JP6651202B1 - A novel lactic acid bacterium belonging to Streptococcus salivarius and its use - Google Patents

Abstract

An object of the present invention is to provide Streptococcus salivarius which has a strong fermenting power for soy milk and solidifies soy milk in a short time. A lactic acid bacterium belonging to Streptococcus salivarius capable of coagulating 5 mL of a 10% soymilk-containing solution at 37 ° C. within 6 hours per 5 × 10 7 bacteria, and a fermentation obtained by fermenting a food material with the lactic acid bacterium. And a food or drink containing the lactic acid bacterium or fermented product, and a food material, wherein the lactic acid bacterium is inoculated and fermented. [Selection diagram] None

Description

  The present invention belongs to Streptococcus salivarius, a novel lactic acid bacterium having strong fermentative power against soy milk, food and drink containing the lactic acid bacterium, fermented product obtained by lactic acid bacterium, and food and drink containing the fermented product, The present invention relates to a method for producing a fermented product using lactic acid bacteria.

  It is said that there are more than 100 trillion bacteria in the human intestine and more than 1000 species. Bacteria that have the effect of maintaining and improving the intestinal environment are lactic acid bacteria called good bacteria. Lactic acid bacteria not only suppress the growth of Escherichia coli and pathogenic bacteria by producing lactic acid in the intestine, but also exhibit physiological effects such as improved bowel movements, protection against infection by increasing immunity, and antiallergic effects.

  With the expectation of such effects, various lactic acid bacteria fermented products have been developed to incorporate these useful lactic acid bacteria, called probiogenics, into the body and maintain daily health.

  The lactic acid bacteria used in such a lactic acid fermentation product are animal lactic acid bacteria suitable for fermentation of milk raw materials, and specifically, Lactococcus lactis, Lactobacillus vulgaris, Lactobacillus acidophilis, Streptococcus thermos. Such as Philis.

  On the other hand, there are people who have allergies to dairy products and indigestion due to dairy products, and it is difficult for such people to effectively ingest lactic acid bacteria from foods. There is a need to develop new lactic acid bacteria strains that can ingest lactic acid bacteria.

  Recently, plant lactic acid bacteria capable of fermenting plant components have attracted attention. It is said that plant lactic acid bacteria not only ferment plant components, but also survive and proliferate in an acidic region, reach the intestine alive, and have an effect of directly maintaining and improving the intestinal environment. So far, lactic acid bacteria capable of fermenting plant components using raw materials other than milk raw materials have been isolated, and lactic acid bacteria belonging to Lactobacillus plantarum are known as plant lactic acid bacteria capable of fermenting soy milk. For example, when Lactobacillus plantarum HOKKAIDO strain is fermented using soymilk as a fermentation substrate, it solidifies to a solid yogurt state, and this yogurt stimulates immune function and reduces stress markers (Non-Patent Document 1). Streptococcus salivarius has been found to be present in the oral cavity to reduce bad breath and prevent the growth of bad bacteria associated with caries and periodontal disease.

Nishimura, et al. J Tradit Complement Med. 2015 Aug 21; 6 (3): 275-80.

  However, Streptococcus salivarius which has a strong fermenting power for soy milk and coagulates soy milk in a short time has not been found.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, lactic acid bacteria belonging to Streptococcus salivarius isolated from cherry blossoms can ferment and coagulate soy milk more quickly than conventional lactic acid bacteria. The inventors have found that the present invention has been completed.

The present invention
It is a lactic acid bacterium capable of coagulating a 10% soymilk-containing solution at a concentration of 5 mL per 5 × 10 ⁷ bacteria at 37 ° C. within 6 hours.

  Further, the present invention is a fermented product obtained by fermenting a food material with the lactic acid bacterium.

  Further, the present invention is a food or drink containing the above fermented product.

  Still further, the present invention is a food or drink containing the lactic acid bacterium.

  Further, the present invention is a method for producing a fermented product, wherein a food material is inoculated with the lactic acid bacterium and fermented.

