TWI551684B - Swiss lactic acid bacteria with high proteolytic activity - Google Patents

Description

Swiss lactic acid bacteria with high protein decomposition activity

The present invention relates to a novel lactic acid bacterium belonging to the Swiss lactic acid bacteria. Further, the present invention relates to a fermented milk obtained by fermenting the lactic acid bacterium, a composition containing the lactic acid bacterium, and a food or drink. Further, the present invention relates to a process for producing an amino acid and/or a peptide using the lactic acid bacteria.

It is known that there are potentially functional peptides in milk proteins or food proteins, and attempts are being made to utilize milk proteins or food proteins by utilizing enzyme decomposition or fermentation. For example, as such a functional peptide, a tripeptide VPP (Valine-Proline-Proline, lysine-proline-proline) and IPP (Isoleucine-Proline-Proline, iso-aracine-proline-- The proline acid has an inhibitory action by angiotensin converting enzyme (ACE) and exhibits a blood pressure lowering action (Patent Documents 1 to 4 and Non-Patent Document 1).

As a functional peptide derived from milk other than VPP and IPP, it has been reported that YP as a dipeptide has a blood pressure lowering action and an anxiolytic effect (Patent Document 5). In addition, a peptide group having ACE inhibitory activity, such as IP, AP, EP, RP, QP, TP, MP, and GP, which are dipeptides, is reported (for example, Patent Document 6).

On the other hand, it has been known that L. helveticus has been used in the manufacture of dairy products from the beginning, and the protein decomposition activity is high. In order to provide a lyophilized milk that is easier to ingest and easy to process, L. helveticus strains having high protein decomposition activity and excellent peptide production properties have been reported (Patent Documents 1 to 4 and 7, Non-Patent Document 1).

Since the protein is composed of an amino acid, it can be further decomposed to become a supply source of an amino acid. Amino acids are known to have various functions in addition to nutrition and flavor. For example, it is known that Branched Chain Amino Acid (BCAA) is contained in proteins constituting muscles, and BCAA in muscles such as exercise is consumed as energy, and therefore supplementation of BCAA is important. Further, alanine (Ala) is a glycogen-based amino acid which is known as an energy source of the liver. Since the NADH (reduced nicotinamide adenine dinucleotide) produced by the decomposition of alcohol is consumed, it is considered that the decomposition reaction in the liver is carried out due to the presence of Ala. Further, it is also known that Ala is delicious in terms of taste, and has a masking effect such as salting, vinegar pickling or irritating taste. It is generally considered that the amino acid which is the most easily deficient among the amino acid (Lys) is an essential amino acid. It must be taken in the form of antibodies or raw materials such as hormones and enzymes. Also, it is reported that Lys is also involved in calcium absorption, collagen formation, and bone tissue production. Serine is important as a precursor to various important substances in living organisms such as phospholipid serine or other amino acids. Further, it is known that it lowers blood cholesterol, prevents brain cell death by inhibiting brain cell death such as activation of neurons in the brain, or becomes a moisturizing component of the skin.

The use of microorganisms is actively carried out for the production of such amino acids. However, there are not many examples of amino acids which can be taken as a fermentation product of microorganisms. In the case of the lactic acid bacteria, the lactic acid bacteria excellent in the decomposition of the amino acid are selected to affect the flavor of the pickles, but it is not known to study the intake of functional amino acids and functional peptides by the lactose itself.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] European Patent No. 1016709B

[Patent Document 2] International Publication WO 01/32836

[Patent Document 3] International Publication WO 2007/096510

[Patent Document 4] European Patent No. 1820850

[Patent Document 5] European Patent No. 0821968

[Patent Document 6] European Patent No. 1661909

[Patent Document 7] Japanese Patent Laid-Open No. Hei 7-274949

[Non-patent literature]

[Non-Patent Document 1] Nakamura, Y. et al., J. Dairy Sci. Vol. 78, pp. 1253-1257, 1995

Accordingly, an object of the present invention is to provide a lactic acid bacterium which has high protein decomposition activity and is excellent in peptide-forming ability and/or amino acid-forming ability. Further, an object of the present invention is to provide a fermented milk which contains an amino acid and/or a peptide at a high concentration and which has a good flavor.

The present inventors have made an effort to solve the above problems, and as a result, the lactic acid strain belonging to the Swiss lactic acid bacteria has been successfully obtained from the lactic acid bacteria of the Swiss lactic acid bacteria, and has excellent ability to produce an amino acid and a functional peptide, and is compatible with the prior lactic acid bacteria. More strains with higher proteolytic activity. Further, the knowledge that the amino acid and the peptide having a high content of amino acids and/or peptides can be obtained by using the strain to produce an amino acid and/or a peptide is obtained, so that the present invention has been completed.

