WO2020013307A1

WO2020013307A1 - Method for producing concentrated yogurt

Info

Publication number: WO2020013307A1
Application number: PCT/JP2019/027634
Authority: WO
Inventors: 公一郎角; 衣代長田; 誠二長岡; 卓也 ▲高▼林; 欣也芦田; 佳久平伊澤; 隆也北村; 真也永渕
Original assignee: 株式会社明治
Priority date: 2018-07-13
Filing date: 2019-07-12
Publication date: 2020-01-16

Abstract

Provided is, inter alia, a method for producing a concentrated yogurt for promoting muscle synthesis. A method for producing a concentrated yogurt, in which a yogurt is concentrated, is used to obtain a yogurt by fermenting a prepared yogurt mix in the presence of lactic acid bacteria, the prepared yogurt mix being prepared such that the non-fat milk solids content is 1-30 mass%.

Description

Method for producing concentrated fermented milk

Reference to related application

The present patent application is based on Japanese Patent Application No. 2018-133445 filed on Jul. 13, 2018 and Japanese Patent Application No. 2019-8041 filed on Jan. 21, 2019. The entire disclosure content of these Japanese applications is incorporated by reference into the disclosure of the present invention.

The present invention relates to a method for producing a concentrated fermented milk in which a prepared fermented milk mix is fermented in the presence of lactic acid bacteria to obtain fermented milk, and the fermented milk is concentrated.

筋肉 Muscle loss associated with sarcopenia has been attracting attention in recent years. Muscle atrophy is said to be a major factor that increases the risk of requiring nursing care and support, and reduces the quality of life and healthy life expectancy of the elderly. Therefore, prevention of muscle loss due to sarcopenia and the like is a major issue for Japan in a super-aging society.

It is known that protein synthesis of skeletal muscle and the like increases up to a certain level in a manner dependent on the amount of protein taken (see Non-Patent Document 1). There is a need for foods that can consume more protein in smaller amounts.

In order to obtain fermented milk that can consume more protein in a small amount, for example, when centrifugal concentration is performed, many free amino acids and peptides are water-soluble small molecules, and they are reduced. There is a concern that the effect of promoting muscle synthesis may be reduced.

However, the present inventors have surprisingly found that the ingestion of concentrated fermented milk produced by a specific production method into a subject can significantly promote the synthesis of the muscle of the subject. The present inventors have also surprisingly found that the ingestion of concentrated fermented milk having a low ratio of free essential amino acids to the subject can significantly promote the synthesis of the muscle of the subject. The present invention is based on these findings.

一つ One object of the present invention is to provide a method for producing concentrated fermented milk or a concentrated fermented milk capable of promoting muscle synthesis.

According to the present invention, the following inventions are provided.
[1] A concentrated fermented milk obtained by fermenting a prepared fermented milk mix having a non-fat milk solid content of 1 to 30% by mass in the presence of lactic acid bacteria to obtain a fermented milk and concentrating the fermented milk. Manufacturing method.
[2] A method for producing a concentrated fermented milk, comprising the following steps:
(1) a step of preparing a fermented milk mix to prepare a prepared fermented milk mix, wherein the non-fat milk solid content in the prepared prepared fermented milk mix is 1 to 30% by mass;
(2) subjecting the prepared fermented milk mix to a sterilization treatment;
(3) a step of adding a lactic acid bacterium starter to the prepared fermented milk mix after the sterilization treatment and fermenting; and (4) a step of concentrating the fermented milk after the fermentation.
[3] The production method according to [1] or [2], wherein the prepared fermented milk mix has a protein mass of 0.5 to 11% by mass.
[4] The method according to any one of [1] to [3], wherein the lactic acid bacterium or the lactic acid bacterium starter is Lactobacillus delbrueckii subsp. Bulgaricus and / or Streptococcus thermophilus.
[5] The production method according to any one of [1] to [4], wherein the step of concentrating the fermented milk is a step of concentrating the fermented milk by centrifugation.
[6] The production method according to any one of [1] to [5], wherein the produced concentrated fermented milk is for promoting muscle synthesis.
[7] A concentrated fermented milk produced by the production method according to any one of [1] to [6].
[8] A concentrated fermented milk for promoting muscle synthesis, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
[9] The concentrated fermented milk for promoting muscle synthesis according to [8], wherein the ratio of the branched-chain amino acid to the total amount of the protein is further 0.1 mass or less.
[10] A food for promoting muscle synthesis, comprising the concentrated fermented milk according to [8] or [9].
[11] A concentrated fermented milk for increasing the amount of amino acids in blood, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
[12] A concentrated fermented milk for promoting an increase in blood amino acid concentration, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
[13] A method for promoting muscle synthesis, comprising causing a subject to ingest a concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
[14] A method for increasing the amount of amino acids in blood, comprising causing a subject to ingest concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
[15] The method according to [14], wherein the daily intake of the concentrated fermented milk to the subject is 50 to 600 g.
[16] A method for promoting an increase in blood amino acid concentration, comprising causing a subject to ingest concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
[17] The method according to [16], wherein the daily intake of the concentrated fermented milk to the subject is 50 to 600 g.
[18] Use of concentrated fermented milk having a ratio of free essential amino acids of 0.2% by mass or less to the total amount of protein for producing concentrated fermented milk for promoting muscle synthesis.
[19] Use of concentrated fermented milk having a ratio of free essential amino acids of 0.2% by mass or less to the total amount of protein for producing concentrated fermented milk for increasing the amount of amino acids in blood.
[20] Use of concentrated fermented milk having a ratio of free essential amino acids of 0.2% by mass or less to the total amount of protein for producing concentrated fermented milk for promoting an increase in blood amino acid concentration.

用いる Use of the production method of the present invention is advantageous in that concentrated fermented milk for promoting muscle synthesis can be provided.

