Novel lactic acid bacteria culture medium

Abstract

To provide a medium that is composed of food material and suitable for proliferation of lactic acid bacteria.SOLUTION: A medium for lactic acid bacteria contains yeast extract and fermented barley extract.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a culture medium for lactic acid bacteria that is suitable for culturing lactic acid bacteria. [Background technology]

[0002] While MRS (deMan, Rogosa, Sharpe) medium is primarily used for culturing lactic acid bacteria (for example, Patent Document 1), MRS medium contains reagents that cannot be used as food ingredients, making it impossible to directly use the cultured lactic acid bacteria for food purposes. Although culture media for lactic acid bacteria composed of food ingredients have been reported, the growth rate of the lactic acid bacteria was sometimes insufficient.

[0003] In response to this, it is conceivable to add other food ingredients as nutrients to promote the growth of lactic acid bacteria. Meanwhile, awareness of food allergies has increased in recent years, and various food-derived allergens are known to cause them. Among these, based on the number of cases and severity, seven items, including eggs, milk, and wheat, are designated as specific raw materials that require labeling on food products. In addition, 21 items, including almonds, squid, and soybeans, are also designated as similar to specific raw materials based on the number of cases and severity, and labeling is recommended.

[0004] Lactic acid bacteria possess various physiological activities in addition to their intestinal regulating effects. These physiological functions are realized through the consumption of foods and beverages containing lactic acid bacteria. However, if the culture medium for lactic acid bacteria contains the specified allergens mentioned above, allergens may remain in the final product. Therefore, consumers with allergies to these allergens may not be able to enjoy the physiological benefits of lactic acid bacteria. For this reason, it is desirable to improve the growth potential of lactic acid bacteria using food ingredients other than the specified allergens. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2019-30292 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a culture medium composed of food materials that exhibits good growth characteristics for lactic acid bacteria. [Means for solving the problem]

[0007] As a result of diligent research to solve the above problems, the inventors of the present invention discovered that by adding yeast extract and fermented barley extract to the culture medium, the growth of lactic acid bacteria is improved, and the number of viable bacteria can be increased to a level that enables mass production, thus completing the present invention.

[0008] In other words, the present invention is a culture medium for lactic acid bacteria containing yeast extract and fermented barley extract.

[0009] Furthermore, the present invention is a method for culturing lactic acid bacteria, characterized by culturing the lactic acid bacteria in the above-mentioned culture medium for lactic acid bacteria.

[0010] Furthermore, the present invention relates to a lactic acid bacteria culture obtained by culturing lactic acid bacteria in the above-mentioned lactic acid bacteria culture medium.

[0011] Furthermore, the present invention relates to a food or beverage containing the above-mentioned lactic acid bacteria culture. [Effects of the Invention]

[0012] The lactic acid bacteria culture medium of the present invention is excellent at promoting the growth of lactic acid bacteria and has a high number of viable bacteria after cultivation. Furthermore, it can maintain a high number of viable bacteria even after freezing, making it excellent for cryopreservation.

[0013] Since the lactic acid bacteria culture medium of the present invention is composed of food materials, it can be used directly in various foods and beverages after cultivation. Furthermore, because lactic acid bacteria proliferate well even without the addition of specific raw materials, it is possible to provide lactic acid bacteria-containing foods and beverages or lactic acid bacteria-fermented foods and beverages that do not use specific raw materials. [Modes for carrying out the invention]

[0014] The lactic acid bacteria culture medium of the present invention (hereinafter referred to as "the culture medium of the present invention") contains yeast extract and fermented barley extract.

[0015] The above-mentioned yeast extract is not particularly limited as long as it is derived from yeast, but examples include yeast autodigestion fluid and yeast extract. The method for obtaining the yeast extract is not particularly limited, and examples include extraction with a solvent such as hot water or alcohol. The yeast extract may be yeast autodigestion fluid, the yeast extract itself, or concentrated liquids or powders thereof. The type of yeast is not particularly limited, and examples include brewer's yeast, baker's yeast, and wine yeast, but brewer's yeast is preferred. In addition, one or more types of these yeast extracts can be used. Among these yeast extracts, brewer's yeast extract is preferred. Examples of commercially available brewer's yeast extracts include Meast P1G (registered trademark, Asahi Beer Foods Co., Ltd.), Meast P2G (registered trademark, Asahi Beer Foods Co., Ltd.), and Yeast Extract B2 (Oriental Yeast Co., Ltd.).

