JP2001120179A - Method for production of gaba-containing fermented milk - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain fermented milk having good flavor and containing a large amount of γ-amino butyric acid(GABA). SOLUTION: This method for producing GABA-containing fermented milk is to process a milk product with a protease, subsequently inoculate lactic bacteria and/or bacteria belonging to the genus Bifidobacterium having glutamic acid decarboxylase-producing performance and culture the milk product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to γ-aminobutyric acid (G
The present invention relates to a method for producing fermented milk containing ABA) and a GABA-containing fermented milk obtained by the method.

[0002]

2. Description of the Related Art In mammals, GABA is an inhibitory neurotransmitter present in the brain and spine. Its physiological effect is to increase the blood flow of the brain and increase the oxygen supply to increase the amount of oxygen. Increases the metabolic function of blood vessels, acts on the vasomotor center of the spine to lower blood pressure, suppresses the secretion of vasopressin, an antidiuretic hormone, dilates blood vessels and lowers blood pressure, necessary for the introduction of TCA cycle Involved in enhancing the activity of hexokinase and promoting glucose metabolism (Eiichi Otomo, a novel brain metabolic activator, edited by Pharmaceutical Journal (1987)), and has been used as a drug for the purpose of activating brain metabolism. I have.

[0003] Therefore, since ingestion of GABA as a food can be expected to improve hypertension and to stabilize the psychiatric effect, GABA can be obtained by applying an appropriate treatment to the food.
Several foods with enhanced BA content have also been proposed. For example, GABA-enriched rice germ (Oriza Yuka Co., Ltd.) (a food product obtained by increasing the GABA content to 0.3% by anaerobic treatment of rice germ); The GABA content is increased by several tens of times to 220 before the treatment.
mg) is known.

On the other hand, as a method for producing GABA-enriched foods using microorganisms, glutamic acid (salt) is added to milk, lactic acid bacteria capable of producing GABA are inoculated and cultured, and fermentation containing GABA is performed. A method for obtaining food (Japanese Patent Application Laid-Open No. 7-227245), and a method in which yeast or chlorella cell lysate is allowed to act on glutamic acid (sodium).
Method for producing food material enriched in BA
No. 38850). Further, JP-A-10-295
No. 394 discloses a method for producing γ-aminobutyric acid by the decarboxylase action of lactic acid bacteria in fermented milk products. However, even with these methods, a satisfactory amount of GABA could not be obtained unless glutamic acid was added. As described above, in the conventional methods for producing GABA-containing foods and drinks using microorganisms, satisfactory effects could not be obtained unless glutamic acid or a salt thereof was added.

[0005] However, the addition of glutamic acid changes the taste of food, and if heat is applied during sterilization, etc., glutamic acid and reducing sugar (sugar) cause an aminocarbonyl reaction and discolor, resulting in poor workability. There were problems such as an increase in manufacturing cost. Glutamic acid has low solubility, so when it is added to liquid fermented milk, workability is usually improved by using sodium glutamate, but in this case sodium remains, and there is a concern that sodium may increase blood pressure. It will be. Further, when glutamic acid is used as a food additive in a product, it is disadvantageous that it gives an unfavorable image to consumers in recent years.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for producing a GABA-containing fermented milk having good flavor and color tone without adding glutamic acid.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, lactic acid bacteria and / or glutamic acid decarboxylase-producing lactic acid and / or glutamic acid decarboxylase-producing ability are obtained after a dairy product as a raw material is treated with protease. It has been found that fermented milk containing GABA abundantly, having good flavor and color tone, and having an increased number of bacteria can be obtained by inoculating and cultivating bacteria of the genus Bifidobacterium, thereby completing the present invention.

[0008] That is, the present invention provides a method for producing GABA-containing fermented milk, which comprises subjecting a dairy product to protease treatment, inoculating with a lactic acid bacterium and / or a Bifidobacterium bacterium capable of producing glutamate decarboxylase, and culturing the resultant. It is intended to provide a manufacturing method.