FIG. 1 shows the free amino acid concentration in each test solution. Each sample is measured three times and displayed as mean ± SD. FIG. 2 shows TAA (total amino acid) (A), EAA (essential amino acid) (B), BCAA (branched chain amino acid) (C), Leu (leucine) (D) in portal vein blood after administration of each test solution. Represents the concentration transition of. The horizontal axis represents time (minute). In addition, there is a significant difference (P <0.05) in the simple main effect test (round slice test at each time point, Tukey-Kramer @ test) between different characters (a, b, c). There is a significant tendency (P <0.1) between upper case and lower case of the same character. FIG. 3 shows FSR (Fractional Synthesis Rate (% / day)) which is an index of skeletal muscle synthesis rate with respect to elapsed time (minutes) before and after administration. FIG. 4 shows the blood total amino acid change concentration (nmol / mL) over time of a subject who has consumed the test fermented milk or the prepared fermented milk mix. The horizontal axis represents time (minute). “**” indicates that there is a significant difference (P <0.01) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. The blood total amino acid change concentration was determined by comparing the blood total amino acid concentration collected before eating (0 minute) with the blood total amino acid concentration after eating (15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, and 120 minutes). , And 180 minutes later), and the difference was determined as the blood total amino acid change concentration. In FIGS. 5 and 6 described below, the total amino acid change concentration in blood was calculated in the same manner as in FIG. 4 to determine AUC and Cmax. FIG. 5 shows AUC (Area under the blood concentration-time curve) (nmol · min / mL) of the blood total amino acid change concentration of the test subject who consumed the test fermented milk or the prepared fermented milk mix. This AUC was calculated by obtaining the area under the curve of the blood amino acid change concentration from before (0 minute) to 180 minutes after eating. FIG. 6 shows the Cmax (maximum blood amino acid change concentration) of the total amino acid change concentration in blood from before (0 minute) to 180 minutes after ingestion of the test subjects who consumed the test fermented milk or the prepared fermented milk mix. / ML). “†” indicates that there is a significant tendency (P <0.1) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. FIG. 7 shows the blood essential amino acid change concentration (nmol / mL) over time of the test subject who consumed the test fermented milk or the prepared fermented milk mix. The horizontal axis represents time (minute). “**” indicates that there is a significant difference (P <0.01) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. The blood essential amino acid change concentration was determined by comparing the blood essential amino acid concentration collected before eating (0 minute) with the blood essential amino acid concentration (15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, and 120 minutes after eating). , And after 180 minutes), and the difference between the essential amino acid concentration in blood was determined, and the difference was defined as the essential amino acid change concentration in blood. In FIGS. 8 and 9 described below, the blood essential amino acid change concentration was calculated in the same manner as in FIG. 7 to determine AUC and Cmax. FIG. 8 shows the AUC (nmol · min / mL) of the essential amino acid change concentration in the blood of the test subject who consumed the test fermented milk or the prepared fermented milk mix. This AUC was calculated by obtaining the area under the curve of the blood amino acid change concentration from before (0 minute) to 180 minutes after eating. “†” indicates that there is a significant tendency (P <0.1) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. FIG. 9 shows Cmax (maximum blood amino acid change concentration) of the essential amino acid change concentration in blood from before (0 minute) to 180 minutes after ingestion of the test subjects who consumed the test fermented milk or the prepared fermented milk mix. / ML). “*” Indicates that there is a significant difference (P <0.05) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. FIG. 10 shows the BCAA change concentration (nmol / mL) in blood over time of a subject who has consumed the test fermented milk or the prepared fermented milk mix. The horizontal axis represents time (minute). “**” indicates that there is a significant difference (P <0.01) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. The blood BCAA change concentration was determined by comparing the blood BCAA concentration collected before eating (0 minute) with the blood BCAA concentration after eating (15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, and The difference from each blood BCAA concentration (after 180 minutes) was determined, and the difference was defined as the blood BCAA change concentration. In FIGS. 11 and 12 described below, similarly to FIG. 10, the blood BCAA change concentration was calculated, and the AUC and Cmax were obtained. FIG. 11 shows the AUC (nmol · min / mL) of the BCAA change concentration in the blood of the test subject who consumed the test fermented milk or the prepared fermented milk mix. This AUC was calculated by obtaining the area under the curve of the blood amino acid change concentration from before (0 minute) to 180 minutes after eating. “*” Indicates that there is a significant difference (P <0.05) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. FIG. 12 shows Cmax (maximum blood amino acid change concentration) of the BCAA change concentration in blood from before (0 minute) to 180 minutes after ingestion of the test fermented milk or the prepared fermented milk mix. mL). “*” Indicates that there is a significant difference (P <0.05) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. FIG. 13 shows the concentration of leucine change (nmol / mL) in blood over time of a test subject fed the test fermented milk or the prepared fermented milk mix. The horizontal axis represents time (minute). “**” indicates that there is a significant difference (P <0.01) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. The change in blood leucine concentration was determined by comparing the blood leucine concentration collected before eating (0 minute) with the blood leucine concentration after eating (15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, and The difference from each blood leucine concentration (after 180 minutes) was determined, and the difference was defined as the blood leucine change concentration. In FIGS. 14 and 15 described below, similarly to FIG. 13, the blood leucine change concentration was calculated, and AUC and Cmax were obtained. FIG. 14 shows the AUC (nmol · min / mL) of the blood leucine change concentration of the test subjects who consumed the test fermented milk or the prepared fermented milk mix. This AUC was calculated by obtaining the area under the curve of the blood amino acid change concentration from before (0 minute) to 180 minutes after eating. “**” indicates that there is a significant difference (P <0.01) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test. FIG. 15 shows Cmax (maximum blood amino acid change concentration) of the leucine change concentration in the blood of the subject who consumed the test fermented milk or the prepared fermented milk mix from before (0 minute) to 180 minutes after ingestion. mL). “*” Indicates that there is a significant difference (P <0.05) between the test fermented milk fed group and the prepared fermented milk mixed feed group. Statistical analysis was performed by paired t-test.

Detailed description of the invention

[Deposit of microorganism]
The Lactobacillus delbrueckii subsp. Bulgaricus OLL200513 strain was obtained on February 3, 2017 (the date of original deposit) by the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (2-5-8 Kazusa Kamazari, Kisarazu-shi, Chiba, Japan). Room No.) under the Budapest Treaty under the accession number NITE BP-02411.

Streptococcus thermophilus OLS3290 strain was transferred to the National Institute of Technology and Evaluation, Patent Organism Depositary (Patent Depositary Center, 2-5-8 Kazusa-Kamashita, Kisarazu-shi, Chiba, Japan, Room 120) on January 19, 2004 (the original deposit date). International Deposit under the Budapest Treaty under accession number FERM @ BP-19638. The deposited strain was transferred from a domestic deposit (original deposit) to an international deposit based on the Budapest Treaty on September 30, 2013 (date of issue) (the transfer request was received on September 6, 2013). .

[Production method of fermented milk]
In the present invention, the term "fermented milk" refers to, for example, "Ministerial Ordinance on Milk and Dairy Product Ingredients" which is a ministerial ordinance based on the Food Sanitation Law of Japan (Ministerial Ordinance on Milk and the like; 52), "fermented milk" and the like. In this Ministerial Ordinance, "fermented milk" refers to "fermented milk or milk containing non-fat milk solids equivalent to or higher than this, fermented with lactic acid bacteria or yeast, made into a paste or liquid, or frozen from them. Is defined. Therefore, in the present invention, the “fermented milk” includes, for example, solid fermented milk such as yogurt (set type yogurt), pasty fermented milk (soft type yogurt), liquid fermented milk (drink type yogurt), and the like. .

において In the present invention, “fermented milk mix” refers to a mixture of raw materials of fermented milk. The fermented milk mix contains at least raw milk.

In the present invention, the raw milk includes milk, pasteurized milk, skim milk, whole fat milk powder, skim milk powder, whole fat concentrated milk, skim concentrated milk, cream, butter, buttermilk, whey, milk protein concentrate (MPC) , Whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg) and the like.

において In the present invention, as other materials for preparing the fermented milk mix, any materials known in the art can be used, and examples thereof include sugar, sweetener, sugar, flavor, and water. If necessary, a gelling agent such as gelatin, agar, pectin, carboxymethylcellulose (CMC), a thickener, a stabilizer and the like may be used.

In the production method of the present invention, the non-fat milk solids (SNF) content in the prepared fermented milk mix prepared is 1 to 30% by mass, preferably 5 to 25% by mass, more preferably 10 to 25% by mass. The content is 20% by mass, more preferably 11 to 20% by mass, and particularly preferably 12 to 16% by mass. When the content of non-fat milk solids (SNF) in the prepared fermented milk mix is 5 to 25% by mass, muscle synthesis can be further promoted, and the amount of amino acids in blood can be increased. It can promote an increase in blood amino acid concentration.

In the production method of the present invention, the protein mass in the prepared fermented milk mix prepared is not particularly limited, but is preferably 0.5 to 11 mass%, more preferably 1.5 to 9 mass%. %, More preferably 3 to 7% by mass, and particularly preferably 4 to 6% by mass.