[0016] The yeast extract content in the culture medium of the present invention is not particularly limited, but for example, it is 0.1 to 10% by mass (hereinafter, unless otherwise specified, "%" means "mass%") as yeast extract powder, preferably 0.5 to 5%.

[0017] The above-mentioned fermented barley extract is not particularly limited as long as it is an extract derived from fermented barley. For example, it could be the residue after distillation of alcoholic beverages such as shochu, which are obtained when fermenting barley to produce alcoholic beverages. Specifically, it could be the residue from barley shochu distillation. One or more types of these fermented barley extracts can be used. Among the fermented barley extracts, the residue from barley shochu distillation is preferred. Examples of commercially available barley shochu distillation residues include Barlex (registered trademark, Sanwa Shurui Co., Ltd.) and Barlex S (registered trademark, Sanwa Shurui Co., Ltd.).

[0018] The content of the fermented barley extract in the medium of the present invention is not particularly limited. For example, as the extract (liquid), it is 0.1 to 15%, preferably 1 to 10%.

[0019] From the viewpoint of the growth properties of lactic acid bacteria and the like, it is preferable to further add an emulsifier to the medium of the present invention. The emulsifier is not particularly limited. For example, polyglycerol fatty acid esters such as decaglycerin monooleate and decaglycerin monostearate can be mentioned. Further, one or more of these emulsifiers can be used. Among the polyglycerol fatty acid esters, hydrophilic polyglycerol fatty acid esters are preferable, and decaglycerin monooleate is particularly preferable. As decaglycerin monooleate, Sansoft Q-17S (registered trademark, Taiyo Chemical Co., Ltd.), NIKKOL DECAGLYN 1-OV (Nikken Chemicals Co., Ltd.) and the like are commercially available.

[0020] The content of the emulsifier in the medium of the present invention is not particularly limited. For example, it is 0.01 to 1%, preferably 0.05 to 0.5%.

[0021] From the viewpoint of the growth properties of lactic acid bacteria and the like, it is preferable to further add saccharides to the medium of the present invention. The saccharides are not particularly limited as long as they can be assimilated by lactic acid bacteria. For example, glucose, fructose, sucrose and the like can be mentioned, and one or more of these saccharides can be used. Among these saccharides, glucose is preferable. As glucose, D-(+)-glucose (Sigma-Aldrich Japan Co., Ltd.) and the like are commercially available.

[0022] The content of the saccharides in the medium of the present invention is not particularly limited. For example, it is 0.01 to 10%, preferably 0.05 to 5%.

[0023] Furthermore, the medium of the present invention may contain mineral components and components that do not inhibit the growth of lactic acid bacteria and improve operability (such as defoaming agents).

[0024] On the other hand, it is preferable that the culture medium of the present invention does not use specified raw materials such as lactose and other milk components, or soybean components such as soy milk, as food ingredients. Since lactic acid bacteria grow well in the culture medium of the present invention even without the use of specified raw materials, it is possible to manufacture food and beverages that do not use specified raw materials by not adding them. In this specification, specified raw materials refer to the seven specified raw materials (shrimp, crab, wheat, buckwheat, egg, milk, peanuts) and the 21 items equivalent to specified raw materials (almonds, abalone, squid, salmon roe, oranges, cashews, kiwifruit, beef, walnuts, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, gelatin) as defined in the Food Labeling Standards based on Article 4, Paragraph 1 of the Food Labeling Act (Act No. 70 of 2013).

[0025] Preferred embodiments of the culture medium of the present invention include the following: Yeast extract 0.1-10%, preferably 0.5-5% Fermented barley extract 0.1-15%, preferably 1-10% Emulsifier 0.01-1%, preferably 0.05-0.5% Sugars 0.01-10%, preferably 0.05-5% Water level remaining

[0026] The most preferred embodiment of the culture medium of the present invention has the following composition, and this culture medium is referred to as MBG medium. Yeast extract 2% Fermented barley extract 5% Decaglyceryl monooleate 0.1% Glucose 2% Water level remaining

[0027] The culture medium of the present invention can be prepared by dissolving the above components in water and performing pH adjustment, sterilization, etc. For pH adjustment, sodium hydroxide or the like may be added, with a preferred pH of 6 to 8 and a more preferred pH of 6.5 to 7.5.

