JP7544461B2 - Composition for improving survival of bifidobacteria - Google Patents

Description

The present invention relates to a composition for improving the survival of bifidobacteria.

Useful microorganisms that can have beneficial effects on the host, such as improving the bacterial flora in the digestive tract, are called probiotic bacteria and have attracted attention. One such useful microorganism is bifidobacteria. However, bifidobacteria with such functionality have been considered to have poor growth and survival in milk. Thus, various solutions have been disclosed so far for culturing probiotic bacteria in milk and improving survival.
Patent Document 1 aims to provide a survival enhancer for lactic acid bacteria and/or bifidobacteria that can improve the survival of probiotic lactic acid bacteria and bifidobacteria in an acidic (low pH) environment, a food composition using the same, and a method for producing the same. As a means to achieve this goal, it discloses a survival enhancer for lactic acid bacteria and/or bifidobacteria that contains an amino acid as an active ingredient.
Patent Document 2 aims to provide a novel substance that promotes the growth of bifidobacteria and lactic acid bacteria, and discloses, as a means for solving this problem, the use of egg white treated with a protease as a growth promoter for bifidobacteria and lactic acid bacteria.

However, the technology described in Patent Document 1 requires the addition of various amino acids to the yogurt mix, and there is a high possibility that these amino acids will affect the flavor of the final product. Patent Document 2 also requires the addition of egg white hydrolysate to the culture medium. In this case as well, there is a possibility that the flavor will be affected, and there is also the possibility that allergies may arise, limiting the number of people who can eat it.

Patent No. 6088821 JP 2000-93166 A

The objective of the present invention is to provide a novel composition for improving the survival rate of bifidobacteria and a novel method for improving the survival rate of bifidobacteria.

In order to solve the above problems, the present invention includes the following configurations.
(1) A composition for improving the survival rate of bifidobacteria, comprising a milk protein hydrolysate;
(2) The composition for improving the viability of bifidobacteria according to (1), which is a medium for culturing bifidobacteria.
(3) The composition for improving the viability of bifidobacteria according to (1) or (2), wherein the peptide concentration in the composition is 1.8 mg/L or more.
(4) The composition for improving the viability of bifidobacteria according to any one of (1) to (3), wherein the total concentration of L-amino acids in the composition is 4.6 mM or more.
(5) The composition for improving the survival rate of bifidobacteria according to any one of (1) to (4), wherein the improvement in survival rate is suppression of a decrease in the number of bifidobacterial cells at 10° C. or lower.
(6) A method for improving the survival rate of bifidobacteria, comprising culturing or fermenting bifidobacteria in a culture medium containing 1.8 mg/L or more of a peptide derived from a milk protein in terms of the medium.
(7) The method for improving the viability of bifidobacteria according to (6), wherein the L-amino acid concentration in the culture medium is 4.6 mM or more.
(8) The method for improving the viability of bifidobacteria according to (6) or (7), wherein the bifidobacteria is Bifidobacterium longum.
(9) A method for improving the viability of bifidobacteria according to any one of (6) to (8), further comprising producing a total of 0.2 mM or more of phenylalanine and serine in the culture medium; and (10) a method for improving the viability of bifidobacteria according to any one of (6) to (9), wherein the improvement in viability is suppression of a decrease in the number of bifidobacterial cells at a temperature of 10°C or less.

The present invention provides a novel composition for improving the survival rate of bifidobacteria and a method for improving the survival rate of bifidobacteria. According to the present invention, it is possible to improve the survival rate of bifidobacteria, particularly during refrigerated storage, without the need to add anything other than milk components from the outside and without affecting the flavor or physical properties of the food.

1 is a graph showing the relative ratio of the number of Bifidobacterium longum bacteria attained in each of 10 types of protease-treated media and a control medium (untreated with protease). 1 is a graph showing peptide concentrations in each of the four types of protease-treated media and in a control medium (not treated with protease). 1 is a graph showing the L-amino acid concentrations in each of the four types of protease-treated media and in a control medium (untreated with protease). Five types of bulk starters were prepared using four types of protease-treated media and a control medium (not treated with protease), and the fermented milk prepared using the five types of bulk starters was stored in a refrigerator. This is a graph showing the survival rate of Bifidobacterium longum when these fermented milks were stored in a refrigerator.