において In this specification, proteins include casein proteins and whey proteins. Examples of the casein protein include α-casein and β-casein, and examples of the whey protein include α-lactalbumin, β-lactoglobulin, and serum albumin.

According to one aspect of the present invention, there is provided a method for producing a concentrated fermented milk, comprising the steps of:
(1) a step of preparing a fermented milk mix to prepare a prepared fermented milk mix, wherein the non-fat milk solid content in the prepared prepared fermented milk mix is 1 to 30% by mass;
(2) subjecting the prepared fermented milk mix to a sterilization treatment;
(3) a step of adding a lactic acid bacterium starter to the prepared fermented milk mix after the sterilization treatment and fermenting; and (4) a step of concentrating the fermented milk after the fermentation.

According to one embodiment of the present invention, the production method of the present invention is a method including (1) a step of preparing a fermented milk mix to prepare a prepared fermented milk mix. The prepared fermented milk mix can be prepared by mixing the fermented milk mix with other ingredients. The mixing conditions are not particularly limited, and may be arbitrarily determined. The prepared fermented milk mix thus prepared may be subjected to a homogenization treatment, if necessary. The conditions for the homogenization treatment are not particularly limited, and may be arbitrarily determined.

製造 The production method of the present invention is a method including (2) a step of subjecting the prepared fermented milk mix to a sterilization treatment. The sterilization treatment is not particularly limited, but is usually performed by heat treatment. The conditions for the heat treatment are not particularly limited, provided that the prepared fermented milk mix is not excessively denatured by heat, and conditions generally selected in the art can be used.

製造 The production method of the present invention is a method including (3) a step of adding a lactic acid bacteria starter to the prepared fermented milk mix after the sterilization treatment and fermenting the mixture.

Examples of the lactic acid bacteria starter used in the production method of the present invention include, for example, Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), Streptococcus thermophilus (Streptococcus Salivarius subsp. Thermophilus), and Lactobacillus acidophilus (L. acidophilus), Lactobacillus amylovorus (L. amylovorus), Lactobacillus brevis (L. brevis), Lactobacillus buchineri (L. buchneri), Lactobacillus casei (L. casei), Lactobacillus casei subspecies・ Lamnosus (L. casei subsp. Rhamnosus), Lactobacillus crispatas (L. crispatus), Lactobacillus delbrucky subspecies Lactis (L. delbrueckii subsp. Lactis), Lactobacillus fermentum (L.Lfermentum) ), Lactobacillus galinarum (L. gallinarum), Lactobacillus gasseri (L. gasseri), Lactobacillus helveticus (L. helveticus), Lactobacillus helveticus subspecies euglutii (L. helveticus subsp. Jugurti) , Lactobacillus johnsonii (L. johnsonii), Lactobacillus kefir (L. kefir), Lactobacillus oris (L. 、 oris), Bacill lactos paracasei subspecies paracasei (L. paracasei subsp. Paracasei), lactobacillus・ Lactobacillus pentosus (L. pentosus), Lactobacillus plantarum (L. plantarum), Lactobacillus reuteri (L. reuteri), Lactobacillus salivaryus (L. salivalius), Lactobacillus zeae, L. B. adolescentis, B. animalis, B. animalis, B. bifidum, B. breve, Bifidobacterium -Catenulatum (B. catenulatum), Bifidobacterium globosum (B. globosum), Bifidobacterium infantis (B. infantis), Bifidobacterium lactis (B. lactis), Bifidobacterium -From lactic acid bacteria and yeasts commonly used in the production of fermented milk, such as longum (B. longum), Bifidobacterium pseudocatenulatum (B. pseudocatenulatum), and Bifidobacterium suis (B. suis) One or more selected ones can be used. As a lactic acid bacterium starter used in the production method of the present invention, Lactobacillus delbrueckii subsp. Bulgaricus and / or Streptococcus thermophilus are preferably used, and more preferably Lactobacillus delbrueckii subsp. Bulgaricus and Streptococcus thermophilus are used in combination. More preferably, the Lactobacillus delbrueckii subsp. Bulgaricus OLL2050013 strain (Accession No .: NITE BP-02411) and the Streptococcus thermophilus ERS32 accession No. FS32 (Accession number: Lactobacillus delbrueckii subsp. -19638) can be used.

The starter can be prepared into a bulk starter and added to the prepared fermented milk mix. The amount of the bulk starter added can be 0.5 to 5%, preferably 1 to 3%.

The bulk starter can be prepared by sterilizing skim milk powder, adding the cells used for the starter, and fermenting. The fermentation temperature is preferably from 37 to 47 ° C, particularly preferably from 40 to 45 ° C. This fermentation is preferably performed until the pH at the end of fermentation becomes 4.0 to 4.7, and more preferably until the pH at the end of fermentation becomes 4.2 to 4.5.

製造 The production method of the present invention is a method including (4) a step of concentrating the fermented milk after the fermentation. The method for concentrating the fermented milk is not particularly limited, and examples thereof include a UF (Ultrafiltration @ Membrane) membrane concentration method using a UF membrane, a whey discharge method by compression, a centrifugal separation method, and the like. Among them, it is preferable to concentrate the fermented milk by a centrifugation method.

The conditions for centrifugation in the centrifugal separation method are not particularly limited, and the centrifugation is performed at 800 G to 10000 G, preferably 3000 G to 8000 G, after separating into a light liquid and a heavy liquid, removing the light liquid and concentrating. Can be. Industrially, a quark separator (for example, manufactured by GEA Westfalia Separator) or the like is suitably used. The heavy liquid is appropriately homogenized according to the intended product design. In the case of a fat-free yogurt, the homogenization condition is 8 to 20 MPa, preferably 12 to 17 MPa when a high-pressure homogenizer (for example, manufactured by Sanwa Engineering Co., Ltd.) is used, and an ultrasonic or polytron homogenizer is used. In this case, the same homogenization can be performed.

発酵 The obtained fermented milk curd can be made into liquid fermented milk by crushing with a filter or homogenization with a homogenizer.

According to a preferred embodiment of the production method of the present invention, the concentrated fermented milk to be produced is a concentrated fermented milk for promoting muscle synthesis, preferably a concentrated fermented milk for promoting skeletal muscle synthesis. Here, “promotion of muscle (skeletal muscle) synthesis” refers to the promotion of muscle (skeletal muscle) synthesis of the present invention as compared to the case where the subject does not take the concentrated fermented milk for promoting muscle (skeletal muscle) synthesis of the present invention. When the subject ingests the concentrated fermented milk for use, if a slight amount of synthesis of muscle (skeletal muscle) is observed, it is considered to have a muscle (skeletal muscle) synthesis promoting effect. Further, according to a more preferred embodiment of the present invention, “promotion of muscle (skeletal muscle) synthesis” means that the fermented milk of the present invention is compared with a case where non-fermented milk having the same milk protein concentration is ingested. It is assumed that the ingestion has a more remarkable muscle (skeletal muscle) synthesis promoting effect when FSR (Fractional Synthesis Rate), which is an index of the skeletal muscle synthesis rate, increases. The skeletal muscle attaches to the skeleton in the muscle and has a role of controlling the movement and posture of the joint, and means striated muscle other than the myocardium.

