Lactic acid bacteria composition and method for producing fermented milk

Abstract

The lactic acid bacterium composition is characterized in that the lactic acid bacterium composition contains lactobacillus delbrueckii subsp. Bulgaricus, streptococcus thermophilus which carries a prtS gene (Streptococcus thermophilus), and streptococcus thermophilus which does not carry a prtS gene (Streptococcus thermophilus), and the lactic acid bacterium composition is also characterized in that the lactic acid bacterium composition is prepared from the following raw materials: lactobacillus delbrueckii subsp. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), streptococcus thermophilus (Streptococcus thermophilus) which carries the prtS gene (Streptococcus thermophilus).

Description

Technical Field

[0001] The present invention relates to a lactic acid bacteria composition and a method for producing fermented milk. Background Art

[0002] Fermented milk is defined in the "Ministerial Ordinance on the Standards of Composition of Milk and Dairy Products (Ministerial Ordinance on Milk, etc.)" of Japan as "a paste-like or liquid-like substance obtained by fermenting milk or milk containing an equal or higher amount of non-fat milk solids with lactic acid bacteria or yeast, or a frozen product thereof". Representative examples of fermented milk include set yogurt (solid fermented milk), soft yogurt (paste-like fermented milk), and drinkable yogurt (liquid fermented milk). It should be noted that, for example, in the Codex Standard (FAO (Food and Agriculture Organization of the United Nations) / World Health Organization (WHO)), a food standard commonly used by the international community, yogurt is defined as "a substance obtained by lactic acid fermentation of two bacteria, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus". Conventionally, yogurt generally refers to a substance obtained by adding these two lactic acid bacteria as starter to raw milk and fermenting lactose in the raw milk, and is characterized in that it has a sour taste mainly due to the lactic acid produced in the above fermentation.

[0003] However, lactic acid bacteria such as thermophilic Streptococcus can also produce lactic acid by fermentation at low temperatures. Therefore, the fermented products after the above fermentation (such as fermented milk such as yogurt) have the following problem: when stored, even under low temperature conditions, the amount of lactic acid produced is too excessive and the acidity increases (that is, the pH decreases), which damages its flavor.

[0004] Therefore, some research has been conducted to suppress the problem of acid generation of such lactic acid bacteria at low temperatures. For example, Japanese Patent Publication No. 7-236416 (Patent Document 1) records: In order to obtain fermented milk with less increase in acidity under low temperature storage, an acid generation inhibition strain of Lactobacillus delbrueckii subspecies bulgaricus and a viscous substance production strain of Streptococcus salivarius thermophilus subspecies are used. In addition, Japanese Patent Publication No. 2000-270844 (Patent Document 2) records: a low-temperature sensitive lactic acid strain belonging to Streptococcus thermophilus, which has excellent acid generation ability in the temperature range of 37 to 43°C and poor acid generation ability in the temperature range of 15 to 25°C. Furthermore, Japanese Patent Application Laid-Open No. 2005-21050 (Patent Document 3) describes a highly acid-producing Streptococcus thermophilus KT01 bacterium that exhibits excellent proliferation in a yogurt mix containing milk as a main raw material and has a final lactic acid acidity of 0.9 mass % or more and less than 1 mass % when cultured at 37° C. for 3 days.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 7-236416

[0008] Patent Document 2: Japanese Patent Application Publication No. 2000-270844

[0009] Patent Document 3: Japanese Patent Application Publication No. 2005-21050 Summary of the invention

[0010] Problem that the invention aims to solve

[0011] In the past, in order to suppress acid production at low temperatures, the method of finding a specific strain and using the strain as mentioned above was mainly adopted, but this method is very laborious. In addition, the flavor and physical properties of fermented milk mainly depend on the characteristics of each lactic acid bacteria. Therefore, the fermented milk that needs to be limited to a combination of specific strains as in the above-mentioned prior art can only obtain fermented milk with limited flavor and physical properties. In addition, the present inventors conducted further research and found that, for example, when using thermophilic streptococci with low acid production, such as in the above-mentioned prior art, there is the following problem: although the acid production of the obtained fermented milk at low temperatures is reduced, the pH drop and the acidity increase are suppressed, it takes time to complete the fermentation (for example, the pH reaches 4.65). In addition, it was found that there is the following problem: when the fermented milk is stored at low temperatures, the number of live bacteria of Lactobacillus delbrueckii subspecies bulgaricus decreases, while simply increasing the number of live bacteria of Lactobacillus delbrueckii subspecies bulgaricus (for example, 1×10 7 cfu / g or more), the acidity of the fermented milk increases and the flavor is impaired.

[0012] The present invention has been made in view of the problems of the above-mentioned prior art, and its object is to provide fermented milk in which the number of live bacteria of Lactobacillus delbrueckii subsp. bulgaricus is sufficiently maintained even when stored at low temperature, the decrease in pH and the increase in acidity during the above-mentioned low-temperature storage are suppressed, and the fermented milk has smooth physical properties, as well as a lactic acid bacteria composition and a method for producing fermented milk that can obtain the fermented milk in a sufficiently short fermentation time.

[0013] Solutions for solving problems

[0014] The present inventors have conducted in-depth research to achieve the above-mentioned purpose, and as a result, it has been found that by combining Lactobacillus delbrueckii subspecies Bulgaria with thermophilic streptococci carrying the prtS gene as thermophilic streptococci and thermophilic streptococci not carrying the prtS gene, the fermented milk obtained can maintain the viable count of Lactobacillus delbrueckii subspecies Bulgaria even when stored at low temperature. It has also been found that even if the viable count of Lactobacillus delbrueckii subspecies Bulgaria is large as described above, the pH drop and acidity increase during the above-mentioned low-temperature storage are suppressed, and smooth physical properties are achieved. It has also been found that according to the combination, by combining two lactic acid bacteria, Lactobacillus delbrueckii subspecies Bulgaria and thermophilic streptococci not carrying the prtS gene, it is also possible to achieve a shortened fermentation time that is difficult to achieve, and obtain the above-mentioned fermented milk by a sufficiently short fermentation time. Moreover, as the above-mentioned three lactic acid bacteria, as long as it is the above-mentioned combination, it is not limited to a specific strain, for example, lactic acid bacteria with various characteristics such as high-polysaccharide-producing lactic acid bacteria can be used, so it is easy to adjust the flavor and physical properties of the fermented milk, thereby completing the present invention.

[0015] The embodiments of the present invention obtained based on the above findings are as follows. [1]

[0017] A lactic acid bacteria composition contains Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus carrying a prtS gene, and Streptococcus thermophilus not carrying a prtS gene. [2]

[0019] The lactic acid bacteria composition according to [1], wherein the content of Lactobacillus delbrueckii subsp. bulgaricus to the total content of Streptococcus thermophilus carrying the prtS gene and Streptococcus thermophilus not carrying the prtS gene is 1:1 to 1:150 in a mass ratio. [3]

[0021] The lactic acid bacteria composition according to [1] or [2], wherein the content of thermophilic Streptococcus carrying the prtS gene and the content of thermophilic Streptococcus not carrying the prtS gene are in a mass ratio of 1:0.05 to 1:10. [4]

[0023] The lactic acid bacteria composition according to any one of [1] to [3], which is a composition that is added to a prepared milk liquid containing raw material milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the number of viable cells of Lactobacillus delbrueckii subspecies bulgaricus in the fermented composition after 30 days of storage reaches 1×10 7 cfu / g or above. [5]

[0025] The lactic acid bacteria composition according to any one of [1] to [4], which is a composition that is added to a prepared milk liquid containing raw milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the number of live cells of Lactobacillus delbrueckii subsp. bulgaricus in the fermented composition after 30 days of storage reaches at least 10% of the number of live cells of Lactobacillus delbrueckii subsp. bulgaricus in the fermented composition after 1 day of storage. [6]

[0027] The lactic acid bacteria composition according to any one of [1] to [5], wherein when added to a prepared milk liquid containing raw material milk and fermented at 43° C., the pH of the prepared milk liquid reaches 4.65 within 5 hours from the start of the fermentation. [7]

[0029] The lactic acid bacteria composition according to any one of [1] to [6], which is a composition as follows: when added to a prepared milk liquid containing raw milk and fermented at 43°C, the time when the pH of the prepared milk liquid reaches 4.65 is less than 99% of the time when the pH of the prepared milk liquid reaches 4.65 when a lactic acid bacteria composition containing the above-mentioned Lactobacillus delbrueckii subsp. bulgaricus and the above-mentioned thermophilic Streptococcus not carrying the prtS gene but not the above-mentioned thermophilic Streptococcus carrying the prtS gene is fermented under the same conditions. [8]

[0031] The lactic acid bacteria composition according to any one of [1] to [7], which is a composition that is added to a prepared milk liquid containing raw milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the pH of the fermented composition after 30 days of storage is 4.1 or above. [9]

[0033] The lactic acid bacteria composition according to any one of [1] to [8], which is a composition that is added to a prepared milk liquid containing raw milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the acidity of the fermented composition after 30 days of storage is less than 1.0%.

[10]

[0035] The lactic acid bacteria composition according to any one of [1] to [9], which is a composition that is added to a prepared milk liquid containing raw material milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the acidity (D1%) of the fermented composition after one day of storage and the acidity (D2%) of the fermented composition after 30 days of storage are 30 %) satisfies the following formula: D 30 -The conditions shown in D1≤0.25.

[11]

[0037] The lactic acid bacteria composition according to any one of [1] to

[10] , which is a composition as follows: when it is added to a modulated milk liquid containing raw milk and fermented at 43°C until the pH of the above-mentioned modulated milk liquid reaches 4.65, when the fermented composition is stored at 10°C for 30 days, the content of Lactobacillus delbrueckii subsp. bulgaricus in the above-mentioned fermented composition stored for 1 to 30 days and the content of thermophilic Streptococcus carrying the prtS gene and thermophilic Streptococcus not carrying the prtS gene in the above-mentioned fermented composition after storage for 1 to 30 days are 1:2 to 1:150 in terms of the ratio of the number of viable bacteria.

[12]

[0039] The lactic acid bacteria composition according to any one of [1] to

[11] is a composition as follows: when it is added to a modulated milk liquid containing raw milk and fermented at 43°C until the pH of the above-mentioned modulated milk liquid reaches 4.65, when the fermented composition is stored at 10°C for 30 days, the content of thermophilic Streptococcus carrying the prtS gene in the above-mentioned fermented composition stored for 1 day to 30 days and the content of thermophilic Streptococcus not carrying the prtS gene in the above-mentioned fermented composition after storage for 1 day to 30 days reaches 1:0.005 to 1:30 in terms of the ratio of the number of viable bacteria.

[13]

[0041] The lactic acid bacteria composition according to any one of [1] to

[12] , wherein Lactobacillus delbrueckii subspecies bulgaricus and / or Streptococcus thermophilus not carrying the prtS gene is a lactic acid bacterium that produces high polysaccharide yields.

[14]

[0043] The lactic acid bacteria composition according to any one of [1] to

[13] , which is a lactic acid bacteria starter seed.

[15]

[0045] The lactic acid bacteria composition according to any one of [1] to

[13] , which is fermented milk.

[16]

[0047] A lactic acid bacteria combination product comprises Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus carrying a prtS gene, and Streptococcus thermophilus not carrying a prtS gene.

[17]

[0049] The lactic acid bacteria combination product according to

[16] is a lactic acid bacteria starter.

[18]

[0051] A method for producing fermented milk, comprising a fermentation step of adding the lactic acid bacteria composition described in any one of [1] to

[15] or the lactic acid bacteria combination described in

[16] or

[17] to a prepared milk liquid containing raw material milk and fermenting the mixture to obtain fermented milk.

[0052] Effects of the Invention

[0053] According to the present invention, there can be provided fermented milk having smooth physical properties, in which the number of live bacteria of Lactobacillus delbrueckii subspecies bulgaricus is sufficiently maintained even when stored at low temperatures (e.g., 10° C.), the decrease in pH and the increase in acidity during the above-mentioned low-temperature storage are suppressed, and a lactic acid bacteria composition and a method for producing fermented milk that can obtain the fermented milk in a sufficiently short fermentation time can be provided. DETAILED DESCRIPTION

[0054] The present invention will be described in detail below based on preferred embodiments of the present invention.

[0055] (Lactic acid bacteria)

[0056] In the present invention, "lactic acid bacteria" is a general term for microorganisms that assimilate glucose and produce lactic acid at a yield of 50% or more relative to sugar, and as physiological properties, they have the following characteristics: they are Gram-positive bacteria, cocci or bacilli, non-motile, in most cases non-spore-forming (there are also lactic acid bacteria with spore-forming ability such as Bacillus coagulans), catalase-negative, etc. The lactic acid bacteria of the present invention are lactic acid bacteria belonging to Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus.

[0057] (Lactobacillus delbrueckii subsp. bulgaricus)

[0058] Lactobacillus delbrueckii subsp. bulgaricus is a subspecies of lactic acid bacteria belonging to the genus Lactobacillus, which belongs to the family Lactobacillaceae. In the present specification, Lactobacillus delbrueckii subsp. bulgaricus may be referred to as "bulgaricus".

[0059] Examples of the Bulgarian bacteria of the present invention include Lactobacillus delbrueckii subspecies bulgaricus OLL1171 specified by accession number NITE BP-01569 (hereinafter sometimes referred to as “OLL1171 strain”), Lactobacillus delbrueckii subspecies bulgaricus OLL1073R-1 specified by accession number FERM BP-10741 (hereinafter sometimes referred to as “OLL1073R-1 strain”), Lactobacillus delbrueckii subspecies bulgaricus OLL205013 specified by accession number NITE BP-02411 (hereinafter sometimes referred to as “OLL205013 strain”), Lactobacillus delbrueckii subspecies bulgaricus OLL1247 specified by accession number NITE BP-01814 (hereinafter sometimes referred to as “OLL1247 strain”), and Lactobacillus delbrueckii subspecies bulgaricus OLL1247 specified by accession number NITE BP-01815 (hereinafter sometimes referred to as “OLL1247 strain”). Strains include, but are not limited to, Lactobacillus delbrueckii subsp. bulgaricus OLL1251 specified in BP-02703 (hereinafter sometimes referred to as "OLL1251 strain") and Lactobacillus delbrueckii subsp. bulgaricus 1589 specified in NITE BP-03716 (hereinafter sometimes referred to as "1589 strain").

