WO2009150888A1 - Milk protein degradation product, manufacturing method for milk protein degradation product and bifidobacterial growth-promoting agent - Google Patents

Abstract

Provided are a milk protein degradation product that has excellent mass producibility and requires no protein source other than a milk-derived starting material that is the main culture starting material for bifidobacterium and can promote growth of bifidobacterium, a method for manufacturing said milk protein degradation product, and a bifidobacterial growth-promoting agent that has said milk protein degradation product as the active ingredient. Disclosed are a milk protein degradation product that is obtained by hydrolyzing milk protein using 1 or more selected from a group consisting of live lactobacillus cells comprising cell wall-localized protease (cell wall-enveloped proteinase, PrtP), disrupted cells of the above lactobacillus, and a fraction of the above enzyme fractionated from the above lactobacillus, a method for manufacturing said milk protein degradation product and a bifidobacterial growth-promoting agent having said milk protein degradation product as the active ingredient.

Description

Milk protein degradation product, milk protein degradation product production method and bifidobacteria growth promoter

The present invention uses a living lactic acid bacterium having a cell wall-enhanced proteinase (cell wall-enveloped proteinase, PrtP), a disrupted microbial cell of the lactic acid bacterium, an enzyme fraction fractionated from the lactic acid bacterium, and the like. The present invention relates to a protein degradation product to be obtained, a method for producing the protein degradation product, and a bifidobacteria growth promoter comprising the protein degradation product as an active ingredient.
This application claims priority based on Japanese Patent Application No. 2008-15951 filed in Japan on June 11, 2008, the contents of which are incorporated herein by reference.

Bifidobacterium spp., That is, bifidobacteria, is one of the dominant fungal species of the intestinal flora formed in the human intestinal tract, the intestinal regulating action to restore the balance of intestinal bacteria, It is known to have an immunopotentiating action, a carcinogenesis suppressing action, and the like.
For this reason, in recent years, the demand for foods containing living bifidobacteria such as fermented bifidobacteria has increased along with an increase in the health orientation of consumers.

Bifidobacteria have poor growth in milky media. For this reason, in order to contain a certain amount of, for example, 1 × 10 ⁷ CFU / mL bifidobacteria in the fermented milk, usually, various growth promoting substances such as yeast extract are added. However, the growth promoting substance is generally expensive and the flavor may be impaired. For this reason, development of a growth promoter for bifidobacteria that is less expensive and has less influence on the flavor has been demanded.

For example, (1) it has been reported that a peptide obtained by pepsin treatment of human milk has a bifidobacteria growth promoting action, and in particular, a pepsin degradation product of human protein lactoferrin has a bifidobacterial growth promoting action. (For example, see Non-Patent Document 1). Also disclosed is an invention relating to a bifidobacterial growth promoter characterized by comprising (2) actomyosin or a peptide-containing fraction obtained by treating a meat protein suspension containing actomyosin with a protease. (For example, refer to Patent Document 1).
Liepke, C., 5 others, European Journal of Biochemistry, 2002, volume 269, pages 712-718. JP 2007-189914 A

However, since the peptide of the above (1) is a protein contained in human milk or human milk, it is difficult to secure the raw material and is not very suitable for industrial use such as addition to food and drink. On the other hand, the bifidobacteria growth promoter of (2) above has no particular problem in securing the raw material because the raw material is meat protein, but since the peptide can be an allergen, it is originally derived from meat such as bifidobacteria fermented milk. It is not preferable to add this bifidobacterial growth promoter to foods and drinks not using protein as a raw material from the viewpoint of food allergy.

The present invention is a milk protein degradation product which is excellent in mass productivity and can promote the growth of bifidobacteria without requiring a protein source other than a milk-derived material which is a main culture raw material of bifidobacteria, and the milk protein It aims at providing the manufacturing method of a degradation product, and the bifidobacteria growth promoter which uses this milk protein degradation product as an active ingredient.

As a result of diligent research to solve the above-mentioned problems, the present inventors have found that a living lactic acid bacterium having a cell wall-localized proteolytic enzyme PrtP represented by Lactococcus lactis or a microbial cell of the lactic acid bacterium. The present inventors have found that a crushed product and a milk protein degradation product from a cell wall-localized proteolytic enzyme PrtP fraction fractionated from the lactic acid bacteria have a bifidobacteria growth-promoting action, thereby completing the present invention.

That is, the present invention provides a living cell of lactic acid bacteria having cell wall-enveloped proteinase (cell wall-enveloped proteinase, PrtP), a crushed cell of the lactic acid bacteria, and the enzyme fraction fractionated from the lactic acid bacteria. The present invention provides a milk protein hydrolyzate obtained by hydrolyzing milk protein using one or more selected from the group consisting of:
The present invention also provides the milk protein degradation product as described above, wherein the lactic acid bacterium is Lactococcus lactis.
The present invention also provides the milk protein degradation product as described above, wherein the Lactococcus lactis does not have xylose utilization and does not produce diacetyl and acetoin.
In addition, the present invention provides the milk protein degradation product according to any one of the above, wherein the milk protein is casein or total milk protein.
In the present invention, the hydrolysis is performed under the conditions of 20 to 37 ° C. and a milk protein concentration of 0.5% by weight or more using a milk protein adjusted to pH 6.0 to 8.0 as a substrate. The milk protein degradation product according to any one of the above-mentioned features is provided.
The present invention also provides a living cell of lactic acid bacteria having cell wall-enhanced proteinase (cell wall-enveloped proteinase, PrtP), a crushed cell of the lactic acid bacteria, and the enzyme fraction fractionated from the lactic acid bacteria. The present invention provides a method for producing a milk protein hydrolyzate, which comprises hydrolyzing milk protein using one or more selected from the group consisting of:
The present invention also provides the method for producing a milk protein degradation product as described above, wherein the lactic acid bacterium is Lactococcus lactis.
Further, the present invention provides the method for producing a milk protein degradation product as described above, wherein the Lactococcus lactis has no xylose utilization property and does not produce diacetyl and acetoin. It is.
The present invention also provides the method for producing a milk protein degradation product according to any one of the above, wherein the milk protein is casein or total milk protein.
In the present invention, the hydrolysis is performed under the conditions of 20 to 37 ° C. and a milk protein concentration of 0.5% by weight or more using a milk protein adjusted to pH 6.0 to 8.0 as a substrate. The present invention provides a method for producing a milk protein degradation product according to any one of the above.
Moreover, this invention provides the bifidobacteria growth promoter which uses the milk protein degradation product in any one of the said description as an active ingredient.
The present invention also provides the aforementioned bifidobacteria growth promoter, wherein the bifidobacteria is Bifidobacterium longum.
In the present invention, the Bifidobacterium longum strain is the Bifidobacterium longum ATCC BAA-999 strain and / or the Bifidobacterium longum type strain ATCC15700 strain. An agent is provided.

