JP2005220065A - Immunopotentiator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an immunopotentiator free from any side effects and having higher immunopotentiating ability. <P>SOLUTION: This immunopotentiator comprises, as active ingredients, a cultured product comprising β-1,3-1,6-glucan that is obtained by culturing fungi belonging to Aureobasidium sp. and lactobacillus. In this immunopotentiator, the fungi belonging to Aureobasidium sp. is preferably Aureobasidium pullulans M-1(FERM P-19213) and it is preferable that the lactobacillus is Enterococcus faecalis and has been subjected to heat sterilization. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to an immunostimulant that can improve immune function by ingestion, and specifically, a culture obtained by culturing a bacterium belonging to the genus Aureobasidium ( Aureobasidium sp.), And a lactic acid bacterium It is related with the immunostimulant which contains a body as an active ingredient.

  The immune system of the living body plays an important role as a defense mechanism against infection by microorganisms such as bacteria and viruses from the outside, and tumors generated in the living body. Since a decrease in immune function induces various diseases, it is considered important to improve the immune function from the viewpoints of disease prevention and health maintenance.

  Conventionally, β-glucan, lactic acid bacteria, and the like have been known as materials having an effect of improving immune function, that is, immunostimulatory activity, and active development of immunostimulants and health foods containing these as active ingredients has been active. Has been done.

For example, as an immunostimulator using β-glucan, the following Patent Document 1 mainly contains β-1,3-linked glucose residues obtained from the culture supernatant of Aureobasidium pullulans IFO4466 strain. As a chain, a highly branched β-glucan having a molecular weight of 10,000 to 5,000,000 having many branched chains of β-1,6-linked glucose residues as side chains is highly antitumor activity and immunostimulatory activity. It is described that it is useful as a medicine, food additive, feed additive and the like.

  Patent Document 2 below describes that an aureobasidium culture solution containing β-1,3-1,6 glucan as a main component can be applied as a medicine for various diseases.

On the other hand, as an immunostimulant using lactic acid bacteria, for example, Patent Document 3 below discloses an immunomodulator mainly composed of dead cells of Enterococcus faecalis AD101 strain.

  Patent Document 4 listed below discloses an immunostimulatory material characterized by using as an active ingredient a cell obtained by culturing lactic acid bacteria and koji molds belonging to the genus Enterococcus by liquid culture.

  Furthermore, as a combination of β-glucan and lactic acid bacteria, for example, the following Patent Document 5 includes a material containing β-glucan and a heat-treated cell of lactic acid-producing bacteria as active ingredients. An infection control composition is disclosed.

In addition, Patent Document 6 listed below promotes the production of cytokines including TNF in the body, enhances its action, and enhances antibody production ability or overall immune action, thereby preventing various infectious diseases and tumors. A useful low molecular weight water-soluble β-glucan is disclosed, and the combination of this β-glucan and lactic acid bacteria is described.
JP-A-6-340701 Japanese Patent Laid-Open No. 2002-204687 JP 2001-48796 A JP 2003-113114 A JP 2003-40785 A JP 2001-323001 A

  However, the conventional immunostimulants as described above have few side effects, but few are satisfactory in their effectiveness.

  Accordingly, an object of the present invention is to provide an immunostimulator having no side effects and having a superior immunostimulatory ability.

In order to achieve the above object, the immunostimulant of the present invention comprises a culture containing β-1,3-1,6-glucan obtained by culturing a bacterium belonging to the genus Aureobasidium ( Aureobasidium sp.), It contains lactic acid bacteria as an active ingredient.

The immunostimulant of the present invention contains a culture containing β-1,3-1,6-glucan obtained by culturing a bacterium belonging to the genus Aureobasidium ( Aureobasidium sp.), And lactic acid bacteria. Thus, an excellent immunostimulatory effect is obtained by the synergistic effect of these components. Β-1,3-1,6-glucan activates intestinal immunity of the small intestine, and lactic acid bacteria promotes the activation of macrophages, T cells, and B cells. It is thought that the activation effect is brought about.

In the immunostimulant of the present invention, the bacterium belonging to the genus Aureobasidium sp. Is preferably Aureobasidium pullulans M-1 (FERM P-19213). According to this aspect, β-1,3-1,6-glucan having higher physiological activity can be obtained.

