Fermented milk and method for producing the same

Abstract

To improve survival property of a Bifidobacterium bacterium, and a specific Lactobacillus bacterium, particularly survival property when storing in refrigeration without affecting a flavor of a fermented milk, and without requiring addition of a material other than a raw material for production of the fermented milk.SOLUTION: The problem can be solved by a fermented milk in which the viable bacteria count of a Bifidobacterium bacterium at a final storage day is 1.0E+7 cfu / g or more, and a viable bacteria count of a Lactobacillus bacterium is 1.0E+7 cfu / g or more when the fermented milk is stored at 1-10°C for 15-24 days where the Lactobacillus bacterium is one or more selected from the group consisting of Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacilus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to fermented milk that retains viable cell counts of Bifidobacterium bacteria and specific Lactobacillus bacteria at 1.0E+7 cfu / g or more and 1.0E+7 cfu / g or more, respectively, on the 15th to 24th days of refrigerated storage. The present invention relates to a method for producing fermented milk in which the viable cell counts of Bifidobacterium bacteria and specific Lactobacillus bacteria are improved on the 15th to 24th days of refrigerated storage. The present invention also relates to a method for improving the viability of Bifidobacterium bacteria and specific Lactobacillus bacteria in fermented milk.

Background Art

[0002] In recent years, the functions of lactic acid bacteria typified by Bifidobacterium bacteria and Lactobacillus bacteria, such as intestinal regulation and immune functions, have been gradually revealed. Improving the viability of Bifidobacterium bacteria and lactic acid bacteria in products has great industrial advantages such as utilization in health functional foods such as foods for specified health use and foods with functional claims. However, fermented milk products during refrigerated storage are not an environment suitable for the survival of Bifidobacterium bacteria and lactic acid bacteria, and various solutions for improving the viability of Bifidobacterium bacteria and lactic acid bacteria in milk have been disclosed so far. Patent Document 1 aims to provide a viability improver for lactic acid bacteria and / or Bifidobacterium bacteria that can improve the viability of probiotic lactic acid bacteria and Bifidobacterium bacteria in an acidic (low pH) environment, a food composition using the same, and a method for producing the same. As a solution, a viability improver for lactic acid bacteria and / or Bifidobacterium bacteria containing an amino acid as an active ingredient is disclosed. Patent Document 2 aims to provide a method for producing a post-fermentation type of fermented milk capable of obtaining fermented milk containing a large amount of Bifidobacterium bacteria. After inoculating Bifidobacterium bacteria into a medium mainly composed of milk and performing primary culture, lactic acid bacteria are inoculated into the obtained culture solution, and after filling into a container, secondary culture is performed. Patent Document 3 aims to provide a highly palatable fermented milk that promotes the fermentation of lactic acid bacteria, shortens the fermentation time, suppresses the increase in acidity during refrigerated storage, and maintains a moderately mild sour taste, and a method for producing the same. In the production of fermented milk using a mixed starter, it discloses a method of blending a single culture solution of Lactobacillus delbrueckii subsp. bulgaricus in raw milk. However, in the technology described in Patent Document 1, it is necessary to add various amino acids to the yogurt mix, and this amino acid is likely to have a significant impact on the flavor of the final product. In Patent Documents 2 and 3, a method for simultaneously improving the viability of not only Bifidobacterium bacteria but also both Bifidobacterium bacteria and lactic acid bacteria is not disclosed.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] The problem of the present invention is to provide a fermented milk that maintains viable cell counts of Bifidobacterium bacteria and specific Lactobacillus bacteria at 1.0E+7 cfu / g or more and 1.0E+7 cfu / g or more, respectively, on the 15th to 24th days of refrigerated storage, and a production method for producing the fermented milk.