[0009] The present invention also relates to a method for treating a dairy product with a protease, treating the dairy product with glutaminase simultaneously with or after the protease treatment, and then producing a lactic acid bacterium and / or a bacterium belonging to the genus Bifidobacterium having glutamate decarboxylase-producing ability. A method for producing a GABA-containing fermented milk characterized by inoculating and culturing. Furthermore, the present invention provides a GABA-containing fermented milk obtained by the above method.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The fermented milk of the present invention is a solution obtained by culturing a lactic acid bacterium and / or a bacterium belonging to the genus Bifidobacterium or a sterilized product thereof in a dairy product as it is or in a solution diluted as necessary. This includes fermented milk specified by a ministerial ordinance such as milk, beverages containing live bacteria such as dairy lactic acid bacteria beverages, and milky beverages obtained by sterilizing them. Here, the dairy product refers to a product containing milk protein, and specifically refers to raw milk such as cow's milk and goat's milk, skim milk powder, whole milk powder, and fresh cream.

In the present invention, first, dairy products are treated with protease to release various peptides and amino acids derived from milk proteins. The dairy product to be used is not particularly limited, but it is preferable to use skim milk powder or whole fat milk powder derived from cows. The amount of the dairy product to be used is not particularly limited, and may be determined in consideration of the form of the final product, adaptation to the ministerial ordinance on the specification of the milk and the components of the dairy product, and the like. The content is preferably 3 to 25% by weight, particularly 4 to 10% by weight in view of taste and physical properties as a final product and standards of ministerial ordinances such as milk. In addition, in this specification,% shows mass% (weight%) except the GABA conversion rate mentioned later.

As the protease used in the treatment, those derived from microorganisms, plants and animals can be suitably used. Specifically, exoproteases such as aminopeptidase which releases amino acids one by one from the N-terminus of a peptide chain, carboxypeptidase which is released from the C-terminus of peptides, and serine proteases and thiol proteases which decompose from the middle of a peptide binding chain And endoproteases such as metalloproteases. These can be used alone or in combination of two or more. Among them, when exoprotease and endoprotease are used in combination, the GABA content in the fermented milk is increased, the survival of lactic acid bacteria is improved, and the flavor is improved. Therefore, it is preferable. The amount of the enzyme used is about 10,000 U / g as the titer, which is 0.5% based on the milk protein as the total amount of the enzyme protein.
-3.0%, especially 0.5% -1.0% is preferable from the viewpoint of the GABA amount in the final product, adjustment cost and the like. Furthermore,
It is preferable that the ratio of both exoproteases be used at a higher titer than the endoprotease in terms of the amount of glutamic acid released.If the titers are similar, the ratio of exoprotease to endoprotease is determined as the amount of enzyme protein. Is preferably 7: 3 to 5: 5, particularly preferably 6: 4 to 5: 5, from the viewpoint of GABA production. Therefore, when the protease is a mixture of an exoprotease and an endoprotease, it is preferable that 50% or more of the protease is an exoprotease.

In the production method of the present invention, it is not preferable to use a pH adjuster when treating the protease. Therefore, since milk is neutral, the protease is preferably a neutral protease,
In order to increase the efficiency of conversion to GABA by the action of lactic acid bacteria, which is the next step, it is preferable to perform treatment in a neutral region.

Examples of such (neutral) proteases for food processing include actinase (manufactured by Kaken Pharmaceutical Co., Ltd.), equine enzyme (manufactured by Amano Pharmaceutical Co., Ltd.), and protease "Amano".
G (manufactured by Amano Pharmaceutical Co., Ltd.), flavorzyme (Novolordix Bio-Industry), punchase NP-2
(Manufactured by Yakult Pharmaceutical Co., Ltd.). Further GA
In order to prepare fermented milk having a high BA content, it is preferred that the dairy product is treated with protease and the dairy product is treated with glutaminase simultaneously with or after the protease treatment. Glutaminase converts glutan released by protease treatment into glutamic acid, which is a precursor of GABA. Examples of such glutaminase for food processing include glutaminase F “Amano” 100, glutaminase (manufactured by Daiwa Kasei Co., Ltd.), and glutaminase 100FG (manufactured by Nagase Seikagaku Corporation). Among these, glutaminase "Amano" 100 is preferable because it most efficiently converts glutamine to glutamic acid.