[0060] The OLL1171 strain is marked with (1) identification: Lactobacillus delbrueckii subsp.bulgaricusOLL1171, (2) deposit number: NITE BP-01569, (3) deposit date: March 13, 2013, the OLL1073R-1 strain is marked with (1) identification: Lactobacillus delbrueckii subsp.bulgaricus OLL1073R-1, (2) deposit number: FERM BP-10741, (3) deposit date: February 22, 1999, and the OLL205013 strain is marked with (1) identification: Lactobacillus delbrueckii subsp.bulgaricus OLL205013, (2) deposit number: NITE BP-02411, (3) Deposit date: February 3, 2017, OLL1247 strain is identified by (1) identification mark: Lactobacillusdelbrueckii subsp.bulgaricus OLL1247, (2) Deposit number: NITE BP-01814, (3) Deposit date: March 6, 2014, OLL1251 strain is identified by (1) identification mark: Lactobacillus delbrueckiisubsp.bulgaricus OLL1251, (2) Deposit number: NITE BP-02703, (3) Deposit date: April 25, 2018, 1589 strain is identified by (1) identification mark: Lactobacillus delbrueckii subsp.bulgaricus 1589, (2) Deposit number: NITE No. BP-03716, (3) Deposit date: August 9, 2022, the strain indicated by "NITE" is deposited at (4) Depository: National Institute of Technology for Evaluation of Products Patent Microorganisms Depository (Zip Code: Room 122, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan 292-0818), and the strain indicated by "FERM" is deposited at (4) Depository: National Institute of Technology for Evaluation of Products Patent Microorganisms Depository (Zip Code: Room 120, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan 292-0818; among them, the OLL1073R-1 strain was deposited at the National Institute of Advanced Industrial Science and Technology Patent Organisms Depository (Zip Code: 305-8566, Tsukuba City, Ibaraki Prefecture, Japan 1-1-1, Higashi-1-chō-dai 6) at the time of deposit. It should be noted that the Bulgarian bacteria specified by these accession numbers may be subculture strains of the strains, or artificial mutants, natural mutants, genetically recombinant strains, or derivatives of the strains or their subcultures within the range that does not inhibit the effects of the present invention.

[0061] In addition, as the Bulgarian bacteria of the present invention, it can be, for example, Bulgarian bacteria isolated from commercially available fermented milk using a known agar medium for Lactobacillus (e.g., MRS agar medium, plate count agar medium with BCP added). As such Bulgarian bacteria, for example, Bulgarian bacteria 2038 strains isolated from "Meiji Bulgarian Yogurt (registered trademark), manufactured by Meiji Co., Ltd." using a plate count agar medium with BCP added to form a spindle-shaped colony are kept by Meiji Innovation Center of Meiji Co., Ltd. (1-29-1, Shichikoku, Hachioji City, Tokyo, Japan, postal code 192-0919).

[0062] 〔High-polysaccharide-producing Bulgarian bacteria〕

[0063] As the Bulgarian bacteria of the present invention, Bulgarian bacteria having various characteristics can be used, and for example, lactic acid bacteria that produce high polysaccharides are preferred from the viewpoint of increasing the viscosity of fermented milk. In the present specification, "lactic acid bacteria that produce high polysaccharides" refers to lactic acid bacteria that produce a large amount of exopolysaccharides (EPS).

[0064] Whether Bulgaria is a high-yield polysaccharide lactic acid bacteria can be confirmed by the method described in the following examples, for example. That is, for example, Bulgaria activated as needed (for example, the culture solution after activating the frozen strain twice in a 10w / v% skim milk powder medium in the form of 1 platinum loop / 5mL, inoculated in a 10w / v% skim milk powder medium in the form of 1w / w%, and cultured at 37°C for 18 hours), the amount of extracellular polysaccharide (EPS) in the obtained culture solution is measured, and the amount can be determined as a high-yield polysaccharide lactic acid bacteria. The amount of extracellular polysaccharide (EPS) can be appropriately measured by existing known methods, for example, the following method can be cited: after dissolving the components in 4mL of the above-mentioned culture solution with trichloroacetic acid, the polysaccharide is recovered by ethanol precipitation, and dialyzed with ultrapure water as needed, and the amount of polysaccharide thus obtained is measured by the phenol·sulfuric acid method. For example, as the amount of polysaccharide, when the amount of polysaccharide (mg / kg) in the culture solution measured by the above method is 70 mg / kg or more, 105 mg / kg or more, 116 mg / kg or more, or 120 mg / kg or more, it can be determined as a high-polysaccharide-producing lactic acid bacteria. In addition, for example, when the amount of polysaccharide (mg / kg) measured by the above method exceeds the amount of extracellular polysaccharide (EPS) in the culture solution obtained by culturing the above-mentioned Bacillus bulgaricus 2038 strain, more specifically, exceeds 1.0 times or 1.1 times the amount of extracellular polysaccharide (EPS) in the culture solution obtained by culturing the above-mentioned Bacillus bulgaricus 2038 strain, it can be determined as a particularly high-polysaccharide-producing lactic acid bacteria.

[0065] Specific examples of Bacillus bulgaricus belonging to such high-polysaccharide-producing lactic acid bacteria include, but are not limited to, OLL1251 strain, OLL1073R-1 strain, and OLL1247 strain.

[0066] As the Bulgarian bacteria of the present invention, one species may be used alone or two or more species may be used in combination.

[0067] (Streptococcus thermophilus)

[0068] Streptococcus thermophilus is a streptococcus also called a thermophilic bacterium, and is a lactic acid bacterium that can produce lactic acid from lactose. In this specification, lactic acid bacteria belonging to Streptococcus thermophilus may be referred to as "Streptococcus thermophilus".

[0069] As the thermophilic Streptococcus of the present invention, it is necessary to combine those carrying the prtS gene and those not carrying the prtS gene. In the present invention, the "prtS gene" refers to a gene encoding a cell wall-bound serine protease that can decompose casein. In addition, in the present invention, whether the thermophilic Streptococcus carries the prtS gene can be confirmed by the method described in the following examples, for example. That is, for example, a highly conservative sequence is selected among the prtS genes of thermophilic Streptococcus that can be obtained from public databases (Genbank, etc.), and a primer set prepared based on the sequence is used to amplify a part of the prtS gene by PCR, and the desired PCR product is obtained to determine. As the above-mentioned primer set, for example, a primer set including the F primer (sequence number: 1) and the R primer (sequence number: 2) described in the following examples can be listed, but it is not limited to this.

[0070] [Thermophilic Streptococcus carrying the prtS gene (prtS+)]

[0071] Examples of thermophilic Streptococcus carrying the prtS gene of the present invention include, but are not limited to, thermophilic Streptococcus OLS4801 specified by Accession No. NITE BP-03504 (hereinafter sometimes referred to as “OLS4801 strain”), thermophilic Streptococcus OLS4802 specified by Accession No. NITE BP-03505 (hereinafter sometimes referred to as “OLS4802 strain”), thermophilic Streptococcus OLS4803 specified by Accession No. NITE BP-03506 (hereinafter sometimes referred to as “OLS4803 strain”), thermophilic Streptococcus OLS4823 specified by Accession No. NITE BP-03507 (hereinafter sometimes referred to as “OLS4823 strain”), and thermophilic Streptococcus OLS4824 specified by Accession No. NITE BP-03508 (hereinafter sometimes referred to as “OLS4824 strain”).

[0072] The OLS4801 strain is marked with (1) identification: Streptococcus thermophilus OLS4801, (2) deposit number: NITE BP-03504, (3) deposit date: August 4, 2021, the OLS4802 strain is marked with (1) identification: Streptococcus thermophilus OLS4802, (2) deposit number: NITE BP-03505, (3) deposit date: August 4, 2021, the OLS4803 strain is marked with (1) identification: Streptococcus thermophilus OLS4803, (2) deposit number: NITE BP-03506, (3) deposit date: August 4, 2021, and the OLS4823 strain is marked with (1) identification: Streptococcus thermophilus OLS4823, (2) deposit number: NITE BP-03507, (3) deposit date: August 4, 2021, OLS4824 strain with (1) identification mark: Streptococcus thermophilus OLS4824, (2) deposit number: NITE BP-03508, (3) deposit date: August 4, 2021, respectively deposited in (4) deposit institution: Patent Microorganism Depository of the Independent Administrative Institution Product Evaluation Technology Foundation (Room 122, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, 292-0818, Japan). It should be noted that the thermophilic Streptococcus specified by these deposit numbers may also be a subculture strain of the strain or an artificial mutant strain, natural mutant strain, genetically recombinant strain or derivative strain of the strain or its subculture strain within the range that does not inhibit the effect of the present invention.

[0073] The thermophilic Streptococcus carrying the prtS gene of the present invention may be, for example, thermophilic Streptococcus isolated as large yellow colonies from commercially available fermented milk using an agar medium for Lactococcus (e.g., fast-slow differential agar (FSDA, hereinafter sometimes referred to as "FS agar medium")). The FS agar medium is preferably a plate medium prepared as follows: Solution A: 3.8 g of disodium glycerophosphate pentahydrate (final concentration 1.9 w / w%), 0.008 g of bromocresol purple (final concentration 0.004 w / w%), 3 g of agar (final concentration 1.5 w / w%), and 120 g of ultrapure water and Solution B: 20 ​​g of skim milk powder (final concentration 10 w / w%), and 80 g of ultrapure water are prepared separately, sterilized (for example, solution A is sterilized by autoclave at 121° C. for 7 minutes, and solution B is sterilized by autoclave at 110° C. for 10 minutes), and then cooled (for example, to about 55° C.), and solution A and solution B are mixed so that the concentration of each component becomes the above-mentioned final concentration. For example, the culture in the FS agar medium is preferably prepared by diluting 10% with physiological saline. 6 100 μL of the sample was applied to the surface and cultured at 37°C for 24 hours.

[0074] As the thermophilic Streptococcus carrying the prtS gene of the present invention, one species may be used alone or two or more species may be used in combination.

[0075] [Thermophilic Streptococcus without the prtS gene (prtS-)]

[0076] Examples of thermophilic Streptococcus that do not carry the prtS gene of the present invention include, for example, thermophilic Streptococcus OLS3078 specified by deposit number NITE BP-01697 (hereinafter sometimes referred to as "OLS3078 strain"), thermophilic Streptococcus OLS3615 specified by deposit number NITE BP-01696 (hereinafter sometimes referred to as "OLS3615 strain"), and thermophilic Streptococcus OLS3290 specified by deposit number FERM BP-19638 (hereinafter sometimes referred to as "OLS3290 strain"), but are not limited to these.

[0077] The OLS3078 strain is marked with (1) identification: Streptococcus thermophilus OLS3078, (2) deposit number: NITE BP-01697, (3) deposit date: August 23, 2013. The OLS3615 strain is marked with (1) identification: Streptococcus thermophilus OLS3615, (2) deposit number: NITE BP-01696, (3) deposit date: August 23, 2013. The OLS3290 strain is marked with (1) identification: Streptococcus thermophilus OLS3290, (2) deposit number: FERM BP-19638, (3) Deposit date: January 19, 2004, strains indicated by "NITE" are deposited at (4) Depository: National Institute for Product Evaluation and Technology, Patent Microorganism Collection Center (Zip Code: 292-0818, Room 122, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan), strains indicated by "FERM" are deposited at (4) Depository: National Institute for Product Evaluation and Technology, Patent Microorganism Collection Center (Zip Code: 292-0818, Room 120, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan). It should be noted that the thermophilic Streptococcus specified by these deposit numbers may be a subculture strain of the strain or an artificial mutant, natural mutant, genetically recombinant strain, or derivative strain of the strain or its subculture within the range that does not inhibit the effect of the present invention.

[0078] In addition, as the thermophilic streptococcus that does not carry the prtS gene of the present invention, it can be, for example, a thermophilic streptococcus isolated in the form of translucent small and flat colonies using agar medium for the genus Lactococcus (e.g., FS agar medium) from commercially available fermented milk. As such a thermophilic streptococcus that does not carry the prtS gene, for example, the thermophilic streptococcus 1131 strain isolated from "Meiji Bulgarian Yogurt (registered trademark) LB81, manufactured by Meiji Co., Ltd." using M17 agar medium, plate count agar with added BCP, or FS agar medium is kept by Meiji Innovation Center of Meiji Co., Ltd. (postal code 192-0919, Shichikoku 1-29-1, Hachioji City, Tokyo, Japan). It should be noted that when isolating the thermophilic streptococcus 1131 strain using a plate count agar medium with added BCP, a spherical colony is isolated.

[0079] As the thermophilic Streptococcus not carrying the prtS gene of the present invention, one species may be used alone or two or more species may be used in combination.

[0080] 〔High-polysaccharide-producing Streptococcus thermophilus〕

[0081] As the thermophilic streptococcus of the present invention, in particular, the thermophilic streptococcus not carrying the prtS gene, thermophilic streptococcus having various characteristics can be used. For example, from the viewpoint of being able to increase the viscosity of fermented milk, the above-mentioned high-polysaccharide-producing lactic acid bacteria are preferred.

[0082] Whether Streptococcus thermophilus is a high-yield polysaccharide lactic acid bacteria can be confirmed by the method described in the following examples, for example. That is, for example, the thermophilic Streptococcus thermophilus activated as needed is cultured (for example, the frozen strain is activated twice in a 10w / v% skim milk powder-0.1w / v% casein decomposition peptide medium in the form of 1 platinum ring / 5mL, and the culture solution is inoculated in a 10w / v% skim milk powder-0.1w / v% casein decomposition peptide medium in the form of 1 platinum ring / 5mL, and is statically cultured at 43°C for 4 hours), and the amount of extracellular polysaccharide (EPS) in the obtained culture solution is measured. If the amount is large, it can be determined as a high-yield polysaccharide lactic acid bacteria. The amount of extracellular polysaccharide (EPS) can be appropriately measured by existing known methods, for example, the following method can be cited: after the components in 10g of the above-mentioned culture solution are dissolved with trichloroacetic acid, the polysaccharide is recovered by ethanol precipitation, and it is dialyzed with ultrapure water as needed, and the amount of polysaccharide thus obtained is measured by the phenol·sulfuric acid method. For example, as the above-mentioned polysaccharide amount, when the polysaccharide amount (mg / kg) in the above-mentioned culture solution measured by the above-mentioned method is 3 mg / kg or more, 12 mg / kg or more, 13 mg / kg or more, or 14 mg / kg or more, it can be judged as a high-polysaccharide-producing lactic acid bacteria. In addition, for example, when the polysaccharide amount (mg / kg) measured by the above-mentioned method exceeds the amount of extracellular polysaccharide (EPS) in the culture solution obtained by culturing the above-mentioned thermophilic Streptococcus 1131 strain, more specifically exceeds 1.0 times or 1.1 times the amount of extracellular polysaccharide (EPS) in the culture solution obtained by culturing the thermophilic Streptococcus 1131 strain, it can be judged as a particularly high-polysaccharide-producing lactic acid bacteria.