The milk protein degradation product of the present invention is obtained by hydrolyzing milk protein with a cell wall-localized proteolytic enzyme PrtP derived from lactic acid bacteria, so that it is excellent in mass productivity and manufactured at a relatively low cost. Is also possible.
Further, the bifidobacteria growth promoter of the present invention is unprecedented and can grow bifidobacteria, particularly Bifidobacterium longum. Moreover, since the milk protein degradation product which is an active ingredient is a milk-derived substance, the bifidobacteria growth promoter of the present invention is also applied to foods and drinks produced by culturing bifidobacteria with milk-derived raw materials such as fermented milk. It can be used without having to consider protein allergies other than milk protein, and is useful in designing dairy products such as fermented milk.

Using the DNA derived from each strain of Lactococcus lactis as a template, the PCR product obtained as a result of PCR using the primer for detecting the PrtP enzyme gene was separated by electrophoresis, and the band pattern detected by staining was detected. FIG.

The milk protein degradation product of the present invention includes a living lactic acid bacterium having a cell wall-localized proteolytic enzyme PrtP (hereinafter sometimes simply referred to as “PrtP enzyme”), a crushed cell product of the lactic acid bacterium, and It is obtained by hydrolyzing milk protein using one or more selected from the group consisting of the enzyme fractions fractionated from the lactic acid bacteria. Since the milk protein degradation product of the present invention has a bifidobacteria growth promoting activity, an excellent bifidobacteria growth promoter having a high bifidobacteria growth promoting effect can be obtained by using the milk protein degradation product of the present invention as an active ingredient. Can be obtained. The reason why the milk protein degradation product of the present invention exerts the bifidobacteria growth-promoting effect is not clear. It is guessed that there is not.

The lactic acid bacterium used in the present invention has a PrtP enzyme. The PrtP enzyme is an enzyme that exists in the cell membrane and has an active site exposed on the cell surface. Examples of lactic acid bacteria having a PrtP enzyme include Lactococcus lactis subspices cremoris and Lactococcus lactis subspice lactos. In the genus, several strains having PrtP have been reported.

To date, PrtP enzymes derived from Lactococcus lactis include P type _I (which does not degrade α-casein very much and β-casein is degraded well from the vicinity of the C-terminal), type P _III (α-casein and β-casein is well resolved from both C-terminus and N-terminus) and its intermediate form (P _I / P _III type) (eg, Reid, JR. et al., Applied and Environmental Microbiology, 1994). (See Years, Volume 60, Issue 3, pages 801-806.)
Specific examples of PrtP enzymes derived from Lactococcus lactis include PrtP enzymes whose gene sequences are registered in NCBI (National Center for Biotechnology Information) as accession numbers AY542690, AY5422691, and the like.

Whether or not a certain lactic acid bacterium is a PrtP enzyme-containing lactic acid bacterium (lactic acid bacterium having a PrtP enzyme) has, for example, a PrtP gene encoding a PrtP enzyme using a gene analysis technique such as PCR (Polymerase Chain Reaction). It can be confirmed by examining whether or not.

Since the PrtP enzyme has an enzyme active site outside the cell, it can degrade proteins in the medium. For example, when a lactic acid bacterium grows in a milky medium, the PrtP enzyme degrades milk protein in the milky medium, and oligopeptides and amino acids necessary for the growth of the lactic acid bacterium are provided. Therefore, Lactococcus lactis with PrtP enzyme grows so that the medium can be solidified when cultured in a 10% (W / W) reduced skim milk medium at a temperature range of 25-30 ° C. for 16 hours. It is fast and has strong fermentability. PrtP enzyme-containing lactic acid bacteria can also be detected by utilizing such characteristics of high growth and fermentability. In addition, PrtP enzyme-containing lactic acid bacteria can also be detected by detecting PrtP enzyme activity.

The lactic acid bacterium used in the present invention is not particularly limited as long as it has a PrtP enzyme, but is preferably a bacterium belonging to the genus Lactococcus, more preferably Lactococcus lactis, and Lactococcus lactis. -Subspecies Cremolis and Lactococcus lactis subspecies lactis are more preferable.
This is because dairy products made from bifidobacteria such as fermented milk have been used as raw materials and are considered to be highly safe. Examples of Lactococcus lactis having PrtP enzyme include Lactococcus lactis subspices cremolith NBRC100676 ^T strain, Lactococcus lactis subspices lactis JCM20101 strain and the like.

In the present invention, the medium used for the preculture of the PrtP enzyme-containing lactic acid bacteria is not particularly limited as long as it is a medium that is usually used for culturing Lactococcus lactis, but Difco (registered trademark) M17 Broth (Becton And a commercially available semi-synthetic medium such as Dickinson) or a dairy medium such as a reduced skim milk medium. In addition, growth promoting substances such as glucose and yeast extract, reducing agents such as L-cysteine, and the like can be added to the medium used for preculture. Moreover, the culture medium used for preculture uses what sterilized. The sterilization treatment can be performed by a commonly used method, for example, by heat treatment at 80 to 122 ° C. for 5 to 40 minutes, preferably 85 to 95 ° C. for 5 to 35 minutes.

In the present invention, the one used for the hydrolysis of milk protein is not particularly limited as long as the enzyme activity of the PrtP enzyme is maintained, but the live cell of the PrtP enzyme-containing lactic acid bacterium, It is preferable that it is the crushed cell body of lactic acid bacteria, and the PrtP enzyme fraction fractionated from the said lactic acid bacteria.