[0015] Enzyme treatment conditions are as described above from the viewpoint of GABA content in the final product, fermented milk product, flavor of the final product, preparation cost, and a series of workability.
Use 3-25% dairy products, with 0.1% milk protein.
Using 5% to 3.0% enzyme protein, 30 to 70 ° C,
Preferably, the treatment is performed for 12 to 24 hours without adjusting the pH. Thereafter, heating at 80 to 100 ° C. for about 3 to 20 minutes to deactivate the enzyme is the most efficient treatment condition. There is no problem even if the preparation is performed outside the range of such conditions, and the amount of glutamic acid released may be adjusted by increasing or decreasing the concentration of the dairy product or the amount of the enzyme. The dairy product after the enzyme treatment thus obtained may be used alone, but is preferably mixed with other dairy products and then subjected to the subsequent fermentation step. The amount used at that time may be appropriately determined according to the GABA amount set in the final product in consideration of the GABA conversion rate of lactic acid bacteria used in the subsequent step, but in the present invention, the amount is adjusted under the above conditions. It is preferable to use the processed dairy product in an amount of 5 to 50%, particularly about 10 to 20% of the whole dairy product, from the viewpoint of flavor, production cost and the like. For example, when dairy products are treated under the above conditions using flavorzymes, about 16% of glutamic acid contained in milk protein is released, and fermented dairy products manufactured using 10% of the total dairy products are The GABA content is high and the flavor is good.

The protease-treated product is inoculated with a lactic acid bacterium and / or a bacterium belonging to the genus Bifidobacterium having the ability to produce glutamate decarboxylase, and cultured.
It can be a GABA-containing fermented milk.

The lactic acid bacterium or Bifidobacterium bacterium capable of producing glutamate decarboxylase used herein is not particularly limited as long as it is a glutamic acid decarboxylase-producing bacterium that can be added to food.
Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbruchy subspecies. Lactobacillus bacteria such as Bulgaricus, Lactobacillus helveticus, Lactobacillus brevis; Streptococcus bacteria such as Streptococcus thermophilus; Lactococcus bacteria such as Lactococcus lactis; Leuconostoc species such as Leuconostoc mesenteritees Bacteria; Enterococcus
Enterococcus bacterium such as B. faecalis; Bifidobacterium genus bacteria such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium addressacetis and the like can be preferably used. . Among them, Lactococcus lactis, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus brevis, Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium -When using one or two or more selected from Longum or Bifidobacterium addresscentis, excellent GABA production and lactic acid bacteria growth ability are obtained,
Lactococcus lactis and Lactobacillus casei are preferred because the flavor of the fermented milk is also improved. Glutamate decarboxylase-producing ability of a lactic acid bacterium or Bifidobacterium bacterium was determined by GABA when cultured at 25 to 40 ° C. for 48 hours in a medium containing 120 mg / 100 ml of glutamic acid or a salt thereof.
The conversion is preferably 20% or more, particularly 70%
Those having the ability to produce above are preferred. If it is less than 20%, to produce GABA to the extent that a physiological effect can be obtained, the cultivation time will be long and the working efficiency will be reduced, and the acid production during that time may deteriorate the flavor of the fermented milk. It is. Since the amount of GABA when 1 g of glutamic acid is converted to 100% is about 0.71 g, the GABA conversion rate referred to in this specification is 1
It refers to a relative value based on 00%. A strain having such a conversion rate includes, for example, Lactococcus lactis YIT2027.