The present invention will be described in detail below. Note that in this specification, all percentages refer to weight percent unless otherwise specified.

[1] Composition for improving the survival rate of bifidobacteria (survival rate improvement)
In the present invention, "improving survival" has two meanings: suppressing the death of bifidobacteria and maintaining the number of surviving bacteria (suppressing decline), and promoting the growth of the bacteria (promoting proliferation).
In addition, in the present invention, "improving survival ability" includes suppressing a decrease in the number of bacteria when stored under specific conditions, for example, at 10°C or lower for a certain period of time.

(Composition for improving the survival rate of bifidobacteria)
The composition for improving the viability of bifidobacteria of the present invention contains a milk protein hydrolysate obtained by treating milk protein with a protease. This milk protein hydrolysate preferably contains a peptide and an L-amino acid having a molecular weight of 10,000 or less.
The peptide may be any peptide derived from a milk protein, but is preferably one or more selected from a peptide derived from casein, a peptide derived from β-lactoglobulin, and a peptide derived from α-lactalbumin, and it is more preferable to adjust the ratio of casein-derived peptide to peptide other than casein to about 75:25 to 85:15.
The concentration of L-amino acids in the composition for improving the viability of bifidobacteria of the present invention is preferably 4.6 mM or more, more preferably 4.65 mM or more, and even more preferably 4.7 mM or more.

(Milk Protein)
The milk protein used in the production of the composition for improving the survival rate of bifidobacteria of the present invention may be any protein contained in animal milk, such as cow, buffalo, sheep, goat, horse, etc. Therefore, in the production of the composition for improving the survival rate of bifidobacteria of the present invention, any milk material containing the above-mentioned protein may be used, and not only milk materials containing a large amount of protein, such as milk protein concentrate (milk protein isolate, milk protein concentrate) and whey protein concentrate (whey protein isolate, whey protein concentrate), but also raw milk, cow's milk, partially skimmed milk, skimmed milk, whole skimmed milk powder, whole milk powder, concentrated milk, skimmed concentrated milk, adjusted milk powder, milk protein, etc., containing milk protein may be used, and these materials may be used alone or in combination of two or more kinds.

(Decomposition of milk proteins)
The composition for improving the viability of bifidobacteria of the present invention can be obtained by preparing an aqueous solution containing about 1 to 10% of the above-mentioned milk protein, and adding a protease described below to the aqueous solution to decompose the milk protein. The proteolysis is preferably carried out so that the molecular weight of the peptide is 10,000 or less and the total amount of phenylalanine and serine generated by the proteolysis is 0.2 mM or more. It is more preferable that the proteolysis is carried out so that the total amount of phenylalanine and serine generated by the proteolysis is 0.2 to 0.5 mM.
The conditions at this time, such as the milk protein concentration, the enzyme used, the reaction temperature, the reaction time, and inactivation, can be appropriately adjusted depending on the milk protein, the enzyme, the equipment, etc. used, so that the molecular weight of the peptide is 10,000 or less and the total amount of phenylalanine and serine generated by protein hydrolysis is 0.2 mM or more.
The resulting solution containing the peptides can be used as it is as a composition for improving the survival rate of bifidobacteria, but it can also be dried and used in the form of a powder, or the peptides can be purified by separation, fractionation, or the like before use.

The proteolytic enzyme may be any food-use proteolytic enzyme derived from a microorganism or the like, without any particular limitation, and may be an endoprotease or an exoprotease. However, as described above, it is preferable to use an enzyme that produces peptides having a molecular weight of 10,000 or less and that can produce a total amount of phenylalanine and serine produced by proteolysis of 0.2 mM or more. A mixture of endoprotease and exoprotease derived from Aspergillus. oryzae (rice koji mold) or an exoprotease derived from Aspergillus. oryzae (rice koji mold) is more preferable, and among the mixture of endoprotease and exoprotease derived from Aspergillus. oryzae (rice koji mold) or an exoprotease derived from Aspergillus. oryzae (rice koji mold), one having an optimum pH of around 7 and an optimum temperature of about 50 to 70° C. is even more preferable. More preferred examples of the enzyme include Denazeme AP (Nagase ChemteX Corporation) and Proteax (Amano Enzyme Inc.).
Other usable enzymes include Sumiteam LP50D (Shin Nippon Chemical Industry Co., Ltd.), Sumiteam FL-G (Shin Nippon Chemical Industry Co., Ltd.), Sumiteam LPL-G (Shin Nippon Chemical Industry Co., Ltd.), Sumiteam DPP-G (Shin Nippon Chemical Industry Co., Ltd.), Sumiteam P (Shin Nippon Chemical Industry Co., Ltd.), Protease A "Amano" SD (Amano Enzyme Co., Ltd.), Protease M "Amano" SD (Amano Enzyme Co., Ltd.), and Panchidase NP-2 (Yakult Pharmaceutical Industry Co., Ltd.).