  The lactic acid bacterium of the present invention can ferment various plant components and soymilk, and can prepare food and drink such as beverages used for probiotics based on various fermentation substrates.

  Hereinafter, embodiments of the present invention will be described. The following embodiments are exemplifications, and the scope of the present invention is not limited to those shown in the following embodiments. In addition, in order to avoid repetitive complications about the same content, the description is omitted.

Definitions For convenience, certain terms used in the present application are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise stated in context, the singular forms “a”, “an”, and “the” include plural references.

  The numerical ranges and parameters given in the present invention are approximations, but the numerical values given in specific examples are described as accurately as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in each test measurement. Also, as used herein, the term "about" generally means within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error, as considered by one skilled in the art.

Lactic Acid Bacteria The lactic acid bacteria of the present embodiment can coagulate 5 mL of a 10% soymilk-containing solution at 37 ° C. within 6 hours per 5 × 10 ⁷ cells. The condition “5 mL of a 10% soymilk-containing solution at 37 ° C. per 5 × 10 ⁷ bacteria count” does not mean that the lactic acid bacterium of the present embodiment can coagulate soymilk only under these conditions. Means having the ability to coagulate the soymilk within 6 hours. Therefore, the coagulation time of the soymilk-containing solution varies depending on the number of lactic acid bacteria used, the concentration and amount of soymilk, and the temperature.

  In the present embodiment, the 10% soymilk-containing solution is an aqueous solution (medium: water) containing 10% (soybean solids) of soymilk. Soy milk is a liquid obtained by grinding soybeans swollen with water and boiled juice. In the present embodiment, “coagulation” refers to a gelled state, for example, 10% of 5 mL at the bottom of a γ-ray sterilized polypropylene centrifuge tube (ECK-15ML-R, manufactured by AS ONE Corporation) having an inner diameter of 15 mm. The state in which the soymilk does not flow out even if the soymilk-containing solution faces the mouth of the centrifuge tube downward.

  In the present embodiment, the lactic acid bacterium of the present embodiment can coagulate the soybean milk within 6 hours under the above conditions, and for example, a time of 30 minutes or more and 6 hours or less (eg, 30 minutes or more, 1 hour or more) , 2 hours or more, or 3 hours or more and 4 hours or less, 5 hours or less, or 6 hours or less).

  In one embodiment, the lactic acid bacterium is of the genus Streptococcus. In another embodiment, the lactic acid bacterium belongs to Streptococcus salivarius. In yet another embodiment, the lactic acid bacterium is a lactic acid bacterium deposited under accession number: NITE P-02785.

Fermented product obtained by fermentation of food material by lactic acid bacteria, food and drink containing the fermented product, and food and beverage containing lactic acid bacteria The fermented product of the present embodiment is obtained by fermentation of food material by the lactic acid bacteria. The food and drink of the present embodiment includes the fermented product. In the present embodiment, the food material may be any food material as long as it can be fermented by the lactic acid bacterium, and examples include soy milk, fruits and vegetables. In the case of producing a fermented soymilk beverage using the lactic acid bacteria, first, soymilk sterilized under conditions suitable for the soymilk to be used is inoculated and cultured with the lactic acid bacteria alone or simultaneously with other microorganisms, and homogenized. To obtain fermented soymilk. In the case of fruits or vegetables, fermented fruit juice or fermented vegetables can be obtained by the same method as for soymilk. These fermented products can be mixed with unfermented vegetable juice, unfermented juice, alcohol, and the like, and further added with various nutrients, vitamins, flavors, and the like to obtain a final product. These are preferable because they contain the lactic acid bacteria in a viable state.