That is, the present invention includes the following.

[1] A lactic acid bacterium belonging to the lactic acid bacteria of Switzerland, characterized by having at least one of the following properties (a) to (d):

(a) producing a free amino acid of 25 μmol/ml or more by fermenting the mammal;

(b) producing a peptide of 8.5 mg/ml or more by fermenting the animal's milk;

(c) producing XP peptide and/or XPP peptide in a total amount of 260 μg/ml or more by fermenting the mammal;

(d) The amount of the IPP peptide and the VPP peptide per unit acid of the fermented milk obtained by fermenting the animal's milk at 30 ° C or more and 34 ° C or less is 6.0 mg / g or more in terms of VPP.

[2] The lactic acid bacterium according to [1], wherein more than 15% of the free amino acid is a branched amino acid.

[3] The lactic acid bacteria according to [1] or [2], wherein the free amino acid contains at least one selected from the group consisting of alanine, lysine, and serine.

[4] The lactic acid bacterium according to any one of [1] to [3] wherein a YP peptide of 60 μg/ml or more is produced.

[5] The lactic acid bacteria according to any one of [1] to [4], which is a mutant strain of the Swiss lactic acid bacteria CM4 strain (Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060).

[6] The lactic acid bacteria according to any one of [1] to [5], which is a Swiss lactic acid bacteria CP3232 strain (Taiwan registered number BCRC 910539, Japanese registered number FERM BP-11271) or a variant thereof.

[7] A lactic acid bacterium which is a Swiss lactic acid bacteria CP3232 strain (Taiwan registration number BCRC 910539, Japanese registration number FERM BP-11271).

[8] A composition for producing an amino acid and/or a peptide from a mammal, characterized in that it contains at least one lactic acid bacterium belonging to the Swiss lactic acid bacteria.

[9] The composition according to [8], which produces a free amino acid of 25 μmol/ml or more by fermenting the mammal.

[10] The composition according to [9], wherein 15% or more of the free amino acid is a branched amino acid.

[11] The composition according to [9] or [10] wherein the free amino acid contains at least one selected from the group consisting of alanine, lysine, and serine.

[12] The composition according to any one of [8] to [11] wherein a peptide of 8.5 mg/ml or more is produced by fermenting the mammal.

[13] The composition according to any one of [8] to [12] wherein a total of 260 μg/ml or more of XP peptide and/or XPP peptide is produced by fermenting the mammal.

[14] The composition according to any one of [8] to [13] wherein a YP peptide of 60 μg/ml or more is produced by fermenting the mammal.

[15] The composition according to any one of [8] to [14] wherein the amount of the IPP peptide and the VPP peptide of the acidity of the fermented milk obtained by fermenting the animal milk at 30 ° C or more and 34 ° C or less is 6.0. Above mg/g.

[16] The composition according to any one of [8] to [15] wherein the lactic acid bacteria are any one of [1] to [7].

[17] A fermented milk obtained by fermenting a mammal with at least one lactic acid bacterium belonging to the Swiss lactic acid bacterium, and comprising at least one of the following (a) to (d):

(a) a free amino acid of 25 μmol/ml or more;

(b) peptides of 8.5 mg/ml or more;

(c) XP peptide and/or XPP peptide in a total amount of 260 μg/ml or more;

(d) VPP conversion amount per unit acidity of IPP peptide and VPP peptide of 6.0 mg/g or more.

[18] The fermented milk of [17], which is a lactic acid bacteria containing at least one lactic acid bacteria belonging to Switzerland.

[19] The fermented milk of [17] or [18], which is at least one lactic acid bacteria according to any one of [1] to [7].

[20] The fermented milk of any one of [17] to [19], wherein more than 15% of the free amino acid is a branched amino acid.

[21] The fermented milk of any one of [17] to [20] wherein the free amino acid contains at least one selected from the group consisting of alanine, lysine, and serine.

[22] The fermented milk of any one of [17] to [21] which contains a YP peptide of 60 μg/ml or more.

[23] The fermented milk of any one of [17] to [22] wherein the fermentation is carried out at a temperature of from 30 ° C to 34 ° C.

[24] The fermented milk of any one of [17] to [23], which is used as a blood pressure lowering agent, a brain function improving agent, an autonomic nervous regulator, a parasympathetic regulating agent, a sympathetic regulating agent, a nutritional adjuvant. , energy supplements, muscle breakdown damage inhibitors, muscle enhancers, fatigue relievers, endurance improvers, flavoring agents or alcohol metabolizing agents.

[25] A food or drink comprising at least one lactic acid bacterium according to any one of [1] to [7] and/or a treatment thereof, and/or any one of [17] to [24] The fermented milk and/or its treatment.