[0083] Specific examples of thermophilic Streptococcus belonging to such high-polysaccharide-producing lactic acid bacteria include OLS3290 strain and OLS3078 strain, but are not limited thereto.

[0084] (Lactic acid bacteria composition, lactic acid bacteria combination product)

[0085] The lactic acid bacteria composition of the present invention contains Bulgaria, Streptococcus thermophilus carrying the prtS gene, and Streptococcus thermophilus not carrying the prtS gene. In addition, when the lactic acid bacteria composition of the present invention is the following lactic acid bacteria fermentation seed, the lactic acid bacteria composition can be a lactic acid bacteria combination product (i.e., a lactic acid bacteria fermentation seed kit) comprising Bulgaria, Streptococcus thermophilus carrying the prtS gene, and Streptococcus thermophilus not carrying the prtS gene. In this case, the combination ratio and total usage of Bulgaria, Streptococcus thermophilus carrying the prtS gene, and Streptococcus thermophilus not carrying the prtS gene are preferably respectively equivalent to the mass ratio or viable count ratio and total content or total viable count in the following lactic acid bacteria composition of the present invention.

[0086] The present inventors speculate that in the lactic acid bacteria composition of the present invention, these three types of lactic acid bacteria (Bulgaricus, thermophilic Streptococcus carrying the prtS gene, and thermophilic Streptococcus not carrying the prtS gene) have acquired a new symbiotic relationship, and the live cell count of Bulgaricus is fully maintained even when stored at low temperature, the pH drop and acidity increase during the above-mentioned low-temperature storage are also suppressed, and fermented milk with smooth physical properties can be obtained in a sufficiently short fermentation time.

[0087] In the lactic acid bacteria composition of the present invention, the content of Bacillus bulgaricus (the content of live bacteria of Bacillus bulgaricus, the same below) is preferably 0.5 to 30% by mass, more preferably 0.8 to 15% by mass, and even more preferably 1 to 10% by mass, relative to the total mass (total mass) of the live bacteria of the three types of lactic acid bacteria.

[0088] In the lactic acid bacteria composition of the present invention, the content of thermophilic Streptococcus carrying the prtS gene (the content of live bacteria of thermophilic Streptococcus carrying the prtS gene, the same below) is preferably 15 to 80 mass %, more preferably 18 to 80 mass %, and further preferably 20 to 75 mass % relative to the total mass (total mass) of the live bacteria of the three types of lactic acid bacteria.

[0089] In the lactic acid bacteria composition of the present invention, the content of thermophilic Streptococcus not carrying the prtS gene (the content of live bacteria of thermophilic Streptococcus not carrying the prtS gene, the same below) is preferably 10 to 75% by mass, more preferably 10 to 72% by mass, and further preferably 10 to 70% by mass, relative to the total mass (total mass) of the live bacteria of the three types of lactic acid bacteria.

[0090] In the lactic acid bacteria composition of the present invention, the content of the above-mentioned Bacillus bulgaricus, the content of Streptococcus thermophilus carrying the prtS gene, and the content of Streptococcus thermophilus not carrying the prtS gene (the content of Bacillus bulgaricus: the content of Streptococcus thermophilus carrying the prtS gene: the content of Streptococcus thermophilus not carrying the prtS gene) can be appropriately combined, for example, with respect to the total mass (total mass) of the live bacteria of the three kinds of lactic acid bacteria, 0.5 to 30 mass %: 15 to The combination of 80 mass %: 10-75 mass %, the combination of 0.8-15 mass %: 18-80 mass %: 10-72 mass %, the combination of 1-10 mass %: 20-75 mass %: 10-70 mass %, the combination of 0.8-15 mass % or 1-10 mass %: 15-48 mass %: 48-75 mass %, the combination of 0.8-15 mass % or 1-10 mass %: 48-75 mass %: 15-48 mass %.

[0091] More preferably, in the lactic acid bacteria composition of the present invention, the ratio of the content of Bacillus bulgaricus to the total content of Streptococcus thermophilus carrying the prtS gene and the content of Streptococcus thermophilus not carrying the prtS gene is preferably 1:1 to 1:150, more preferably 1:2 to 1:120, and even more preferably 1:5 to 1:100 in terms of mass ratio (mass of live bacteria of Bacillus bulgaricus: total mass of live bacteria of Streptococcus thermophilus).

[0092] Furthermore, in the lactic acid bacteria composition of the present invention, the ratio of the content of thermophilic Streptococcus carrying the prtS gene to the content of thermophilic Streptococcus not carrying the prtS gene, measured as a mass ratio (mass of live bacteria of thermophilic Streptococcus carrying the prtS gene: mass of live bacteria of thermophilic Streptococcus not carrying the prtS gene), is preferably 1:0.05 to 1:10, more preferably 1:0.075 to 1:8, and even more preferably 1:0.1 to 1:6.

[0093] The lactic acid bacteria composition of the present invention may further contain other ingredients in addition to the three lactic acid bacteria of the present invention. The above-mentioned other ingredients are not particularly limited and include, for example, solvents such as water; cultures such as culture supernatants and culture medium components after activation and culture of lactic acid bacteria; fermented compositions after fermentation of the lactic acid bacteria composition; concentrates, dilutions, dried products, frozen products, etc. of the above-mentioned cultures and fermented compositions, which may be one or a combination of two or more of these.

[0094] In addition, when being a lactic acid bacteria combination product, as 3 kinds of lactic acid bacteria of the present invention, it can be a composition containing 1 or 2 kinds of these, in which case, other components other than the lactic acid bacteria can also be contained in the above-mentioned composition. As the above-mentioned other components, other components similar to those mentioned above can be enumerated.

[0095] 〔Fermentation time〕

[0096] The lactic acid bacteria composition of the present invention (comprising the following lactic acid bacteria starter and fermented milk, preferably the lactic acid bacteria starter) preferably satisfies the following condition (b):

[0097] Condition (b): when added to a prepared milk liquid containing raw milk and fermented at 43°C, the time at which the pH of the prepared milk liquid reaches 4.65 is 99% or less of the time at which the pH of the prepared milk liquid reaches 4.65 when a lactic acid bacteria composition containing Bacillus bulgaricus and thermophilic Streptococcus not carrying the prtS gene but not containing thermophilic Streptococcus carrying the prtS gene is fermented under the same conditions (hereinafter referred to as the fermentation time using two types of lactic acid bacteria);

[0098] More specifically, the following conditions are preferably satisfied: regarding the time when the pH of the prepared milk liquid reaches 4.65 when added to the prepared milk liquid containing raw milk and fermented at 43°C, when the fermentation time with the two lactic acid bacteria is 6 hours or more, it is 75% or less, when the fermentation time with the two lactic acid bacteria is 4 hours or more and less than 6 hours, it is 90% or less, and when the fermentation time with the two lactic acid bacteria is 3 hours or more and less than 4 hours, it is 99% or less. In addition, the following conditions are more preferably satisfied:

[0099] (a) When added to a prepared milk liquid containing raw material milk and fermented at 43° C., the time when the pH of the prepared milk liquid reaches 4.65 is within 5 hours from the start of the fermentation.

[0100] 〈Activation Culture〉

[0101] In conditions (a) to (b) and the following conditions (c) to (i), it is preferred to first perform activation culture on the lactic acid bacteria composition (or each lactic acid bacteria constituting the lactic acid bacteria composition) after low temperature (e.g., below 10°C) or frozen storage. As the activation culture, it is preferred to culture in a skim milk powder medium at 37°C under aerobic conditions for 16 to 18 hours (preferably 16 hours). At this time, as the amount of lactic acid bacteria provided for activation culture, it is preferred that the amount of each lactic acid bacteria is 1 platinum ring / 5mL relative to the above-mentioned skim milk powder medium, more specifically, it is preferred that the number of viable bacteria reaches 1×10 2 ~2×10 8 The amount of cfu / g is more preferably 1×10 4 ~1×10 6 The amount of cfu / g.

[0102] It should be noted that, in the present invention, the determination of the number of viable cells can be determined, for example, as follows: a liquid (e.g., a culture solution of activated culture (lactic acid bacteria and skimmed milk powder culture medium)) containing an appropriately diluted lactic acid bacteria composition (or each lactic acid bacteria constituting the lactic acid bacteria composition) is appropriately diluted, smeared on a suitable agar medium (e.g., in the case of Bulgarian bacteria, it is a statutory culture medium prescribed by the provincial ordinance of milk, i.e., a plate count agar medium with BCP added; in the case of thermophilic streptococci, it is a FS agar medium) and cultured, and the number of colonies that appear is counted, thereby determining. At this time, Bulgarian bacteria form spindle-shaped colonies in the above-mentioned plate count agar medium with BCP added, thermophilic streptococci carrying the prtS gene form large yellow colonies in the above-mentioned FS agar medium, and thermophilic streptococci that do not carry the prtS gene form translucent small and flat colonies in the above-mentioned FS agar medium, so they can be distinguished.

[0103] In the present invention, when simply referred to as "skim milk powder culture medium", it refers to a culture medium containing 10 w / w% skim milk powder and 0.1 w / w% yeast extract in ultrapure water. The skim milk powder of the present invention refers to skim milk obtained by removing fat components from animal milk (preferably cow's milk) and drying it into a powder form, and examples thereof include those having a non-fat milk solid content of 95.0% or more and a water content of 5.0% or less as specified in Japan's "Ministerial Ordinance on the Standards of Composition of Milk and Dairy Products (hereinafter sometimes referred to as "Ministerial Ordinance on Milk, etc.")". As the composition of such skim milk powder, for example, a composition including the composition recorded in "Japanese Food Standard Composition Table 2015 Edition (Seventh Revision)" can be cited, that is, in 100g, energy 300-400 (preferably 359) kcal, water 3-5 (preferably 3.8) g, protein 30-40 (preferably 34.0) g, lipid 0.2-2 (preferably 1.0) g, carbohydrate (including lactose) 45-60 (preferably 53.3) g, calcium 900-2000 (preferably 1100) mg, vitamin A 3-10 (preferably 6) μg, vitamin B2 0.5-2 (preferably 1.60) mg, cholesterol 10-30 (preferably 25) mg, and salt equivalent 0.5-2 (preferably 1.4) g. The pH of such skim milk powder medium is usually 6-7.

[0104] The skim milk powder medium may further contain other components (e.g., peptides) in addition to the skim milk powder and yeast extract. In this case, the content of the other components is more preferably 0.01% by mass or less. In addition, when the activation culture is performed twice or more, the skim milk powder medium used in the second and subsequent activation cultures may contain part or all of the culture solution after the previous culture. The skim milk powder medium is preferably sterilized or disinfected.

[0105] The activation culture method is not particularly limited, and static culture under conditions known as aerobic culture conditions for lactic acid bacteria can be cited. In the present invention, the culture solution after the above culture can be inoculated again in the above skim milk powder medium in a manner such that the culture solution after the culture is 1w / w%, and the activation culture can be repeated multiple times (preferably twice).

[0106] 〈Fermentation〉

[0107] Under conditions (a) to (b) and the following conditions (c) to (i), the lactic acid bacteria composition of the present invention after the above-mentioned activation culture or the lactic acid bacteria composition of the present invention mixed with the above-mentioned lactic acid bacteria after the above-mentioned activation culture is added to the modulated milk liquid, and fermented at 43°C until the pH of the above-mentioned modulated milk liquid reaches 4.65 to prepare a fermented composition.

[0108] The amount of lactic acid bacteria used for fermentation under conditions (a) to (b) and the following conditions (c) to (i) is preferably 1×10 5 ~5×10 9 The amount of cfu / g is more preferably 1×10 6 ~2×10 9 The amount of cfu / g.

[0109] In the present invention, the "modulated milk liquid" refers to a liquid containing raw milk. As the raw milk, preferably lactose-containing milk, for example, raw milk (e.g., milk of cows, buffaloes, sheep, goats, etc.), sterilized milk, whole milk, skim milk, whey, and processed products thereof (e.g., whole milk powder, whole fat concentrated milk, skim milk powder, skim concentrated milk, condensed milk, whey powder, buttermilk, butter, cream, cheese, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg)), may be one of these, or a mixture of two or more thereof.

[0110] The modulated milk liquid of the present invention may contain only the raw milk mentioned above, or may be an aqueous solution, dilution or concentrated solution of the raw milk mentioned above. In addition to the raw milk mentioned above, other ingredients may be contained as needed. The modulated milk liquid of conditions (a) to (b) and the following conditions (c) to (i) preferably contains milk, skim milk powder and ion exchange water. Furthermore, the modulated milk liquid of the above conditions preferably has a fat content of 0 to 4.0 w / w% (preferably 3.05 w / w%) and a non-fat milk solid content of 8.0 to 12.0 w / w% (preferably 9.7 w / w%). The pH of such a modulated milk liquid is usually 6 to 7.

[0111] The above-mentioned prepared milk liquid can be prepared by mixing the above-mentioned components while heating and / or homogenizing as needed. In addition, the above-mentioned prepared milk liquid is preferably sterilized or disinfected.

[0112] The fermentation method is not particularly limited, and examples thereof include static culture under conditions conventionally known as fermentation conditions using lactic acid bacteria.

[0113] In the fermentation, when the lactose contained in the prepared milk liquid is metabolized by the lactic acid bacteria to produce lactic acid, the lactic acid lowers the pH of the prepared milk liquid. In the fermentation under conditions (a) to (b) and the following conditions (c) to (i), the pH of the prepared milk liquid (i.e., the pH of the prepared milk liquid containing the lactic acid bacteria composition) reaches 4.65.