  The food and drink of the present embodiment may be any form that can be orally ingested, such as a solution, a solid, and a powder, and is not particularly limited. Specific examples include drinks (lactic acid drinks, fruit drinks, soy milk drinks, vegetable drinks, tea drinks, carbonated drinks, nutritional drinks, sports drinks, coffee drinks, soups, alcoholic drinks, etc.), dairy products (yogurt, cheese, butter, Ice cream, etc.), flour products (bread, noodles, cake mix, etc.), confectionery (chocolate, cookies, candy, caramel, jelly, gum, Japanese confectionery, etc.), oil and fat foods (dressing, mayonnaise, cream, etc.), seasonings (source) , Tomato ketchup, vinegar, flavor seasoning, soup base, etc., instant food (instant noodles, instant soup, miso soup, canned food, retort food, etc.), supplements (tablets, syrups, granules, capsules, quick disintegrants) Etc.).

  The lactic acid bacterium of the present embodiment is used for various uses similarly to the lactic acid bacterium used in the conventional probiogenics, and by ingesting the lactic acid bacterium, a physiological effect such as an intestinal regulating action and an antibacterial action can be expected.

When the lactic acid bacterium of the present embodiment is consumed by humans or animals, the amount is not strictly limited, but a suitable intake is preferably 10 ⁶ CFU to 10 ¹⁴ CFU per serving as the number of viable bacteria. In addition, it is preferable that the lactic acid bacteria of the present embodiment be continuously taken, but the present invention is not limited to this. For example, the lactic acid bacteria may be taken every other week, every other day, or may be taken for a short period.

  In the food and drink and fermented products, various nutrients, various vitamins, various minerals, sweeteners, stabilizers such as emulsifiers, thickeners, dietary fiber, optional components such as flavors can be blended,

  Nutrients include DHA, EPA, acetylglucosamine, catechin, turmeric, propolis, agarisk, β-cryptoxanthin, quercetin, anthocyanins and the like. Examples of various vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, folic acid, nicotinamide, biotin, vitamin K, and the like. Various minerals include magnesium, calcium, zinc, manganese, iron, sodium, potassium and the like. Sweeteners include sucrose, glucose, fructose, trehalose, xylose, lactose, palatinose, fructose-glucose liquid sugar, maltose, fructose, honey and other sugars, sorbitol, xylitol, erythritol, lactitol, palatinit, aspartame, sucralose, stevia, acesulfame. Sweeteners with a high degree of sweetness such as K. Acidulants include citric acid, lactic acid, acetic acid, malic acid, tartaric acid, butyric acid and the like. Flavors include orange, citrus, berry, apple, mint, grape, apricot, perilla, lemon, grapefruit, peach, banana, tropical, herbal, coffee, and tea. And the like.

  In addition, when producing the fermented product or food or drink of the present embodiment, bacteria other than the lactic acid bacteria of the present embodiment can be used in combination. Examples of such fungi include Bifidobacterium genus bacteria such as Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus Brevis, Lactobacillus kefir, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus bulgaricus, Lactobacillus delbruchy, Lactobacillus pentosus, Lactobacillus plantarum, etc. Streptococcus bacteria such as Lactobacillus bacteria, Streptococcus thermophilis, Streptococcus salivarius, Lactococcus lactis subsp. Practices, Lactococcus bacteria such as Lactococcus lactis, subsp. Cremoris, Enterococcus bacteria such as Enterococcus faecalis, Saccharomyces cerevisiae, Saccharomyces genus such as Candida, kefir, yeast belonging to the genus Candida, and the like.

  Hereinafter, the content of the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Isolation and Culture Conditions of Lactic Acid Bacteria in Examples A novel lactic acid bacterium belonging to Streptococcus salivarius was isolated using the following sake cake filtration medium. Add 800 g of sake lees to 800 mL of distilled water, heat over low heat, stir well and dissolve by boiling. The mixture was removed from the heat, cooled to about 50 ° C., filtered with kitchen paper, adjusted to a Brix of 5.0, autoclaved at 121 ° C. for 15 minutes, and used as a sake cake filtration medium. Fungi adhering to the flowers of Yaezakura were brought into contact with a sake lees filtration medium, cultured at 30 ° C. for 24 hours, and the grown bacteria were isolated. After the isolation, bacteria that could be cultured in a Lactobacilli MRS Broth (manufactured by Difco) medium autoclaved at 121 ° C. for 15 minutes at 30 ° C. for 24 hours were used in subsequent experiments. Such bacteria formed circular white colonies in the Lactobacilli MRS Broth (manufactured by Difco) medium. Under microscopic observation, the bacterium was a staphylococci, had no motility, and did not form spores (not shown). The above bacterium showed a positive result in the Gram stain test, showed no activity in the catalase activity test, and did not observe gas generation (not shown).