(Method of using the composition for improving the survival rate of bifidobacteria)
The composition for improving the viability of bifidobacteria can be added to a desired medium, food, etc. so that the peptide concentration is 1.8 mg/mL or more, preferably 1.85 mg/mL or more, and more preferably 1.90 mg/mL or more. Examples of usage modes include the following.
1) A composition for improving the survival rate of bifidobacteria is added to a medium in which bifidobacteria are used so that the peptide concentration is 1.8 mg/mL or more, and the medium is sterilized. Bifidobacteria are inoculated into the sterilized medium and cultured at a desired temperature for a desired time. The bifidobacteria culture thus obtained can be used as a raw material for foods such as fermented milk.
2) A protease is added to a medium containing milk so that the peptide concentration is 1.8 mg/mL or more, and the mixture is incubated to prepare a composition (medium) for improving the survival rate of bifidobacteria. The composition (medium) for improving the survival rate of bifidobacteria is sterilized. Bifidobacteria are inoculated into the sterilized composition (medium) for improving the survival rate of bifidobacteria, and the bifidobacteria are cultured at a desired temperature and time. The culture of bifidobacteria obtained here can be used as a raw material for foods such as fermented milk. For example, it can be added to skim milk powder or the like as a bulk starter to obtain fermented milk.
3) A protease is added to a medium containing milk so that the peptide concentration is 1.8 mg/mL or more, and the medium is incubated to prepare a composition (medium) for improving the viability of bifidobacteria. This composition (medium) for improving the viability of bifidobacteria is sterilized. A bulk starter consisting of bifidobacteria is added to the sterilized composition (medium) for improving the viability of bifidobacteria, and fermentation is carried out at a desired temperature and time to obtain fermented milk.

In the present invention, as in the above-mentioned mode 2), if bifidobacteria are cultured in the presence of a composition for improving the survival rate of bifidobacteria, the effect of high survival rate of bifidobacteria can be obtained even if the bifidobacteria are subsequently cultured in a medium that does not contain the composition for improving the survival rate of bifidobacteria of the present invention. The reason for this is not clear at present, but can be presumed as follows. However, the present invention is not limited to the following presumption.

It is believed that placing bifidobacteria in nutrient-rich conditions during the initial stages of cultivation contributes to subsequent good growth. Therefore, it is believed that the decrease in bacterial cell count can be suppressed even under harsh conditions such as low temperatures.

(Target bifidobacteria)
The bifidobacteria targeted by the composition for improving the survival rate of bifidobacteria are not particularly limited as long as they are bacteria of the genus Bifidobacterium, and examples thereof include Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium animalis, Bifidobacterium alesentis, Bifidobacterium lactis, Bifidobacterium catenulatum, Bifidobacterium dentium, etc. Examples of the bacterial strain include the Bifidobacterium longum SBT2928 strain and the Bifidobacterium pseudolongum SBT2908 strain.

The composition for improving the viability of bifidobacteria of the present invention may contain externally added substances for the growth of bifidobacteria, such as peptone, yeast extract, and amino acids. In addition, these substances may be added as externally added substances in order to adjust the peptide concentration, L-amino acid concentration, and serine and phenylalanine concentrations. However, taking into consideration the flavor, it is preferable that these externally added substances are not substantially contained, and more preferably, are not contained at all. Note that "substantially not contained" means that they are not contained in an amount that affects the flavor of the final product.

(Method of measuring peptides)
The peptide concentration can be determined by any known method (colorimetric methods such as the Lowry method, the Bradford method, and the BCA method). It may also be measured using a commercially available protein concentration measurement reagent (product name: DC Protein Assay Kit, manufactured by BIO-RAD).