The live cell of the PrtP enzyme-containing lactic acid bacterium used in the present invention may be a lactic acid bacterium cultured by a conventional method as it is, or may be a dry bacterium powder prepared by a conventional method such as lyophilization. The cultured lactic acid bacteria may be used together with the medium used for the culture, or may be one obtained by collecting only the cells from the medium by centrifugation or the like and then suspending them in an appropriate buffer or the like.
Since it is possible to remove excess medium and the concentration can be adjusted by concentration or dilution, it is preferable to use a suspension of cells recovered from the medium in a buffer.

The buffer for suspending the cells recovered from the medium is not particularly limited as long as it is a solvent that can suspend lactic acid bacteria in a live state, and is usually used for suspending lactic acid bacteria. Although a buffer etc. can be used, it is preferable that it is a buffer containing calcium ion. The pH of the buffer used for the suspension is preferably 4.5 to 8.0. Examples of the buffer include MES buffer, HEPES buffer, and phosphate buffer. In addition, physiological saline may be used.

The cell disruption product of the PrtP enzyme-containing lactic acid bacterium used in the present invention is not particularly limited as long as it is obtained by crushing the lactic acid bacterium without impairing the enzyme activity of the PrtP enzyme. Examples of such a crushing method include a crushing process using ultrasonic waves, a glass bead crushing process, and a crushing process using osmotic shock. These crushing treatments are preferably performed at a low temperature of 50 ° C. or lower, preferably room temperature or lower, more preferably 10 ° C. or lower. This is because the PrtP enzyme is deactivated by heating.

If the PrtP enzyme fraction fractionated from the PrtP enzyme-containing lactic acid bacterium used in the present invention is obtained by fractionating the PrtP enzyme from the lactic acid bacterium without impairing the enzyme activity of the PrtP enzyme, It is not particularly limited. For example, after culturing the lactic acid bacteria collected from the culture medium after suspending in a buffer, removing the bacterial cells by centrifugation or the like, the supernatant containing the PrtP enzyme released from the cell surface of the lactic acid bacteria, It can be obtained as a PrtP enzyme fraction. The addition of a chelating agent such as EDTA to the buffer facilitates the release of the PrtP enzyme from the cell surface of lactic acid bacteria. Therefore, it is preferable to use a buffer to which a chelating agent such as EDTA is added. As a buffer for obtaining such a supernatant, for example, a buffer obtained by adding a chelating agent such as EDTA to the above-mentioned buffers that can be used for suspending viable cells can be used. The temperature at which lactic acid bacteria are retained in the buffer is not particularly limited as long as it is less than 50 ° C., but is preferably about 4 to 37 ° C., more preferably about 30 ° C. The holding time can be appropriately determined in consideration of the holding temperature, the type of buffer, etc., but is preferably within 1 hour, more preferably 5 to 30 minutes, and 5 to 15 minutes. More preferably it is.

Note that the chelating agent can be removed from the PrtP enzyme fraction by dialysis of the supernatant thus obtained. Furthermore, the fractionated PrtP enzyme fraction can be concentrated by a known technique such as freeze concentration without impairing the PrtP enzyme activity. In addition, it may be a PrtP enzyme fraction obtained by fractionating a crushed bacterial body of lactic acid bacteria using a known fractionation means such as ammonium sulfate precipitation method or chromatography method.

The milk protein used in the present invention is not particularly limited as long as it is a milk-derived protein collected from animals such as cows, goats, horses and sheep (excluding humans), and is usually used for food. Milk-derived protein can be appropriately selected and used. In this invention, it is preferable that it is the milk protein contained in the milk with high industrial utility value.
The milk protein may be casein, whey protein (protein contained in whey), or total milk protein.
The milk protein used in the present invention is preferably casein derived from milk or total milk protein, and more preferably casein derived from milk. This is because a milk protein degradation product having a higher bifidobacteria growth promoting effect can be obtained.
In addition, as for milk proteins such as casein, total milk protein, and whey protein, those prepared by a conventional method can be used. Moreover, you may use what is marketed.

The milk protein degradation product of the present invention is one or more selected from the group consisting of a milk protein, a living cell of a PrtP enzyme-containing lactic acid bacterium, a crushed cell of the lactic acid bacterium, and a PrtP enzyme fraction fractionated from the lactic acid bacterium. It is obtained by adding and hydrolyzing. The added PrtP enzyme-containing lactic acid bacteria live cells, crushed cell bodies, and PrtP enzyme fractions are preferably added as prepared by appropriately freeze-concentrating or lyophilizing those prepared as described above. The hydrolysis reaction conditions are not particularly limited as long as the enzymatic reaction can be performed by the PrtP enzyme, and may be appropriately determined in consideration of the type of PrtP enzyme, the type of milk protein, and the like. it can. For example, the PrtP enzyme can be expected to have enzyme activity under conditions that are substantially equivalent to the environment in which the derived lactic acid bacteria can grow (proliferate).

In the present invention, the reaction temperature in the hydrolysis is preferably less than 50 ° C., more preferably 10 to 42 ° C., further preferably 20 to 37 ° C., and about 30 ° C. Particularly preferred.
The milk protein to be subjected to hydrolysis is preferably adjusted to pH 6.0 to 8.0, more preferably adjusted to 6.0 to 7.5. More preferably, it is adjusted to around 5. When the pH of the milk protein that is the substrate is 6.0 or more, protein aggregation hardly occurs when the obtained milk protein degradation product is heat-sterilized. On the other hand, when the pH of milk protein is 8.0 or less, protein denaturation hardly occurs when the obtained milk protein degradation product is heat sterilized.

In the present invention, the milk protein concentration in the reaction system in the hydrolysis can be appropriately determined in consideration of the desired milk protein degradation product concentration, the amount of PrtP enzyme to be added, the reaction time, and the like. In the present invention, the milk protein concentration in the hydrolysis is preferably 0.5% by weight or more, and more preferably 1% by weight or more. This is because a milk protein degradation product with more excellent bifidobacteria growth promoting ability can be obtained.