According to another preferred embodiment of the production method of the present invention, the concentrated fermented milk to be produced is a concentrated fermented milk for increasing the amount of amino acids in blood. According to another preferred embodiment of the production method of the present invention, the concentrated fermented milk to be produced is a concentrated fermented milk for promoting an increase in blood amino acid concentration. Concentrated fermented milk for increasing the amount of amino acids in blood is preferably concentrated fermented milk for increasing the amount of essential amino acids in blood, more preferably concentrated fermented milk for increasing the amount of branched-chain amino acids in blood, and even more preferably the amount of leucine in blood It is concentrated fermented milk for increasing. Further, the concentrated fermented milk for blood amino acid concentration increase promotion is preferably concentrated fermented milk for blood essential amino acid concentration increase promotion, more preferably concentrated fermented milk for blood branched chain amino acid concentration increase promotion, and further more preferably. Is concentrated fermented milk for promoting the increase of blood leucine concentration.

[Fermented milk]
According to one aspect of the present invention, it is possible to provide concentrated fermented milk produced by the above-described production method of the present invention.

According to one embodiment of the present invention, the concentrated fermented milk for promoting muscle synthesis, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less) (preferably, (For promoting skeletal muscle synthesis). The type of free essential amino acid is not particularly limited, but is preferably a branched-chain amino acid (BCAA, Branched Chain Amino Acid), and more preferably leucine. Here, free essential amino acids refer to valine, leucine, isoleucine, methionine, phenylalanine, threonine, tryptophan, lysine, and histidine, and branched-chain amino acids refer to valine, leucine, and isoleucine. Here, the amino acid means an L-amino acid (L-form), but may include a D-amino acid (D-form) or a DL-amino acid (DL-form). The definition and the like of “promotion of muscle (skeletal muscle) synthesis” are the same as in the above-mentioned production method of the present invention.

According to a preferred embodiment of the present invention, the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less), and the ratio of branched-chain amino acids is further 0.1% by mass. Concentrated fermented milk for promoting muscle synthesis (preferably 0.06% by weight or less, more preferably 0.05% by weight or less, still more preferably 0.03% by weight or less) (For promotion) can be provided.

According to a more preferred embodiment of the present invention, the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less), and the ratio of branched-chain amino acids is further 0.1% by mass. Or less (preferably 0.06% by weight or less, more preferably 0.05% by weight or less, and still more preferably 0.03% by weight or less) and a leucine ratio of 0.05% by weight or less (preferably 0% by weight or less). 0.025% by mass or less, more preferably 0.02% by mass or less, and still more preferably 0.01% by mass or less can provide concentrated fermented milk for promoting muscle synthesis (preferably for promoting skeletal muscle synthesis).

According to another aspect of the present invention, there is provided concentrated fermented milk for increasing the amount of amino acids in blood, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). it can.

According to a preferred embodiment of the present invention, the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less), and the ratio of branched-chain amino acids is further 0.1% by mass. It is possible to provide concentrated fermented milk for increasing the amount of amino acids in blood of not more than 0.06% by mass (preferably not more than 0.06% by mass, more preferably not more than 0.05% by mass, and still more preferably not more than 0.03% by mass).

According to a more preferred embodiment of the present invention, the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less), and the ratio of branched-chain amino acids is further 0.1% by mass. Or less (preferably 0.06% by weight or less, more preferably 0.05% by weight or less, and still more preferably 0.03% by weight or less) and a leucine ratio of 0.05% by weight or less (preferably 0% by weight or less). 0.025% by mass or less, more preferably 0.02% by mass or less, and still more preferably 0.01% by mass or less).

According to another aspect of the present invention, concentrated fermented milk for promoting an increase in the concentration of amino acids in blood, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less), Can be provided.

According to a preferred embodiment of the present invention, the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less), and the ratio of branched-chain amino acids is further 0.1% by mass. It is possible to provide a concentrated fermented milk for promoting an increase in the concentration of amino acids in blood of not more than 0.06% by mass, preferably not more than 0.05% by mass, more preferably not more than 0.03% by mass.

[Food]
According to one embodiment of the present invention, a concentrated fermented milk produced by the production method of the present invention or a muscle containing a concentrated fermented milk for promoting muscle synthesis wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less. A food for promoting synthesis is provided. Here, the food may be in any form as long as it can contain the concentrated fermented milk of the present invention, and may be a solution, a suspension, an emulsion, a powder, a paste, a semi-solid molded product. , Solid molded products and the like are not particularly limited as long as they can be orally ingested. For example, instant foods such as instant noodles, retort foods, canned foods, microwave foods, instant soups / miso soups, freeze-dried foods, etc .; Beverages such as drinks, fruit drinks, vegetable drinks, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, alcoholic drinks; flour products such as bread, pasta, noodles, cake mixes, crumbs; candy, caramel, chewing gum , Chocolates, cookies, biscuits, bars, cakes, pies, snacks, crackers, Japanese sweets, mousses, desserts and other confectionery; sauces, tomato seasonings Flavor seasonings, cooking mixes, sauces, dressings, soups, curry and stew ingredients, etc .; oils and fats such as processed fats and oils, butter, margarine, mayonnaise; milk drinks, yogurt, lactic acid bacteria drinks, ice Dairy products such as creams and creams; agricultural products such as canned agricultural products, jams and marmalades, and cereals; frozen foods, liquid foods, and the like. Here, the food for promoting muscle synthesis may be a food composition for promoting muscle synthesis.

According to another aspect of the present invention, concentrated fermented milk produced by the production method of the present invention or concentrated fermented milk for increasing the amount of amino acids in blood, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less. A food for increasing the amount of amino acids in blood is provided. According to another aspect of the present invention, there is provided a concentrated fermented milk produced by the production method of the present invention or a concentrate for promoting an increase in blood amino acid concentration wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less. A food for promoting an increase in blood amino acid concentration, including fermented milk is provided. Here, the blood amino acid level increasing food or the blood amino acid level increase promoting food may be a blood amino acid level increasing food composition or a blood amino acid level increasing promoting food composition, respectively.

Foods include health foods, functional foods, dietary supplements, functionally labeled foods, foods for specified health use, foods for the sick, infant formulas, maternal or nursing formulas, or muscle synthesis promotion, blood Also included are foods with a label indicating that they are used to increase the amount of medium amino acids or to promote an increase in blood amino acid concentration. In the present invention, food is a concept including beverages.

According to another embodiment of the present invention, a method for promoting muscle synthesis (preferably, comprising ingesting a concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less to a subject is included. , A method for promoting skeletal muscle synthesis). According to another preferred embodiment of the present invention, a method for promoting muscle synthesis, comprising causing a subject to ingest concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (provided that , Except for medical practice on humans). Here, the “medical act for humans” means an act of ingesting (administering) a medicinal product to a human, which requires a prescription by a doctor or the like. Further, in the above embodiment, the subject preferably needs exercise for promoting health such as athletes, sports enthusiasts (athletes), those who need exercise for improving lifestyle-related diseases, and the elderly. Or those who need to build muscle during the development / growth process, such as infants and / or children, or patients who have muscle-lowering disease (eg, sarcopenia) and need to promote muscle synthesis by exercising And those who need exercise to prevent such diseases. The method for promoting muscle synthesis of the present invention can be carried out according to the method described in the present specification for the method for producing fermented milk of the present invention.

Here, the target is preferably a target that requires promotion of muscle synthesis, and is expected to have a muscle synthesis promotion effect, a muscle decomposition suppressing effect, an anti-fatigue / fatigue recovery effect, a muscle pain suppressing effect, and a reserve capacity improving effect. Or the object which needs it is more preferable. For example, for the purpose of promoting health, improving athletic ability, and relieving potential or overt fatigue, elderly people, malnourished people, those who are ill or ill, and those who are exercising can be mentioned. The subject may be an animal other than a human (a domestic animal such as a horse or a cow, a pet animal such as a dog or a cat, an ornamental animal bred in a zoo or the like), but is preferably a human.