Means for Solving the Problems

[0005] To solve the above problems, the present invention includes the following configurations. <1>When stored at 1 to 10 °C until the 15th to 24th day, on the last day of storage, the viable count of Bifidobacterium bacteria is 1.0E+7 cfu / g or more, and the viable count of Lactobacillus bacteria is 1.0E+7 cfu / g or more. The fermented milk, wherein the Lactobacillus bacteria are one or more selected from the group consisting of Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii. <2>The fermented milk according to <1>, wherein the Bifidobacterium bacteria are Bifidobacterium longum and the Lactobacillus bacteria are Lactobacillus gasseri. <3>(A) A step of adding Bifidobacterium bacteria to the raw material mix; (B) A step of fermenting the raw material mix to obtain a fermented milk mix; (C) A step of adding a bulk starter of Lactobacillus bacteria to the fermented milk mix; (D) A step of refrigerating and storing the fermented milk mix to obtain fermented milk. A method for producing fermented milk, comprising: The Lactobacillus bacteria are one or more selected from the group consisting of Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii. The production method. <4>The method for producing fermented milk according to <3>, wherein the Bifidobacterium bacteria are Bifidobacterium longum and the Lactobacillus bacteria are Lactobacillus gasseri. <5>In the step (A), in addition to the Bifidobacterium bacteria, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are added. The method for producing fermented milk according to <3> or <4>. <6>The method for producing fermented milk according to any one of <3> to <5>, wherein the step (D) is carried out within 6 hours after the step (C). <7>The method for producing fermented milk according to any one of <3> to <6>, including the step of cooling (E) the fermented milk mixture to 20°C or lower between steps (B) and (C). <8>(A) A step of adding Bifidobacterium bacteria to the raw material mixture (B) A step of fermenting the raw material mixture to obtain a fermented milk mixture (C) A step of adding a bulk starter of Lactobacillus bacteria to the fermented milk mixture (D) A step of refrigerating and storing the fermented milk mixture to obtain fermented milk A method for improving the viability of Bifidobacterium bacteria and Lactobacillus bacteria in fermented milk, including: The method for improving viability, wherein the Lactobacillus bacteria are one or more selected from the group consisting of Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii. <9>The method for improving the viability of Bifidobacterium bacteria and Lactobacillus bacteria in fermented milk according to <8>, wherein the Bifidobacterium bacteria is Bifidobacterium longum and the Lactobacillus bacteria is Lactobacillus gasseri. <10>The method for improving the viability of Bifidobacterium bacteria and Lactobacillus bacteria in fermented milk according to <8> or <9>, wherein step (D) is carried out within 6 hours after step (C). <11>The method for improving the viability of Bifidobacterium bacteria and Lactobacillus bacteria in fermented milk according to any one of <8> to <10>, including the step of cooling (E) the fermented milk mixture to 20°C or lower between steps (B) and (C).

Advantages of the Invention

[0006] The present invention provides a fermented milk that retains viable cell counts of Bifidobacterium bacteria and specific Lactobacillus bacteria at 1.0E+7 cfu / g or more and 1.0E+7 cfu / g or more, respectively, on the 15th to 24th days of refrigerated storage. According to the present invention, it is not necessary to add anything other than the raw materials for producing the fermented milk, and it is possible to improve the viability of Bifidobacterium bacteria and specific Lactobacillus bacteria, particularly their viability during refrigerated storage, without affecting the flavor of the fermented milk.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

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Figure 6

Embodiments for Carrying Out the Invention

[0008] 1. Fermented milk (Bifidobacterium bacteria) As used herein, the term "Bifidobacterium bacterium" means a bacterium belonging to the genus Bifidobacterium. The Bifidobacterium bacterium is not particularly limited as long as it belongs to the genus Bifidobacterium, and examples thereof include Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium catenulatum, and Bifidobacterium dentium. Among these, Bifidobacterium longum is mentioned as a preferred example. In addition, as strains, Bifidobacterium longum SBT2928 strain (Deposit number: FERM P-10657, Deposit date: April 13, 1989, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary) and Bifidobacterium pseudolongum SBT2908 strain (Deposit number: FERM P-10138, Deposit date: July 20, 1988, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary) can be exemplified.

[0009] (Lactobacillus bacterium) As used herein, the term "Lactobacillus bacterium" means a bacterium belonging to the genus Lactobacillus. The Lactobacillus bacterium used in the present invention is Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii. In the present specification, these six types of Lactobacillus bacteria may be referred to as "specific Lactobacillus bacteria". Among the Lactobacillus bacteria, Lactobacillus gasseri is most preferred. In addition, as a strain of Lactobacillus gasseri, Lactobacillus gasseri SBT2055 strain (Deposit number: FERM BP-10953, Deposit date: February 26, 2008, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary) can be exemplified.