(Method of measuring L-amino acids)
The L-amino acid concentration can be determined by any known method (a method using an enzyme for quantifying amino acids, a method using high performance liquid chromatography or an automatic amino acid analyzer), or it may be measured using a commercially available L-amino acid concentration measuring reagent (trade name: L-Amino Acid Quantitation Kit, manufactured by Bio Vision).

[2] Method for improving the viability of bifidobacteria In the method for improving the viability of bifidobacteria of the present invention, bifidobacteria are cultured or fermented in a culture medium containing 1.8 mg/L or more of a peptide derived from a milk protein in terms of the medium. The peptide derived from a milk protein may be added from outside, but in consideration of flavor, it is preferable that the peptide is produced within the medium by adding a protease and that no peptide is added from outside.

The method of the present invention for improving the survival rate of bifidobacteria preferably further includes producing phenylalanine and serine in a total amount of 0.2 mM or more in the culture medium. Although "producing" includes adding phenylalanine and serine from the outside, it is preferable that, taking into consideration the flavor, they are produced within the medium by adding a protease, and do not include anything added from the outside.

The bifidobacteria obtained by the method for improving the survival rate of bifidobacteria of the present invention may be used as a starter, or may be used for preparing final products such as fermented milk. As described above, when used as a starter, the effects of the present invention can be obtained even if the medium to which it is added does not contain the composition for improving the survival rate of bifidobacteria of the present invention.

The following describes in detail the examples of the present invention, but the present invention is not limited to these.

Example 1
(1) Preparation of protease-treated milk medium and cultivation of Bifidobacterium longum
A 15% reconstituted skim milk medium was prepared, and 10 types of food-compatible protease were added at 0.001% to the medium, thoroughly stirred, and then allowed to stand for 1 hour for the enzyme reaction to occur at approximately the enzyme's optimal temperature (the reaction temperature was set based on the temperature listed on the data sheet issued by each manufacturer). Table 1 shows each protease and its enzyme reaction temperature.

After the enzyme reaction was completed, the enzyme was inactivated and the medium was sterilized by heating at 95°C for 30 minutes. Concentrated cells of Bifidobacterium longum SBT2928 (Accession Number: FERM P-10657, Date of Deposit: April 13, 1989, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) were added to the medium at a final concentration of 1%, and the medium was cultured at 36°C for 16 hours.

(2) Confirmation of the number of bacteria reached in milk medium After the culture, the bacterial solution was serially diluted with modified A dilution water (0.6% NaHPO4, 0.45% KH2PO4, 0.05% L-cysteineHCl-H2O, 0.05% agar, sterilized at 121°C for 15 minutes after dissolution) and mixed with TOS mupirocin medium, and cultured anaerobically at 37°C for 72 hours using an anaerobic culture system (product name: Anaeropack, manufactured by Mitsubishi Gas Chemical Co., Ltd.). After the culture was completed, the viable number of Bifidobacterium longum was measured by the plate count method. The relative ratio of the number of bacteria reached in the milk medium containing the survival improvement substance obtained by the protease treatment, with the number of bacteria reached after 16 hours of culture in the control (medium not treated with protease) set to 1, is shown in Figure 1.

As a result, compared to the number of bacteria achieved by Bifidobacterium longum in the control, the number of bacteria achieved in the milk medium containing the viability-improving substance obtained by the proteolytic enzyme treatment of the present invention was increased by 1.4 to 3.6 times. The growth of Bifidobacterium longum was improved by all enzyme treatments, but the growth-promoting effect varied depending on the enzyme. The milk medium treated with Proteax showed the greatest growth-promoting effect.

From the above, it was confirmed that the survival rate of Bifidobacterium longum, a species of bifidobacterium used in food as a probiotic that has beneficial effects on humans, is improved.

Table 1. Proteolytic enzymes and enzyme reaction temperatures

Example 2
(1) Preparation of protease-treated milk medium and bulk starter
A 15% reconstituted skim milk medium was prepared, and 0.001% of four food-safe proteolytic enzymes (Sumiteam LP50D, Proteax, Sumiteam FL-G, Denatteam AP) were added to the medium, thoroughly stirred, and the enzyme reaction was carried out by leaving it to stand for 1 hour at near the optimal temperature for the enzyme (the reaction temperature was set based on the temperature listed on the data sheet issued by each manufacturer) (same temperature conditions as in Table 1). After the enzyme reaction was completed, the enzyme was inactivated and the medium was sterilized by heating at 95 degrees for 30 minutes.