[0114] In condition (a), the time to complete the above-mentioned fermentation (that is, the pH of the above-mentioned modulated milk liquid reaches 4.65) needs to be within 5 hours from the start of fermentation, more preferably within 4.5 hours, and further preferably within 4 hours. When the time to complete the above-mentioned fermentation exceeds the above-mentioned upper limit, when used for the manufacture of fermented milk, the manufacture consumes time, so it is not preferred in terms of manufacturing efficiency. There is no particular restriction on the lower limit of the time to complete the above-mentioned fermentation, for example, it is preferably more than 1 hour, and more preferably more than 2 hours. It should be noted that the conventional lactic acid bacteria composition sometimes also satisfies condition (a) and / or the following condition (b), but in this case, for example, the number of live bacteria of Bulgarian bacteria decreases due to low-temperature storage (not satisfying conditions (c) and / or (d)) or the pH decreases or the acidity increases due to low-temperature storage (not satisfying at least any of conditions (e) to (g)).

[0115] In condition (b), the time for completing the above-mentioned fermentation (i.e., the pH of the above-mentioned prepared milk liquid reaches 4.65) needs to be 99% or less of the time for completing the fermentation (fermentation time with two types of lactic acid bacteria) of the lactic acid bacteria composition in which the thermophilic Streptococcus carrying the prtS gene is removed from the lactic acid bacteria composition of the present invention (i.e., a lactic acid bacteria composition containing Bacillus bulgaricus and thermophilic Streptococcus not carrying the prtS gene and not containing thermophilic Streptococcus carrying the prtS gene, the same hereinafter). More specifically, the time for completing the above-mentioned fermentation is more preferably 75% or less of the time for completing the fermentation when the fermentation time with the two types of lactic acid bacteria is 6 hours or more, 90% or less of the time for completing the fermentation when the fermentation time with the two types of lactic acid bacteria is 4 hours or more and less than 6 hours, and 99% or less of the time for completing the fermentation when the fermentation time with the two types of lactic acid bacteria is 3 hours or more and less than 4 hours. In particular, when the time required to complete fermentation of the lactic acid bacteria composition in which the thermophilic Streptococcus carrying the prtS gene is removed from the lactic acid bacteria composition of the present invention is long (for example, 7 hours or more), the lactic acid bacteria composition of the present invention can shorten the time to 70% or less, and further to 65% or less.

[0116] [The number of viable bacteria of Bulgarian bacteria]

[0117] The lactic acid bacteria composition of the present invention (comprising the following lactic acid bacteria starter and fermented milk, preferably the lactic acid bacteria starter) preferably satisfies at least one of the following conditions (c) to (d), and more preferably satisfies two of the following conditions:

[0118] (c) added to a prepared milk liquid containing raw material milk and fermented at 43°C until the pH of the prepared milk liquid reached 4.65, and the fermented composition was stored at 10°C for 30 days. The number of viable Bacillus subtilis in the fermented composition after 30 days of storage was 1×10 7 cfu / g or more

[0119] (d) added to a prepared milk liquid containing raw milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the number of live bacteria of Bacillus bulgaricus in the fermented composition after 30 days of storage is at least 10% of the number of live bacteria of Bacillus bulgaricus in the fermented composition after 1 day of storage.

[0120] <save>

[0121] In conditions (c) to (d) and the following conditions (e) to (i), the fermented composition after the fermentation is completed (i.e., after the pH reaches 4.65) is cooled and stored. The conditions for the storage are not particularly limited, and examples thereof include methods known as methods for storing fermented milk, such as a method of sealing the culture solution in a container (preferably sealed) and leaving it to stand.

[0122] It is preferred that the conditions (c) to (d) and the following conditions (e) to (i) do not include a step of suddenly lowering the pH. The step of suddenly lowering the pH may be, for example, artificially adding an acid (eg, lactic acid).

[0123] In addition, the storage temperature of conditions (c) to (d) and the following conditions (e) to (i) needs to be low temperature, more specifically, 10°C. It should be noted that an error of ±1°C is acceptable. In addition, the storage period under these conditions is 30 days. Even if the lactic acid bacteria composition of the present invention is stored for a long time under low temperature conditions, the number of viable cells of Bacillus bulgaricus is fully maintained.

[0124] In condition (c), the number of viable Bacillus subtilis in the fermented composition after storage at 10°C for 30 days must be 1×10 7 cfu / g or more, more preferably 2×10 7 cfu / g or more, more preferably 2.5×10 7 cfu / g or above.

[0125] In condition (d), the number of viable bacteria of Bacillus subtilis in the fermented composition after storage at 10°C for 30 days needs to be 10% or more of the number of viable bacteria of Bacillus subtilis in the fermented composition after storage for 1 day, more preferably 15% or more, further preferably 20% or more, further more preferably 23% or more, and particularly preferably 30% or more.

[0126] 〔pH and acidity〕

[0127] The lactic acid bacteria composition of the present invention (comprising the lactic acid bacteria starter and fermented milk described below, preferably the lactic acid bacteria starter) preferably satisfies at least one of the following conditions (e) to (g), more preferably satisfies two or more conditions, and further satisfies all of the conditions:

[0128] (e) adding to a prepared milk liquid containing raw material milk and fermenting at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the pH of the fermented composition after 30 days of storage is 4.1 or more

[0129] (f) adding to a prepared milk liquid containing raw material milk and fermenting at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the acidity of the fermented composition after 30 days of storage is 1.0% or less

[0130] (g) When the fermented composition is added to a prepared milk liquid containing raw material milk and fermented at 43° C. until the pH of the prepared milk liquid reaches 4.65, and the fermented composition is stored at 10° C. for 30 days, the acidity (D1%) of the fermented composition after 1 day of storage and the acidity (D2%) of the fermented composition after 30 days of storage are 30 %) is the following formula: D 30 -The conditions shown in D1≤0.25.

[0131] Lactic acid bacteria (particularly thermophilic streptococci) can usually produce the above-mentioned lactic acid even at low temperatures. Therefore, as mentioned above, when the fermented composition after fermentation is stored for a long time, even under low temperature conditions, the pH of the fermented composition will further decrease as the amount of lactic acid produced increases, and the acidity will further increase. In contrast, the lactic acid bacteria composition of the present invention fully suppresses the pH decrease and acidity increase during the above-mentioned low-temperature storage.

[0132] In condition (e), the pH of the post-fermentation composition (i.e., the pH of the prepared milk liquid containing the lactic acid bacteria composition) when stored at 10°C for 30 days needs to be 4.1 or higher, more preferably 4.12 or higher, and even more preferably 4.14 or higher.

[0133] In addition, in condition (f), the acidity of the above-mentioned post-fermentation composition (i.e., the lactic acid acidity (w / v%) of the prepared milk liquid containing the lactic acid bacteria composition, the same below) when stored at 10°C for 30 days needs to be 1.0% or less, more preferably 0.98 or less, and even more preferably 0.96 or less.

[0134] In condition (g), the acidity (D1%) of the fermented composition after storage at 10°C for 1 day and the acidity (D 30 %) need to satisfy the following formula: D 30 -D1≤0.25, as the above formula: D 30 -D1 is more preferably 0.23 or less, further preferably 0.20 or less, and further preferably 0.19 or less.

[0135] In conventional lactic acid bacteria compositions, for example, when lactic acid bacteria known as low-temperature-sensitive lactic acid bacteria are contained, the pH of the composition may not decrease after fermentation even when stored at low temperatures for a long period of time. However, in this case, for example, it takes time to complete the fermentation (not satisfying conditions (a) and / or (b)) or the low-temperature storage causes a decrease in the number of viable cells of Bacillus bulgaricus (not satisfying conditions (c) and / or (d)).

[0136] 〔Viable count of thermophilic streptococci〕

[0137] The lactic acid bacteria composition of the invention (comprising the lactic acid bacteria starter and fermented milk described below, preferably the lactic acid bacteria starter) preferably satisfies at least one of the following conditions (h) to (i), and more preferably satisfies two of the following conditions:

[0138] (h) adding to a prepared milk liquid containing raw milk and fermenting at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the content of Bacillus bulgaricus in the fermented composition stored for 1 to 30 days and the content of Streptococcus thermophilus carrying the prtS gene and Streptococcus thermophilus not carrying the prtS gene in terms of viable cell count ratio is 1:2 to 1:150

[0139] (i) added to a prepared milk liquid containing raw milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the content of thermophilic Streptococcus carrying the prtS gene in the fermented composition stored for 1 to 30 days and the content of thermophilic Streptococcus not carrying the prtS gene in the fermented composition is 1:0.005 to 1:30 in terms of the ratio of viable bacteria.

[0140] Under condition (h), the ratio of the number of live bacteria of Bacillus bulgaricus in the above-mentioned fermented composition stored at 10°C for 1 day to 30 days to the total number of live bacteria of Streptococcus thermophilus carrying the prtS gene and Streptococcus thermophilus not carrying the prtS gene needs to be 1:2 to 1:150, more preferably 1:3 to 1:140, further preferably 1:5 to 1:140, further preferably 1:10 to 1:130, and further preferably 1:3 to 1:60.

[0141] In condition (i), the ratio of the number of viable thermophilic Streptococci carrying the prtS gene to the number of viable thermophilic Streptococci not carrying the prtS gene in the above-mentioned fermented composition stored at 10°C for 1 day to 30 days needs to be 1:0.005 to 1:30, more preferably 1:0.010 to 1:20, further preferably 1:0.015 to 1:10, and also preferably 1:0.03 to 1:15.

[0142] 〔Lactic acid bacteria ferment〕

[0143] The lactic acid bacteria composition and lactic acid bacteria combination of the present invention satisfy at least any of the above conditions (a) to (i) (preferably two conditions of (c) or (d) and (e) or (f) or (g); more preferably four conditions of (a) or (b), (c) or (d), (e) or (f) or (g) and (h) or (i); further preferably at least five conditions of (c) to (g); further more preferably all of (a) to (i)), and therefore, for example, can be preferably used as a lactic acid bacteria starter in the method for producing fermented milk of the present invention described below. As a result, fermented milk can be obtained in a short period of time in which the number of live bacteria of Bulgarian bacteria is fully maintained even when stored at low temperatures, the decrease in pH and the increase in acidity during the above-mentioned low-temperature storage are suppressed, and the fermented milk also has smooth physical properties.

[0144] As the lactic acid bacteria ferment of the present invention, as long as the effect of the present invention is not inhibited, it can be a composition or combination product that also contains other lactic acid bacteria and yeast. As the above-mentioned other lactic acid bacteria and yeast, lactic acid bacteria and yeast that have been known to be contained in fermented milk can be listed. For example, as the above-mentioned other lactic acid bacteria, lactic acid bacteria of the genus Lactobacillus other than Bulgarian bacteria (for example, Lactobacillus delbrueckii subspecies delbrueckii, Lactobacillus delbrueckii lactic subspecies, Lactobacillus delbrueckii subspecies indica, Lactobacillus delbrueckii subspecies mulberry, Lactobacillus delbrueckii subspecies Jacobsen, etc. other than Bulgarian bacteria; Lactobacillus helveticus; Lactobacillus acidophilus; Lactobacillus johnsonii; Lactobacillus gasseri; Lactobacillus paragasseri; Lactobacillus amylovora; Lactobacillus crispatus, etc.), lactic acid bacteria of the genus Lactaseibacillus, lactic acid bacteria of the genus Lactiplantibacillus, and lactic acid bacteria of the genus Liquidilactobacillus can be listed. The lactic acid bacteria may be selected from the group consisting of Lactobacillus, Latilactobacillus, Ligilactobacillus, Limosilactobacillus, Lentilactobacillus, Levilactobacillus, Pediococcus, Leuconostoc and Lactococcus. The lactic acid bacteria may be selected from the group consisting of Lactobacillus, Latilactobacillus, Ligilactobacillus, Limosilactobacillus, Lentilactobacillus, Levilactobacillus, Pediococcus, Leuconostoc and Lactococcus.

[0145] The lactic acid bacteria starter of the present invention may be a liquid, or a solid such as a frozen state or a dry powder, and may further contain other components. Other components that may be contained in the lactic acid bacteria starter include, for example, fermentation-promoting substances (formic acid, nucleic acid, etc.); protective agents (sugars); culture medium components (milk or whey, etc.), which may be one of these or a combination of two or more.

[0146] When the lactic acid bacteria composition of the present invention is a lactic acid bacteria starter, the total content of the three lactic acid bacteria of the present invention is preferably 0.01 to 100 mass%, more preferably 0.1 to 90 mass%. In addition, the total amount of the viable count of the three lactic acid bacteria of the present invention is preferably 1×10 6 cfu / g or more, more preferably 1×10 6 ~2×10 11 cfu / g, more preferably 1×10 7 ~1×10 11 cfu / g.

[0147] As the lactic acid bacteria starter of the present invention, for example, as described above, it is possible to prepare a combination product including Bulgarian bacteria or a composition containing it, thermophilic Streptococcus carrying the prtS gene or a composition containing it, and thermophilic Streptococcus not carrying the prtS gene or a composition containing it, such as a lactic acid bacteria starter kit. In addition, in this case, for example, the above-mentioned kit may contain additives for producing fermented milk (the above-mentioned fermentation-promoting substances, protective agents, etc.), containers, instructions for use of lactic acid bacteria starter, etc.

[0148] 〔Fermented milk〕

[0149] The lactic acid bacteria composition of the present invention also includes fermented milk. The fermented milk of the present invention can be obtained in the form of the above-mentioned fermented composition by, for example, fermentation under the above-mentioned conditions (a) to (i) (i.e., added to the above-mentioned modulated milk liquid, and fermented at 43°C until the pH of the above-mentioned modulated milk liquid reaches 4.65), preferably satisfying at least any one of the conditions (a) to (i). The fermented milk of the present invention maintains the viable count of Bacillus bulgaricus even when stored at low temperature, suppresses the decrease in pH and the increase in acidity during the above-mentioned low-temperature storage, and also has smooth physical properties. In addition, the fermented milk of the present invention can be used as the above-mentioned lactic acid bacteria starter seed in the method for producing fermented milk of the present invention described below.

[0150] As the fermented milk of the present invention, for example, fermented milk that meets the standard of "fermented milk" stipulated in the above-mentioned provincial ordinance on milk, etc. can be cited (more specifically, fermented milk with a non-fat milk solid content of 8.0% or more and a lactic acid bacteria count or yeast count (preferably a lactic acid bacteria count (more preferably a total count of the three types of lactic acid bacteria of the present invention), the same below) of 10 million / mL or more). In addition, the fermented milk of the present invention also includes fermented milk that meets the standard of "dairy lactic acid bacteria beverage" stipulated in the above-mentioned provincial ordinance on milk, etc. (more specifically, fermented milk with a non-fat milk solid content of 3.0% or more and a lactic acid bacteria count or yeast count of 10 million / mL or more); fermented milk that meets the standard of "lactic acid bacteria beverage" (more specifically, fermented milk with a non-fat milk solid content of less than 3.0% and a lactic acid bacteria count or yeast count of 1 million / mL or more). It should be noted that the non-fat milk solids refer to the components remaining after subtracting the fat component from the total milk solids (mainly protein, lactose, minerals, etc.), and the lactic acid bacteria count and yeast count are the live bacteria counts measured by the inspection methods specified in the provincial ordinances on milk, etc.