According to a preferred embodiment of the present invention, the method comprises ingesting a concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). A method for increasing the amount of amino acids in blood is provided. According to another preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). A method for increasing the amount of amino acids in blood (excluding medical practice on humans), comprising: Here, “medical action for humans” is used in the same meaning as described above. In this embodiment, the subject may be the same as the above-mentioned method for promoting muscle synthesis, and may be bred in animals other than humans (livestock such as horses and cows, companion animals such as dogs and cats, zoos, etc.). Animals, etc.), but humans are preferred. The method for increasing the amount of amino acids in blood of the present invention can be carried out according to the method described in the present specification for the method for producing fermented milk of the present invention.

According to a more preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). And the area under the curve (AUC) of the total amino acid change concentration in the blood from immediately after ingestion of the concentrated fermented milk to 180 minutes is 82,000 nmol · min / mL or more (preferably, 82,000 to 120,000 nmol · min / mL). A method for increasing the amount of amino acids is provided, wherein AUC can be determined by, for example, collecting blood from a subject over time and calculating the amino acid concentration in blood, as described in the Examples below.

According to a more preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). And the area under the curve (AUC) of the essential amino acid change concentration in the blood from immediately after ingestion of the concentrated fermented milk to 180 minutes is 45,000 nmol · min / mL or more (preferably, 45,000 to 65000 nmol · min / mL). A method for increasing the amount of amino acids is provided.

According to a more preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). And the area under the curve (AUC) of the blood BCAA (branched chain amino acid) change concentration from immediately after ingestion of the concentrated fermented milk to 180 minutes is 25,000 nmol · min / mL or more (preferably, 25,000 to 35,000 nmol · min / mL). A method for increasing the amount of amino acids in blood is provided.

According to a more preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). Blood amino acid having an area under the curve (AUC) of the leucine change concentration in blood from immediately after ingestion of the concentrated fermented milk to 180 minutes or more (preferably 9000 to 14000 nmol · min / mL). A volume increase method is provided.

According to a preferred embodiment of the present invention, there is provided a method for promoting an increase in blood amino acid concentration, which comprises inducing a subject to take in a concentrated fermented milk in which the ratio of free essential amino acids to the total amount of proteins is 0.2% by mass or less. Provided. According to another preferred embodiment of the present invention, the promotion of an increase in the concentration of amino acids in blood comprising causing a subject to ingest concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less. A method is provided, except for medical practice on humans. Here, “medical action for humans” is used in the same meaning as described above. In this embodiment, the subject may be the same as the above-mentioned method for promoting muscle synthesis, and may be bred in animals other than humans (livestock such as horses and cows, companion animals such as dogs and cats, zoos, etc.). Animals, etc.), but humans are preferred. The method for promoting an increase in blood amino acid concentration of the present invention can be carried out according to the contents described in the present specification for the method for producing fermented milk of the present invention.

According to a more preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). And the Cmax (maximum blood amino acid change concentration) of the total amino acid change concentration in blood from immediately after ingestion of the concentrated fermented milk to 180 minutes is at least 1200 nmol / mL (preferably 1200 to 1700 nmol). / ML) is provided.

According to a more preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). And the Cmax (maximum blood amino acid change concentration) of the essential amino acid change concentration in blood from immediately after ingestion of the concentrated fermented milk to 180 minutes is 600 nmol / mL or more (preferably, 600 to 1000 nmol). / ML) is provided.

According to a more preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). And the Cmax (maximum blood amino acid change concentration) of the blood BCAA (branched chain amino acid) change concentration from immediately after ingestion of the concentrated fermented milk to 180 minutes from the concentrated fermented milk intake target is 350 nmol / mL or more (preferably , 350-500 nmol / mL).

According to a more preferred embodiment of the present invention, the subject is ingested with concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less (preferably 0.1% by mass or less). And the Cmax (maximum blood amino acid change concentration) of the leucine change concentration in blood from immediately after ingestion of the concentrated fermented milk to 180 minutes is 130 nmol / mL or more (preferably, 130 to 200 nmol / min). mL) of the blood amino acid concentration.

The above-mentioned method for promoting an increase in blood amino acid concentration may be a method for promoting muscle synthesis (preferably, a method for promoting skeletal muscle synthesis).

In the muscle synthesis promoting method, the blood amino acid amount increasing method, or the blood amino acid concentration increasing promoting method of the present invention, the concentrated fermented milk of the present invention (for example, the ratio of free essential amino acids to the total amount of protein is 0.2% by mass). % (Preferably 0.1% by mass or less) of the concentrated fermented milk is not particularly limited as long as the effects of the present invention are exhibited. And more preferably 80 to 150 g per serving.

According to a preferred embodiment of the present invention, the concentrated fermented milk of the present invention is in a unit package form per serving, and the unit package form contains 50 to 200 g (preferably 80 to 150 g) of the concentrated fermented milk. Here, `` consisting of a unit packaging form per meal '' means a form in which the amount of intake per meal is predetermined, for example, as a food that can be orally ingested a specific amount, not only general food, but also beverage (Such as drinks), health supplements, health functional foods, and supplements. In the "unit packaging form per serving", for example, in the case of liquid food, gel, paste, or paste jelly, powder, granule, capsule, or block-shaped solid food, etc. , Metal cans, glass bottles (bottles, etc.), plastic containers (pet bottles, etc.), packs, pouches, film containers, packaging boxes such as paper boxes, etc., in a form that can specify a specific amount (dose), or intake per serving (usage , Dosage) can be specified on a packaging container, a homepage, or the like to specify a specific amount. Alternatively, a form in which a measuring instrument such as a spoon that can measure the intake per meal is attached.

In the muscle synthesis promoting method, blood amino acid amount increasing method, or blood amino acid concentration increasing promoting method of the present invention, the concentrated fermented milk of the present invention (for example, a free essential amino acid with respect to the total amount of protein, Of the concentrated fermented milk having a ratio of 0.2% by mass or less (preferably 0.1% by mass or less) is preferably 50 to 600 g of the concentrated fermented milk of the present invention. , And more preferably 80 to 450 g.

The time of ingesting (administering) the concentrated fermented milk of the present invention to a subject is not particularly limited, but ingestion in a state where the amino acid concentration in the blood is low is considered to be particularly effective. It can be used preferably during evening meals, between meals, or after exercise.

According to another aspect of the present invention, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention for promoting muscle synthesis (preferably promoting skeletal muscle synthesis) is 0.1%. Use of concentrated fermented milk for promoting muscle synthesis of 2% by mass or less is provided.

According to another aspect of the present invention, for increasing the amount of amino acids in blood, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention is 0.2% by mass or less. Use of concentrated fermented milk for increasing the amount of medium amino acids is provided.

According to another aspect of the present invention, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention for promoting an increase in blood amino acid concentration is 0.2% by mass or less. Use of concentrated fermented milk for promoting an increase in blood amino acid concentration is provided.

In these uses, according to one preferred embodiment of the present invention, the use of the present invention is a non-therapeutic use.

According to another aspect of the present invention, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention for promoting muscle (skeletal muscle) synthesis is 0.2% by mass or less. The present invention provides a concentrated fermented milk for promoting muscle synthesis.

According to another aspect of the present invention, in order to increase the amount of amino acids in blood, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention is 0.2% by mass or less. A concentrated fermented milk for increasing the amount of amino acids in blood is provided.