[0010] (Viable cell count) The fermented milk of the present invention, when stored at 1 to 10 °C, preferably at 5 to 10 °C, more preferably at 8 to 10 °C until the 15th to 24th day, on the last day of storage, the viable count of bacteria belonging to the genus Bifidobacterium and the viable count of specific bacteria belonging to the genus Lactobacillus are each 1.0E+7 cfu / g or more and 1.0E+7 cfu / g or more. In this specification, the description of a specific temperature, for example, "10 °C", is a concept that allows a slight temperature change. For example, it is allowed that the storage or refrigeration temperature is "10 ± 1 °C". The day when the storage of the fermented milk of the present invention is started in a refrigerator or the like is the 0th day of refrigerated storage, and the day following the start of storage is the 1st day of refrigerated storage. "1.0E+7 cfu / g" means 1.0×10 7 cfu / g. The storage from 1 to 10 °C until the 15th to 24th day can be carried out in a container having oxygen permeability such as plastic in a refrigerator or the like. The upper limit of the viable count of bacteria belonging to the genus Bifidobacterium on the last day of storage is not particularly limited as long as the effects of the present invention can be obtained, but can be 1.0E+11 cfu / g, 1.0E+10 cfu / g, 1.0E+9 cfu / g, 5.0E+8 cfu / g, 1.5E+8 cfu / g, or 1.4E+8 cfu / g. The upper limit of the viable count of specific bacteria belonging to the genus Lactobacillus on the last day of storage is not particularly limited as long as the effects of the present invention can be obtained, but can be 1.0E+11 cfu / g, 1.0E+10 cfu / g, 1.0E+9 cfu / g, 5.0E+8 cfu / g, 1.0E+8 cfu / g, 3.0E+7 cfu / g, or 2.3E+7 cfu / g. The viable count of bacteria belonging to the genus Bifidobacterium and the viable count of specific bacteria belonging to the genus Lactobacillus can be measured by the plate count method. Specifically, the fermented milk is serially diluted using sterilized dilution water (0.6% disodium hydrogen phosphate, 0.45% potassium dihydrogen phosphate, 0.05% L-cysteine hydrochloride monohydrate, 0.05% Tween 80, 0.05% agar). For measuring the viable count of Bifidobacterium bacteria, the diluted sample is mixed using mupirocin-added TOS propionate agar medium, and for measuring the viable count of Lactobacillus bacteria, the diluted sample is spread on a plate medium of MRS / CL / CIP agar medium, and the viable count in the serially diluted fermented milk is measured. As the MRS / CL / CIP agar medium, clindamycin is added to the sterilized MRS medium at 0.1 μg / mL and ciprofloxacin at 10 μg / mL, and a plate medium poured and solidified in a petri dish is used. Anaerobic culture is performed at 37°C for 72 hours using an anaerobic culture system. After the culture is completed, the number of colonies is counted to measure the viable count. In this specification, "when stored at 1 to 10°C until the 15th to 24th day, on the last day of storage, the viable count of Bifidobacterium bacteria is 1.0E+7 cfu / g or more, and the viable count of a specific Lactobacillus bacteria is 1.0E+7 cfu / g or more" means that when stored at 1 to 10°C until the 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, or 24th day, on each of the 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, or 24th day, the viable count of Bifidobacterium bacteria is 1.0E+7 cfu / g or more, and the viable count of a specific Lactobacillus bacteria is 1.0E+7 cfu / g or more.

[0011] When the fermented milk of the present invention is stored at 1 to 10°C until the 15th to 24th day, on the last day of storage, the viable count of Bifidobacterium bacteria and the viable count of a specific Lactobacillus bacteria are preferably 1.2E+7 cfu / g or more and 1.2E+7 cfu / g or more, and more preferably 1.5E+7 cfu / g or more and 1.5E+7 cfu / g or more.

[0012] When the fermented milk of the present invention is stored at 1 to 10 °C until the 21st to 24th day, on the last day of storage, the viable count of bacteria belonging to the genus Bifidobacterium and the viable count of specific bacteria belonging to the genus Lactobacillus are 1.0E+7 cfu / g or more and 1.0E+7 cfu / g or more, preferably 1.2E+7 cfu / g or more and 1.2E+7 cfu / g or more, and more preferably 1.5E+7 cfu / g or more and 1.5E+7 cfu / g or more.

[0013] In the fermented milk of the present invention, when stored at 1 to 10 °C until the 24th day, on the last day of storage, the viable count of bacteria belonging to the genus Bifidobacterium is preferably 10.0% or more, more preferably 10.5% or more, based on the viable count on the first day from the start of storage. In the fermented milk of the present invention, when stored at 1 to 10 °C until the 24th day, on the last day of storage, the viable count of specific bacteria belonging to the genus Lactobacillus is preferably 80% or more, more preferably 82% or more, based on the viable count on the first day from the start of storage.

[0014] The fermented milk of the present invention may also contain other bacteria as the fermentation starter lactic acid bacteria, such as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0015] The container for filling the fermented milk of the present invention is not particularly limited as long as the effects of the present invention can be obtained, and examples include plastic containers and paper containers. The fermented milk of the present invention is preferably fermented milk in a container.

[0016] (Fermented milk) In this specification, "fermented milk" refers to fermented milk in the Ministerial Ordinance (Ordinance on Milk etc.) regarding the ingredient standards of milk and dairy products, and examples include so-called stirred yogurt, set yogurt, pre-fermented yogurt, post-fermented yogurt, soft yogurt, and hard yogurt.