[0151] The fermented milk of the present invention preferably has a viable cell count of 1×10 7 cfu / g or more, more preferably 2×10 7 cfu / g or more, more preferably 2.5×10 7 cfu / g or above.

[0152] In addition, in the fermented milk of the present invention, the ratio of the content of Bacillus bulgaricus to the total content of Streptococcus thermophilus carrying the prtS gene and Streptococcus thermophilus not carrying the prtS gene is preferably 1:1 to 1:150 in terms of viable cell count ratio (viable cell count of Bacillus bulgaricus: total viable cell count of Streptococcus thermophilus), more preferably 1:2 to 1:150, more preferably 1:5 to 1:140, and even more preferably 1:10 to 1:130.

[0153] Furthermore, in the fermented milk of the present invention, the ratio of the content of thermophilic Streptococcus carrying the prtS gene to the content of thermophilic Streptococcus not carrying the prtS gene is preferably 1:0.005 to 1:30 in terms of viable cell count ratio (viable cell count of thermophilic Streptococcus carrying the prtS gene:viable cell count of thermophilic Streptococcus not carrying the prtS gene), more preferably 1:0.010 to 1:20, further preferably 1:0.015 to 1:10, and further preferably 1:0.05 to 1:10.

[0154] Furthermore, the fermented milk of the present invention preferably has a pH of 4.1 or higher, more preferably 4.12 or higher, and even more preferably 4.14 or higher. Furthermore, the fermented milk of the present invention preferably has a lactic acid acidity of 1.0% or lower, more preferably 0.98 or lower, and even more preferably 0.96 or lower.

[0155] The fermented milk of the present invention has a smooth physical property, and the smooth physical property is preferably maintained even after storage at a low temperature (for example, 10° C.) for a long period of time (for example, 20 days or 30 days).

[0156] In the present invention, "smooth physical properties" refers to a uniform touch of the tongue and no roughness in sensory evaluation. For example, when the tongue feels less roughness than fermented milk obtained under the same conditions using the above-mentioned Bacillus bulgaricus 2038 strain and the above-mentioned OLS4802 strain carrying the prtS gene, it can be judged to be smooth. In addition, in the present invention, "smooth physical properties" can also be confirmed by, for example, sufficiently low water separation rate. That is, for example, when fermented milk (preferably fermented milk immediately after fermentation) is stored at 10°C for 20 days, the water separation rate of the fermented milk after 5 days of storage (S5%) and the water separation rate of the fermented milk after 20 days of storage (S 20 %) satisfies the following formula: S 20 -S5≤6.5 (preferably S 20 -S5≤6.0), it can be judged as smooth. In the present invention, "water separation rate" refers to the value (%) calculated by the following formula: weight of precipitate (g) / 40g×100 when 40g of fermented milk is weighed, centrifuged at 1610×g, 25°C, and 10 minutes, and the weight of the precipitate is measured after removing the supernatant.

[0157] The fermented milk of the present invention may be the fermented composition itself, or may be obtained by concentrating, diluting, drying, freezing, or the like.

[0158] The fermented milk of the present invention comprises components from the above-mentioned raw milk (preferably a modulated milk liquid). In addition, other components listed in the above-mentioned lactic acid bacteria composition and lactic acid bacteria fermentation species, as well as other lactic acid bacteria and yeast, can be further contained within the scope that does not inhibit the effect of the present invention. Furthermore, various components that can be contained in food and beverages can be contained. As further components that can be contained in fermented milk, there are no particular restrictions, and examples thereof include sugars, sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, organic acids, pH regulators, starch and modified starch, dietary fiber, fruits / vegetables and processed products thereof, animal and plant herbal medicine extracts, natural source polymers (collagen, hyaluronic acid, chondroitin, etc.), oils and fats, thickeners, emulsifiers, solvents, surfactants, gelling agents, stabilizers, buffers, suspending agents, thickeners, excipients, disintegrating agents, binders, fluidizing agents, preservatives, colorants, spices, flavoring agents, sweeteners, etc., which can be one of these or a combination of two or more.

[0159] As such fermented milk, yogurt, cheese, fermented cream, fermented butter, etc. are preferred, and yogurt is particularly preferred. As the above-mentioned yogurt, specifically, coagulated yogurt (solid fermented milk) such as coagulated yogurt, soft yogurt (paste-like fermented milk) and drinkable yogurt (liquid fermented milk) can be mentioned. It can be frozen yogurt using these as materials. In addition, the fermented milk of the present invention can also be used as a material for fermented foods such as cheese, fermented cream, fermented butter, kefir yogurt, etc.

[0160] (Method for producing fermented milk)

[0161] The method for producing fermented milk of the present invention (sometimes referred to as "the production method of the present invention" in this specification) includes the following fermentation step: adding the above-mentioned lactic acid bacteria composition or lactic acid bacteria combination of the present invention to a modulated milk liquid containing raw milk and fermenting it to obtain fermented milk. According to the production method of the present invention, by using the lactic acid bacteria composition or lactic acid bacteria combination of the present invention to ferment raw milk, it is possible to produce fermented milk in which the pH drop and the acidity increase during low-temperature storage are suppressed, and the number of live bacteria of Bulgarian bacteria contained in the fermented milk is sufficiently maintained even during the above-mentioned low-temperature storage, and the physical properties are smooth. The smooth physical properties tend to be maintained even after long-term storage (for example, 20 days, 30 days) at low temperatures (for example, 10°C). Furthermore, the production method of the present invention can also sufficiently shorten the time required for fermentation to be completed.

[0162] 〔Preparing emulsion liquid〕

[0163] In the production method of the present invention, the above-mentioned modulated milk liquid may be, for example, the modulated milk liquid of conditions (a) to (i) in the above-mentioned lactic acid bacteria composition. The modulated milk liquid of the production method of the present invention is not limited thereto, and any conventionally known modulated milk liquid for producing fermented milk may be appropriately used.

[0164] For example, the above-mentioned modulated milk liquid may also contain other ingredients. Examples of such other ingredients include water; soy milk, sugars represented by sugar, sweeteners, spices, fruit juice, pulp, vitamins, minerals, oils, ceramides, collagen, milk lecithin, polyphenols and other foods, food ingredients or food additives; pectin, soybean polysaccharides, CMC (carboxymethyl cellulose), agar, gelatin, carrageenan, gums and other stabilizers, thickeners, gelling agents, etc. It may be one of these or a mixture of two or more.

[0165] 〔Fermentation〕

[0166] In the manufacturing method of the present invention, as a fermentation step of adding the lactic acid bacteria composition or lactic acid bacteria combination of the present invention to the above-mentioned modulated milk liquid and fermenting it, fermentation under conditions (a) to (i) using the above-mentioned lactic acid bacteria composition can be listed, but the fermentation of the manufacturing method of the present invention is not limited to this, and the existing known fermentation method in the production of fermented milk can be appropriately adopted.

[0167] For example, the amount of the lactic acid bacteria composition or lactic acid bacteria combination of the present invention added to the above-mentioned prepared milk liquid can be appropriately set according to the amount of lactic acid bacteria starter used in the conventionally known method for producing fermented milk. For example, the total viable count of the three types of lactic acid bacteria of the present invention relative to the above-mentioned prepared milk liquid is 1×10 5 ~5×10 9 The range of cfu / g is preferably 1×10 6 ~5×10 9 cfu / g, more preferably 1×10 7 ~5×10 9 cfu / g, more preferably 1×10 8 ~2×10 9 cfu / g. In addition, the above-mentioned other lactic acid bacteria and yeast may be further added to the lactic acid bacteria composition or lactic acid bacteria combination product of the present invention to the above-mentioned prepared milk liquid.

[0168] In addition, the fermentation conditions of the manufacturing method of the present invention can be appropriately selected according to the growth conditions of each lactic acid bacteria added, the amount of the above-mentioned modulated milk liquid, etc., without particular restrictions. For example, it is preferably preferably at a temperature of 35 to 45°C, more preferably at a temperature of 38 to 43°C and under aerobic or anaerobic conditions, usually standing or stirring (preferably standing) for 2 to 16 hours, more preferably 2.5 to 8 hours, and further preferably 3 to 6 hours to ferment until the pH of the modulated milk liquid to which the lactic acid bacteria composition of the present invention is added is 4.7 or less, more preferably 4.45 to 4.65. According to the present invention, the time required for fermentation can be sufficiently shortened. In addition, when the above-mentioned anaerobic conditions are adopted, for example, fermentation under the condition of passing nitrogen can be adopted.

[0169] Fermented milk can be obtained by the above-mentioned fermentation. The fermented product after the above-mentioned fermentation process can be directly used as fermented milk or can be concentrated, diluted, dried or frozen as needed to make fermented milk. As the obtained fermented milk, it is preferred to be fermented milk as the lactic acid bacteria composition of the present invention. As the fermented milk obtained by the production method of the present invention, due to the use of conditions known as the production method of fermented milk, such as the addition of the above-mentioned other components, fermentation conditions, processing, etc., it is also possible not to form fermented milk as the lactic acid bacteria composition of the present invention. In addition, in the production method of the present invention, fermented milk can also be made by crushing or heat-treating the lactic acid bacteria in the above-mentioned fermented product, or by concentrating, diluting, drying or freezing it as needed. Therefore, as the production method of fermented milk of the present invention, it can also include these steps (concentration step, dilution step, drying step, freezing step, crushing step, heating treatment step, etc.).

[0170] Example

[0171] The present invention will be described in more detail below based on Examples and Comparative Examples, but the present invention is not limited to the following Examples.

[0172] <Lactic acid bacteria>

[0173] The lactic acid strains used in the following examples and comparative examples are as follows.

[0174] (Bulgarian bacteria strain 2038)

[0175] Lactobacillus delbrueckii subsp. bulgaricus stored at Meiji Innovation Center of Meiji Co., Ltd. (1-29-1 Shichikoku, Hachioji-shi, Tokyo, Japan, zip code 192-0919). It is Lactobacillus delbrueckii subsp. bulgaricus isolated from "Meiji Bulgarian Yogurt (registered trademark), manufactured by Meiji Co., Ltd." using agar medium for Lactobacillus.

[0176] (Bulgarian bacteria 1589 strain)

[0177] Lactobacillus delbrueckii subspecies bulgaricus specified by the deposit number NITE BP-03716 is not a lactic acid bacterium that produces high polysaccharides.

[0178] (Bulgarian bacteria OLL205013 strain)

[0179] Lactobacillus delbrueckii subspecies bulgaricus specified by the deposit number NITE BP-02411 is not a lactic acid bacterium that produces high polysaccharides.

[0180] (Bulgarian bacteria OLL1073R-1 strain)

[0181] Lactobacillus delbrueckii subsp. bulgaricus specified by the accession number FERM BP-10741 is a high-polysaccharide-producing lactic acid bacterium having a higher polysaccharide content as measured by the following method than Lactobacillus delbrueckii 2038 strain.

[0182] (Bulgarian bacteria OLL1251 strain)

[0183] Lactobacillus delbrueckii subsp. bulgaricus specified by the deposit number NITE BP-02703 is a high-polysaccharide-producing lactic acid bacterium having a higher polysaccharide content than that of Lactobacillus delbrueckii 2038 strain as measured by the following method.

[0184] (Bulgarian bacteria OLL1171 strain)

[0185] Lactobacillus delbrueckii subspecies bulgaricus specified by the deposit number NITE BP-01569 is not a lactic acid bacterium that produces high polysaccharide.

[0186] (Bulgarian bacteria OLL1247 strain)

[0187] Lactobacillus delbrueckii subsp. bulgaricus specified by the deposit number NITE BP-01569 is a high-polysaccharide-producing lactic acid bacterium having a higher polysaccharide content as measured by the following method than Lactobacillus delbrueckii 2038 strain.

[0188] (Streptococcus thermophilus OLS4801 strain)

[0189] Streptococcus thermophilus specified by the accession number NITE BP-03504. Carrying the prtS gene. Not a lactic acid bacterium that produces high polysaccharide.

[0190] (Streptococcus thermophilus OLS4802 strain)

[0191] Streptococcus thermophilus specified by the deposit number NITE BP-03505. Carrying the prtS gene. Not a lactic acid bacterium that produces high polysaccharide.

[0192] (Streptococcus thermophilus OLS4803 strain)

[0193] Streptococcus thermophilus specified by accession number NITE BP-03506. Carrying prtS gene. Not a lactic acid bacterium that produces high polysaccharide.

[0194] (Streptococcus thermophilus strain 1131)

[0195] Lactobacillus delbrueckii subsp. bulgaricus kept at Meiji Innovation Center of Meiji Co., Ltd. (1-29-1 Shichikoku, Hachioji, Tokyo, Japan, zip code 192-0919). It is a thermophilic Streptococcus isolated from "Meiji Bulgarian Yogurt (registered trademark) LB81, manufactured by Meiji Co., Ltd." using agar medium for Lactococcus. It does not carry the prtS gene.

[0196] (Streptococcus thermophilus OLS3290 strain)

[0197] Streptococcus thermophilus specified by the accession number FERM BP-19638. It does not carry the prtS gene. It is a high-polysaccharide-producing lactic acid bacterium that produces more polysaccharides than Streptococcus thermophilus 1131 strain as measured by the following method.

[0198] (Streptococcus thermophilus OLS3078 strain)

[0199] Thermophilus Streptococcus specified by the accession number: NITE BP-01697. It does not carry the prtS gene. It is a high-polysaccharide-producing lactic acid bacterium that produces more polysaccharides than the thermophilus Streptococcus 1131 strain as measured by the following method.

[0200] (Streptococcus thermophilus OLS3615 strain)

[0201] Thermophilic Streptococcus as specified by NITE BP-01696. Does not carry the prtS gene. Not a high-polysaccharide-producing lactic acid bacterium.