[0028] Furthermore, the culture medium of the present invention can also be packaged as a culture medium kit by sealing all components other than water together. When using this kit, simply dissolve it in water as needed.

[0029] The culture medium of the present invention described above can be used for culturing lactic acid bacteria. The culture conditions for lactic acid bacteria using the culture medium of the present invention are not particularly limited, and ordinary culture conditions for lactic acid bacteria can be applied. The types of lactic acid bacteria that can be cultured in the culture medium of the present invention are not particularly limited, and examples include lactic acid bacteria of the genera Lactobacillus, Enterococcus, Lactococcus, Streptococcus, and Bifidobacterium. Among these lactic acid bacteria, Lactobacillus is preferred, and among Lactobacillus, Lactobacillus casei ( Lactobacillus cheese ), Lactobacillus crispatus ( Lactobacillus curly ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus delbrueckii subspecies bulgaricus ( Lactobacillus delbrueckii subsp. Bulgarian ), Lactobacillus helveticus ( Lactobacillus Swiss ), Lactobacillus delbrueckii subspecies lactis ( Lactobacillus delbrueckii subsp. milk ), Lactobacillus plantarum ( Lactobacillus plants ), Lactobacillus gasseri ( Lactobacillus gas station ), Lactobacillus assidypisis ( Lactobacillus sour fish ), Lactobacillus brevis ( Lactobacillus short ), Lactobacillus coliniformis ( Lactobacillus coryniform ), Lactobacillus delbrookii subspecies delbrookii ( Lactobacillus delbrueckii subsp. delbrueckii ), Lactobacillus kefiri ( Lactobacillus kefiri), Lactobacillus kephyranofaciens subspecies kephyranofaciens ( Lactobacillus kefir-making subsp. kefir-making ), Lactobacillus kephyranofaciens subspecies kephyrigranum ( Lactobacillus saffron-making subsp. kefir grain ), Lactobacillus nodensis ( Lactobacillus knotted ), Lactobacillus parabrevis ( Lactobacillus brief ), Lactobacillus paracasei ( Lactobacillus cheese ), Lactobacillus parakefiri ( Lactobacillus parakefir ), Lactobacillus pentosa ( Lactobacillus penitent ), Lactobacillus perolens, Lactobacillus rhamnosus ( Lactobacillus buckthorn ), Lactobacillus salivarius ( Lactobacillus salivary ), Lactobacillus tucceti, Lactobacillus curvatus ( Lactobacillus bent ), Lactobacillus johnsonii ( Lactobacillus johnson's ), Lactobacillus fermentum ( Lactobacillus leaven ), Lactobacillus mari ( Lactobacillus evil ) are more preferable, and one or more of these can be cultured. Among these, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus brevis, Lactobacillus plantarum, etc. are even more preferable, and Lactobacillus casei is particularly preferred. Examples of Lactobacillus casei include Lactobacillus casei YIT9029 and Lactobacillus casei YIT0180. TThese are some examples, with Lactobacillus casei YIT9029 being particularly preferred. Lactobacillus casei YIT9029 was deposited by the applicant as FERM BP-1366 on May 18, 1987, at the National Institute of Microbial Science, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (Note that the aforementioned National Institute of Microbial Science is now the Patent Organism Depositary Center of the National Institute of Technology and Evaluation, and has moved to a new address of Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818).

[0030] A lactic acid bacteria culture can be obtained by culturing lactic acid bacteria in the culture medium of the present invention. The culture temperature can be set appropriately depending on the type of lactic acid bacteria, but 30 to 37°C is preferred. Since the culture medium of the present invention is composed of food materials, this lactic acid bacteria culture can be incorporated into food and beverages, etc., either as is or after processing such as concentration, dilution, or drying.