  Moreover, it is preferable to contain 5-80 mass% of said cultures in conversion of (beta) -1,3-1,6-glucan and 10-80 mass% of said lactic acid bacteria in solid content.

  Furthermore, it is preferable that the culture has a solid concentration of 0.5 to 5% by mass and contains β-1,3-1,6-glucan in the solid content of 10% by mass or more.

Furthermore, the lactic acid bacterium is preferably Enterococcus faecalis .

  According to these aspects, a synergistic immunostimulatory effect by β-1,3-1,6-glucan and lactic acid bacteria can be expected more.

  Furthermore, it is preferable that the lactic acid bacteria are heat-sterilized. According to this aspect, an immunostimulant that can be widely added to foods and drinks that require heat treatment, has high storage stability, and is extremely safe when used as a raw material for foods and drinks or pharmaceuticals. Can be provided.

The immunostimulant of the present invention contains a culture containing β-1,3-1,6-glucan obtained by culturing a bacterium belonging to the genus Aureobasidium ( Aureobasidium sp.), And lactic acid bacteria. Thus, an excellent immunostimulatory effect is obtained by the synergistic effect of these components.

In the present invention, a culture obtained by culturing a bacterium belonging to the genus Aureobasidium ( Aureobasidium sp.) (Hereinafter simply referred to as culture) belongs to the genus Aureobasidium ( Aureobasidium sp.), Β- A culture solution in which a bacterium capable of producing 1,3-1,6-glucan is cultured, a concentrated solution of the culture solution, or a solid obtained by removing water from the culture solution can be used. The solution itself or a concentrated solution of the culture solution is used. In this case, the solid content concentration is preferably 0.5 to 5% by mass, and more preferably 1 to 3% by mass.

As a bacterium belonging to the genus Aureobasidium sp., For example, the strains described in JP-A-57-149301, JP-B-5-4063, JP-A-2002-335926 and the like are used. However, in the present invention, Aureobasidium pullulans M-1 (independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary No. FERM P-19213) is preferably used. In the present invention, β-1,3-1,6-glucan means one having a structure in which glucose is branched by a β-1,6 bond from a main chain in which glucose is β-1,3 bonded. .

Cultivation of the bacterium belonging to the genus Aureobasidium sp. Can be performed according to a known method (see JP-A-57-149301, etc.). That is, a medium (pH 5.2-6.0) containing 0.5 to 5.0% by mass of carbon source (sucrose), 0.1% by mass of N source, and other trace substances (for example, vitamins and inorganic substances). The inoculum is inoculated and aerated culture is performed at a temperature of 20 to 30 ° C. for 2 to 14 days, preferably aerated and stirred. As β-1,3-1,6-glucan is produced, the viscosity of the culture solution increases and becomes a highly viscous gel. The culture broth thus obtained usually contains 0.6 to 1.8% by mass of solid content, and 5-1, β-1,3-1,6-glucan is contained in the solid content. -80 mass% is contained. In addition to β-1,3-1,6-glucan, other useful components such as phosphorus, potassium, magnesium, and vitamin C, which are components that help the action of the glucan, are also included. The physiologically active effect of -1,3-1,6-glucan can be efficiently exhibited.

  In the present invention, a culture containing 10% by mass or more of β-1,3-1,6-glucan in the solid content is preferably used, and β-1,3-1,6-glucan in the solid content of 20%. A culture containing at least mass% is more preferably used. If the β-1,3-1,6-glucan concentration in the culture is too low, the bioactive effect of the glucan cannot be sufficiently expected.

Note that β-1,3-1,6-glucan can be quantified, for example, by the following method. That is, the culture solution is subjected to an enzyme treatment using amylase, amyloglucosidase, protease, or the like, and an α-glucan such as protein or pullulan is removed, followed by ethanol precipitation. Further, it is filtered through a glass filter to obtain a polymer sample. At this time, in order to remove low-molecular substances containing monosaccharides, the cells are thoroughly washed with 80% ethanol. The washed polymer sample is further washed with acetone, and sulfuric acid is added for hydrolysis. After hydrolysis, the solution is neutralized, the filtrate is collected, glucose is quantified by the glucose oxidase method, and the value calculated based on Equation 1 below is used as the glucan amount.
Formula 1: β-glucan (g / 100 g) = glucose (g / 100 g) × 0.9