[26] A method for producing an amino acid and/or a peptide, characterized by using at least one lactic acid bacterium according to any one of [1] to [7] or any one of [8] to [16] The composition ferments the animal's milk or milk protein and extracts the amino acid and/or peptide from the obtained extract.

[27] The method of [26], wherein the amino acid is a branched amino acid.

[28] The method according to [26] or [27] wherein the amino acid contains at least one selected from the group consisting of alanine, lysine, and serine.

[29] The method according to any one of [26] to [28] wherein the peptide is at least one selected from the group consisting of XP peptide and XPP peptide.

[30] The method according to any one of [26] to [29] wherein the fermentation is carried out at a temperature of 30 ° C or more and 34 ° C or less.

[31] A method for obtaining a lactic acid bacterium belonging to a Swiss lactic acid bacterium having proteolytic activity, and comprising the steps of:

(a) a step of preparing a variant strain of the Swiss lactic acid bacteria CM4 strain (Taiwan registered number CCRC 910111, Japanese registered number FERM BP-6060) or the Swiss lactic acid bacteria CP3232 strain (Taiwan registered number BCRC 910539, Japanese registered number FERM BP-11271);

(b) a step of selecting a strain having at least one of the following properties (a) to (d) from the mutant strain:

(a) producing a free amino acid of 25 μmol/ml or more by fermenting the animal's milk,

(b) Producing a peptide of 8.5 mg/ml or more by fermenting the animal's milk,

(c) Producing a total of 260 μg/ml of XP peptide and/or XPP peptide by fermenting the mammalian milk,

(d) The amount of the IPP peptide and the VPP peptide per unit acid of the fermented milk obtained by fermenting the animal's milk at 30 ° C or more and 34 ° C or less is 6.0 mg / g or more in terms of VPP.

[32] A method for producing a functional food or drink, comprising the steps of: preparing at least one lactic acid bacterium according to any one of [1] to [7] and/or a treatment thereof, and/or as in [17] [24] The step of fermenting the milk and/or the processed product thereof, and the step of preparing the lactic acid bacteria and/or the processed material thereof and/or the fermented milk and/or the processed material thereof into the food or beverage.

According to the present invention, there is provided a lactic acid bacterium belonging to a Swiss lactic acid bacterium which has high proteolytic activity and is excellent in peptide-forming ability and/or amino acid-forming ability. The amino acid and/or peptide system produced by the present lactic acid bacteria has various functions, and peptides such as IPP, VPP, and YP have blood pressure lowering effects. Further, by using the lactic acid bacteria in the fermentation, it is possible to obtain a fermented milk containing a plurality of kinds of amino acids and/or peptides having various functions at a high concentration. Since the amount of IPP and VPP peptide per unit acidity of the obtained fermented milk is high, there is no problem that the acidity caused by the lactic acid which is formed by the progress of the fermentation is high. In other words, since a fermented milk containing a large amount of amino acid and/or a peptide with respect to the amount of lactic acid produced can be produced, a small amount of fermented milk can be used, and a product having a good flavor and easy to drink in the case of maintaining functionality can be easily produced. And available to consumers.

The present invention will be described in detail below. The present application claims the priority of Japanese Patent Application No. 2010-250754, filed on Nov. 9, 2010, which is incorporated herein by reference.

The lactic acid bacteria belonging to the lactic acid bacteria (Lactobacillus helveticus) of the present invention are one type of lactic acid bacteria which produce lactic acid from sugar by fermentation, and are also referred to as "lactic acid bacteria of the present invention" in the present specification. The lactic acid bacterium of the present invention is characterized by having high protein decomposition activity and excellent in amino acid-forming ability and/or functional peptide-producing ability. Specifically, it has at least one of the following properties (a) to (d):

(a) producing a free amino acid of 25 μmol/ml or more, preferably 30 μmol/ml or more, by fermenting the mammal;

(b) producing a peptide of 8.5 mg/ml or more, preferably 10 mg/ml or more, by fermenting the mammal;

(c) producing XP peptide and/or XPP peptide in a total amount of 260 μg/ml or more, preferably 280 μg/ml or more, and more preferably 300 μg/ml or more by fermenting the mammal;

(d) The amount of the IPP peptide and the VPP peptide per unit acid of the fermented milk obtained by fermenting the animal's milk at 30 ° C or more and 34 ° C or less is 6.0 mg / g or more in terms of VPP.