[0202] (1) prtS gene

[0203] In this specification, whether each thermophilic Streptococcus carries the prtS gene was confirmed by the following method. That is, first, the prtS gene sequences of 5 thermophilic Streptococcus strains with known genome sequences were obtained from the NCBI database, and a primer set (F primer: sequence number 1, R primer: sequence number 2) was prepared based on the highly conservative sequence. In addition, genomic DNA was extracted from the M17 culture solution of the above 5 strains using InstaGeneMatrix (manufactured by BioRad). 0.5 μL of the extracted genomic DNA (template), 1 μL of each prepared substrate (5 μM), 0.1 μL of Phusion high-fidelity DNA polymerase, 2 μL of 5×HF buffer, 0.8 μL of 2.5 mM dNTP, and 4.6 μL of ultrapure water were mixed (a total of 10 μL), and the following conditions were set: 30 seconds at 98°C; 30 cycles of 5 seconds at 98°C, 20 seconds at 63°C, and 20 seconds at 72°C; standing at 72°C for 5 minutes; standing at 4°C, thereby performing PCR. The obtained PCR products were subjected to agarose gel electrophoresis, and strains with a band at the position of 684 bp were determined to carry the prtS gene, and strains without such a band were determined to not carry the prtS gene. In the following, thermophilic Streptococcus carrying the prtS gene is sometimes represented as "prtS+", and thermophilic Streptococcus not carrying the prtS gene is sometimes represented as "prtS-".

[0204] (2) Lactic acid bacteria with high polysaccharide production

[0205] In this example, whether Lactobacillus delbrueckii subsp. bulgaricus (bulgaricus) and Streptococcus thermophilus (thermophilus) are lactic acid bacteria that produce high polysaccharides was confirmed by the following methods.

[0206] (Bulgarian bacteria)

[0207] For Bulgaria bacteria, the frozen strain was first inoculated in a 10w / v% skim milk powder medium (10w / v% skim milk powder was added to ultrapure water and sterilized by autoclave at 121°C for 7 minutes, pH: 6-7, the same below) at 1 platinum loop / 5mL, and cultured at 37°C under aerobic conditions for 16 hours. The culture solution was inoculated again in a 10w / v% skim milk powder medium at 1w / w%, and cultured under the same conditions to activate it. Next, the culture solution of the activated Bulgaria bacteria was inoculated in a 1w / w% manner in 4mL of skim milk powder medium in a 15mL conical tube, and cultured at 37°C for 18 hours.

[0208] To 4 mL of the culture solution after cultivation, 400 μL (final concentration 10%) of 100% trichloroacetic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after stirring until there was no viscosity, the supernatant was recovered by centrifugation (12000 g, 4°C, 20 minutes), and all of it was transferred to a new 15 mL conical tube. Ultrapure water was added to the recovered supernatant, and after the volume was fixed to 4 mL according to the scale of the conical tube, 8 mL (2 times the amount) of cold ethanol was slowly added and mixed thoroughly, and then left to stand at 4°C overnight to precipitate the polysaccharide. The polysaccharide was recovered in the form of a precipitate by centrifugation, and 4 mL of ultrapure water was added to completely dissolve it. The obtained polysaccharide aqueous solution was added to a dialysis container (96-well dispo DIALYZER: 250-300μ; MWCO5000; HARVARD 74-0902) at 180 μL / well, and dialyzed against 5 L of ultrapure water for 2 days (the ultrapure water was replaced every day). After the dialysis, the volume of the polysaccharide aqueous solution was measured and the polysaccharide concentration was measured by the phenol-sulfuric acid method to calculate the amount of polysaccharide (mg / kg) in the culture medium. In this embodiment, the Bulgarian bacteria having a polysaccharide amount greater than that of the Bulgarian bacteria 2038 strain and a polysaccharide amount of 105 mg / kg or more were used as high-polysaccharide-producing lactic acid bacteria.

[0209] (Streptococcus thermophilus)

[0210] For Streptococcus thermophilus, the frozen strain was first inoculated in a 10 w / v% skim milk powder-0.1 w / v% casein decomposition peptide medium (obtained by adding 10 w / v% skim milk powder and 0.1 w / v% casein decomposition peptide (CE90GMM, manufactured by Nippon Shinyaku Co., Ltd.) to ultrapure water and sterilizing at a temperature of 95°C, pH: 6-7, the same below), and then statically cultured at 37°C under aerobic conditions for 16 hours. The culture solution was then inoculated again in a 10 w / v% skim milk powder-0.1 w / v% casein decomposition peptide medium and cultured under the same conditions to activate it. Next, the culture solution of Streptococcus thermophilus after activation was inoculated in a 15 mL peptide-supplemented medium in a 50 mL conical tube in a 1 w / w% manner, and statically cultured at 43°C for 4 hours.

[0211] Weigh 10 g of the culture after cultivation, add 1 mL of 100% trichloroacetic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (final concentration 10%), stir until there is no viscosity, and then recover the supernatant by centrifugation (12000g, 4°C, 20 minutes), and transfer all of it to a new 50 mL conical tube. Slowly add 2 times the amount of cold ethanol to the recovered supernatant and mix thoroughly, then let it stand overnight at 4°C to precipitate the polysaccharide. Recover the polysaccharide in the form of a precipitate by centrifugation, and add 10 mL of ultrapure water to completely dissolve it. The obtained polysaccharide aqueous solution is added to a dialysis container (MWCO: 6000-8000; SPECTRUM) and dialyzed against 100 times the amount of ultrapure water for 2 days (the ultrapure water is replaced every day). After the dialysis is completed, the volume of the polysaccharide aqueous solution is measured and the polysaccharide concentration is measured by the phenol-sulfuric acid method to calculate the amount of polysaccharide in the culture (mg / kg). In this example, the Streptococcus thermophilus strain having a polysaccharide production of 12 mg / kg or more, which is higher than that of the Streptococcus thermophilus strain 1131, was used as a high-polysaccharide-producing lactic acid bacterium.

[0212] <Example 1>

[0213] (1) Yogurt preparation

[0214] First, frozen strains of Bacillus bulgaricus 2038, Streptococcus thermophilus OLS4802 carrying the prtS gene, and Streptococcus thermophilus 1131 not carrying the prtS gene were inoculated in a skim milk powder medium (obtained by adding 10 w / w% skim milk powder and 0.1 w / w% yeast extract to ultrapure water and sterilizing by autoclave at 121°C for 7 minutes, pH: 6-7, the same below) at 1 platinum loop / 5 mL, and after static culture at 37°C under aerobic conditions for 16 hours, the culture solution was again inoculated in a skim milk powder medium at 1 w / w% and cultured under the same conditions to perform activation.

[0215] Next, in a prepared milk liquid (prepared by mixing milk, skim milk powder and ion exchange water to a fat content of 3.05 w / w %, a non-fat milk solid content of 9.7 w / w %, and sterilizing by autoclave at 75° C. for 15 minutes, pH: 6-7, the same below), a total mass of activated lactic acid bacteria of 3 w / w % (Bulgaricus 2038 strain: 0.3 w / w % (viable cell count: 3.0×10 5 ~9.0×10 7 cfu / g), Streptococcus thermophilus OLS4802 strain: 2.1w / w% (viable count: 2.1×10 6 ~6.3×10 8 cfu / g), Streptococcus thermophilus 1131 strain: 0.6w / w% (viable count: 6.0×10 5 ~1.8×10 8 cfu / g)) and allowed to stand at 43°C for fermentation. The time required from the inoculation to the pH drop to 4.65 (fermentation time) was measured. The fermentation time was 3 hours and 24 minutes. Furthermore, the fermentation was terminated (standing at 43°C was terminated, the same applies below) when the pH reached 4.65, and yogurt was prepared.

[0216] (2) Yogurt analysis

[0217] The yogurt prepared in the above (1) was sealed so as not to enclose air, and was stored at 10°C. The pH (10°C pH) and acidity (lactic acid: % (w / v%, hereinafter the same)) were measured by the following methods at 1 day and 30 days after the start of storage, and the number of viable bacteria (cfu / g) of Bacillus bulgaricus and the number of viable bacteria (cfu / g) of each thermophilic streptococcus were measured. The results are shown in Table 1 below.

[0218] (pH)

[0219] The pH was measured by a glass electrode method using a pH meter HM-30R (manufactured by Mettler Toledo Co., Ltd.).

[0220] (acidity)

[0221] 9.0 g of each yogurt after storage was collected, 500 μL of phenolphthalein indicator was added, and the mixture was measured by neutralization titration using 0.1 N-NaOH. The point at which the light red color did not disappear within 30 seconds was used as the limit.

[0222] (Viable count of Bulgarian bacteria)

[0223] Each yogurt after storage was cultured at 37°C under aerobic conditions for 72 hours using a plate count agar flat medium supplemented with BCP (manufactured by Eiken Chemical Co., Ltd.), which is a legal medium for measuring the number of lactic acid bacteria, and the number of colonies formed was counted. It should be noted that spindle-shaped colonies in the agar medium were identified as colonies of Bacillus bulgaricus.

[0224] (Viable count of Streptococcus thermophilus)

[0225] First, a solution A was prepared according to the following composition: 3.8 g of disodium glycerophosphate pentahydrate, 0.008 g of bromocresol purple, 3 g of agar, and 120 g of ultrapure water. In addition, a solution B was prepared according to the following composition: 20 g of skim milk powder and 80 g of ultrapure water. Next, solution A was sterilized by autoclave at 121° C. for 7 minutes, and solution B was sterilized by autoclave at 110° C. for 10 minutes, and after cooling to 55° C., solution A and solution B were mixed so that the concentrations of the components were as follows: 1.9 w / w% of disodium glycerophosphate pentahydrate, 0.004 w / w% of bromocresol purple, 1.5 w / w% of agar, and 10 w / w% of skim milk powder, and the mixture was dispensed into a plate to prepare a FS agar medium.

[0226] The yogurts were diluted with saline for 10 6 100 μL of the sample obtained was smeared on the surface of the FS agar medium, and after culturing at 37° C. for 24 hours, the number of colonies formed was counted. It should be noted that the yellow large colonies in the agar medium were determined to be colonies of thermophilic Streptococcus carrying the prtS gene, and the translucent small and flat colonies were determined to be colonies of thermophilic Streptococcus not carrying the prtS gene.

[0227]

Table 1

[0228]

[0229] <Examples 11 to 16>

[0230] As Bacillus bulgaricus, Streptococcus thermophilus carrying the prtS gene (prtS+), and Streptococcus thermophilus not carrying the prtS gene (prtS-), each strain shown in Table 2 below was used for combination, and each yogurt was prepared in the same manner as in Example 1, and yogurt analysis (measurement of pH and acidity after 1 day and 30 days from the start of storage, and measurement of the number of live cells of Bacillus bulgaricus and the number of live cells of each Streptococcus thermophilus) was performed. The results are shown in Table 2 below.

[0231]

Table 2

[0232]

[0233] <Comparative Example 1>

[0234] First, the Bulgarian bacteria 2038 strain and the thermophilic Streptococcus 1131 strain not carrying the prtS gene were activated in the same manner as in Example 1. Next, the total mass of the activated lactic acid bacteria was 3 w / w% (Bulgarian bacteria 2038 strain: 0.3 w / w% (viable bacteria count: 3.0×10 5 ~9.0×10 7 cfu / g), Streptococcus thermophilus 1131 strain: 2.7w / w% (viable count: 2.7×10 6 ~8.1×10 8 cfu / g)) was inoculated, and fermented at 43°C as in Example 1, and the time required from the inoculation to the pH of the prepared milk liquid dropping to 4.65 (fermentation time) was measured. The fermentation time was 3 hours and 28 minutes. Furthermore, the fermentation was terminated at the time when the pH reached 4.65 to prepare yogurt.

[0235] The prepared yogurt was stored in the same manner as in Example 1, and the pH (10°C pH) and acidity (lactic acid content: %) were measured 1 day and 30 days after the start of storage, and the number of viable Bacillus bulgaricus cells (cfu / g) was measured. The results are shown in Table 3 below.

[0236]

Table 3

[0237]

[0238] <Comparative Examples 11 and 12>

[0239] As Bacillus bulgaricus and Streptococcus thermophilus (prtS-) not carrying the prtS gene, the strains shown in Table 4 below were used in combination, and each yogurt was prepared in the same manner as in Comparative Example 1, and yogurt analysis (measurement of acidity after 1 day and 30 days from the start of storage and measurement of the number of viable cells of Bacillus bulgaricus) was performed. The results are shown in Table 4 below.

[0240]

Table 4

[0241]

[0242] As shown in Tables 3 and 4, in the case of the combination of two lactic acid bacteria (Comparative Examples 1, 11-12), when stored at low temperature, the pH dropped after 30 days, the acidity also increased significantly, and the number of viable cells of Bacillus bulgaricus also dropped to 10 1 ~10 2On the other hand, as shown in Tables 1 and 2, it can be confirmed that the combination of the three lactic acid bacteria of the present invention (Examples 1, 11 to 16) can fully suppress the decrease in pH and the increase in acidity even when stored at low temperatures for 30 days, and surprisingly, the number of viable bacteria of Bulgarian bacteria is also fully maintained at 1×10 7 As shown in Tables 2 and 4, this tendency was also the same when other strains were used in combination as Bacillus bulgaricus, Streptococcus thermophilus carrying the prtS gene (prtS+), and Streptococcus thermophilus not carrying the prtS gene (prtS-).

[0243] That is, the combination of three lactic acid bacteria, namely, the bulgaricus strain 2038, the OLS4802 strain carrying the prtS gene, and the 1131 strain not carrying the prtS gene, achieved the suppression of pH drop and acidity increase during low-temperature storage, which was difficult to achieve when the two lactic acid bacteria, namely, the 2038 strain and the 1131 strain, were combined, and the number of viable cells of the bulgaricus strain (1×10 7 cfu / g or more). Specifically, in the case of a combination of two lactic acid bacteria, the acidity D after 30 days of storage at 10°C was 30 =1.05, Bulgarian bacteria count 3.5×10 6 cfu / g, in contrast, for the combination of three lactic acid bacteria, D 30 =0.96, viable count 2.6×10 7 cfu / g. In addition, compared with the combination of two lactic acid bacteria, the fermentation time was further shortened, specifically, in the case of the combination of two lactic acid bacteria, it was shortened to 3 hours and 28 minutes (less than 98% of the combination of two lactic acid bacteria). It can be said that this shortening is sufficient for the fermentation time of 3 to 4 hours.