  Further, β-1,3-1,6-glucan can be quantified according to the method described in Japanese Examined Patent Publication No. 3-48201. That is, after completion of the culture, the culture broth was sterilized and centrifuged to remove the cells, and 10% (v / v) chloroform / butanol mixture was added to the resulting solution and shaken (Sevage method). Thereafter, the mixture is centrifuged to remove chloroform and insoluble matters. After repeating this operation twice, the precipitate is recovered by ethanol precipitation and dissolved in distilled water, the pullulan is decomposed by enzyme treatment, dialyzed in distilled water, and the dialysate is ethanol precipitated to precipitate. The product (β-1,3-1,6-glucan) may be recovered to obtain the yield.

In the present invention, the culture solution obtained as described above may be used as it is by sterilization by heating or pressure heating, or may be used after sterilization after separating and removing cells by centrifugation or the like. Moreover, what was concentrated as needed and also what was dried can also be used. In addition, the culture of the bacterium belonging to the genus Aureobasidium sp. Is used as a food additive such as a thickening stabilizer and has high safety.

On the other hand, the lactic acid bacteria used in the present invention, can be used without any particular limitation as long as the lactic acid bacteria that can be used in food, specifically, Enterococcus faecalis (Enterococcus faecalis), Enterococcus faecium (Enterococcusfaecium), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacilluscasei), Streptococcus cremoris (Streptococcus cremoris), Streptococcus lactis (Streptococcuslactis), Streptococcus thermophilus (Streptococcus thermophilus), Bifidobacterium longum (BifidobacteriumLongum), Bifido bacterium breve (Bifidobacterium breve), Bifidobacterium bifidum (Bifidobacteriumbifidum) and the like. The said lactic acid bacteria may be used independently and may use 2 or more types together.

In addition, Enterococcus faecalis ( Enterococcus faecalis ) and Enterococcus faecium ( Enterococcus faecium ) are lactic acid bacteria used for lactic acid bacteria preparations, etc., Lactobacillus casei ( Lactobacillus casei), Lactobacillus acidophilus ( Lactobacillus acidophilus ), cheese is a lactic acid bacteria fermented milk, yogurt, have been used in lactic acid bacteria beverages, and the like, Streptococcus cremoris (Streptococcuscremoris), Streptococcus lactis (Streptococcus lactis), Streptococcus thermophilus (Streptococcusthermophilus) is, cheese, lactic acid bacteria are used in yogurt, etc. Bifidobacterium Longum, Bifidobacterium breve , Bifidobacterium bifidum ( Bifido bacteriumbifidum ) is a lactic acid bacterium used in fermented milk. Therefore, any of these lactic acid bacteria can be easily obtained by those skilled in the art.

In the present invention, among the above lactic acid bacteria, Enterococcus faecalis is particularly preferably used. Enterococcus faecalis (Enterococcus faecalis) are known to have a strong immunostimulatory activity, by combination with the above culture, synergistic with beta-1,3-1,6-glucan An immunostimulatory effect can be expected.

  In the present invention, the lactic acid bacteria are preferably heat-sterilized. Thereby, it can be widely added to foods and drinks that require heat treatment, has high storage stability, and is extremely safe when used as a raw material for foods and drinks and pharmaceuticals.

  The lactic acid bacteria may be cultured according to a conventional method. For example, cells are collected from a culture obtained by culturing the lactic acid bacteria according to a conventional method by a method such as filtration or centrifugation, washed with water, It is sufficient to suspend it in a heat treatment at 80 to 115 ° C. for 30 minutes to 3 seconds. The heat-sterilized lactic acid bacteria may be used after being concentrated and dried as necessary.

The immunostimulant of the present invention can be obtained, for example, by sterilizing a culture solution of a bacterium belonging to the above genus Aureobasidium sp. And mixing and dispersing the sterilized bacterial body of the lactic acid bacterium. it can. Moreover, it can also be set as various forms, such as a tablet, a capsule, a powder, a granule, a liquid form, a paste form, and a jelly form, as needed.