[0017] The milk raw materials used for the production of the fermented milk of the present invention may be those used in the production of ordinary fermented milk, and are not particularly limited. For example, raw milk, cow milk, special milk, raw goat milk, pasteurized goat milk, raw ewe milk, adjusted milk, low-fat milk, non-fat milk, processed milk, cream, butter, butter oil, cheese, concentrated whey, ice creams, condensed milk, skim condensed milk, unsweetened condensed milk, unsweetened skim condensed milk, sweetened condensed milk, sweetened skim condensed milk, whole milk powder, skim milk powder, cream powder, whey powder, protein concentrated whey powder, buttermilk powder, sweetened milk powder, whey protein concentrate (WPC), whey protein isolate (WPI), milk protein concentrate (MPC), milk protein isolate (MPI), total milk protein (TMP), de-lactosed permeate powder (ultrafiltration membrane permeate component of milk), microparticulated whey, acid casein, rennet casein, sodium caseinate, potassium caseinate, etc. can be mentioned. Any of these milk raw materials can be used, and those obtained by combining two or more of these milk raw materials can also be used. Further, fats and oils, oligosaccharides, sweeteners, acidulants, flavors, fruit juices, fruit pulps, emulsifiers, etc. may be blended.

[0018] The pH of the fermented milk of the present invention is preferably 4.00 to 4.80, more preferably 4.20 to 4.60.

[0019] 2. Method for producing fermented milk (Step (A)) The method for producing fermented milk of the present invention includes a step of adding bacteria belonging to the genus Bifidobacterium to a raw material mix. The raw material mix is a mix suitable for culturing bacteria belonging to the genus Bifidobacterium, bacteria belonging to the genus Lactobacillus, etc., including the above-described milk raw material. The method for dissolving the milk raw material in a solvent to form a solution is not particularly limited and can be carried out by a commonly used method. The raw material mix is preferably sterilized by batch sterilization or the like at the time of adding the bacteria belonging to the genus Bifidobacterium.

[0020] The bacteria belonging to the genus Bifidobacterium are not particularly limited as long as they are bacteria belonging to the genus Bifidobacterium, and examples include Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium catenulatum, Bifidobacterium dentium, etc. Among these, as a preferred example, Bifidobacterium longum can be mentioned. Also, as strains, Bifidobacterium longum SBT2928 strain and Bifidobacterium pseudolongum SBT2908 strain can be exemplified. When using Bifidobacterium longum SBT2928 strain and / or Bifidobacterium pseudolongum SBT2908 strain, the bacteria belonging to the genus Lactobacillus are preferably Lactobacillus gasseri, and more preferably Lactobacillus gasseri SBT2055 strain.

[0021] The bacteria belonging to the genus Bifidobacterium are preferably added in the form of a bulk starter. In the present specification, the "bulk starter" is a starter prepared through intermediate fermentation by culturing a cell sample such as a concentrated cell mass or a culture in a medium. That is, the bulk starter includes a cell mass and a culture medium. On the other hand, in the present specification, a lactic acid bacteria starter that is the cell sample itself such as a concentrated cell mass or a culture is referred to as a "cell starter". When adding Bifidobacterium bacteria as a bulk starter, it can be added in an amount of 0.10 to 20%, preferably 0.50 to 15%, more preferably 1.0 to 10%, and even more preferably 2.0 to 8.0% based on the raw material mix standard. When adding Bifidobacterium bacteria as a bulk starter, considering that the change in viable cell count is small during cultivation in the raw material mix, the viable cell count of Bifidobacterium bacteria can be added so as to be 1.0E+8 cfu / g to 1.0E+9 cfu / g, more preferably 1.2E+8 cfu / g to 5.0E+8 cfu / g, and even more preferably 1.4E+8 cfu / g to 3.0E+8 cfu / g based on the raw material mix standard. The bulk starter is not particularly limited as long as the effects of the present invention can be obtained, and a bulk starter prepared by adding a cell sample to a culture medium can be used. When preparing a bulk starter of Bifidobacterium bacteria, external additive substances for the growth of Bifidobacterium bacteria such as yeast extract, peptone, and amino acids can be added as necessary.

[0022] In the method for producing fermented milk of the present invention, in addition to Bifidobacterium bacteria, it is preferable to add Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus as fermented starter lactic acid bacteria. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are preferably added in the form of a cell starter. It is more preferable to add Bifidobacterium bacteria as a bulk starter and further add Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the form of a cell starter. The dosage of the cell starter is 0.010 to 10%, preferably 0.010 to 3.0%, more preferably 0.020 to 1.0%, and most preferably 0.050 to 0.50% based on the raw material mix standard. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus may be added alone or may be added after being premixed. It is preferable to promptly start step (B) after the completion of step (A).

[0023] (Process (B)) In the method for producing fermented milk of the present invention, after adding bacteria belonging to the genus Bifidobacterium to the raw material mix, fermentation is carried out to obtain a fermented milk mix. In this specification, fermentation means incubating the raw material mix at an appropriate temperature to grow the bacteria contained in the raw material mix.

[0024] The fermentation temperature is not particularly limited as long as the effects of the present invention can be obtained, and for example, it can be carried out at 30 to 50°C, preferably 35 to 45°C. The fermentation time is about 3 to 24 hours, preferably about 3 to 12 hours.

[0025] Process (B) can preferably be carried out until the acidity of the raw material mix reaches 0.75, preferably 0.80.