Bacterial species identification using 16S rDNA nucleotide sequence The bacterial species of the above bacteria was identified by the following method based on the 16S rDNA nucleotide sequence. Using a DNA extracted from a cell pellet obtained by centrifuging a culture solution for culture cultured at 30 ° C. for 24 hours in an MRS medium as a template, the full length of the 16S rDNA sequence is amplified by PCR, and the nucleotide sequence of the amplified product is determined by Dye. It was determined by the Terminator method. The nucleotide sequence is shown in SEQ ID NO: 1. The obtained nucleotide sequence was searched in a database to identify the bacterial species. As a result, the 16S rDNA nucleotide sequence of the lactic acid bacterium was confirmed to be 99% identical to the 16S rDNA nucleotide sequence of Streptococcus salivarius and Streptococcus thermophilis, but Streptococcus salivarius differs from Streptococcus thermophilis, Since the milk could not be fermented, the bacterium was identified as a new strain of lactic acid bacterium belonging to Streptococcus salivarius and named as Streptococcus salivarius sakura 2 (hereinafter referred to as lactic acid bacterium in Examples) on September 19, 2018. Was deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center, located at 2-5-8 Kazusa-Kamashita, Kisarazu-shi, Chiba, Japan. The accession number is NITE P-02785.

  Table 1 shows the sugar assimilation properties of the lactic acid bacteria of the examples.

Culture conditions of lactic acid bacteria of comparative example Lactobacillus plantarum (Comparative Example 1), Lactobacillus sakei (Comparative Example 2), Leuconostoc mesenteroides (Comparative Example 3) and Streptococcus salivarius (Comparative Example) as lactic acid bacteria of Comparative Example 4), Streptococcus thermophilis (Comparative Example 5) was used. The lactic acid bacteria of Comparative Examples 1 to 3 were cultured at 30 ° C. for 24 hours using a Lactobacilli MRS Broth (manufactured by Difco) medium autoclaved at 121 ° C. for 15 minutes. The lactic acid bacteria of Comparative Examples 4 and 5 were cultured at 37 ° C. for 24 hours using a trypticase soy yeast extract medium recommended by NITE Biological Resource Center (NBRC) autoclaved at 121 ° C. for 15 minutes.

Number of bacteria used in soymilk fermentation test The culture solution of lactic acid bacteria cultured at 37 ° C for 24 hours is appropriately diluted with a sterilized diluent (0.1% yeast extract solution), applied to an MRS agar plate medium by a spiral system, and subjected to live bacteria. The number was measured. CFU / ml, which is a unit indicating the number of viable cells, shows the ability to form colonies when 1 ml of the culture plate is inoculated. The lactic acid bacteria cultured during that time were stored at 4 ° C., from which 5 × 10 ⁷ lactic acid bacteria were used as a single dose for fermenting soymilk.

Comparison of soymilk fermentation time between lactic acid bacteria of Example and lactic acid bacteria of comparative example (1) Strains 10 strains (3074 strains (Comparative Example 1-1) belonging to Lactobacillus plantarum of NBRC as lactic acid bacteria of Comparative Example 1 (control group), 14712 strains (Comparative Example 1-2), 101972 strains (Comparative Example 1-3), 15891 strains (Comparative Example 1-4), 3070 strains (Comparative Example 1-5), 12006 strains (Comparative Example 1-6), 14713 strains (Comparative Example 1-7), 101973 strains (Comparative Example 1-8), 101975 strains (Comparative Example 1-9), 109604 strains (Comparative Example 1-10), two strains belonging to Lactobacillus plantarum ( Lactobacillus plantarum HOKKAIDO (Comparative Example 1-11) and Lactobacillus plantarum SN13T (Comparative Example 1-12) were used.