Further, in the present invention, the amount of the PrtP enzyme-containing lactic acid bacterium live cells, crushed cells, and PrtP enzyme fraction added to the reaction system in the hydrolysis is determined in consideration of the amount of milk protein to be added and the like. Although it can determine suitably, it is preferable to add so that it may become 1 Unit / ml or more as a unit of the PrtP enzyme activity mentioned later. The hydrolysis reaction time can be appropriately determined in consideration of the reaction temperature, milk protein concentration, the amount of PrtP enzyme to be added, and the like. For example, the addition amount of live cells of PrtP enzyme-containing lactic acid bacteria will be described later. When the unit of PrtP enzyme activity is 3.5 Unit / ml and the reaction temperature is 30 ° C., it is preferably 5 hours or longer.

In the present invention, the milk protein degradation product obtained by hydrolysis may use the reaction solution after the reaction as it is, but after the reaction, the PrtP enzyme in the reaction solution is inactivated by heat treatment or the like. It is preferable.

The bifidobacteria growth promoter of the present invention contains the milk protein degradation product produced as described above as an active ingredient. Thus, in order to use milk-derived ingredients as active ingredients, food and drink produced by culturing bifidobacteria with milk-derived raw materials such as fermented milk should be used without considering protein allergies other than milk proteins. Can do.

The amount of the milk protein degradation product of the present invention contained in the bifidobacteria growth promoter of the present invention is not particularly limited as long as the bifidobacteria growth promoting effect is obtained, but it may be 10% by weight or more. It is preferably 15% by weight or more, more preferably 20% by weight or more, and particularly preferably 40% by weight or more.

The dosage form of the Bifidobacterium growth promoter of the present invention is not particularly limited, and may be a powder such as a lyophilized product, a tablet, a liquid, or a paste. There may be. Moreover, the bifidobacteria growth promoter of this invention may contain the other component, unless the bifidobacteria growth promotion effect by the milk protein degradation product of this invention is inhibited. Examples of other components include commonly added adjuvants and stabilizers such as starch and lactose. In addition to the milk protein degradation product of the present invention, a known substance having a bifidobacteria growth promoting action may be added.

Specifically, the bifidobacteria growth promoter of the present invention can exert a bifidobacteria growth promoting effect by adding it to a culture medium of bifidobacteria. The amount to be added to the culture medium is not particularly limited as long as the effect of promoting the growth of bifidobacteria is obtained. The amount of milk protein degradation product in the bifidobacteria growth promoter, the composition of the culture medium of bifidobacteria, and the starter It can be determined as appropriate in consideration of the inoculation amount of a certain bifidobacteria, the type of bifidobacteria and the like. The bifidobacteria growth promoter of the present invention is preferably added so as to be 0.01 to 10 (V / V)%, for example, 0.1 to 5 (V / V)% is particularly preferable.

The culture medium for bifidobacteria to which the agent for promoting growth of bifidobacteria of the present invention is added is not particularly limited as long as it is a commonly used medium. You can choose. For example, the fermenting base used for producing bifidobacteria fermented milk is not particularly limited as long as it is a base usually used for producing fermented milk. The base is, for example, milk, skim milk, fresh cream, butter, whole milk powder, skim milk powder, sweeteners such as sucrose, pectin, fruit, fruit juice, agar, gelatin, oil, fat, flavor, coloring It can be prepared by blending a material, a stabilizer, a reducing agent, etc., and sterilizing, homogenizing, cooling and the like according to a conventional method.

The bifidobacteria to which the bifidobacteria growth promoter of the present invention is added can be those pre-cultured by a conventional method. The culture medium used for pre-culture of bifido is not particularly limited as long as it is a commonly used medium, but is preferably a milky medium. A reduced skim milk medium is particularly preferred because of easy handling. The concentration of the reduced skim milk medium is preferably 3% (W / W) or more, particularly preferably 8% (W / W) or more. In addition, growth-promoting substances such as yeast extract, reducing agents such as L-cysteine, and the like can be added to the medium used for preculture. In particular, since bifidobacteria have a low growth ability in a milky medium, it is preferable to use a medium to which a growth promoting substance is added. For example, a medium containing 0.1 to 1% (W / W) yeast extract can be used. Moreover, the culture medium used for preculture uses what sterilized. The sterilization treatment can be performed by a commonly used method, for example, by heat treatment at 80 to 122 ° C. for 5 to 40 minutes, preferably 85 to 95 ° C. for 5 to 35 minutes.

The growth target bacterium of the Bifidobacterium growth promoter of the present invention is not particularly limited as long as it is a Bifidobacterium, but is preferably Bifidobacterium longum. This is because the growth promoting effect of the milk protein degradation product of the present invention can be obtained more significantly. In particular, Bifidobacterium longum ATCC BAA-999 strain and Bifidobacterium longum type strain ATCC15700 strain are preferable.

Further, since the bifidobacteria growth promoter of the present invention is capable of growing bifidobacteria, particularly Bifidobacterium longum, which has never existed before, the use of the bifidobacteria growth promoter of the present invention makes it more effective in regulating the intestine. It is possible to produce foods and beverages containing bifidobacteria such as bifidobacteria fermented milk, which are high in health and useful for health care.

Hereinafter, the production method of the milk protein degradation product and the bifidobacteria growth promoting action of the present invention will be described in more detail.
1. Detection of PrtP enzyme retention in strains of Lactococcus lactis Lactococcus lactis subspecies Cremolis NBRC100676 ^T (ATCC19257 ^T ) strain, Lactococcus lactis subspices cremolith ATCC-9625 strain, Lactococcus lactis subs It was confirmed whether Spices lactis NBRC12007 (NCDO 497) strain, Lactococcus lactis subspices lactis JCM20101 strain, and Lactococcus lactis subspices lactis JCM20128 strain possessed PrtP enzyme.
Specifically, 3% of each strain was inoculated into Difco (registered trademark) M17 Broth (Becton, manufactured by Dickinson) supplemented with 0.5% lactose and glucose, and cultured at 30 ° C. for 16 hours. Bacteria were obtained by centrifugation, DNA was extracted using DNeasy Blood and Tissue kit (manufactured by QIAGEN), and the possession of the PrtP gene was confirmed by PCR. PCR was performed according to the method described in the reference (Journal of Applied Microbiology, 2006, Vol. 100, pp. 1307-1317). The primer used was a primer set of forward primer GBf (GCAAATACGGTGACGGCTGGCGA) and reverse primer GB2r (TGAGCATTATAATAGGTCTTTCTTCC), or forward primer GHf (CAAATACGGTGACGCGCTGCTAAA) and reverse primer GH2r (TAGCATTATATAGTGTG).