[0026] In the method for producing fermented milk of the present invention, after process (B), it is preferable to include (E) a step of cooling the fermented milk mix to 20°C or lower. By cooling the fermented milk mix, it is possible to more effectively prevent the presence of Lactobacillus gasseri or bacteria belonging to the genus Bifidobacterium from being affected by the presence of the other. The fermented milk mix is more preferably cooled to 15°C or lower, and even more preferably to 10 ± 5°C.

[0027] (Process (C)) In the method for producing fermented milk of the present invention, a bulk starter of specific Lactobacillus bacteria is added to the fermented milk mix after step (B) (see Figure 1). The bulk starter of specific Lactobacillus bacteria is cooled to 20°C or lower, preferably 15°C or lower, more preferably 10 ± 5°C, and then added to the fermented milk mix. The Lactobacillus bacteria used in the present invention are Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii. Among them, Lactobacillus gasseri is most preferred. As a strain of Lactobacillus gasseri, Lactobacillus gasseri SBT2055 can be exemplified. When using Lactobacillus gasseri SBT2055, the Bifidobacterium bacteria are preferably Bifidobacterium longum, and more preferably Bifidobacterium longum SBT2928 and / or Bifidobacterium pseudolongum SBT2908.

[0028] In step (C), a bulk starter of specific Lactobacillus bacteria is added. The bulk starter can be added in an amount of 0.010 to 10%, preferably 0.10 to 5.0%, more preferably 0.20 to 3.0%, still more preferably 0.30 to 2.0% based on the fermented milk mix. Also, the bulk starter of specific Lactobacillus bacteria can be added so as to be 2.0E+7 cfu / g to 1.0E+9 cfu / g, 2.0E+7 cfu / g to 5.0E+8 cfu / g, 2.0E+7 cfu / g to 1.0E+8 cfu / g based on the fermented milk mix. The bulk starter is not particularly limited as long as the effects of the present invention can be obtained, and a bulk starter prepared by adding a cell sample to a culture medium can be used. When preparing a bulk starter of Lactobacillus bacteria, external additives for the growth of Lactobacillus bacteria such as yeast extract, peptone, and amino acids can be added as necessary.

[0029] (Step (D)) In the method for producing fermented milk of the present invention, after adding a bulk starter of specific Lactobacillus bacteria to the fermented milk mix, the fermented milk mix is refrigerated and stored. After adding the bulk starter of specific Lactobacillus bacteria to the fermented milk mix, it is preferably refrigerated and stored promptly. Specifically, the fermented milk mix is refrigerated and stored within 6 hours, preferably within 5 hours, more preferably within 3 hours, and most preferably within 1 hour after the addition.

[0030] In this specification, "refrigeration" means storing the fermented milk mix at a temperature of 10°C or lower in a refrigerator or the like. The fermented milk mix is preferably stored at 1°C to 10°C, more preferably at 5°C to 10°C, and even more preferably at 8 to 10°C.

[0031] In the method for producing fermented milk of the present invention, when stored from the start of step (D) to the 15th to 24th day (or when stored to the 21st to 24th day), on the last day of storage, the viable count of Bifidobacterium bacteria and the viable count of specific Lactobacillus bacteria are preferably 1.0E+7 cfu / or more and 1.0E+7 cfu / or more, respectively. In the method for producing fermented milk of the present invention, compared with the case where a cell starter of Lactobacillus bacteria is administered in step (A) instead of step (C), the survival rate of Bifidobacterium bacteria can be 1.5 times or more, preferably 2 times or more, and the survival rate of specific Lactobacillus bacteria can be 5 times or more, preferably 8 times or more.

[0032] (Other steps) In the method for producing fermented milk of the present invention, as long as the effects of the present invention can be obtained, it may include the step of adding (F) stabilizers, additives, flavors, fruits, fruit sauces, etc. and stirring, and the step of filling (G) into containers. The step of adding and stirring (F) stabilizers, additives, flavors, fruits, fruit sauces, etc. and the step of filling (G) into containers are preferably included between step (C) and step (D). In the method for producing fermented milk of the present invention, it preferably includes the step of filling (G) into containers between step (C) and step (D), and step (D) is carried out in a state where the container is filled. The container is not particularly limited as long as the effects of the present invention can be obtained, and examples include plastic containers and paper containers.

[0033] 3. Method for improving viability (Improving viability) "Improving viability" in the present invention means suppressing the death of bacterial cells and maintaining the number of surviving bacterial cells (suppressing decrease). In the present invention, "improving viability" includes suppressing the decrease in the number of bacterial cells when stored for a certain period under specific conditions, for example, storage conditions of 1 to 10 °C, preferably 5 to 10 °C, more preferably 8 to 10 °C. In the present invention, "improving viability" means that when stored at 1 to 10 °C until the 15th to 24th day (or when stored until the 21st to 24th day), on the last day of storage, the viable count of Bifidobacterium bacteria and the viable count of specific Lactobacillus bacteria are, respectively, for example, 1.0E+7 cfu / g or more and 1.0E+7 cfu / g or more, more preferably 1.2E+7 cfu / g or more and 1.2E+7 cfu / g or more, and most preferably 1.5E+7 cfu / g or more and 1.5E+7 cfu / g or more.