According to another aspect of the present invention, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention is 0.2% by mass in order to promote an increase in amino acid concentration in blood. The following concentrated fermented milk for promoting an increase in blood amino acid concentration is provided.

According to another aspect of the present invention, for producing concentrated fermented milk for promoting muscle synthesis, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention is 0.2% by mass. There is provided the use of concentrated fermented milk as follows.

According to another aspect of the present invention, for producing concentrated fermented milk for increasing the amount of amino acids in blood, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention is 0.2%. There is provided the use of concentrated fermented milk which is less than or equal to% by weight.

According to another aspect of the present invention, the ratio of free essential amino acids to the total amount of concentrated fermented milk or protein produced by the production method of the present invention for producing concentrated fermented milk for promoting an increase in blood amino acid concentration is 0. Provided is the use of concentrated fermented milk that is less than 2% by weight.

濃縮 The concentrated fermented milk and the like used in the muscle synthesis promoting method and the like of the present invention, the concentrated fermented milk and the like contained in the food of the present invention, and the like may be the same as the above-described production method of the present invention.

Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples.

Example 1 Preparation of Each Test Solution (Test Milk) and Measurement of Each Amino Acid Concentration The test solution used was “Skim Milk Q” (manufactured by Meiji Co., Ltd.) as a raw material (fermented milk mix) and had a protein concentration of about 6. A material prepared according to the following procedure so as to be about 6.4% by volume when considering the specific gravity at 0 to 6.4% by mass. Table 1 shows the properties of each test milk after preparation. FIG. 1 shows the results of measuring the total free amino acid (TAA), non-essential amino acid (NEAA), essential amino acid (EAA), and branched chain amino acid (BCAA) concentrations in each sample by LC-MSMS.
(1) Skim milk: prepared by mixing with water so that the SNF (non-fat milk solid content) content is 16% by mass. After preparation, the mixture was sterilized at a temperature of 95 ° C., homogenized (15 MPa), and heated at 65 ° C. for 30 minutes.
(2) Control fermented milk: A fermented milk mix was prepared by mixing with water so that the SNF content was 15.7% by mass. After the preparation, the mixture was sterilized at a temperature of 95 ° C. and fermented with a fermentation starter (Lactobacillus delbrueckii subsp. 0.3% of a strain of Streptococcus thermophilus OLS3290 (accession number: FERM BP-19638) was added, followed by fermentation at 42 ° C. (pH at the end of fermentation is 4.4 to 4.3). It was cooled and homogenized (15 MPa) and sterilized (65 ° C., 30 minutes).
(3) Test fermented milk: A fermented milk mix was prepared by mixing with water so that the SNF content was 15.7% by mass. After preparation, the mixture was sterilized at a temperature of 95 ° C., a fermentation starter was added, and fermented at 42 ° C. (pH at the end of fermentation is 4.4 to 4.3). After cooling and homogenization (15 MPa), the mixture was concentrated by centrifugation (manufactured by HITACHI) to a protein concentration of 12% by mass (approximately twice that of the prepared fermented milk mix). Thereafter, the mixture was diluted with water so that the protein concentration became 6.4% by volume, and sterilized (65 ° C., 30 minutes).

After preparation, each test solution (test milk) was stored at −20 ° C., thawed immediately before administration, and used at room temperature.

によ According to FIG. 1, free amino acids are increased in the control fermented milk compared to the raw material skim milk (fermented milk mix). However, in the test fermented milk concentrated by the centrifugation step (the test fermented milk of the present invention), the free essential amino acids are transferred to the removed light liquid (the part removed by centrifugation in the production process of the test fermented milk), Free essential amino acids are lower than control fermented milk. However, since the protein administration dose is designed to be about 6.4% by volume, the protein content in the test fermented milk is not necessarily reduced, and the ratio of free essential amino acids to the total protein is low. (In the test fermented milk of the present invention, the ratio of free essential amino acids to the total amount of protein is about 0.1% by mass, the ratio of branched-chain amino acids is about 0.05% by mass, and the ratio of leucine is about 0.1% by mass. 02% by mass).

Example 2: Evaluation of muscle synthesis enhancing action of each test solution by rats (FSR evaluation)
For each test solution used in this example, skim milk, control fermented milk, and test fermented milk prepared in Example 1 were used.

The details of the experiment are described below.
A total of 136 7-week-old male SD rats were purchased from CLEA Japan, and after completion of the breeding, rats subjected to all tests were fasted for about 18 hours. On the day of the FSR evaluation test, the rats were divided into the following 16 groups by 8 rats after excluding 4 cases whose body weights deviated from the average of the whole (only 12 groups were administered with “none”). Excluded rats were euthanized by carbon dioxide inhalation.

Group 1: administration: none, dissection time: after fasting for about 18 hours (0 minutes)
Group 2: administration: skim milk, dissection time: 30 minutes after administration 3 groups: administration: skim milk, dissection time: 60 minutes after administration 4 groups: administration: skim milk, dissection time: 90 minutes after administration, 5 groups : Administration: skim milk, dissection time: 6 groups 120 minutes after administration: administration: skim milk, dissection time: 7 groups 240 minutes after administration: administration: control fermented milk, dissection time: 8 groups 30 minutes after administration: Dose: Control fermented milk, dissection time: 9 groups 60 minutes after administration: Dose: Control fermented milk, necropsy time: 10 groups 90 minutes after administration: Dose: Control fermented milk, dissection time: 11 groups 120 minutes after administration : Dosage: Control fermented milk, dissection time: 12 groups 240 minutes after administration: Dosage: Test fermented milk, dissection time: 13 minutes after administration 13 groups: Dosage: Test fermented milk, dissection time: 14 minutes after administration 60 minutes Group: administration: test fermented milk, dissection time: 90 minutes after administration 15 groups: administration: test fermented milk, dissection time: administration 12 Min After 16 group: administration thereof: test fermented milk, dissection time: administered 240 min after

The test solution was orally administered to rats other than the first group, and all rats were dissected at the above timing (dissection time). Fifteen minutes before the dissection, deuterium-labeled phenylalanine (D ₅ -Phe) (manufactured by CIL), a tracer for measuring the rate of skeletal muscle synthesis, was injected from the tail vein (45 mg / kg BW). Dissection was performed under isoflurane anesthesia. After partial collection of portal vein blood, whole blood was collected from the abdominal vena cava and euthanized. Immediately after excision of the triceps surae, they were frozen with liquid nitrogen.

Evaluation items Portal vein amino acid concentration and plantar muscle FSR (Statistical analysis was performed by two-way analysis of variance with time and group as factors)
The concentration of amino acids in blood and free amino acids in test milk was determined by LC / MS / MS using an Intrada Amino Acid column (manufactured by Intact) after removing proteins from each sample by treating with 3% sulfosalicylic acid. Amino acids were quantified by the external standard method.

FSR analysis method (1) Homogenization of skeletal muscle Transfer the whole cryopreserved right plantar muscle (approximately 250-300 mg) to a homogenized tube (CK Mix Kit Tube 7 mL) containing beads, and ice-cool 3 mL of 0.3 M perchloric acid solution. , And homogenized using a high-speed cell disruption device Precelllys Evolution (manufactured by M & S) under air cooling with Cryolys (manufactured by M & S, air flow cooling device using liquid nitrogen and dry ice). Crushing conditions were as follows.
・ 4500rpm 20sec → 7500rpm 20sec × 2 → 8500rpm 20sec × 2
An air cooling time of 30 seconds was provided between each crushing process.