[0244] The combination of three lactic acid bacteria, namely, the 1589 strain of Bacillus bulgaricus, the OLS4801 strain carrying the prtS gene, and the OLS3290 strain not carrying the prtS gene, achieved the suppression of pH drop and acidity increase during low-temperature storage, which was difficult to achieve when the 1589 strain and the OLS3290 strain were combined, and the number of viable cells of Bacillus bulgaricus (1×10 7 cfu / g or more). Specifically, in the case of a combination of two lactic acid bacteria, the acidity D after 30 days of storage at 10°C was 30 =1.02, viable count of Bulgarian bacteria 5.0×10 6 cfu / g, in contrast, for the combination of three lactic acid bacteria, D 30 =0.98, viable count 3.6×10 7 cfu / g.

[0245] The combination of three lactic acid bacteria, namely, the 1589 strain of Bacillus bulgaricus, the OLS4802 strain carrying the prtS gene, and the OLS3290 strain not carrying the prtS gene, achieved the suppression of pH drop and acidity increase during low-temperature storage, which was difficult to achieve when the 1589 strain and the OLS3290 strain were combined, and the number of viable cells of Bacillus bulgaricus (1×10 7 cfu / g or more). Specifically, in the case of a combination of two lactic acid bacteria, the acidity D after 30 days of storage at 10°C was 30 =1.02, viable count of Bulgarian bacteria 5.0×10 6 cfu / g, in contrast, for the combination of three lactic acid bacteria, D 30 =0.98, viable count 4.3×10 7 cfu / g.

[0246] The combination of three lactic acid bacteria, namely, the 1589 strain of Bacillus bulgaricus, the OLS4803 strain carrying the prtS gene, and the OLS3290 strain not carrying the prtS gene, achieved the suppression of pH drop and acidity increase during low-temperature storage, which was difficult to achieve when the 1589 strain and the OLS3290 strain were combined, and the number of viable cells of Bacillus bulgaricus (1×10 7 cfu / g or more). Specifically, in the case of a combination of two lactic acid bacteria, the acidity D after 30 days of storage at 10°C was 30 =1.02, viable count of Bulgarian bacteria 5.0×10 6 cfu / g, in contrast, for the combination of three lactic acid bacteria, D 30 =0.99, viable count 6.4×10 7 cfu / g.

[0247] The combination of three lactic acid bacteria, namely, the bulgaricus 2038 strain, the OLS4801 strain carrying the prtS gene, and the OLS3290 strain not carrying the prtS gene, achieved the suppression of pH drop and acidity increase during low-temperature storage, which was difficult to achieve when the 2038 strain and the OLS3290 strain were combined, and the number of viable cells of the bulgaricus strain (1×10 7 cfu / g or more). Specifically, in the case of a combination of two lactic acid bacteria, the acidity D after 30 days of storage at 10°C was 30 =1.03, Bulgarian bacteria count 9.5×10 6 cfu / g, in contrast, for the combination of three lactic acid bacteria, D 30 =0.98, viable count 4.2×10 7 cfu / g.

[0248] The combination of three lactic acid bacteria, namely, the bulgaricus 2038 strain, the OLS4802 strain carrying the prtS gene, and the OLS3290 strain not carrying the prtS gene, achieved the suppression of pH drop and acidity increase during low-temperature storage, which was difficult to achieve when the 2038 strain and the OLS3290 strain were combined, and the number of viable cells of the bulgaricus (1×10 7 cfu / g or more). Specifically, in the case of a combination of two lactic acid bacteria, the acidity D after 30 days of storage at 10°C was 30 =1.03, Bulgarian bacteria count 9.5×10 6 cfu / g, in contrast, for the combination of three lactic acid bacteria, D 30 =0.97, viable count 4.5×10 7 cfu / g.

[0249] The combination of three lactic acid bacteria, namely, the 2038 strain of Bacillus bulgaricus, the OLS4803 strain carrying the prtS gene, and the OLS3290 strain not carrying the prtS gene, achieved the suppression of pH drop and acidity increase during low-temperature storage, which was difficult to achieve when the 2038 strain and the OLS3290 strain were combined, and the number of viable cells of Bacillus bulgaricus (1×10 7 cfu / g or more). Specifically, in the case of a combination of two lactic acid bacteria, the acidity D after 30 days of storage at 10°C was 30 =1.03, Bulgarian bacteria count 9.5×10 6 cfu / g, in contrast, for the combination of three lactic acid bacteria, D 30 =0.98, viable count 4.5×10 7 cfu / g.

[0250] <Example 2>

[0251] First, the Bulgarian bacteria OLL205013 strain, the thermophilic Streptococcus OLS4802 strain carrying the prtS gene, and the thermophilic Streptococcus OLS3290 strain not carrying the prtS gene were activated in the same manner as in Example 1. Next, the total mass of the activated lactic acid bacteria was 3 w / w% (Bulgarian bacteria OLL205013 strain: 0.3 w / w% (viable cell count: 3.0×10 5 ~9.0×10 7 cfu / g), Streptococcus thermophilus OLS4802 strain: 0.6w / w% (viable count: 6.0×10 5 ~1.8×10 8 cfu / g), Streptococcus thermophilus OLS3290 strain: 2.1w / w% (viable count: 2.1×10 6 ~6.3×10 8cfu / g)) and allowed to ferment at 43°C as in Example 1, and the time (fermentation time) required from the inoculation to the pH drop to 4.65 was measured. Then, the fermentation was terminated at the time when the pH reached 4.65 to prepare yogurt. The prepared yogurt was stored in the same manner as in Example 1, and the number of viable bacteria (cfu / g) of Bacillus bulgaricus and the number of viable bacteria (cfu / g) of each thermophilic streptococcus were measured after one day from the start of storage. The results are shown in Table 5 below.

[0252]

Table 5

[0253]

[0254] <Examples 21 to 27>

[0255] As Bacillus bulgaricus and thermophilic streptococci (prtS-) not carrying the prtS gene, the strains shown in Table 6 below were used in combination, and fermentation was carried out in the same manner as in Example 2, and the fermentation time was measured. In addition, the number of viable bacteria (cfu / g) of Bacillus bulgaricus and the number of viable bacteria (cfu / g) of each thermophilic streptococcus after 1 day from the start of storage were measured for each yogurt in the same manner as in Example 2. The results are shown in Table 6 below.

[0256]

Table 6

[0257]

[0258] <Comparative Example 2>

[0259] First, the Bulgarian bacteria OLL205013 strain and the thermophilic Streptococcus OLS3290 strain not carrying the prtS gene were activated in the same manner as in Example 1. Next, the total mass of the activated lactic acid bacteria was 3 w / w% (Bulgarian bacteria OLL205013 strain: 0.3 w / w% (viable cell count: 3.0×10 5 ~9.0×10 7 cfu / g), Streptococcus thermophilus OLS3290 strain: 2.7w / w% (viable count: 2.7×10 6 ~8.1×10 8 cfu / g)) was inoculated, and fermented at 43°C as in Example 1, and the time (fermentation time) required from the inoculation to the pH drop to 4.65 of the prepared milk liquid was measured. Furthermore, the fermentation was terminated at the time when the pH reached 4.65 to prepare yogurt. The prepared yogurt was stored in the same manner as in Example 1, and the number of viable bacteria (cfu / g) of Bulgarian bacteria was measured after one day from the start of storage. The results are shown in Table 7 below.

[0260]

Table 7

[0261]

[0262] <Comparative Examples 21 to 23>

[0263] As Bacillus bulgaricus and thermophilic streptococci (prtS-) not carrying the prtS gene, the strains shown in Table 8 below were used in combination, and fermentation was carried out in the same manner as in Comparative Example 2, and the fermentation time was measured. In addition, the number of viable bacteria (cfu / g) of Bacillus bulgaricus and the number of viable bacteria (cfu / g) of each thermophilic streptococcus after 1 day from the start of storage were measured for each yogurt in the same manner as in Comparative Example 2. The results are shown in Table 8 below.

[0264]

Table 8

[0265]

[0266] As shown in Table 5 and Table 7, relative to the combination of two kinds of lactic acid bacteria with a fermentation time of more than 7 hours (Comparative Example 2), by setting it as the combination of three kinds of lactic acid bacteria of the present invention (Example 2), the fermentation time is about 4 hours, which is greatly shortened to about 0.6 times, and the shortening of the fermentation time that is difficult to achieve when the two kinds of lactic acid bacteria are combined is achieved. Furthermore, in the combination of three kinds of lactic acid bacteria of the present invention, even if the fermentation time is shortened in this way, the number of viable bacteria of Bulgaria after fermentation can also achieve the same number as the combination of two kinds of lactic acid bacteria with a long fermentation time. In addition, as shown in Table 6 and Table 8, when various combinations are taken as Bulgaria, thermophilic streptococcus carrying the prtS gene, and thermophilic streptococcus not carrying the prtS gene, compared with the combination of two kinds of lactic acid bacteria, the shortening of the fermentation time is also achieved, and the number of viable bacteria of Bulgaria after fermentation is also fully maintained.

[0267] <Example 3>

[0268] First, yogurt was prepared in the same manner as in Example 1 using Bacillus bulgaricus 2038 strain, Streptococcus thermophilus OLS4802 strain carrying the prtS gene, and Streptococcus thermophilus 1131 strain not carrying the prtS gene. The prepared yogurt was immediately stirred and cooled to 10°C, and then lactic acid (food additive) was added and stirred to mix, thereby artificially adjusting the pH to 4.5. The yogurt of pH 4.5 was sealed in a manner to avoid air being enclosed, and was stored at 10°C. The number of live bacteria (cfu / g) of Bacillus bulgaricus at each time of pH 4.5, pH 4.3, pH 4.1, and pH 4.0 was measured by the method described in Example 1. The number of live bacteria (cfu / g) of Bacillus bulgaricus at pH 4.5 was set to 1.0, and the relative number of live bacteria (cfu / g) of Bacillus bulgaricus at each pH time is shown in Table 9 below.

[0269]

Table 9

[0270]

[0271] <Comparative Example 3>

[0272] Yogurt was prepared in the same manner as in Comparative Example 1 using 2038 strains of Bulgaria and 1131 strains of thermophilic Streptococcus that do not carry the prtS gene. The prepared yogurt was immediately stirred and cooled to 10°C, and then lactic acid (food additive) was added and stirred to mix, thereby artificially adjusting the pH to 4.5. The yogurt of pH 4.5 was sealed in a manner to avoid enclosing air, and was stored at 10°C. The number of live bacteria (cfu / g) of Bulgaria at each time of pH 4.5, pH 4.3, pH 4.1, and pH 4.0 was measured by the method described in Example 1. The number of live bacteria (cfu / g) of Bulgaria at pH 4.5 was set to 1.0, and the relative number of live bacteria (cfu / g) of Bulgaria at each pH time is shown in Table 10 below.

[0273]

Table 10

[0274]

[0275] As shown in Tables 9 and 10, it can be confirmed that according to the combination of three lactic acid bacteria of the present invention (Example 3), even if the pH is artificially lowered to 4.5 and stored at low temperature, and even if the pH is further lowered to 4.0, the number of viable bacteria of Bacillus bulgaricus is maintained at a higher level compared with the combination of two lactic acid bacteria at the same pH (Comparative Example 3).

[0276] <Example 4>

[0277] (4-1~4-3)

[0278] First, the Bulgarian 2038 strain, the thermophilic Streptococcus OLS4802 strain carrying the prtS gene, and the thermophilic Streptococcus 1131 strain not carrying the prtS gene were activated in the same manner as in Example 1. Next, the total mass of the activated lactic acid bacteria was 3 w / w% (Bulgarian 2038 strain: 0.3 w / w% (viable count: 3.0×10 5 ~9.0×10 7 cfu / g), Streptococcus thermophilus OLS4802 strain: 1.35w / w% (viable count: 1.4×10 6 ~4.2×10 8 cfu / g), Streptococcus thermophilus 1131 strain: 1.35w / w% (viable count: 1.4×10 6 ~4.2×10 8cfu / g)) and allowed to stand at 43°C for fermentation in the same manner as in Example 1. The fermentation was terminated when the pH of the prepared milk liquid dropped to 4.65, thereby obtaining yogurt 4-1.

[0279] Yogurt 4-2 was prepared in the same manner as Yogurt 4-1 except that Bacillus bulgaricus 2038 strain, Streptococcus thermophilus OLS4802 strain, and Streptococcus thermophilus OLS3290 strain, which does not carry the prtS gene and is a lactic acid bacterium that produces high polysaccharides in this example, were used. Yogurt 4-3 was prepared in the same manner as Yogurt 4-1 except that Bacillus bulgaricus OLL1251 strain, Streptococcus thermophilus OLS4802 strain, and Streptococcus thermophilus OLS3290 strain, which are lactic acid bacteria that produce high polysaccharides in this example, were used. Table 11 below shows the combinations of lactic acid bacteria in Yogurts 4-1 to 4-3, respectively.

[0280]

Table 11

[0281] yogurt Bulgarian bacteria prtS+ prtS- 4-1 2038 strains OLS4802 strain 1131 strains 4-2 2038 strains OLS4802 strain OLS3290 strain 4-3 OLL1251 strain OLS4802 strain OLS3290 strain

[0282] A total of 10 panelists were asked to conduct a sensory evaluation of the viscosity of the prepared yogurts 4-1 to 4-3. The panelists were all skilled personnel (sensory evaluation experts) who had received sensory evaluation training at Meiji Co., Ltd., which manufactures and sells fermented milk, and had more than 10 years of experience in conducting sensory evaluation tests in their daily work. The results of evaluating yogurt 4-1 and yogurt 4-2 by the 2-point test method according to JIS Z 9080 were that 10 of the 10 panelists evaluated that yogurt 4-2 had strong viscosity, and the panelists determined that it could be significantly distinguished at a significance level of 5%. Furthermore, the results of evaluating yogurt 4-2 and yogurt 4-3 by the above-mentioned 2-point test method were that 10 of the 10 panelists evaluated that yogurt 4-3 had strong viscosity, and the panelists determined that it could be significantly distinguished at a significance level of 5%. This confirmed that the viscosity of yogurt can be easily adjusted by using high-polysaccharide-producing lactic acid bacteria that produce more polysaccharides as Bacillus bulgaricus and / or Streptococcus thermophilus that does not carry the prtS gene among the combination of three lactic acid bacteria of the present invention.

[0283] (4-4~4-6)

[0284] Each yogurt was prepared in the same manner as in yogurts 4-1 to 4-3 except that the strains shown in Table 12 below were used in combination as Bacillus bulgaricus, Streptococcus thermophilus carrying the prtS gene (prtS+), and Streptococcus thermophilus not carrying the prtS gene (prtS-).