[0034] In the present invention, "improvement in viability" preferably means that when stored at 1 to 10°C until the 24th day, the viable cell count of Bifidobacterium bacteria on the last day of storage is 5.5% or more relative to the viable cell count on the first day of storage, and when stored at 1 to 10°C until the 24th day, the viable cell count of specific Lactobacillus bacteria on the last day of storage is 30% or more relative to the viable cell count on the first day of storage. In the present invention, "improvement in viability" more preferably means that when stored at 1 to 10°C until the 24th day, the viable cell count of Bifidobacterium bacteria on the last day of storage is 6% or more relative to the viable cell count on the first day of storage, and when stored at 1 to 10°C until the 24th day, the viable cell count of specific Lactobacillus bacteria on the last day of storage is 50% or more relative to the viable cell count on the first day of storage. In the present invention, "improvement in viability" even more preferably means that when stored at 1 to 10°C until the 24th day, the viable cell count of Bifidobacterium bacteria on the last day of storage is 10% or more relative to the viable cell count on the first day of storage, and when stored at 1 to 10°C until the 24th day, the viable cell count of specific Lactobacillus bacteria on the last day of storage is 80% or more relative to the viable cell count on the first day of storage.

[0035] (Function) The fermented milk of the present invention is characterized by changes in the addition form and addition timing of specific Lactobacillus bacteria during production. By changing the addition timing, co-cultivation of Bifidobacterium bacteria and specific Lactobacillus bacteria can be avoided. Also, avoiding co-cultivation of specific Lactobacillus bacteria and lactic acid bacteria serving as a fermentation starter and culturing them individually is considered to have a favorable effect on the viability of specific Lactobacillus bacteria. The reason for the effect obtained in the present invention can be inferred as follows. However, the present invention is not limited by the following explanation. When Lactobacillus gasseri and Bifidobacterium bacteria are mixed, it is thought that the presence of one affects the presence of the other, and the growth of either one or both is inhibited. By changing the administration form and administration timing of Lactobacillus bacteria, it is considered that the growth stages in which Lactobacillus bacteria and Bifidobacterium bacteria significantly affect each other can be shifted. In view of the above effects, it is considered that not only Lactobacillus gasseri but also Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii, which are related species of Lactobacillus gasseri and have similar biological properties, will also show effects.

Example

[0036] Hereinafter, examples of the present invention will be described in detail, but the present invention is not limited thereto. Unless otherwise specified in this specification, the % representation indicates % by weight.

[0037] 〔Example 1〕 (1) Preparation and culture of bulk starter culture of Bifidobacterium longum A 15% reduced skim milk medium supplemented with 0.5% yeast extract was prepared, and the medium was sterilized by heat treatment at 95°C for 30 minutes. Then, a cell starter of Bifidobacterium longum SBT2928 (Accession No.: FERM P-10657, Deposit Date: April 13, 1989, Patent Biological Deposit Center, National Institute of Advanced Industrial Science and Technology) was added at 1% based on the reduced skim milk medium, and cultured at 36°C for 16 hours. By this operation, a bulk starter with a viable cell count of about 1.0E+9 cfu / g was obtained.

[0038] (2) Preparation and culture of bulk starter culture of Lactobacillus gasseri A 12% reduced skim milk medium supplemented with 0.50% yeast extract was prepared and sterilized by heat treatment at 95°C for 30 minutes. Then, a cell starter of Lactobacillus gasseri SBT2055 (Accession No.: FERM BP-10953, Deposit Date: February 26, 2008, Patent Biological Deposit Center, National Institute of Advanced Industrial Science and Technology) was added at 1% based on the reduced skim milk medium and cultured at 36°C for 16 hours. By this operation, a bulk starter with a viable cell count of about 1.0E+9 cfu / g was obtained.

[0039] (3) Preparation and storage of fermented milk 12% skim milk powder and 2% unsalted butter were mixed and dissolved, heated at 60°C, homogenized, held at 95°C for 5 minutes for heat sterilization, and cooled to 40°C to prepare a raw material mix. After sterilizing the raw material mix, the prepared bulk starter of Bifidobacterium longum was added at 4% based on the raw material mix. Further, cell starters of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus as fermentation starter lactic acid bacteria were added at 0.1% based on the raw material. To the control raw material mix, in addition to this, a cell starter of Lactobacillus gasseri was added at 0.005% based on the raw material mix. After addition, fermentation was carried out at 39°C until the acidity reached 0.8.