  The immunostimulant of the present invention contains 5 to 80% by mass of the above culture in terms of β-1,3-1,6-glucan and 10 to 80% by mass of the above lactic acid bacteria in the solid content. More preferably, the culture is contained in an amount of 25 to 70% by mass in terms of β-1,3-1,6-glucan, and more preferably 20 to 70% by mass of the lactic acid bacteria. Most preferably, the culture contains 30-60% by mass in terms of β-1,3-1,6-glucan, and 30-60% by mass of the lactic acid bacteria. In addition to the above basic components, fragrances, sweeteners, vitamins, minerals, oligosaccharides, thickening polysaccharides, dextrins, plant extracts, other plant components, and the like can be included as appropriate.

  The effective intake of the immunostimulant of the present invention is 0.02-0.50 g of the culture in terms of β-1,3-1,6-glucan and 0.10 of the lactic acid bacteria per adult day. To 0.90 g, preferably 0.06 to 0.40 g in terms of β-1,3-1,6-glucan and 0.15 to 0.45 g of the lactic acid bacteria. .

  Moreover, the immunostimulant of this invention can also be mix | blended with various food / beverage products, such as a soft drink, jelly drink, fruit juice drink, vegetable juice, soup, miso soup, frozen food, and other processed foods, for example. The addition amount of the present immunostimulant in each of the foods and drinks may be set based on the effective daily intake per adult, but is usually 0.02 in terms of β-1,3-1,6-glucan. 0.50 mass% is preferable and 0.06-0.40 mass% is more preferable. Moreover, about lactic acid bacteria, 0.10-0.90 mass% is preferable, and 0.15-0.45 mass% is more preferable. In addition, there is no restriction | limiting in particular in the addition method, It can also add from the beginning with the other raw material used for each food-drinks.

(1) Cultivation of Aureobasidium Aureobasidium pullulans M-1 (FERM P-19213) pre-cultured solution was prepared with 1% sucrose, 0.2% ascorbic acid, 0.2% rice bran An appropriate amount was inoculated into a liquid medium (pH 5.3) containing 2%, and aerated and agitated culture was performed at 25 ° C. for 2 days. After completion of the culture, this culture solution was sterilized at 121 ° C. for 15 minutes. This culture solution had a solid content of 1% by mass, and contained 35% by mass of β-1,3-1,6-glucan in the solid content.

(2) Enterococcus faecalis (Enterococcus faecalis) culturing Enterococcus faecalis (Enterococcus faecalis, IFO 16803) and, 37 ° C. in Rogosa medium, the preculture was incubated for 24 hours, yeast extract 4%, polypeptone 3%, lactose 10 An appropriate amount was inoculated into a liquid medium containing 1%, and neutralization culture was performed at 37 ° C. for 22 to 24 hours while adjusting with a sodium hydroxide aqueous solution to pH 6.8 to 7.0 using a pH stat.

After completion of the culture, the cells were separated and collected with a continuous centrifuge, then diluted with water to the original liquid volume, and again separated and collected with a continuous centrifuge. This operation was repeated a total of 4 times to wash the cells. Next, the washed cells are suspended in an appropriate amount of water, sterilized at 100 ° C. for 30 minutes, and then dried using a spray dryer to prepare a heat-sterilized cell powder (5 × 10 ¹² cfu / g). did.

(3) Preparation of immunostimulatory agent 1 L of aureobasidium culture obtained in (1) above is added with 10 g of heat-sterilized bacterial cells of lactic acid bacteria obtained in (2) above, and mixed uniformly to immunostimulate. An agent was obtained.

  Using the immunostimulant obtained in Example 1, the initial infection protective effect was examined by the following method.

(1) Measurement of survival rate 28 BALB / c mice (7 weeks old, female) that had been preliminarily bred for 1 week (purchased from Nippon SLC Co., Ltd.) were divided into 4 groups (7 mice for each group). The following test substances were orally administered to the group in an amount of 200 μl per mouse once daily during the test period.

Test group: The above immunostimulant Comparative group 1: Aureobasidium culture solution (obtained in Example 1 (1))
Comparative group 2: Heat-sterilized bacterial cells of lactic acid bacteria (obtained in Example 1 (2)) suspended in PBS (1 g bacterial cells / 100 ml)
Control group: PBS
Listeria monocytogenes (EGD strain) 5.4 × 10 ⁴ cfu / 200 μl / mouse (inside the tail vein of each group of mice administered orally for 1 week) 2 × LD ₅₀ ), followed by follow-up for 2 weeks, and the survival rate and average survival time of each group of mice were determined.