  As lactic acid bacteria of Comparative Example 2 (control group), three strains belonging to Lactobacillus sakei of NBRC (3541 strains (Comparative Example 2-1), 107130 strains (Comparative Example 2-2), and 15893 strains (Comparative Example 2-3) )It was used.

  As lactic acid bacteria of Comparative Example 3 (control group), 7 strains (100495 strains (Comparative Example 3-1), 3832 strains (Comparative Example 3-2), 12060 strains (Comparative Example 3-3) belonging to NBRC Leuconostoc, 3349 strains (Comparative Example 3-4), 100496 strains (Comparative Example 3-5), 102481 strains (Comparative Example 3-6), and 102475 strains (Comparative Example 3-7) were used.

  As a lactic acid bacterium of Comparative Example 4 (control group), one strain belonging to Streptococcus salivarius of NBRC (13956 strain (Comparative Example 4)) was used.

  As lactic acid bacteria of Comparative Example 5 (control group), two strains belonging to Streptococcus thermophilis of NBRC (13957 strains (Comparative Example 5-1) and 111149 strains (Comparative Example 5-2)) were used.

(2) Inoculum and culture Lactobacillus MRS Broth (manufactured by Difco) sterilized at 121 ° C. for 15 minutes was subjected to γ-ray sterilization in a polypropylene centrifuge tube (ECK-15ML-R, manufactured by ASONE Corporation). Was inoculated and cultured at 30 ° C. for 24 hours until a steady state was reached. The lactic acid bacteria of Comparative Examples 4 to 5 were inoculated into a trypticase soybean extract medium which had been autoclaved at 121 ° C. for 15 minutes and dispensed into a γ-ray sterilized polypropylene centrifuge tube (ECK-15ML-R, manufactured by AS ONE). Then, the cells were cultured at 37 ° C. for 24 hours until a steady state was reached. The lactic acid bacterium (Streptococcus salivarius Sakura 2) of Example 1 was prepared by dissolving a Lactobacilli MRS Broth (manufactured by Difco) medium autoclaved at 121 ° C. for 15 minutes into a γ-ray-sterilized polypropylene centrifuge tube (ECK-15ML-R, Asone Corporation). Was inoculated and cultured at 30 ° C. for 24 hours until a steady state was reached.

(3) Fermentation of soy milk Commercially available 10% soy milk (manufactured by Sujata Meiraku) is sterilized at 90 ° C. for 30 minutes, and dispensed into a γ-ray sterilized polypropylene centrifuge tube (ECK-15ML-R, manufactured by AS ONE Corporation) in 5 ml portions. Then, 5 × 10 ⁷ lactic acid bacteria (lactic acid bacteria of Example 1 and Comparative Examples 1 to 5) were respectively added thereto, and the mixture was kept at 37 ° C. Thereafter, the coagulation time during which the soymilk fermented and solidified over time was examined. A total of five experiments were performed, three for each experiment, and the results for each of the 15 specimens are shown in Table 2.

  As shown in Table 2, the lactic acid bacterium of Example 1 (Leuconostoc) was the lactic acid bacterium of Comparative Example 3 having the strongest fermentative power in less than half the time of the lactic acid bacterium of Comparative Example 4 (conventional Streptococcus salivarius). (Mesenteroides) can be fermented and coagulated in about half the time, which proves that it has a very strong fermenting power for soymilk.

Claims (4)

Accession number: Lactic acid bacterium belonging to Streptococcus salivarius, deposited under NITE P-02785. Fermenting a food material with the lactic acid bacterium according to claim 1 ,
A step of including the fermented food material in food and drink,
A method for producing a food or drink containing a fermented product , comprising:   A food or drink containing the lactic acid bacterium according to claim 1.   A method for producing a fermented product, wherein a food material is inoculated with the lactic acid bacterium according to claim 1 and fermented.