(2) Separation of supernatant and washing of muscle homogenized sample After 1 mL of muscle homogenized sample was centrifuged (4 ° C, 8000 g, 15 min), the supernatant was separated. The supernatant was filtered through a 0.2 μm filter and used for “(5) Determination of phenylalanine by LC / MS / MS” (sample for quantifying muscle supernatant).
The muscle homogenized sample from which the supernatant was separated was washed twice with 1 mL of milliQ, and then suspended in 1 mL of 0.1 N HCl.

(3) Protein hydrolysis of muscle homogenized sample 100 μL of washed muscle homogenized sample was added to 900 μL of 6N HCl (1% phenol), and nitrogen replacement was performed using a PICO-TAG workstation (manufactured by Nippon Waters Co., Ltd.). It sealed under reduced pressure. The mixture was heated at 150 ° C. for 1 hour using a heat block to hydrolyze the muscle protein (muscle hydrolyzate).

(4) Preparation of muscle hydrolyzate quantitative sample 200 μL of muscle hydrolyzate was concentrated using a centrifugal evaporator. The concentrate was dissolved in 80 μL of 10% acetonitrile containing 25 uM internal standard (stock solution), and diluted 20-fold with 10% acetonitrile containing 25 uM internal standard (20-fold diluted solution). Each of the stock solution and the 20-fold diluted solution was filtered through a 0.2 μm filter to obtain a sample for quantifying muscle hydrolyzate (stock solution, 20-fold diluted solution).
13C9, 15N, α, β1, β2, 2, 3, 4, 5, 6-D8-Phe (IC-Phe, manufactured by Sigma) was used as the internal standard.

(5) Quantification of phenylalanine by LC / MS / MS Using LC / MS / MS (manufactured by Waters), a sample for quantifying muscle supernatant and a sample for quantifying muscle hydrolyzate (stock solution, 20-fold diluted solution) The enrichment of phenylalanine (Phe) and deuterium labeled phenylalanine (Phe (Ring-D5)) was measured. The analysis conditions were as follows.

<LC conditions>
・ LC column: ACQUITYUPLCBEHC181.7um
・ Mobile phase A: 0.05% TFA / milliQ
-Mobile phase B: 0.05% TFA / acetonitrile-Mobile phase flow rate: 0.3 mL / min
・ Column temperature: 40 ℃
・ Sample injection volume: 3 μL
・ Mobile phase ratio
0 ~ 3.4min ： A100% → A85% B15% (Gradient)
3.4 ~ 4.5min ： A70% B30%
4.5 ~ 6.0min ： A50% B50%
6.0 ~ 8.0min ： A20% B80%
8.0 ~ 10.5min: A100% (Total running time: 10.5min)

<MS / MS conditions>
・ Fragment ion
Phe: m / z166.19> 120.10
Phe (Ring-D5): m / z 171.19> 125.10
IC-Phe: mz184.17> 137.17
・ CapillaryVoltage: 3000V
・ SourceTemperature: 120 ℃
・ DesolvationTemperature: 400 ℃
・ DesolvationGasFlow: 849L / h
・ ConeGasFlow: 48L / h
・ ConeVoltage: 25V
・ ConeEnergy: 15eV

・ Enrichment measurement
D5-Phe enrichment = Res (D5-Phe) / (Res (D5-Phe) + Res (Phe))
Res (D5-Phe) = relative peak intensity of D5-Phe relative to IC-Phe Res (Phe) = relative peak intensity of Phe relative to IC-Phe

(6) Calculation of skeletal muscle synthesis rate (FSR) Based on the enrichment of plantar muscle protein and free Phe (Ring-D5) calculated by LC / MS / MS, FSR) was calculated (A. Kanda et al., Br J Nutr, Feb. 7, 1-7, 2013).

FSR (% / day) = (Eb × 100) / (Ea × T)
Ea: Enrichment of phenylalanine present in free state in skeletal muscle (= enrichment in sample for quantification of muscle supernatant)
Eb: Enrichment of phenylalanine present in skeletal muscle assimilated to protein (= enrichment in muscle hydrolyzate quantification sample)
T: Time from the administration of Phe (Ring-D5) via the tail vein until the cryopreservation of the extracted skeletal muscle (days)
Enrichment = Phe (Ring-D5) / (Phe + Phe (Ring-D5))

<Statistical analysis method>
Statistical methods performed hierarchical analysis. That is, two-way analysis of variance using skim milk, control fermented milk, test fermented milk group (Food), post-administration time (Time: not repeated measurement), and Food × Time was performed, and the main effect of Food or Food was performed. When the interaction of xTime was significant, pairwise comparison between groups was performed by the following procedure. First, when the main effect of Food was significant and the interaction of Food × Time was not significant, a variance analysis model of Food and Time was reconstructed to perform pair comparison. When the interaction of Food × Time was significant, a simple main effect test was performed, and a pair comparison was performed at the time when a significant difference was observed. Tukey-Kramer multiple comparison was performed for the pair comparison, and a case where the P value was less than 0.05 was regarded as significant. Furthermore, in order to examine at which Time point each group had a difference from the case without administration (baseline), a Dunnett test for each of the non-administration group, skim milk, control fermented milk and test fermented milk groups was performed. Carried out. JMP11 (SAS Institute Inc.) was used for statistical analysis.

Fig. 2 shows the results of changes in the concentration of amino acids in portal blood. According to FIG. 2, in the case of the test fermented milk group (concentrated fermented milk of the present invention), the control fermented milk group has significantly more amino acid absorption than the skim milk group at 30 minutes after administration, and the light liquid is removed. Further, it was found that the amount of amino acid absorption at 30 minutes was increased as compared with the non-fat milk group and the control fermented milk group. That is, the concentrated fermented milk of the present invention has not only a non-concentrated fermented milk (control fermented milk group) but also a non-concentrated fermented milk (control fermented milk group) as well as a raw material skim milk (a non-condensed milk). It turned out to be suitable for raising.

結果 The results of plantar muscle FSR are shown in FIG. According to FIG. 3, the control fermented milk group and the test fermented milk group increase the FSR of plantar muscles at an early point after administration. Furthermore, in the case of the test fermented milk from which the light liquid has been removed, not only the timing at which the FSR increases but also the time during which the FSR increases (e.g., significantly higher than without administration even after 240 minutes) as well as the control fermentation milk as well as the non-fat milk group. It became clear that the FSR was significantly higher than that of the milk group. That is, after fermentation, the concentrated fermented milk of the present invention that has undergone a concentration step (for example, a centrifugation step) has a uniform protein content, compared with skim milk as a raw material and ordinary non-concentrated fermented milk. Thus, it was found that the composition was excellent from the viewpoint of promoting muscle synthesis, particularly skeletal muscle synthesis.

Example 3 Human Protein Absorbing Effect Evaluation Test Fermented milk was prepared by using the following procedure so that the protein concentration was about 10.2% by mass using defatted concentrated milk and milk protein as raw materials (fermented milk mix). Was used.
Test fermented milk: A raw material (fermented milk mix) was mixed with water so as to have a protein content of about 5.1% by mass to prepare a prepared fermented milk mix (non-fat milk solid content: 11.2% by mass). . After the preparation, the mixture was sterilized at a temperature of 95 ° C. and fermented with a fermentation starter (Lactobacillus delbrueckii subsp. Streptococcus thermophilus) OLS3290 strain (combination of accession number: FERM BP-19638) was added at 3% and fermented at 42 ° C. After cooling, the mixture was concentrated to a protein concentration of 10.2% by mass (approximately twice as much as that of the prepared fermented milk mix) by centrifugation (manufactured by HITACHI).