FIG. 1 is a diagram showing band patterns obtained by separating PCR products by electrophoresis and detecting them by staining. In FIG. 1, “1” is a lane in which a molecular weight marker is passed, “2” is a PCR product of Lactococcus lactis subspecies Cremolis NBRC100676 ^T strain, and “3” is Lactococcus lactis subspices lactis. PCR product of JCM20101 strain, “4” is a PCR product of Lactococcus lactis subspecies lactis NBRC 12007 strain, “5” is a PCR product of Lactococcus lactis subspecies lactis JCM20128 strain, “6” is a lactococcus -Lanes in which PCR products of Lactis subspecies Cremolis ATCC-9625 strain were respectively flowed. As a result, it was confirmed that Lactococcus lactis sub-species Cremolis NBRC100676 ^T strain and Lactococcus lactis sub-species lactis JCM20101 strain possessed the PrtP gene. On the other hand, Lactococcus lactis subspecies Cremolis ATCC-9625 strain, Lactococcus lactis subspices lactis NBRC12007 strain and Lactococcus lactis subspices lactis JCM20128 strain each possesses Not found out.

2. Unit Definition of PrtP Enzyme Activity First, 0.4 ml of 30 mM MES-NaOH buffer (pH 6.5) containing 0.02% FTC-casein was added to 0.1 ml of a commercial proteolytic enzyme trypsin solution (manufactured by Sigma, enzyme activity). 11,100 Unit / mg) was added and reacted at 30 ° C. for 1 hour. 1.2 ml of 5% trichloroacetic acid was added, mixed by vortexing, and allowed to stand at room temperature for 15 minutes. 0.3 ml of the supernatant obtained by centrifugation at 15000 × g for 5 minutes was mixed with 2 ml of 500 mM Tris-HCl (pH 8.5), and the fluorescence intensity was measured. The measurement was performed using an MTP-800AFC / Lab microplate reader (Corona Electric Co., Ltd.) under conditions of an excitation wavelength of 490 nm and a fluorescence wavelength of 530 nm.
On the other hand, instead of the trypsin solution, a living cell body of Lactococcus lactis having a PrtP enzyme gene prepared by the method described later, a crushed cell body product, or a PrtP enzyme fraction was added, and a decomposition reaction was performed in the same manner. As a result, the fluorescence intensity value obtained by decomposing one unit of trypsin was converted into a PrtP enzyme in the case of degrading with a live cell of Lactococcus lactis having a PrtP enzyme gene, a crushed cell or a PrtP enzyme fraction. One enzyme unit of activity was defined.

3. Preparation of milk protein degradation product First, a dairy medium containing 10% (W / W) reduced skim milk powder containing 70 mM disodium β-glycerophosphate was sterilized at 95 ° C. for 30 minutes, and the seed culture of each strain was determined. Inoculated with 3% and cultured at 30 ° C. for 16 hours. At this time, 0.5% glucose and 0.05% yeast extract were added to the medium as required by the strain. Thereafter, the pH was adjusted to 6.8 using 1% trisodium citrate and an aqueous sodium hydroxide solution, and the cells were obtained by centrifugation. Next, the cells were washed twice with 30 mM MES-NaOH buffer (pH 6.5) containing 10 mM calcium chloride, suspended in the same buffer, and washed cells (live cells) concentrated 10 times. Moreover, the heat processing for 10 minutes were performed at 90 degreeC with respect to a part of microbial cell.
Next, 1% of viable cells or heat-treated cells was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein, and the mixture was maintained at 30 ° C. for 24 hours with gentle stirring. After the reaction, it was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected solution was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/50 volume by freeze concentration.

4). Measurement of bifidobacteria growth-promoting activity 4-1 Measurement of the number of bifidobacteria The medium containing total milk protein 5% (W / W) and lactose 5% (W / W) was sterilized at 90 ° C. for 10 minutes, as described above. A fixed amount of casein degradation product of each prepared strain of Lactococcus lactis was added, inoculated with 0.1% culture of Bifidobacterium longum ATCC BAA-999 strain, and cultured at 37 ° C. for 16 hours. . After cultivation, the number of bifidobacteria was measured by a serial dilution method using a TOS propionic acid agar medium (manufactured by Yakult Pharmaceutical Co., Ltd.).

4-2 Measurement of pH Fluctuation In the culture test of 4-1, it is known that lactic acid and acetic acid are produced with the growth of bifidobacteria and the pH of the medium is lowered. For this reason, the degree of growth of bifidobacteria can be determined from the degree of decrease in the pH of the medium. The pH was measured using a pH meter (manufactured by HORIBA). The bifidobacteria growth-promoting activity of the milk protein degradation product of the present invention was judged based on a difference in pH decrease of 0.3 or more compared to the control without addition.

5). First, a culture of Bifidobacterium longum ATCC BAA-999 strain was prepared as described in Example 1 below.
Next, a medium containing 5% (W / W) total milk protein and 5% (W / W) lactose was sterilized at 90 ° C. for 10 minutes, and the bacterial cells of each strain of Lactococcus lactis prepared as described above. 1% of the casein degradation product and 0.1% culture of Bifidobacterium longum ATCC BAA-999 strain were inoculated and cultured at 37 ° C. for 16 hours. The culture was rapidly cooled, and the number of bifidobacteria and pH were measured. The measurement results are shown in Table 1.