One or two selected from lactic acid bacteria or bacteria of the genus Bifidobacterium having the ability to produce glutamate decarboxylase are added to the protease-treated dairy product.
When inoculating and cultivating more than one species, culture conditions suitable for the used bacterium may be appropriately selected. In general, culture may be performed at about 30 to 37 ° C. for about one day to one week. More specifically, for example, Lactococcus lactis may be cultured at about 30 ° C. to 37 ° C. and at a pH of about 4.3 to 4.5 after inoculation of a dairy product, and for bacteria of the genus Bifidobacterium, At about 37 ° C. for about 20 to 22 hours, pH 4.5
What is necessary is just to culture to about 4.7. As a culture method, a method suitable for culture of microorganisms such as stationary culture, stirring culture, shaking culture, and aeration culture may be appropriately selected and used.

By culturing such bacteria, in a dairy product which has been treated with protease, in addition to directly converting the released glutamic acid to GABA, various free peptides and amino acids (excluding glutamic acid) are also indirectly converted. It is considered that it is converted to GABA.

The thus obtained fermented milk of the present invention may contain other food materials, that is, various sugars, emulsifiers, thickeners, sweeteners, acidulants, fruit juices and the like. Specifically, sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose and xylose; sorbitol, xylitol, erythritol, lactitol, palatinit, sugar alcohols such as reduced syrup, reduced maltose syrup; sucrose fatty acid esters Emulsifiers such as glycerin fatty acid esters and lecithin; thickening (stabilizing) agents such as agar, gelatin, carrageenan, guar gum, xanthan gum, pectin, locust bean gum; yogurt, berry, orange, quince, perilla,
Flavors such as citrus, apple, mint, grape, apricot, pair, custard cream, peach, melon, banana, tropical, herb, tea, coffee, etc. In addition, even if various vitamins such as vitamin A, vitamin B, vitamin C and vitamin E and minerals such as calcium, iron, manganese and zinc are blended, fermented milk with excellent flavor can be obtained. it can.

The fermented milk product of the present invention may be used to produce a fermented milk product by a conventional method. For example, first, fermented milk is obtained as described above. Next, a syrup solution containing various sweetening ingredients may be added and mixed, and a flavor may be added to finish the final product. Alternatively, various thickeners such as agar and gelatin may be prepared in a syrup solution or separately as a thickener, and added to obtain a solid fermented milk.

The fermented milk of the present invention can be made into any type of product such as plain type, flavored type, fruit type, sweet type, etc. Also, plain type, soft type, drink type, solid ( Any type of product, such as a hard type or a frozen type, can be used. Further, after the fermentation is completed, the fermented milk may be subjected to a sterilization treatment at a stage such as before mixing with the syrup liquid, to obtain a product containing dead bacteria.

[0023]

The present invention provides a lactic acid bacterium or a bifidobacterium having a glutamate decarboxylase-producing ability by releasing various peptides and glutamic acid from milk protein by subjecting a dairy product to protease treatment. GABA by the action of genus bacteria
Is converted to By performing such a treatment, it is not necessary to add glutamic acid as a food additive, so that the taste and color tone do not change and fermented milk having a high GABA content can be obtained at low cost. In addition, the peptides produced by the protease treatment are considered to be diverse, including those that are undesirable in flavor such as bitterness, but in fact, protease-treated dairy products are fermented with lactic acid bacteria. By doing so, the slightly present bitterness and astringency disappear and fermented milk having a round and good flavor can be obtained.

[0024]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
“%” Indicates “% by weight”.