[0031] The foods and beverages that can be formulated with the above-mentioned lactic acid bacteria culture are not particularly limited, but examples include processed meat products such as ham and sausage, processed seafood products such as kamaboko and chikuwa, bread, confectionery, butter, yogurt and fermented milk, soft drinks, dairy lactic acid bacteria beverages, and lactic acid bacteria beverages. The food and beverages can be in the form of commonly used foods and beverages, such as solids like powders and granules, pastes, and liquids. They may also be processed into tablets, powders, chewable tablets, hard capsules, soft capsules, pills, gums, etc. Furthermore, they can be used as oral compositions in mouthwashes, toothpastes, toothpaste powders, toothpaste liquids, oral ointments, gels, tablets, granules, fine granules, gummy jellies, lozenges, tablets, capsules, candies, chewing gums, etc., and can also be used in pet food, etc.

[0032] The cultured lactic acid bacteria can be dispersed in a cryoprotective agent and frozen for storage. For example, they can be stored as frozen cells such as glycerol stocks, which are frozen after being mixed with a glycerin solution, or as freeze-dried cells. Lactic acid bacteria cultured using the culture medium of the present invention do not experience a decrease in viable cell count even after freezing, and have a high survival rate, making them suitable for use as a starter in the production of various lactic acid bacteria fermented foods and beverages. For example, by culturing them in a culture medium containing milk components such as skim milk powder or soy milk, fermented milk beverages and fermented soy milk can be produced. Furthermore, even in culture media that do not use these milk or soy components, good growth is observed, making it possible to produce lactic acid bacteria fermented foods and beverages that are free of milk and / or soy components. Moreover, by culturing them in a culture medium that does not contain specific raw materials, not limited to milk and soy components, it becomes possible to produce lactic acid bacteria fermented foods and beverages that do not use specific raw materials. Examples of culture media other than specific raw materials include pineapple juice, mango juice, mandarin orange juice, tomato juice, green juice, and carrot juice.

[0033] The method for producing frozen or freeze-dried lactic acid bacteria cells is not particularly limited, and known methods can be used as appropriate. However, a preferred method involves dispersing lactic acid bacteria cells cultured using the culture medium of the present invention into a dispersion medium, followed by freezing or freeze-drying.

[0034] The dispersion medium used to disperse the lactic acid bacteria cells can be appropriately selected according to the type of lactic acid bacteria, but it is preferable to use an aqueous solution containing a cryoprotectant, for example. The method of dispersing the lactic acid bacteria cells in the dispersion medium is not particularly limited; the lactic acid bacteria cells or culture can be added to the dispersion medium and stirred, etc. Also, the amount of lactic acid bacteria cells to be dispersed in the dispersion medium is not particularly limited, but for example, 1.0 × 10⁻⁶ 5 ~1.0×10 10 The concentration is approximately (cfu / ml). While the lactic acid bacteria dispersed are primarily live cells, dead cells may also be included.

[0035] The cryoprotectant used in the dispersion medium is not particularly limited and includes, for example, glycerin, glutamic acid, glutamic acid salts such as sodium glutamate and potassium glutamate, disaccharides such as trehalose, sucrose, lactose and maltose, maltodextrin, cyclodextrin, potato starch, dairy products, DMSO, etc. One or more of these cryoprotectants can be used, and the use of glycerin is preferred. The content of the cryoprotectant in the dispersion medium is not particularly limited, but the final concentration is preferably 1 to 25%, more preferably 5 to 20%, and particularly preferably 8 to 15%.

[0036] Antioxidants can be added to the dispersion medium, such as ascorbic acid, ascorbic acid salts such as sodium ascorbate and calcium ascorbate, vitamin E, catechin, glutathione, and astaxanthin. One or more of these antioxidants can be used. The content of the antioxidant in the dispersion medium is not particularly limited, but for example, 0.01 to 5% is preferred, and 0.05 to 1% is more preferred.

[0037] As described above, frozen lactic acid bacteria can be obtained by dispersing the lactic acid bacteria cells or culture in a dispersion medium and then rapidly freezing them, for example, in a dry ice ethanol bath.