  The results are shown in Table 1 and FIGS. During the test period, water and feed (trade name “Powdered Feed CRF-1”, manufactured by Oriental Yeast Co., Ltd.) were freely ingested.

  As can be seen from Table 1 and FIGS. 1-3, in the test group, only one case died (6 days after inoculation), and the survival rate at the end of the test was 85.7%. In the control group, all cases died (4 days after bacterial inoculation) and the survival rate at the end of the study was 0%. In Comparative Group 1, 2 cases died (1 case on the 6th day after inoculation and 1 case on the 7th day), and the survival rate at the end of the test was 71.4%. In Comparative Group 2, 5 cases died (3 cases on the 4th day after bacterial inoculation and 2 cases on the 5th day), and the survival rate at the end of the test was 28.6%. When the significant difference between the test group and other groups was tested using the Mann-Whitney U test, the test group was significantly different from the control group and the comparison group 2 with a risk rate of less than 1%.

  The mean survival time was 12.7 days in the test group, whereas it was 3.0 days in the control group, 11.5 days in the comparison group 1, and 6.4 days in the comparison group 2. It was. When the significant difference between the test group and other groups was tested using the Mann-Whitney U test, the test group was significantly different from the control group and the comparison group 2 with a risk rate of less than 1%.

  From these results, it was suggested that oral ingestion of this immunostimulant enhances infection resistance of the host in bacterial infection.

(2) Measurement of the number of bacteria in the organ In the above (1), it was suggested that the present immunostimulant acts on the infection resistance against Listeria, so that The change was analyzed.

Thirty BALB / c mice (7 weeks old, female) that had been bred for one week were used per group, and each test substance was administered in the same manner as described above. In addition, 2.7 × 10 ³ cfu / 200 μl / mouse of Listeria monocytogenes (EGD strain), which is an intracellular parasitic bacterium, is placed in the tail vein of each group of mice that have been orally administered each test substance for one week. (1/10 × LD ₅₀ ) and 5 mice in each group were sacrificed every day on the 1st, 3rd, 5th, 7th and 10th days after bacterial inoculation The spleen was collected.

  The collected spleen was ground with a blender and resuspended in 5 ml of PBS to obtain a stock solution. This stock solution was diluted by a 10-fold serial dilution method, 100 μl was taken from the stock solution and each dilution series solution, smeared on TSA medium, and cultured in a 37 ° C. incubator for 16 hours. The number of bacterial colonies grown on the TSA medium was measured, and the number of bacteria in the organ was calculated. For the number of bacteria in the organ, an average value and a standard error were calculated for each group. The results are shown in Table 2 and FIG.

  As can be seen from Table 2, in the control group, the number of bacteria in the spleen peaked on the third day after bacterial inoculation, and then gradually decreased, and near the detection limit (3.75 ± 7.5 CFU / day) on the tenth day after inoculation. Spleen). On the other hand, in the test group, an increase in the number of bacteria was observed from the first day after the inoculation, but the number of bacteria on the third day of the inoculation was almost the same as that on the first day, and then decreased and 27 days on the tenth day of the inoculation. ± 25 CFU / spleen. In Comparative Group 1 and Comparative Group 2, an increase in the bacteria was observed from the first day after the inoculation, the peak of the number of bacteria in the spleen was observed on the third day after the inoculation, and then the bacteria gradually decreased. Was below the detection limit (<3.33 CFU / spleen).

  From this result, the present immunostimulant acts on the immune mechanism in the early stage of infection, and the enhancement of infection resistance is nonspecific by immature T cells or phagocytic cells rather than the aggressive immune mechanism by mature T cells. It was speculated that it might be carried out by the immune system.

(3) Analysis of cell surface molecule In (1) and (2) above, since differences in survival rate, survival period, and number of bacteria in the organ were observed, the flow of living body cells involved in infection resistance to Listeria Analysis with a cytometer was performed.