[0040] After fermentation, in the fermented milk mix for preparing the fermented milk of the present invention, a bulk starter of Lactobacillus gasseri was added at 1.0% (an amount adjusted so that the cell starter of 0.005% added to the control and the cell count at the end of cultivation were about the same) based on the fermented milk mix. The fermented milk was dispensed into a storage container having oxygen permeability and sealed with an aluminum lid. It was stored refrigerated (10°C) until the 24th day.

[0041] (4) Confirmation of viable cell count and viability The fermented milk was serially diluted using sterilized dilution water (0.6% disodium hydrogen phosphate, 0.45% potassium dihydrogen phosphate, 0.05% L-cysteine hydrochloride monohydrate, 0.05% Tween 80, 0.05% agar). For the viable cell count measurement of Bifidobacterium longum, the diluted sample was mixed with mupirocin-added TOS propionate agar medium. For the viable cell count measurement of Lactobacillus gasseri, the diluted sample was spread on the plate medium of MRS / CL / CIP agar medium, and the viable cell count in the serially diluted fermented milk was measured. The MRS / CL / CIP agar medium was prepared by adding clindamycin at 0.1 μg / mL and ciprofloxacin at 10 μg / mL to the sterilized MRS medium, and the plate medium solidified by pouring into a petri dish was used. Anaerobic culture was performed at 37°C for 72 hours using an anaerobic culture system (trade name: AnaeroPack, Mitsubishi Gas Chemical Company, Inc.). After the completion of the culture, the viable cell count was measured by the plate count method, and the viable cell count on the 24th day of refrigerated storage was divided by the viable cell count on the 1st day, and the quotient was converted to % to obtain the survival rate. The results are shown in Figures 3 and 4, Tables 1 and 2. The transition of the viable cell count during refrigerated storage is shown in Figures 5 and 6. In each of Figures 5 and 6, the viable cell counts plotted are for the 1st, 14th, 21st, and 24th days of refrigerated storage.

[0042] As a result, regarding Bifidobacterium longum, while the viable cell count on the 1st day of refrigerated storage of the control fermented milk was 1.4E+8 cfu / g and the viable cell count on the 24th day of refrigerated storage was 7.0E+6 cfu / g, in the fermented milk of the present invention, the viable cell count on the 1st day of refrigerated storage was 1.5E+8 cfu / g and the viable cell count on the 24th day of refrigerated storage was 1.6E+7 cfu / g (Figure 3). The survival rate on the 24th day of refrigerated storage was 5.0% for the control fermented milk, whereas it was 10.7% for the fermented milk of the present invention (Table 1).

[0043] Regarding Lactobacillus gasseri, the viable cell count on the first day of refrigerated storage of the control fermented milk was 3.0E+7 cfu / g, and the viable cell count on the 24th day of refrigerated storage was 2.6E+6 cfu / g. In contrast, for the fermented milk of the present invention, the viable cell count on the first day of refrigerated storage was 2.3E+7 cfu / g, and the viable cell count on the 24th day of refrigerated storage was 1.9E+7 cfu / g (Figure 4). The survival rate on the 24th day of refrigerated storage was 8.7% for the control fermented milk, while it was 82.6% for the fermented milk of the present invention (Table 2).

[0044] As described above, for both Bifidobacterium longum and Lactobacillus gasseri, the viable cell count and survival rate on the 24th day of refrigerated storage were higher for the fermented milk of the present invention. Also, the viable cell counts of Bifidobacterium longum and Lactobacillus gasseri on the 24th day of refrigerated storage exceeded 1.0E+7 cfu / g and 1.0E+7 cfu / g, respectively. Moreover, in the fermented milk of the present invention, for both Bifidobacterium longum and Lactobacillus gasseri, the counts exceeded 1.0E+7 cfu / g and 1.0E+7 cfu / g throughout the entire period from the 15th to the 24th day of refrigerated storage (Figures 5 and 6). On the other hand, in the control fermented milk, the viable cell count of Lactobacillus gasseri dropped below 1.0E+7 cfu / g on the 15th day of storage.

[0045] [Table 1]

[0046] [Table 2]

[0047] [Comparative Example 1] Instead of adding 1.0% of the bulk starter of Lactobacillus gasseri based on the fermented milk mix after fermentation, 0.5% of the cell starter of Lactobacillus gasseri was added based on the fermented milk mix after fermentation (adjusted so that the number of bacteria of Lactobacillus gasseri on the first day of refrigerated storage after fermentation was 1.0E+7 cfu / g or more). Except for this, the same operations as in Example 1 were performed to produce fermented milk. Regarding the produced fermented milk, the viable count of Lactobacillus gasseri on the 24th day of refrigerated storage was measured. As a result, the viable count of Lactobacillus gasseri on the 24th day of refrigerated storage was less than 1.0E+3 cfu / g, and the viability of Lactobacillus gasseri was very low.