Compared with the additive-free control, the casein degradation product of the Lactococcus lactis subspices cremolith NBRC100676 strain and the Lactococcus lactis subspices lactis JCM20101 strain having the PrtP enzyme gene has a pH of 5. The number decreased to 0 or less, and the number of bifidobacteria reached around 3 × 10 ⁸ CFU / g. In contrast, each of Lactococcus lactis subspecies Cremolis ATCC-9625 strain, Lactococcus lactis subspices lactis NBRC12007 strain, and Lactococcus lactis subspices lactis strain JCM20128 not having a PrtP enzyme gene When the casein degradation product was added by viable cells, the pH was 5.5 or more and the number of bifidobacteria was 5 × 10 ⁷ CFU / g or less, which was not significantly different from the non-treated control. On the other hand, the casein degradation product by heat-treated cells did not show the bifidobacteria growth promoting activity.
That is, it is clear that the milk protein degradation product by the living strain of Lactococcus lactis having the PrtP enzyme gene has growth promoting ability against Bifidobacteria.
Since the casein degradation product of Lactococcus lactis subspecies lactis JCM20101 strain showed stronger bifidobacteria growth promoting activity, the degradation conditions were examined using the JCM20101 strain.

6). Comparison with Other Commercial Enzymes As described in 3 above, washed cells (PrtP enzyme-containing living cells) of Lactococcus lactis subspecies lactis JCM20101 strain were obtained.
Next, 3.5 unit / ml of PrtP enzyme-containing living cells as a PrtP enzyme unit was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein, and gently stirred at 30 ° C. for 24 hours. Retained. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
On the other hand, instead of the above living cells, a commercial proteolytic enzyme trypsin (manufactured by Sigma) was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein at a rate of 3.5 Units / ml. went. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. The results are shown in Table 2. Compared with the commercially available proteolytic enzyme trypsin, the casein degradation product of the living cells of Lactococcus lactis subspecies lactis JCM20101 strain showed strong bifidobacteria growth promoting activity.

7). Effect of Milk Protein Degradation Time As described in 3 above, washed bacterial cells (PrtP enzyme-containing live cells) of Lactococcus lactis subspecies lactis JCM20101 strain were obtained.
Next, 3.5 unit / ml of PrtP enzyme-containing living cells as a PrtP enzyme unit was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein, and gently stirred at 30 ° C. for 5 to Hold for 24 hours. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. The results are shown in Table 3. In the range of 5 hours to 24 hours after degradation, stronger bifidobacterial growth promoting activity was observed when the degradation time was longer. That is, in the case of the PrtP enzyme activity of 3.5 Units / ml according to the present invention, a strong bifidobacteria growth promoting action was observed if the degradation was 5 hours or longer. In addition, when the PrtP enzyme activity was strengthened, a degradation product having a sufficient bifidobacteria growth promoting action could be obtained even with a shorter degradation time.

8). Effect of Milk Protein Decomposition Temperature As described in 3 above, washed cells (PrtP enzyme-containing living cells) of Lactococcus lactis subspecies lactis JCM20101 strain were obtained.
Next, 3.5 unit / ml of PrtP enzyme-containing living cells as a PrtP enzyme unit was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein at 10 to 50 ° C. with gentle stirring. Hold for 24 hours. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. The results are shown in Table 4. Bifidobacteria growth promoting activity was observed at a decomposition temperature of less than 50 ° C. The bifidobacteria growth promoting activity was weak at 10 to 42 ° C., but strong activity was observed at 20 to 37 ° C., and stronger activity was observed particularly at around 30 ° C.

9. Dependence on PrtP enzyme activity As described in 3 above, washed cells (PrtP enzyme-containing live cells) of Lactococcus lactis sub-species lactis JCM20101 strain were obtained.
Next, 0.01 to 10 Units / ml of PrtP enzyme-containing living cells as a PrtP enzyme unit was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein at 30 ° C. with gentle stirring. Hold for 24 hours. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation.
The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. The results are shown in Table 5. The bifidobacteria growth-promoting action by each degradation product became stronger depending on the PrtP enzyme activity, and a stronger bifidobacteria growth-promoting activity was observed with an enzyme activity of 1 Units / ml or more.

10. Effect of Substrate Concentration on Milk Protein Degradation As described in 3 above, a washed cell (PrtP enzyme-containing live cell) of Lactococcus lactis sub-species lactis JCM20101 strain was obtained.
Next, 3.5 unit / ml of PrtP enzyme-containing living cells as a PrtP enzyme unit was added to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1-10% casein at 30 ° C. with gentle stirring. Hold for 24 hours. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. The results are shown in Table 6. The bifidobacteria growth-promoting action of each degradation product was observed to be bifidobacteria growth-promoting activity at a casein protein concentration of 0.5% or more, and particularly stronger at a concentration of 1% or more.

11. Effect of pH on Milk Protein Degradation As described in 3 above, a washed cell (PrtP enzyme-containing live cell) of Lactococcus lactis subspecies lactis JCM20101 strain was obtained.
Next, PrtP enzyme-containing living cells were added to a 50 mM Tris-HCl buffer (pH 6.0 to 8.0) solution containing 1% casein as a PrtP enzyme unit at 3.5 Units / ml, and the mixture was gently stirred for 30 minutes. Hold at 24 ° C. for 24 hours. After the reaction, the mixture was sterilized by heating at 90 ° C. for 10 minutes, adjusted to pH 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation.
The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/20 volume by freeze concentration.
Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. The results are shown in Table 7. Bifidobacteria growth-promoting activity was observed in the range of pH 6.0 to 8.0, and stronger activity was observed particularly around pH 6.5.

12 Degradation activity by PrtP enzyme-containing disrupted cells As described in 3 above, washed cells of Lactococcus lactis sub-species lactis JCM20101 strain were obtained. Subsequently, the washed cells are subjected to ultrasonic crushing treatment for 10 minutes or more using an ultrasonic crusher (BRANSON SOFIER 450) to crush the cells, and then centrifuged (5000 × g, 10 minutes). Unbroken cells were removed to obtain crushed cells. In addition, it was confirmed by microscopic observation and colony culture that there were no living cells in the crushed cells.
Next, 1 Units / ml of the cell disruption was added as a PrtP enzyme unit to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein, and the mixture was kept at 30 ° C. for 16 hours with gentle stirring. After the reaction, heat treatment was performed at 90 ° C. for 10 minutes, the pH was adjusted to 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/50 volume by freeze concentration.
Each of the obtained casein degradation products was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the number of bifidobacteria after culture and the pH were measured. In the control without addition, Bifidobacterium was 1.5 × 10 ⁷ CFU / ml and the pH was 6.18, whereas in the casein addition of the casein degradation product by the crushed cell body, Bifidobacterium was 2.8 ×. 10 ⁸ CFU / ml, pH dropped to 5.31. From these results, it is clear that the disrupted bacterial cell of the present invention has a growth promoting property against Bifidobacterium.