The mesenteric lymph node (MLN) was collected simultaneously with the measurement of the number of bacteria in the organ in (2) above. The collected MLN was crushed with a slide glass, excess tissue pieces were removed with a stainless mesh, and lymphocytes were resuspended in Hank's solution for FACS so that the concentration was 1 × 10 ⁶ / ml. Lymphocytes were stained with the four types of staining patterns shown in the following (a) to (d), and cell (T cell center) surface molecules were analyzed using a flow cytometer (Epics-XL; Beckman Coulter). . The results are shown in Table 3 and FIG.
(A) Cy-chrome (Cy) -labeled anti-CD3 mAb (T cell-specific recognition marker) / FITC-labeled anti-TCRαβ mAb (T-cell type-specific recognition marker) / PE-labeled anti-CD4 mAb / biotin-labeled anti-TCRγδ (T-cell type-specific recognition marker) mAb
(B) Cy-labeled anti-CD3 mAb / FITC-labeled anti-TCRαβ mAb / PE-labeled anti-CD4 mAb / biotin-labeled anti-CD69 mAb (early activation marker)
(C) Cy-labeled anti-CD3 mAb / FITC-labeled anti-TCRαβ mAb / PE-labeled anti-CD4 mAb / biotin-labeled anti-CD25 mAb (IL-2Rα; activation marker)
(D) Cy-labeled anti-CD3 mAb / FITC-labeled anti-TCRαβ mAb / PE-labeled anti-CD122 mAb (IL-2Rβ; activation marker) / biotin-labeled anti-CD4 mAb

  As shown in Table 3 and FIG. 5, the test group and comparative groups 1 and 2 showed an increase in CD4 positive αβ type T cells as compared to the control group. In particular, the rate of increase was higher in the test group than in Comparative Groups 1 and 2. In addition, in 3 days after fungal infection, all groups showed almost the same ratio. On the other hand, regarding the activation marker in CD4 positive cells, all of CD25 molecule (IL-2Rα), CD122 molecule (IL-2Rβ) and CD69 molecule (early activation marker) have almost the same expression level regardless of each group. there were.

  From this result, since no significant change in CD4 positive αβ type T cells was observed before and after infection in the test group, the enhancement of infection resistance by the immunostimulatory agent was due to the enhancement of the function of a cell group with low differentiation It was speculated.

  The cytokines in the blood were measured using a commercially available mouse interferon-γ (IFN-γ) measurement kit (manufactured by Genzyme, USA). As shown in Table 4, in the test group, IFN was observed on the third day of infection. -Γ amount showed a high value. Since no change in CD4 positive αβ type T cells was observed, it was considered that the cells producing IFN-γ could be macrophages.

  Since the immunostimulant of the present invention uses food-derived ingredients as active ingredients, it is highly safe and can be used not only as a pharmaceutical product but also as a health food material.

It is a graph which shows the relationship between the survival rate after Listeria monocytogenes intake, and elapsed days. It is a graph which shows the influence of the to-be-tested substance on the survival rate after inoculating Listeria monocytogenes. It is a graph which shows the relationship between the average survival days after Listeria monocytogenes intake, and a test substance. It is a graph which shows the influence of the to-be-tested substance on the time-dependent change of the number of bacteria in a spleen after listeria bacteria ingestion. It is a graph which shows the result of having analyzed the cell surface molecule | numerator using the flow cytometer after Listeria monocytogenes ingestion.

Claims (6)

It contains a culture containing β-1,3-1,6-glucan obtained by culturing bacteria belonging to the genus Aureobasidium ( Aureobasidium sp.) And lactic acid bacteria as active ingredients. Immunostimulator. The immunostimulant according to claim 1, wherein the bacterium belonging to the genus Aureobasidium sp. Is Aureobasidium pullulans M-1 (FERM P-19213).   The solid content contains 5 to 80% by mass of the culture in terms of β-1,3-1,6-glucan, and 10 to 80% by mass of the lactic acid bacteria. The immunostimulant described.   The culture according to claim 1, wherein the culture has a solid concentration of 0.5 to 5% by mass, and the solid content contains β-1,3-1,6-glucan of 10% by mass or more. The immunostimulant according to any one of the above. The lactic acid bacterium is Enterococcus faecalis (Enterococcus faecalis), immunostimulant according to any one of claims 1 to 4.   The immunostimulant according to any one of claims 1 to 5, wherein the lactic acid bacteria are heat-sterilized.