The peptide concentration in the milk medium treated with the protease was 1.9-2.0 mg/mL (Figure 2), and the L-amino acid concentration was 4.7-5.0 mM (Figure 3). Since the amino acid concentration in the control medium not treated with the protease was 4.5 mM, it was considered that 0.2-0.5 mM of amino acids was produced in the milk medium treated with the protease.
The amino acids produced were phenylalanine and serine, and no increase in other amino acids was observed after the enzyme treatment.

The peptide concentration and L-amino acid concentration were measured, and the free amino acid composition was analyzed by the following methods.
First, the milk medium after enzyme inactivation and sterilization was centrifuged at 15,000 rpm for 10 minutes. The resulting supernatant was ultrafiltered using a centrifugal filter (product name: Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-3 membrane, 10 kD MWCO, Millipore) to remove proteins with a molecular weight of 10 kDa or more. The permeated fraction was diluted with ion-exchanged water to an appropriate concentration and then subjected to measurement of peptide and L-amino acid concentrations, respectively.

Peptide concentration was measured according to the Lowry method using a commercially available kit (product name: DC Protein Assay Kit, manufactured by BIO-RAD). After adding the reagents according to the kit's protocol, the color reaction was carried out at room temperature for 15 minutes, and then the absorbance at 750 nm was measured. The peptide concentration was calculated based on the obtained absorbance and a previously prepared calibration curve (using bovine serum albumin).

L-amino acid concentrations were measured using a commercially available kit (L-Amino Acid Quantitation Kit, Bio-Vision). After adding the reagents according to the kit's protocol, a color reaction was carried out at 37°C for 30 minutes, and the absorbance at 570 nm was measured. The L-amino acid concentrations were calculated based on the absorbance obtained and a calibration curve (standards included in the kit were used).
The free amino acid composition analysis was carried out by the following methods: tryptophan was measured by high performance liquid chromatography, and free amino acids other than tryptophan were measured by an automatic analysis method.

After enzyme inactivation and sterilization, concentrated cells of Bifidobacterium longum SBT2928 (Accession Number: FERM P-10657, Date of Deposit: April 13, 1989, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) were added at 1% to the milk medium and cultured at 36°C for 16 hours. After 16 hours of culture, the bulk starter was promptly cooled to 4°C to prepare the bulk starter. A significant positive correlation was observed between the number of bacteria reached in the bulk starter and the peptide concentration and L-amino acid concentration (p<0.01, Spearman's rank correlation coefficient).

(2) Preparation and storage of fermented milk 12% skim milk powder and 2% unsalted butter were mixed and dissolved, heated to 60°C in a hot water bath, homogenized, then held at 95°C for 5 minutes for heat sterilization, and cooled to 40°C to prepare a fermented milk base mix. After sterilization of the fermented milk base mix, 4% of a bulk starter of Bifidobacterium longum prepared in a medium containing the protease-treated product of the present invention was inoculated. Furthermore, 0.1% of a mixed concentrated bacterial cell of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus was inoculated. After inoculation, the mixture was fermented at 39°C until the acidity reached 0.8. After fermentation, each sample of fermented milk was dispensed into a storage container and sealed with an aluminum lid. The mixture was stored in a refrigerator (10°C) for 24 days.

(3) Confirmation of viability in fermented milk After incubation, the bacterial solution was serially diluted with modified A dilution water (0.6% NaHPO4, 0.45% KH2PO4, 0.05% L-cysteineHCl-H2O, 0.05% agar, sterilized at 121°C for 15 minutes after dissolution) and mixed with TOS mupirocin medium, and incubated anaerobically at 37°C for 72 hours using an anaerobic incubation system (product name: Anaeropack, Mitsubishi Gas Chemical Co., Ltd.). After incubation, the viable cell count of Bifidobacterium longum was measured by plate counting, and the viable cell count on the 24th day of storage was divided by the viable cell count on the 1st day, and the quotient was converted to a percentage to determine the survival rate. The results are shown in Figure 4.