Example 1 To a 30% skim milk solution was added 1% of an enzyme protein based on milk protein, and the mixture was kept at 50 ° C. for 24 hours. Using a 20% nonfat dry milk solution to which 10% of this protease-treated solution was added as a medium, heat sterilization was performed, Lactococcus lactis YIT2027 was inoculated, cultured at 30 ° C. for 72 hours, and the GABA content in the fermented milk was measured. For the measurement of GABA and glutamic acid, an automatic amino acid analyzer (manufactured by JEOL Ltd.) was used. As the enzyme, a neutral protease in which two kinds of enzymes, exoprotease and endoprotease, were combined was used. Specifically, flavorzyme (manufactured by Novo Nordisk Bio-Industry) or equine enzyme (manufactured by Amano Pharmaceutical) having a high ratio of exoprotease, and actinase A having a high ratio of endoprotease are used.
S (manufactured by Kaken Pharmaceutical Co., Ltd.) and protease A "Amano" G (manufactured by Amano Pharmaceutical Co., Ltd.) were used.

[0026]

[Table 1]

[0027] As a result, all of those treated with the enzyme showed a high GABA content (Table 1). Also, under the above conditions,
When endoprotease (manufactured by Sigma) and exoprotease (manufactured by Sigma) were used alone as enzymes, the amount of GABA in the fermented milk was larger than that of untreated but smaller than that of the combination.

Example 2 To a 30% skim milk solution, a protease having an enzyme protein content of 0.1% or 1% relative to milk protein was added, and further, 1/5 of the protease was added to 0.02% or 0.1%. 2%
Was added and incubated at 50 ° C. for 48 hours. Protease used was equine enzyme (manufactured by Amano Pharmaceutical), protease A "Amano" G (manufactured by Amano Pharmaceutical) or flavorzyme (manufactured by Novo Nordisk Bioindustry), and glutaminase was glutaminase F Amano 100 (manufactured by Amano Pharmaceutical). Manufactured). Solution treated with this protease and glutaminase is 10%
Using the added 20% skim milk solution as a medium, the mixture was sterilized by heating, inoculated with Lactococcus lactis YIT2027, cultured at 30 ° C. for 72 hours, and the GABA content in the fermented milk was measured. Table 2 shows the results.

[0029]

[Table 2]

As a result, by using milk in which glutaminase was used in combination with protease, GAB was further improved.
It was found that the A concentration increased.

Example 3 1% flavorzyme and 0.2% glutaminase F "Amano" 100 are added to a 30% -concentrated skim milk solution with respect to protein in terms of enzyme protein, and a pH adjuster is added. The reaction was continued at 50 ° C. for 24 hours. 90 ° C
For 5 minutes to inactivate the enzyme,
10% was added to the skim milk solution. This substrate is 120 mg /
It contained 100 ml of glutamic acid and no GABA. The substrate was inoculated with various lactic acid bacteria shown in Table 3 and 3
The fermented milk was prepared by culturing at 0 to 37 ° C for 72 hours. GABA content in the obtained fermented milk and GABA of lactic acid bacteria used
The conversion was measured. After homogenizing these fermented milks, they were mixed with a 30% syrup solution and filled in a glass bottle to produce a fermented milk product. Specialized panelists 5
Sensory evaluation was performed by name, and flavors were compared. In addition, the index | index of a flavor evaluation is as follows. Table 3 shows the results. +2: good taste, +1: good taste, 0: normal,
-1: slightly bad taste, -2: bad taste

[0032]

[Table 3]

As a result, Lactococcus lactis Y
When IT2027 and Lactobacillus casei C-3 were used, both GABA production ability and flavor were good, and Lactococcus lactis YIT2027 was particularly good.

Example 4 Fermented milk was produced by the following three methods. Example 1; 30% -concentrated skim milk solution, 1% flavorzyme and 0.2% enzyme as protein relative to protein
Was added and the reaction was continued at 50 ° C. for 24 hours without adding a pH adjuster. The enzyme was inactivated by heating at 90 ° C. for 5 minutes, and this treated solution was added to a 20% skim milk solution by 10%. The glutamic acid concentration at this time was 120 mg / 100 ml. 12
After heat sterilization at 1 ° C. for 3 seconds, 1% of Lactococcus lactis YIT2027 was inoculated and cultured at 30 ° C. for 72 hours. Comparative product 1: 20% skim milk solution was added with sodium glutamate in an amount of 120 mg / 100 ml as glutamate, sterilized by heating, inoculated with 1% Lactococcus lactis YIT2027, and cultured at 30 ° C. for 72 hours. . Comparative product 2: 20% skim milk solution was heat-sterilized, and then inoculated with 1% Lactococcus lactis YIT2027.
C. for 72 hours. The GABA content, flavor, and viable cell count of each of the fermented milks thus obtained were compared. The GABA content and the viable cell count were measured in the same manner as in Example 1. Table 4 shows the results.