13. Degradation activity by PrtP enzyme fraction As described in 3 above, washed bacterial cells of Lactococcus lactis subspecies lactis JCM20101 strain were obtained. Subsequently, the washed cells were suspended in 30 mM MES-NaOH buffer (pH 6.5) containing 10 mM EDTA · 2Na and kept at 30 ° C. for 10 minutes. Thereafter, it was rapidly cooled to obtain a supernatant containing PrtP enzyme released by centrifugation at 4 ° C. (8500 × g, 10 minutes). This PrtP enzyme fraction was dialyzed overnight against 30 mM MES-NaOH buffer (pH 6.5) containing 10 mM calcium chloride, and then concentrated to 1/50 volume by freeze concentration, and this was used as the PrtP enzyme fraction.
Next, 1 unit / ml of this PrtP enzyme fraction was added as a PrtP enzyme unit to a 50 mM Tris-HCl buffer (pH 6.5) solution containing 1% casein, and the mixture was kept at 30 ° C. for 16 hours with gentle stirring. After the reaction, heat treatment was performed at 90 ° C. for 10 minutes, the pH was adjusted to 4.6 with hydrochloric acid, and the supernatant was collected by centrifugation. The collected supernatant was adjusted to pH 6.5 with an aqueous sodium hydroxide solution and concentrated to 1/50 volume by freeze concentration.
Each obtained casein degradation product was subjected to a bifidobacteria growth promotion test by the method described in 5 above, and the pH after the culture was measured. In the control without addition, Bifidobacteria was 1.75 × 10 ⁷ CFU / ml and pH was 6.17, whereas in the case of the casein degradation product by the PrtP enzyme fraction, Bifidobacteria was 3.8 ×. 10 ⁸ CFU / ml, pH dropped to 5.34. From these results, it is clear that the PrtP enzyme fraction of the present invention has growth promoting properties against Bifidobacteria.

As described above, the living cells of the Lactococcus lactis subspices cremolith NBRC100676 strain and Lactococcus lactis subspices lactis JCM20101 strain having the PrtP enzyme gene, and milk from the PrtP enzyme fraction It was revealed that the protein degradation product has a growth promoting activity against bifidobacteria. Moreover, it turned out that the activity is stronger than the decomposition product by a general commercial proteolytic enzyme.

Moreover, since Lactococcus lactis having these PrtP enzyme genes does not produce diacetyl and acetoin, it can be expected that a savory fermented product can be produced by using these lactic acid bacteria.
Furthermore, in order to ensure a high number of Bifidobacterium, a Bifidobacterium starter is added to a living cell of Lactococcus lactis having these PrtP enzyme genes, a crushed cell of a cell, or a milk protein degradation product from a PrtP enzyme fraction. And may be fermented.

Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

[Example 1] Preparation 1 of casein degradation product using live cells containing PrtP enzyme
First, 100 L of Difco (registered trademark) M17 Broth (Becton, Dickinson) added with 0.5% lactose and glucose was sterilized for 15 minutes at 121 ° C. 3 L of culture was inoculated and cultured at 30 ° C. for 16 hours. Subsequently, live cells containing PrtP enzyme were obtained by centrifugation (8500 × g, 10 minutes, 4 ° C.).
Next, the obtained PrtP enzyme-containing viable cells were added to 100 L of a 10% casein (fontera) solution, and kept at 30 ° C. for 24 hours with gentle stirring. After the reaction, heat sterilization treatment was performed at 90 ° C. for 10 minutes, and 10.5 kg of casein decomposition product was obtained by freeze-drying.

[Example 2] Preparation 2 of casein degradation product using live cells containing PrtP enzyme
11 kg of casein degradation product was used in the same manner as in Example 1 except that the seed culture of Lactococcus lactis sub-species Lactis JCM20101 strain was used instead of the seed culture of Lactococcus lactis sub-species Cremolis NBRC100676 strain. Obtained.

[Example 3] Preparation 1 of casein degradation product using PrtP enzyme-containing crushed cells
First, 100 L of Difco (registered trademark) M17 Broth (Becton, Dickinson) added with 0.5% lactose and glucose was sterilized for 15 minutes at 121 ° C. 3 L of culture was inoculated and cultured at 30 ° C. for 16 hours. Subsequently, the cells were obtained by centrifugation (8500 × g, 10 minutes, 4 ° C.). Next, the cells were crushed by ultrasonic treatment (BRANSON SOFIER 450, 10 minutes crushing) for 30 minutes, and then centrifuged (8500 × g, 10 minutes, 4 ° C.) to remove unbroken cells. The PrtP enzyme-containing microbial cell disruption product was obtained.
Next, the obtained PrtP enzyme-containing microbial cell disruption was added to 100 L of 10% casein (Fontera) solution, and kept at 30 ° C. for 24 hours with gentle stirring. After the reaction, heat sterilization treatment was performed at 90 ° C. for 10 minutes, and 10 kg of casein decomposition product was obtained by freeze-drying.

[Example 4] Preparation 2 of casein degradation product using crushed cell containing PrtP enzyme
9. Casein degradation product in the same manner as in Example 3 except that the seed culture of Lactococcus lactis subspices lactis JCM20101 strain was used instead of the seed culture of Lactococcus lactis subspecies Cremolis NBRC100676 strain. 1 kg was obtained.