The fermented milk prepared using a bulk starter of Bifidobacterium longum prepared in a medium containing a protease-treated product showed an increased survival rate compared to the control fermented milk.
The survival rate of the fermented milk prepared using the bulk starter of Bifidobacterium longum prepared in a medium containing a protease-treated product was calculated relative to the survival rate of the control, which was set at 1. As a result, the survival rate of the fermented milk stored in a refrigerator was improved by 1.8 to 2.1 times in the fermented milk prepared using the bulk starter of Bifidobacterium longum prepared in a medium containing a protease-treated product of the present invention, compared to the control.
Furthermore, the prepared fermented milk had a good flavor and was not different in flavor from the control example.

Example 3
1) 12% skim milk powder and 2% unsalted butter were mixed and dissolved, heated to 60°C, and subjected to homogenization treatment.
2) The homogenized fermented milk base was adjusted to 65°C, 0.001% Proteax was added, and the mixture was allowed to stand for 1 hour.
3) After standing, the mixture was kept at 95°C for 15 minutes to sterilize the mixture by heat and inactivate the enzyme.
4) The enzyme-inactivated fermented milk base was adjusted to 40°C and inoculated with 4% of a bulk starter of Bifidobacterium longum.Furthermore, a mixed concentrated cell mass of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus was inoculated at 0.1%.
5) After inoculation, the mixture was fermented at 39 degrees until the acidity reached 0.8. After fermentation, each sample of fermented milk was dispensed into a storage container and sealed with an aluminum lid. The containers were stored in a refrigerator (10 degrees) for 24 days.
In addition to the above-mentioned examples, fermented milk prepared under the same conditions except for 2) was prepared as a control.

(1) Confirmation of viability in fermented milk The viable cell count of Bifidobacterium longum was measured by the plate count method in the same manner as in Example 2. The viable cell count on the 24th day of storage was divided by the viable cell count on the 1st day, and the quotient was converted into a percentage to determine the survival rate. When the survival rate of the control was taken as 1, the survival rate of bifidobacteria was improved by 2.1 times in the product of the embodiment. In addition, the prepared fermented milk had a good flavor, and there was no difference in flavor from the control embodiment.

As described above, the present invention provides a novel composition for improving the viability of bifidobacteria that can be used in foods without the need to add additional components such as peptides. This viability improving composition can significantly improve the growth of bifidobacteria during culture and the viability of bifidobacteria during refrigerated storage. This allows the activity of bifidobacteria to be maintained at a high level for a long period of time, making it possible to reduce the amount of bifidobacteria used (the amount added to the food composition), which is expected to reduce costs. In addition, it allows for a stable supply of food compositions containing bifidobacteria and maintain their quality.
In addition, since the viability improving composition of the present invention is obtained by treating a dairy material with a very small amount of enzymes that can be used in food, it is possible to ensure the original flavor of milk without affecting the flavor and physical properties of food compositions such as fermented milk. It is extremely effective for all foods containing bifidobacteria.

Claims (4)

A composition for improving the survival rate of bifidobacteria, comprising a milk protein hydrolysate and serving as a medium for culturing bifidobacteria,
The total concentration of L-amino acids in the composition is 4.6 mM or more ;
The peptide concentration in the composition is 1.8 mg/L or more ;
The composition, wherein the improved survival rate is achieved by promoting the growth of bifidobacteria in the above-mentioned culture medium and inhibiting a decrease in the number of bifidobacterial cells in fermented milk at a temperature of 10° C. or lower. In a culture medium containing 1.8 mg/L or more of a milk protein-derived peptide in terms of the medium,
A method for improving the survival rate of bifidobacteria, comprising culturing bifidobacteria,
the L-amino acid concentration in the culture medium is 4.6 mM or more;
The method as described above, wherein the improvement in survival rate is the promotion of the proliferation of bifidobacteria in the above culture medium and the suppression of a decrease in the number of bifidobacterial cells in fermented milk at a temperature of 10° C. or lower. The method for improving the survival rate of bifidobacteria according to claim 2, wherein the bifidobacteria is Bifidobacterium longum. 4. The method for improving the viability of bifidobacteria according to claim 2 or 3, further comprising producing a total of 0.2 mM or more of phenylalanine and serine in the culture medium.

Images