[0048] [Comparative Example 2] Instead of adding 1.0% of the bulk starter of Lactobacillus gasseri based on the fermented milk mix after fermentation, 0.05% of the bulk starter of Lactobacillus gasseri was added based on the raw material mix immediately after adding the fermentation starter lactic acid bacteria (adjusted so that the number of bacteria of Lactobacillus gasseri on the first day of refrigerated storage after fermentation was 1.0E+7 cfu / g or more). Except for this, the same operations as in Example 1 were performed to produce fermented milk. Regarding the produced fermented milk, the viable counts of Bifidobacterium longum and Lactobacillus gasseri on the 24th day of refrigerated storage were measured. As a result, the viable count of Lactobacillus gasseri on the 24th day of refrigerated storage was 2.1E+6 cfu / g, and the viability of Lactobacillus gasseri was low. Also, the viable count of Bifidobacterium longum on the 24th day of refrigerated storage was 9.9E+6 cfu / g, and no improvement in the viability of Bifidobacterium longum was confirmed.

[0049] Therefore, changing either the administration form or the administration timing of Lactobacillus gasseri could not improve the viability of Lactobacillus gasseri, and it was necessary to change both the administration form and the administration timing from the control.

Industrial Applicability

[0050] According to the present invention, it is not necessary to add anything other than the raw materials for producing fermented milk, and it is possible to improve the viability of Bifidobacterium bacteria and specific Lactobacillus bacteria, particularly the viability during refrigerated storage, without affecting the flavor of the fermented milk.

Claims

1. When stored at 1 - 10°C until the 15th to 24th day, on the last day of storage, the viable count of Bifidobacterium bacteria is 1.0E+7 cfu / g or more and 1.0E+11 cfu / g or less, and the viable count of Lactobacillus bacteria is 1.0E+7 cfu / g or more and 1.0E+11 cfu / g or less, and the fermented milk, wherein the Lactobacillus bacteria is one or more selected from the group consisting of Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii (however, excluding fermented milk with a lactose content of 45 mM or less).

2. The fermented milk according to claim 1, wherein the Bifidobacterium bacteria is Bifidobacterium longum and the Lactobacillus bacteria is Lactobacillus gasseri.

3. (A) A step of adding Bifidobacterium bacteria to the raw material mix; (B) A step of fermenting the raw material mix to obtain a fermented milk mix; (C) A step of adding 0.01 - 10% of a bulk starter of Lactobacillus bacteria to the fermented milk mix after the fermentation; (D) A step of refrigerating and storing the fermented milk mix to obtain fermented milk; A method for producing fermented milk, comprising: The method according to the above, wherein the Lactobacillus bacteria is one or more selected from the group consisting of Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii (however, excluding a production method including a step of fermenting until the lactose content after fermentation is 45 mM or less).

4. The method for producing fermented milk according to claim 3, wherein the Bifidobacterium bacteria is Bifidobacterium longum and the Lactobacillus bacteria is Lactobacillus gasseri.

5. The method for producing fermented milk according to claim 3 or 4, wherein in step (A), in addition to the Bifidobacterium bacteria, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are added.

6. The method for producing fermented milk according to any one of claims 3 - 5, wherein step (D) is carried out within 6 hours after step (C).

7. The method for producing fermented milk according to any one of claims 3 to 6, comprising a step (E) of cooling the fermented milk mixture to 20°C or lower between steps (B) and (C).

8. (A) A step of adding Bifidobacterium bacteria to a raw material mixture, (B) A step of fermenting the raw material mixture to obtain a fermented milk mixture, (C) A step of adding 0.01 to 10% of a bulk starter of Lactobacillus bacteria to the fermented milk mixture after fermentation, (D) A step of refrigerating and storing the fermented milk mixture to obtain fermented milk, which is a method for improving the viability of Bifidobacterium bacteria and Lactobacillus bacteria in fermented milk, wherein the Lactobacillus bacteria are one or more selected from the group consisting of Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, and Lactobacillus johnsonii (however, excluding the method for improving viability including a step of fermenting until the lactose content after fermentation is 45 mM or less).

9. The method for improving the viability of Bifidobacterium bacteria and Lactobacillus bacteria in fermented milk according to claim 8, wherein the Bifidobacterium bacteria are Bifidobacterium longum and the Lactobacillus bacteria are Lactobacillus gasseri.

10. The method for improving the viability of Bifidobacterium bacteria and Lactobacillus bacteria in fermented milk according to claim 8 or 9, wherein step (D) is carried out within 6 hours after step (C).

11. The method for improving the viability of Bifidobacterium bacteria and Lactobacillus bacteria in fermented milk according to any one of claims 8 to 10, comprising a step (E) of cooling the fermented milk mixture to 20°C or lower between steps (B) and (C).

12. Fermented milk produced by the production method according to any one of claims 3 to 7.

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