[Example 5] Preparation 1 of casein degradation product by PrtP enzyme fraction 1
First, 100 L of Difco (registered trademark) M17 Broth (Becton, Dickinson) added with 0.5% lactose and glucose was sterilized for 15 minutes at 121 ° C. 3 L of culture was inoculated and cultured at 30 ° C. for 16 hours. Subsequently, the cells were obtained by centrifugation (8500 × g, 10 minutes, 4 ° C.).
Next, this microbial cell was suspended in 30 mM MES-NaOH buffer (pH 6.5) containing 5 L of 10 mM EDTA · 2Na and kept at 30 ° C. for 10 minutes. Thereafter, it was rapidly cooled, and a supernatant liquid containing PrtP enzyme released by centrifugation (8500 × g, 10 minutes, 4 ° C.) was obtained. The supernatant was dialyzed overnight against 30 mM MES-NaOH buffer (pH 6.5) containing 10 mM calcium chloride, and concentrated to 2 L by freeze concentration to obtain a PrtP enzyme fraction.
Next, the obtained PrtP enzyme fraction was added to 100 L of 10% casein (Fontera) solution, and the mixture was kept at 30 ° C. for 24 hours with gentle stirring. After the reaction, sterilization was performed at 90 ° C. for 10 minutes, and freeze-dried to obtain 10.1 kg of casein degradation product.

[Example 6] Preparation 2 of casein degradation product by PrtP enzyme fraction 2
9. Casein degradation product in the same manner as in Example 5 except that the seed culture of Lactococcus lactis subspices lactis JCM20101 strain was used instead of the seed culture of Lactococcus lactis subspecies Cremolis NBRC100676 strain. 1 kg was obtained.

[Example 7] Preparation of bifidobacteria starter by casein degradation product from PrtP enzyme-containing living cells First, the casein degradation product prepared by the method described in Example 1 is 0.1% (W / W). 1000 mL of the medium containing the added 10% (W / W) nonfat dry milk was sterilized at 90 ° C. for 30 minutes. To this sterilized medium, 100 mL of a seed culture of Bifidobacterium longum ATCC BAA-999 strain was inoculated and cultured at 37 ° C. for 5 hours to obtain a culture. The pH of the obtained culture was 4.7, and the number of bifidobacteria was 1.2 × 10 ⁹ CFU / g.

[Example 8] Preparation of an intestinal preparation by casein degradation product using PrtP enzyme-containing living cells [0109] 1 kg of casein degradation product prepared by the method described in Example 2 above was added to 59 kg of dry sterilized starch and 40 kg of sugar, and mixed uniformly. As a result, about 100 kg of an intestinal preparation was obtained.

[Example 9] Preparation of beverage by casein degradation product using PrtP enzyme-containing living cells 1 kg of casein degradation product prepared by the method described in Example 1 above was added to 100 kg of apple juice, and sterilized at 140 ° C for 2 seconds. Filled into a PET bottle, the milk protein decomposed product-containing beverage of the present invention was obtained. This beverage is a beverage that can be expected to have a bifidobacteria growth action.

[Example 10] Preparation of feed by casein degradation product with live PrtP enzyme-containing cells 1 kg of casein degradation product prepared by the method described in Example 2 above is added to 100 kg of skim milk powder and mixed uniformly. About 100 kg was obtained as a feed containing a milk protein degradation product.

Example 11 Preparation of Total Milk Protein Degradation Product Using PrtP Enzyme-Containing Live Bacteria In the same manner as in Example 1, instead of the seed culture of Lactococcus lactis subspecies cremolith NBRC100676 strain, Lactococcus lactis PrtP enzyme-containing viable cells were obtained using the seed culture of Subspecies lactis JCM20101 strain.
Next, the obtained PrtP enzyme-containing viable cells were added to 100 L of a 10% total milk protein (Mirai) solution, and kept at 30 ° C. for 24 hours with gentle stirring. After the reaction, heat sterilization treatment was performed at 90 ° C. for 10 minutes, and 10.2 kg of total milk protein degradation product was obtained by lyophilization.

Since the milk protein degradation product of the present invention has bifidobacteria growth-promoting activity, it can be used in the field of manufacturing foods and beverages containing bifidobacteria such as fermented milk containing bifidobacteria. In addition, since the milk protein degradation product of the present invention uses milk protein such as cow milk as a raw material, the use of the milk protein degradation product of the present invention makes it possible to design a bifidobacteria-containing product using only a milk product. Therefore, it can be used particularly in the field of dairy products.

Claims (13)

Selected from the group consisting of living lactic acid bacteria having cell wall-localized protease (PrtP), disrupted lactic acid bacteria, and enzyme fractions fractionated from the lactic acid bacteria A milk protein degradation product obtained by hydrolyzing milk protein using one or more. The milk protein degradation product according to claim 1, wherein the lactic acid bacterium is Lactococcus lactis. The milk protein degradation product according to claim 2, wherein the Lactococcus lactis does not have xylose utilization and does not produce diacetyl and acetoin. The milk protein degradation product according to any one of claims 1 to 3, wherein the milk protein is casein or total milk protein. The hydrolysis is performed under the conditions of 20 to 37 ° C and a milk protein concentration of 0.5% by weight or more using a milk protein adjusted to pH 6.0 to 8.0 as a substrate. The milk protein degradation product according to any one of 1 to 4. Selected from the group consisting of living lactic acid bacteria having cell wall-localized protease (PrtP), disrupted lactic acid bacteria, and enzyme fractions fractionated from the lactic acid bacteria A method for producing a milk protein degradation product, comprising hydrolyzing milk protein using one or more. The method for producing a milk protein degradation product according to claim 6, wherein the lactic acid bacterium is Lactococcus lactis. The method for producing a milk protein degradation product according to claim 7, wherein the Lactococcus lactis does not have xylose utilization and does not produce diacetyl and acetoin. The method for producing a milk protein degradation product according to any one of claims 6 to 8, wherein the milk protein is casein or total milk protein. 7. The hydrolysis is performed under the conditions of 20 to 37 ° C. and a milk protein concentration of 0.5% by weight or more using a milk protein adjusted to pH 6.0 to 8.0 as a substrate. 10. The method for producing a milk protein degradation product according to any one of 1 to 9. A bifidobacteria growth promoter comprising the milk protein degradation product according to any one of claims 1 to 5 as an active ingredient. The bifidobacteria growth promoter according to claim 11, wherein the bifidobacteria is Bifidobacterium longum. The Bifidobacterium longum strain according to claim 12, wherein the strain of Bifidobacterium longum is Bifidobacterium longum ATCC BAA-999 and / or Bifidobacterium longum type strain ATCC15700.

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