As a comparative control, the same fermented milk mix (non-fat milk solid content: 11.2% by mass) that had been used as a raw material of the test fermented milk and that had not been subjected to a fermentation or concentration step after sterilization was used.

The test fermented milk and the prepared fermented milk mix described in Table 2 below were mixed with 12 males aged from 20 to 30 and having a BMI of 18.5 to 25.0 kg / m ² (average age: 23.6). Aged, average BMI: 20.6).

The study design was performed by a randomized crossover test. Specifically, subjects are randomly divided into two groups (Group A and Group B), Group A is ingested with the test fermented milk, and Group B is ingested with the comparative control prepared fermented milk mix. After the ingestion, a washout period of 7 days is provided, after which the group A is ingested with the prepared fermented milk mix of the control and the group B is ingested with the test fermented milk. The washout period was set to 7 days as a period sufficient for eliminating the influence of the test fermented milk or the prepared fermented milk mix taken in advance. During the washout period, you will live a normal life, and your diet will prohibit excessive dietary and overeating that greatly deviates from your daily range. The use and consumption of inhibitors, digestive enzyme preparations, quasi-drugs (gastric medicine, digestive medicines), supplements containing proteins and amino acids, and health foods were banned.

The test was performed before (0 minutes) and after (15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, and 120 minutes after ingestion of the test fermented milk or the comparative control prepared fermented milk mix. After 180 minutes), blood was collected from the arm vein and analyzed for the following primary and secondary endpoints. The measurement of the blood amino acid concentration was performed as follows.

The amino acid concentration in blood was measured by a high performance liquid chromatography mass spectrometer (LC / MS) (model: LC20 series LC-MS2020, manufactured by Shimadzu Corporation). The plasma was gradually protein-treated and used as a measurement sample. According to the following reaction formula, the mixture was heated and mixed with a reaction reagent (APDS: 3-aminopyridyl-N-hydroxysuccinimidyl Carbamate) inside the HPLC to induce a free amino acid as a target component. The derivatized sample was separated by an ODS column (Inertosyl ODS-3 2.1 × 100 mm (GL Science)), and the measurement was performed by detecting the mass-to-charge ratio of each amino acid by MS. As measurement reagents, an automatic amino acid analysis for APDS Tagwako, an eluent for APDS Tagwako, a borate buffer for APDS Tagwako, and acetonitrile (for LC / MS) were used (all Fujifilm Wako Pure Chemical Industries, Ltd.) Made).

(1) Main evaluation items AUC (area under the curve of blood amino acid change concentration): The area under the curve of blood amino acid change concentration from before (0 minute) to 180 minutes after eating was calculated.
Here, the amino acid change concentration in blood is the blood amino acid concentration collected before ingestion (0 minute) and the blood amino acid concentration after ingestion (15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes). After and 180 minutes later), and the difference was determined as the blood amino acid change concentration. The blood amino acid change concentration and the maximum blood amino acid change concentration of the following secondary evaluation items were similarly calculated.
(2) Secondary evaluation items The blood amino acid change concentration and its Cmax (every 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, and 180 minutes) after each lapsed time. The highest blood amino acid change concentration) was calculated.

4) The results of the above-mentioned main evaluation items and secondary evaluation items are shown in FIGS. From these results, the intake of the concentrated fermented milk (test fermented milk) produced by the production method of the present invention was compared with the case of ingesting the “prepared fermented milk mix” containing the same amount of the same milk protein (comparative control). Thus, it was found that the blood total amino acid concentration, essential amino acid concentration, BCAA concentration and leucine concentration were further increased in young men (see the results of Cmax in FIGS. 6, 9, 12, and 15). In addition, the AUC between BCAA and leucine before feeding (0 minute) to 180 minutes after eating has a significantly higher value in the fermented milk of the present invention (test fermented milk) than in the prepared fermented milk mix. (See the AUC results in FIGS. 11 and 14). AUC tended to be high for essential amino acids between before (0 min) and 180 min after eating (see AUC results in FIG. 8).

From the above results, the concentrated fermented milk (test fermented milk) produced by the production method of the present invention has an increased amount of amino acids in the blood as compared to the comparative fermented milk mix that has not undergone the fermentation or concentration step. Was found to be able to promote the increase in blood amino acid concentration. Therefore, the concentrated fermented milk produced by the production method of the present invention is considered to contribute to the promotion of muscle synthesis, particularly skeletal muscle synthesis, because it is "easy to be absorbed" in humans.

NITE BP-02411
FERM BP-19638

Claims

A method for producing concentrated fermented milk by fermenting a prepared fermented milk mix having a nonfat milk solid content of 1 to 30% by mass in the presence of lactic acid bacteria to obtain fermented milk and concentrating the fermented milk. .
A method for producing a concentrated fermented milk, comprising the following steps:
(1) a step of preparing a fermented milk mix to prepare a prepared fermented milk mix, wherein the non-fat milk solid content in the prepared prepared fermented milk mix is 1 to 30% by mass;
(2) subjecting the prepared fermented milk mix to a sterilization treatment;
(3) a step of adding a lactic acid bacterium starter to the prepared fermented milk mix after the sterilization treatment and fermenting; and (4) a step of concentrating the fermented milk after the fermentation.
The method according to claim 1 or 2, wherein the mass of the protein in the prepared fermented milk mix is 0.5 to 11% by mass.
The method according to any one of claims 1 to 3, wherein the lactic acid bacterium or the lactic acid bacterium starter is Lactobacillus delbrueckii subsp. Bulgaricus and / or Streptococcus thermophilus.
The method according to any one of claims 1 to 4, wherein the step of concentrating the fermented milk is a step of concentrating the fermented milk by centrifugation.
(6) The production method according to any one of (1) to (5), wherein the produced concentrated fermented milk is for promoting muscle synthesis.
濃縮 Concentrated fermented milk produced by the production method according to any one of claims 1 to 6.
A concentrated fermented milk for promoting muscle synthesis, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
The concentrated fermented milk for promoting muscle synthesis according to claim 8, wherein the ratio of the branched-chain amino acid to the total amount of protein is further 0.1% by mass or less.
食品 A food for promoting muscle synthesis, comprising the concentrated fermented milk according to claim 8 or 9.
A concentrated fermented milk for increasing the amount of amino acids in blood, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
A concentrated fermented milk for promoting an increase in the concentration of amino acids in blood, wherein the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
(5) A method for promoting muscle synthesis, comprising causing a subject to ingest concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
A method for increasing the amount of amino acids in blood, comprising causing a subject to ingest concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
(15) The method according to (14), wherein the daily intake of the concentrated fermented milk to the subject is 50 to 600 g.
A method for promoting an increase in blood amino acid concentration, comprising causing a subject to ingest concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less.
17. The method according to claim 16, wherein the daily intake of the concentrated fermented milk to the subject is 50 to 600 g.
(4) Use of concentrated fermented milk having a ratio of free essential amino acids of 0.2% by mass or less to the total amount of protein for producing concentrated fermented milk for promoting muscle synthesis.
(4) Use of concentrated fermented milk in which the ratio of free essential amino acids to total protein is 0.2% by mass or less for producing concentrated fermented milk for increasing the amount of amino acids in blood.
(4) Use of concentrated fermented milk in which the ratio of free essential amino acids to the total amount of protein is 0.2% by mass or less for producing concentrated fermented milk for promoting an increase in blood amino acid concentration.