[0038] On the other hand, freeze-dried bacterial cells can be prepared by dispersing lactic acid bacteria cells in a dispersion medium, then performing a freeze treatment at, for example, -35°C to -45°C for 6 to 48 hours, followed by a drying treatment at 12°C to 35°C for 40 to 90 hours to perform freeze-drying. Examples of freeze-dryers include the TF20-80TANNS (manufactured by Takara Seisakusho Co., Ltd.). Flash freezing can also be performed using a dry ice ethanol bath or freezing blocks.

[0039] The lactic acid bacteria frozen or freeze-dried cells thus obtained can maintain a high number of viable cells even after storage. For example, it is preferable that the survival rate, calculated as (number of viable cells after freezing / number of viable cells before freezing) × 100, be 75% or higher, and more preferably 90% or higher. [Examples]

[0040] The present invention will be described in detail below with reference to examples, but the present invention is not limited in any way to these examples.

[0041] Example 1 Preparation of culture media and cultivation of lactic acid bacteria: The components listed in Table 1 below were mixed and dissolved in water, then the pH was adjusted to 6.7 with sodium hydroxide, and the mixture was sterilized to prepare a culture medium (hereinafter referred to as "MBG medium"). To this medium, 1% of cryopreserved Lactobacillus casei YIT9029 (hereinafter referred to as "LcS") was added (resulting in a viable cell count of 1.0 × 10⁶). 5 ~1.0×10 7 The cells were inoculated with approximately (cfu / ml) and incubated at 37°C for 24 hours. The results of measuring the viable cell count (cfu / ml) after incubation are shown in Table 1. As a criterion for good growth, a viable cell count of 5.0 × 10⁶ was used. 8 (cfu / ml) or higher.

[0042] [Table 1]

[0043] MBG medium containing yeast extract and fermented barley extract showed good growth potential, and the number of viable cells after culturing reached high levels.

[0044] Example 2 Preparation and evaluation of glycerol stocks: (1) Preparation of glycerol stock of bacterial cells or culture medium The culture medium of LcS pre-cultured in MBG medium was inoculated at 0.1% into MBG medium and incubated at 37°C for 24 hours (main culture). The culture medium was then centrifuged, the supernatant was removed to obtain bacterial cells, and these cells were suspended in sterile glycerol to a final concentration of 12.5% ​​(bacterial cell sample). Furthermore, the ice-cooled culture medium was collected and mixed with sterilized glycerol to a final concentration of 12.5% ​​(culture medium sample). These were dispensed into 1 ml portions into cryotubes and rapidly frozen in a dry ice ethanol bath. The resulting glycerol stock was stored at -70°C.

[0045] (2) Preservation test The viable cell count was measured for the two types of glycerol stocks obtained in (1). The viable cell count was measured after diluting the glycerol stocks with physiological saline (0.85% NaCl solution) and plating them on MRS agar (BD, Difco). TM Lactobacilli (MRS agar) was coated using a spiral plater EDDY JET2 (IUL Instruments) and cultured aerobically at 37°C for at least 2 days. The number of colonies obtained was measured using a colony counter (Qcount). The number of viable cells after this culture and before freezing was measured in the same manner. The survival rate (%) was calculated using the following formula. (survival rate) Survival rate (%) = (Number of viable bacteria after freezing / Number of viable bacteria before freezing) × 100

[0046] [Table 2]

[0047] The growth of LcS in this culture was good. Furthermore, no decrease in viable cell count was observed in the glycerol stock after freezing, confirming a high survival rate.

[0048] Implementation Example 3 Preparation and evaluation of glycerol stocks using bacterial species other than Lcs: (1) Preparation of glycerol stock of bacterial cells In the same manner as in Example 2, Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus brevis ( Lactobacillus short ), Lactobacillus plantarum ( Lactobacillus plants A glycerol stock of the bacterial cells was prepared.

[0049] (2) Preservation test Using the same method as in Example 2, the number of viable cells in glycerol stocks of bacterial cells before and after freezing was examined, and the survival rate was determined. Only Lactobacillus gasseri was cultured under anaerobic conditions.

[0050] [Table 3]

[0051] Glycerol stocks of bacterial species other than LcS also showed no significant decrease in viable cell count after freezing, confirming a high survival rate.