[0035]

[Table 4]

The GABA content of the actual product was at least 10 times that of the comparative product 2, and the number of viable bacteria important as probiotics was 3 times that of the comparative product 1. However, the flavor was mellow and good.

Example 5 7 kg of fermented milk prepared as Example 1 of Example 4 was mixed with 150 kg of fermented milk.
After homogenization at kg / cm ² , the mixture was mixed with 3 kg of a solution containing 10% sucrose and 0.02% vitamin C, which were separately heat-sterilized, and 0.1% of yogurt flavor (Yakult Material Co., Ltd.) was added. This was filled in a polystyrene solution to produce a drink-type fermented milk product. This fermented milk product has a mellow and good flavor,
No sedimentation, separation, etc. were observed even after standing for a week, stable physical properties were maintained, and the GABA content did not decrease.

Example 6 To a solution of skim milk powder having a concentration of 30% was added 1.0% of equine enzyme and 0.2% of glutaminase F "Amano" 100 as an enzyme protein based on protein, and a pH adjuster was added. The reaction was continued at 45 ° C. for 24 hours without any treatment. 80 ℃
For 10 minutes to inactivate the enzyme.
10% to the 10% skim milk solution. The glutamic acid concentration at this time was 110 mg / 100 ml. 3 at 121 ° C
After heat sterilization for 2 seconds, Lactobacillus casei C-3 was added to 0.
5% was inoculated and cultured at 37 ° C. for 72 hours. After 7 kg of the fermented milk thus obtained was homogenized at 150 kg / cm ² , it was mixed with 3 kg of a 10% glucose solution which had been separately sterilized by heating, and an agar solution (agar 1.5%) was added thereto, followed by vanilla flavor (Yakult Material). (Manufactured by Co., Ltd.) was added to a paper container. The container was covered and solidified at a low temperature to produce a hard yogurt type fermented milk product. This fermented milk product has a mellow and good flavor,
No sedimentation, separation, etc. were observed even after standing for a week, and stable physical properties were maintained. In addition, the growth and survival of the bacteria were good without reducing the GABA content.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Keisuke Matsumoto 1-1-1 Higashi-Shimbashi, Minato-ku, Tokyo F-term (reference) 4B001 AC06 AC26 AC30 AC31 BC14 EC05

Claims (5)

[Claims] 1. A method for producing fermented milk containing γ-aminobutyric acid, which comprises subjecting a dairy product to protease treatment and inoculating a lactic acid bacterium and / or a bacterium belonging to the genus Bifidobacterium having a glutamate decarboxylase-producing ability. Method. 2. A dairy product is treated with a protease, the dairy product is treated with glutaminase simultaneously with or after the protease treatment, and then inoculated with a lactic acid bacterium and / or a Bifidobacterium bacterium capable of producing glutamate decarboxylase. A method for producing fermented milk containing γ-aminobutyric acid, which comprises culturing. 3. A lactic acid bacterium and / or a bacterium belonging to the genus Bifidobacterium having a glutamate decarboxylase-producing ability, wherein Lactococcus lactis, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus brevis, It is one or more kinds selected from Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium addressacetis. 3. The production method according to 1 or 2. 4. The protease is a mixture of an exoprotease and an endoprotease, wherein 5
4. The method according to claim 1, wherein 0% or more is exoprotease. 5. A γ-aminobutyric acid-containing fermented milk obtained by the production method according to any one of claims 1 to 4.