[0285]

Table 12

[0286] yogurt Bulgarian bacteria prtS+ prtS- 4-4 1589 strains OLS4802 strain OLS3078 strain 4-5 1589 strains OLS4802 strain OLS3290 strain 4-6 OLL1073R-1 strain OLS4802 strain OLS3290 strain

[0287] A total of 5 panelists were asked to conduct a sensory evaluation of the viscosity of the prepared yogurts 4-4 to 4-6. The panelists were all skilled personnel (sensory evaluation experts) who had received sensory evaluation training at Meiji Co., Ltd., which manufactures and sells fermented milk, and had more than 10 years of experience in conducting sensory evaluation tests in their daily work. The results of the evaluation of yogurts 4-4 and yogurts 4-5 by the 2-point test method according to JIS Z 9080 were that 5 of the 5 panelists evaluated that the viscosity of yogurt 4-5 was strong, and the panelists determined that it could be significantly distinguished at a significance level of 5%. Furthermore, the results of the evaluation of yogurts 4-5 and yogurts 4-6 by the above-mentioned 2-point test method were that 5 of the 5 panelists evaluated that the viscosity of yogurt 4-6 was strong, and the panelists determined that it could be significantly distinguished at a significance level of 5%. This confirmed that when using other strains, in the combination of three lactic acid bacteria of the present invention, by using high-polysaccharide-producing lactic acid bacteria that produce more polysaccharides as Bacillus bulgaricus and / or Streptococcus thermophilus that does not carry the prtS gene, the viscosity of yogurt can be easily adjusted.

[0288] <Example 5>

[0289] First, the Bulgarian 2038 strain, the thermophilic Streptococcus OLS4802 strain carrying the prtS gene, and the thermophilic Streptococcus OLS3290 strain not carrying the prtS gene were activated in the same manner as in Example 1. Next, the total mass of the activated lactic acid bacteria was 3 w / w% (Bulgarian 2038 strain: 0.3 w / w% (viable cell count: 3.0×10 5 ~9.0×10 7 cfu / g), Streptococcus thermophilus OLS4802 strain: 1.35w / w% (viable count: 1.4×10 6 ~4.2×10 8 cfu / g), Streptococcus thermophilus OLS3290 strain: 1.35w / w% (viable count: 1.4×10 6 ~4.2×10 8 cfu / g)) and allowed to stand at 43°C for fermentation in the same manner as in Example 1. The fermentation was terminated when the pH of the prepared milk liquid dropped to 4.65, thereby obtaining Yogurt 5-1.

[0290] Yogurt 5-2 was prepared in the same manner as Yogurt 5-1 except that B. bulgaricus strain 2038, Streptococcus thermophilus strain OLS4802 carrying the prtS gene, and Streptococcus thermophilus strain 1131 not carrying the prtS gene were used. Yogurt 5-3 was prepared in the same manner as Yogurt 5-1 except that B. bulgaricus strain 2038, Streptococcus thermophilus strain OLS4802 carrying the prtS gene, and Streptococcus thermophilus strain OLS3078 not carrying the prtS gene were used.

[0291] The prepared yogurt was stored in the same manner as in Example 1. 40 g of the yogurt was weighed at 5 days and 20 days from the start of storage, respectively, and centrifuged at 1610 × g, 25° C., and 10 minutes. The weight of the precipitate was measured after the supernatant was removed, and the water separation rate (%) was calculated by the following formula: weight of the precipitate (g) / 40 g×100. The combinations of lactic acid bacteria and the water separation rates of yogurts 5-1 to 5-3 are shown in Table 13 below.

[0292] <Comparative Example 5>

[0293] First, the Bulgarian 2038 strain and the thermophilic Streptococcus OLS4802 strain carrying the prtS gene were activated in the same manner as in Example 1. Next, the total mass of the activated lactic acid bacteria was 3 w / w% (Bulgarian 2038 strain: 0.3 w / w% (viable cell count: 3.0×10 5 ~9.0×10 7 cfu / g), Streptococcus thermophilus OLS4802 strain: 2.7w / w% (viable count: 2.7×10 6 ~8.1×10 8 cfu / g)) and allowed to stand at 43°C for fermentation in the same manner as in Example 1. The fermentation was terminated when the pH of the prepared milk liquid dropped to 4.65 to prepare yogurt. For the prepared yogurt, the water separation rate (%) after 5 days and 20 days from the start of storage was calculated in the same manner as in Example 5. The combination of lactic acid bacteria of the prepared yogurt and the water separation rate of each are also shown in Table 13 below.

[0294]

Table 13

[0295]

[0296] As shown in Table 13, it can be confirmed that the combination of three lactic acid bacteria of the present invention (Example 5, yogurt 5-1 to 5-3) has a low water separation rate even when stored at low temperature for 20 days, compared with the combination of two lactic acid bacteria (Comparative Example 5). In order to confirm this by sensory evaluation, a total of 10 or 8 panelists were asked to evaluate the smoothness of the prepared yogurts 5-1 to 5-3 and the yogurt of Comparative Example 5. The panelists were all skilled persons (sensory evaluation experts) who had received sensory evaluation training at Meiji Co., Ltd., which manufactures and sells fermented milk, and had more than 10 years of experience in conducting sensory evaluation tests in their daily work. For the yogurt of Comparative Example 5 and yogurt 5-1, the result of the evaluation by the 2-point recognition method according to JIS Z 9080 was that 10 of the 10 panelists evaluated that yogurt 5-1 was smooth, and the panelists determined that it could be significantly distinguished at a significant level of 5%. In addition, for the yogurt of Comparative Example 5 and Yogurt 5-2, the result of evaluation by the above-mentioned two-point identification method was that 8 out of 8 panelists evaluated that Yogurt 5-2 was smooth, and the panelists determined that it could be significantly distinguished at a significant level of 5%. In addition, for the yogurt of Comparative Example 5 and Yogurt 5-3, the result of evaluation by the above-mentioned two-point identification method was that 10 out of 10 panelists evaluated that Yogurt 5-3 was smooth, and the panelists determined that it could be significantly distinguished at a significant level of 5%. It was thus confirmed that according to the combination of the three lactic acid bacteria of the present invention, a yogurt (fermented milk) having a smooth physical property can be obtained.

[0297] <Examples 51 to 57, Comparative Examples 51 to 57>

[0298] Each yogurt of Examples 51 to 57 was prepared in the same manner as the yogurt 5-1 of Example 5, except that the strains shown in Table 14 below were used in combination as Bacillus bulgaricus, Streptococcus thermophilus carrying the prtS gene (prtS+), and Streptococcus thermophilus not carrying the prtS gene (prtS-). In addition, each yogurt of Comparative Examples 51 to 57 was prepared in the same manner as the yogurt of Comparative Example 5, except that the strains shown in Table 14 below were used in combination as Bacillus bulgaricus and Streptococcus thermophilus carrying the prtS gene (prtS+). For the prepared yogurt, the water separation rate (%) after 5 days and after 20 days from the start of storage was calculated in the same manner as in Example 5. The combination of lactic acid bacteria and each water separation rate of each prepared yogurt are shown in Table 14 below.

[0299]

Table 14

[0300]

[0301] As shown in Table 14, it can be confirmed in the same manner as in Example 5 that when using other strains, the combination of three lactic acid bacteria of the present invention (Examples 51 to 57) has a low water separation rate even when stored at low temperatures for 20 days, compared with the combination of two lactic acid bacteria (Comparative Examples 51 to 57). In addition, a total of five panelists were asked to evaluate the smoothness in the same manner as above, and as a result, five of the five panelists evaluated that the yogurts of Examples 51 to 57 were smoother than the yogurts of Comparative Examples 51 to 57, respectively, and the panelists determined that they could be significantly distinguished at a significant level of 5%. It should be noted that the panelists were all skilled persons (sensory evaluation experts) who had received sensory evaluation training at Meiji Co., Ltd., which manufactures and sells fermented milk, and had more than 10 years of experience in conducting sensory evaluation tests in their daily work. It was thus confirmed that when using other strains, a yogurt (fermented milk) with smooth physical properties can be obtained according to the combination of three lactic acid bacteria of the present invention.

[0302] Industrial Applicability

[0303] As described above, according to the present invention, there can be provided fermented milk in which the number of live bacteria of Lactobacillus delbrueckii subspecies bulgaricus is sufficiently maintained even when stored at low temperatures, the decrease in pH and the increase in acidity during the above-mentioned low-temperature storage are suppressed, and the fermented milk has smooth physical properties, as well as a lactic acid bacteria composition and a method for producing fermented milk that can obtain the fermented milk in a sufficiently short fermentation time.

[0304]

Accession number

[0306] (1) Identification mark: Lactobacillus delbrueckii subsp.bulgaricus OLL1171

[0307] (2) Accession number: NITE BP-01569

[0308] (3) Deposit date: March 13, 2013

[0309] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 2.

[0311] (1) Identification mark: Lactobacillus delbrueckii subsp.bulgaricus OLL1073R-1

[0312] (2) Accession number: FERM BP-10741

[0313] (3) Deposit date: February 22, 1999

[0314] (4) Depository: Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (currently Patent Organism Depository of the National Institute of Product Evaluation and Technology) 3.

[0316] (1) Identification mark: Lactobacillus delbrueckii subsp.bulgaricus OLL205013

[0317] (2) Accession number: NITE BP-02411

[0318] (3) Deposit date: February 3, 2017

[0319] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 4.

[0321] (1) Identification mark: Lactobacillus delbrueckii subsp.bulgaricus OLL1247

[0322] (2) Accession number: NITE BP-01814

[0323] (3) Deposit date: March 6, 2014

[0324] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 5.

[0326] (1) Identification mark: Lactobacillus delbrueckii subsp.bulgaricus OLL1251

[0327] (2) Accession number: NITE BP-02703

[0328] (3) Deposit date: April 25, 2018

[0329] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 6.

[0331] (1) Identification mark: Streptococcus thermophilus OLS4801

[0332] (2) Accession number: NITE BP-03504

[0333] (3) Storage date: August 4, 2021

[0334] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 7.

[0336] (1) Identification mark: Streptococcus thermophilus OLS4802

[0337] (2) Accession number: NITE BP-03505

[0338] (3) Storage date: August 4, 2021

[0339] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 8.

[0341] (1) Identification mark: Streptococcus thermophilus OLS4803

[0342] (2) Accession number: NITE BP-03506

[0343] (3) Storage date: August 4, 2021

[0344] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 9.

[0346] (1) Identification mark: Streptococcus thermophilus OLS4823

[0347] (2) Accession number: NITE BP-03507

[0348] (3) Storage date: August 4, 2021

[0349] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 10.

[0351] (1) Identification mark: Streptococcus thermophilus OLS4824

[0352] (2) Accession number: NITE BP-03508

[0353] (3) Storage date: August 4, 2021

[0354] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 11.

[0356] (1) Identification mark: Streptococcus thermophilus OLS3078

[0357] (2) Accession number: NITE BP-01697

[0358] (3) Deposit date: August 23, 2013

[0359] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 12.

[0361] (1) Identification mark: Streptococcus thermophilus OLS3615

[0362] (2) Accession number: NITE BP-01696

[0363] (3) Deposit date: August 23, 2013

[0364] (4) Depository: Patent Microorganism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 13.

[0366] (1) Identification mark: Streptococcus thermophilus OLS3290

[0367] (2) Accession number: FERM BP-19638

[0368] (3) Deposit date: January 19, 2004

[0369] (4) Depository: Patent Organism Depository of the Institute for Product Evaluation and Technology, an independent administrative institution 14.

[0371] (1) Identification mark: Lactobacillus delbrueckii subsp.bulgaricus 1589

[0372] (2) Accession number: NITE BP-03716

[0373] (3) Storage date: August 9, 2022

[0374] (4) Depository: Patent Microorganism Depository Center of the National Institute for Product Evaluation and Technology, an independent administrative institution.

Claims

1. A lactic acid bacteria composition comprising Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus carrying a prtS gene, and Streptococcus thermophilus not carrying a prtS gene.

2. The lactic acid bacteria composition according to claim 1, wherein The mass ratio of the content of Lactobacillus delbrueckii subspecies bulgaricus to the total content of Streptococcus thermophilus carrying the prtS gene and Streptococcus thermophilus not carrying the prtS gene is 1:1 to 1:

150.

3. The lactic acid bacteria composition according to claim 1, wherein The mass ratio of the content of thermophilic Streptococcus carrying the prtS gene to the content of thermophilic Streptococcus not carrying the prtS gene is 1:0.05 to 1:

10.

4. The lactic acid bacteria composition according to claim 1, wherein the composition is added to a prepared milk liquid containing raw material milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the number of viable cells of Lactobacillus delbrueckii subspecies bulgaricus in the fermented composition after 30 days of storage reaches 1×10 7 cfu / g or above.

5. The lactic acid bacteria composition according to claim 1, which is a composition that is added to a modulated milk liquid containing raw milk and fermented at 43°C until the pH of the modulated milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the acidity of the fermented composition after 30 days of storage is 1.0% or less.

6. The lactic acid bacteria composition according to claim 1, wherein the composition is added to a prepared milk liquid containing raw milk and fermented at 43°C until the pH of the prepared milk liquid reaches 4.65, and when the fermented composition is stored at 10°C for 30 days, the acidity (D1%) of the fermented composition after 1 day of storage and the acidity (D2%) of the fermented composition after 30 days of storage are 30 %) satisfies the following formula: D 30 -The conditions shown in D1≤0.

25.

7. The lactic acid bacteria composition according to claim 1, wherein Lactobacillus delbrueckii subsp. bulgaricus and / or Streptococcus thermophilus without the prtS gene are lactic acid bacteria that produce high polysaccharide. The lactic acid bacteria composition according to claim 1 , which is a lactic acid bacteria starter. The lactic acid bacteria composition according to claim 1, which is fermented milk.

10. A lactic acid bacteria composition comprising Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus carrying a prtS gene, and Streptococcus thermophilus not carrying a prtS gene. The lactic acid bacteria composition according to claim 10 , which is a lactic acid bacteria starter.

12. A method for producing fermented milk, characterized in that: The method comprises a fermentation step of adding the lactic acid bacteria composition according to any one of claims 1 to 9 or the lactic acid bacteria combination according to claim 10 or 11 to a prepared milk liquid containing raw material milk and fermenting the mixture to obtain fermented milk.

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