[0052] Implementation Example 4 Evaluation of the culture properties of glycerol stock as a starter culture: A 10% skim milk powder medium was prepared using skim milk powder (Snow Brand Co., Ltd.). A soy milk medium was also prepared by diluting adjusted soy milk (Yakult Honsha Co., Ltd.) three-fold and adding 1% glucose (Fuso Pharmaceutical Industries, Ltd.). The glycerol stock of the bacterial cells and culture solution prepared in Example 2 was dissolved at room temperature, inoculated at 0.1% into the skim milk powder medium or soy milk medium, stirred, and cultured at 37°C. The culture was performed in two parallel cultures (Culture (1) and (2)). The culture time was 48 hours for the skim milk powder medium and 24 hours for the soy milk medium. After culturing and after storage at 4°C for a predetermined number of days, the number of viable cells, acidity, and pH were measured as follows. The results for the skim milk powder medium are shown in Tables 3 (after culturing) and 4 (after 14 days of storage at 4°C), and the results for the soy milk medium are shown in Tables 5 (after culturing) and 6 (after 10 days of storage at 10°C). (Measurement of viable bacteria count) The culture was stirred well, diluted with physiological saline, and then spread on MRS agar medium (manufactured by BD) using a spiral plater EDDY JET2 (IUL Instruments). This was cultured aerobically at 37°C for 2 days or more. The obtained colonies were measured using a colony counter (Qcount), and the number of colonies per milliliter (cfu) was determined. The criteria for good or poor growth were 1.0×10 9 (cfu / ml) or more in skim milk powder medium and 1.0×10 9 (cfu / ml) or more in soy milk medium. (Acidity measurement) After stirring each sample well, 6 g was taken and RO water (27.3 ml) was added to prepare a sample for acidity measurement (solid content concentration 18.02%). Using an acidity titration device (COM-1700, manufactured by Hiranuma Sangyo), the amount (ml) of 0.1 N NaOH used until reaching pH 8.5 was measured. The obtained value was converted to per 9 g of the sample and taken as the acidity (ml / 9 g). The criteria for good or poor growth were 8.5 (ml / 9 g) or more in skim milk powder medium and 8.5 (ml / 9 g) or more in soy milk medium. (pH measurement) After stirring the culture well, it was measured using a portable pH meter LaquaAct (manufactured by HORIBA).

[0053] Skim milk powder medium, after culture

Table 4

[0054] Skim milk powder medium, after storage at 4°C for 14 days

Table 5

[0055] Soy milk medium, after culture

Table 6

[0056] Soy milk medium, after storage at 4°C for 10 days

Table 7

[0057] The glycerol stocks of the bacterial cells and culture medium showed good culture characteristics in both skim milk powder medium and soy milk medium, demonstrating their suitability as starters. Similar good culture characteristics were observed when using media containing pineapple juice or mandarin orange juice instead of skim milk powder medium. [Industrial applicability]

[0058] The culture medium of the present invention is suitable for culturing lactic acid bacteria and can be used in various foods and beverages containing lactic acid bacteria.

Claims

1. A culture medium for lactic acid bacteria containing yeast extract, fermented barley extract, decaglycerin monooleate, and glucose, wherein the lactic acid bacteria are Lactobacillus casei or Lactobacillus gasseri.

2. The culture medium for lactic acid bacteria according to claim 1, wherein the yeast extract is brewer's yeast extract.

3. The culture medium for lactic acid bacteria according to claim 1 or 2, wherein the fermented barley extract is the residual liquid from the distillation of barley shochu.

4. A culture medium for lactic acid bacteria according to any one of claims 1 to 3, which does not contain milk components.

5. A culture medium for lactic acid bacteria according to any one of claims 1 to 4, which does not contain soybean components.

6. A culture medium for lactic acid bacteria according to any one of claims 1 to 5, for the cryopreservation of lactic acid bacteria.

7. A method for culturing lactic acid bacteria, characterized by culturing lactic acid bacteria in a lactic acid bacteria culture medium according to any one of claims 1 to 6.

8. A lactic acid bacteria culture obtained by culturing lactic acid bacteria in a lactic acid bacteria culture medium according to any one of claims 1 to 6.

9. Food and beverages containing the lactic acid bacteria culture described in claim 8.

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