weight markers, Amersham Bioscience markers (phosphorylase b: 94.0 kDa, bovine serum albmin: 67.0 kDa, ovalbumin: 43.0 kDa, carbonic anhydrase: 30.0 kDa, soybean trypsin inhibitor: 20.1 kDa and α-lactalbumin: 14.4 kDa) was used.

  The results are also shown in FIG.

  As is apparent from FIG. 7 showing the results of SDS-PAGE, the fraction showing IgM production promoting activity contains a single protein with a molecular weight of 19.0 kDa, which is a protein newly produced by fermentation of milk. And is considered an active ingredient substance of the present invention.

Thermal stability The fermented milk emulsion (test solution 1) described in Example 1 (1) is maintained for 30 minutes at 20 ° C., 37 ° C., 50 ° C., 65 ° C., 80 ° C. and 100 ° C., respectively. After returning to room temperature, the respective IgM production promoting effects were measured in the same manner as in Example 1.

  The obtained results are shown in FIG. 8 (horizontal axis: treatment temperature (° C.), vertical axis: relative activity (%)).

In addition, relative activity% shows the percentage of IgM production of the fermented milk maintained under each temperature condition, where the IgM production of the fermented milk maintained at about 4 ° C. in an ice bath is taken as 100.
From the results shown in FIG. 8, the IgM production promoting action (activity) of fermented milk is thermally stable and is not reduced even by heat treatment at 65 ° C., but rather is promoted 2.5 times. This activity was completely inactivated by trypsin treatment as a result of a separate test.

Preparation of the immunopotentiating composition of the present invention (tablet form) After sterilizing skim milk, it was inoculated with a predetermined amount of starters 1, 2 and 3 prepared in Reference Example 1, and in the case of fermented milk 4 described in Table 1, Similarly, fermented milk (ripened complex fermented milk card) was produced by culturing for 2 weeks.

The resulting card is homogenized in water (using a high-pressure homogenizer, homogenization conditions: 100 kg / cm ² )
The obtained liquid was vacuum freeze-dried (using a vacuum freeze dryer, freeze-drying conditions: degree of vacuum = 0.5 torr or less, drying time: 24 hours), and then pulverized to obtain a freeze-dried powder.

  Subsequently, this powder was granulated (granulated), added with excipients, and tableted to prepare the composition of the present invention in tablet form.

  Table 4 below shows the results of analyzing the analytical value of this product in the same manner as in Reference Example 1.

  In addition, Table 5 below shows the results of determining the nutritional components (per 100 g) and energy of the obtained composition (tablet) of the present invention. The nutrient component was measured by the method described above and the following method.

Carbohydrate: 100- (water + protein + lipid + ash)
Non-fat milk solids: protein x 2.82
Minerals (sodium, potassium, calcium, iron): atomic absorption method,
Phosphorus: absorptiometry,
Vitamins: High performance liquid chromatographic method,
Cholesterol: gas chromatographic method

Preparation of lactic acid bacteria beverage After homogenizing fermented milk obtained by allowing complex lactic acid bacteria to act on skim milk (aged complex fermented milk card) in the same manner as in Example 5, appropriate amounts of sugar and flavor are obtained in the obtained homogeneous liquid Was added, and the preparation was sterilized to prepare a lactic acid fermented beverage (concentrated product diluted to be a beverage).

Preparation of hard yogurt Fermented milk (aged combined fermented milk card) obtained by allowing complex lactic acid bacteria to act on skim milk in the same manner as in Example 5, sterilized after adjusting pH, and yogurt mix was added to the obtained fermented milk Appropriate amounts of raw milk (including dairy products and stabilizers) were added, and the preparation was homogenized and sterilized as in Example 5 and inoculated with lactic acid bacteria starter to prepare hard yogurt.

  The present invention provides a composition having an IgM production promoting effect and a composition exhibiting an IFN-γ production promoting effect, and the composition of the present invention is unique not only to pharmaceuticals but also to food materials. It is useful in a wide range of fields.

2 is a graph showing the IgM production promoting effect of the composition of the present invention (fermented milk) in the test according to Example 1. FIG. 2 is a graph showing the IgM production promoting effect (time course) of the composition of the present invention (fermented milk) in the test according to Example 1. FIG. 2 is a graph showing the IgG production promoting effect of the composition of the present invention (fermented milk) in the test according to Example 1. 2 is a graph showing the IgG production promoting effect (time course) of the composition of the present invention (fermented milk) in the test according to Example 1. FIG. 3 is a graph showing the IFN-γ production promoting effect of the composition of the present invention (fermented milk) in the test according to Example 2. 4 is a graph showing the IFN-γ production promoting effect (time course) of the composition of the present invention (fermented milk) in the test according to Example 2. FIG. 4 is a diagram showing the results of SDS-PAGE analysis of the active ingredients of the composition of the present invention (fermented milk) in the test according to Example 3. 6 is a graph showing the thermal stability of the composition of the present invention (fermented milk) in the test according to Example 4.

Claims (2)

An IgM production promoter characterized by containing fermented milk as an active ingredient, wherein fermented milk individually inoculates raw milk with a plurality of types of lactic acid bacteria, and the inoculated one is the optimum growth temperature for each fungus after each individually appropriate incubation time in an environment, resulting to the culture solution individually appropriate amount collected and appropriate incubation time at a plurality of temperature environment different those inoculated collectively milk with yeast After that, each starter, which is a culture of lactic acid bacteria and yeast inoculated into milk in batches obtained for each temperature , is added to the milk at a predetermined ratio and cultured for a certain period of time in a constant temperature environment. It is intended to be extracted from,
The fermented milk is Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Cremoris, Streptococcus lactis subsp. Cremoris Streptococcus thermophillus, Leuconostoc cremoris, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus delbrucilii subsp. Lactobacillus casei), Lactobacillus helveticus, Bich An IgM production promoter, which is a culture product obtained by culturing milk using lactic acid bacteria and yeast consisting of Bifidobacterium longum and Saccharomyces cerevisiae . The IgM production promoter according to claim 1, wherein the fermented milk contains a 19 kDa protein that is a degradation product of milk protein.

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