The properties of the above (a) to (d) can be determined by the methods known in the art, for example, as described in the following examples. For simple explanation, the lactic acid bacteria belonging to the Swiss lactic acid bacteria can be firstly fermented for 10 to 30 hours at 30 to 45 ° C, preferably 30 to 34 ° C, using milk protein (for example, animal milk) as a substrate. The properties of the above (a) to (d) were determined by measuring the amount of the free amino acid or peptide contained in the obtained milk protein mash and the acidity.

Regarding (a), the amount of the amino acid can be measured using a well-known amino acid analyzer such as a high performance liquid chromatography. The lactic acid bacteria of the present invention are produced by the fermentation of animal milk to produce a free amino acid of 25 μmol/ml or more, preferably 30 μmol/ml or more. Further, it is preferred to form a branched amino acid of 5 μmol/ml or more, more preferably 6 μmol/ml or 7 μmol/ml or more, and more than 15%, more preferably 19, of the free amino acid formed. More than or more than 20% is a branched amino acid (proline, leucine, isoleucine). Further, the free amino acid preferably contains at least one selected from the group consisting of alanine, lysine, and serine at 2000 μg/ml or more, and more preferably at least two or more amino acids each at 1500 μg/ml.

With regard to (b), the amount of the peptide can be determined by a method known in the art, such as the OPA (O-Phthaldialdehyde, phthalaldehyde) method (Church et al., J. Dairy Sci. 1983. 66: 1219-1227). The lactic acid bacteria of the present invention produce a peptide of 8.5 mg/ml or more, preferably 10 mg/ml or more, by fermentation of animal milk.

Regarding (c), the XP peptide refers to a peptide comprising Xaa-Pro (Xaa is any amino acid), and the XP peptide contains, for example, YP (Tyr-Pro, tyrosine-proline), VP (Val-Pro) , valine-proline, IP (Ile-Pro, isoleucine-proline), AP (Ala-Pro, alanine-proline), EP (Glu-Pro, glutamic acid - Proline), RP (Arg-Pro, arginine-proline), QP (Gln-Pro, glutamine-proline), TP (Thr-Pro, threonine-proline) , MP (Met-Pro, methionine-proline) and GP (Gly-Pro, glycine-proline). Further, the XPP peptide refers to a peptide comprising Xaa-Pro-Pro (Xaa is any amino acid), and the XPP peptide contains, for example, VPP (Val-Pro-Pro, valine-proline-proline), IPP (Ile-Pro-Pro, isoleucine-proline-proline) and LPP (Leu-Pro-Pro, leucine-proline-proline). In addition, in the present specification, these XP peptides and XPP peptides are also referred to as dipeptides and tripeptides, respectively, and are also collectively referred to as "functional peptides". The amount of XP peptide and/or XPP peptide can be measured by a known LC/MS (Liquid chromatography/Mass spectrometry) apparatus. The lactic acid bacteria of the present invention produce a total of 260 μg/ml or more, preferably 280 μg/ml or more, more preferably 300 μg/ml or more of XP peptide and/or XPP peptide by fermentation of animal milk. More preferably, it is a YP peptide which is 60 μg/ml or more, more preferably 65 μg/ml or more, and still more preferably 70 μg/ml or more.

Regarding (d), the acidity (acid concentration in the fermented milk) can be measured by a known method and a titration method using, for example, a known automatic titrator. Further, the IPP peptide and the VPP peptide can be measured by the method described in (c). In the lactic acid bacteria of the present invention, the amount of the IPP peptide and the VPP peptide per unit acid of the fermented milk obtained by fermenting the animal milk at 30 ° C or more and 34 ° C or less is 5.5 mg/ml or more, preferably 6.0 mg in terms of VPP. /ml or more. Further, the above amount was calculated by setting the specific gravity of the fermented milk to 1.02. In addition, the amount of the IPP peptide and the VPP peptide described in the present specification is a VPP conversion amount based on the ACE inhibitory activity, and can be obtained by the following formula:

(VPP conversion amount μg/ml)=IPP amount (μg/ml)×1.7+VPP amount (μg/ml)

The lactic acid bacteria of the present invention has at least one of the above properties (a) to (d), but preferably one, three or all of the above-described properties are lactic acid bacteria. For example, the lactic acid bacteria of the present invention are as follows: having the properties of (a), (b), (c) or (d), or having (a) and (b), (a) and (c), (a) And (d), (b) and (c), (b) and (d), or (c) and (d), or (a) and (b) and (c), (a) and The nature of (b) and (d), (a) and (c) and (d), (b) and (c) and (d), or the nature of (a) to (d).

In the present invention, a lactic acid bacterium belonging to the Swiss lactic acid bacteria which exhibits at least one of the above properties is included in the lactic acid bacterium of the present invention. The preferred strain of the lactic acid bacterium of the present invention is the mutant strain obtained by the present inventors from the lactic acid bacteria CM4 strain (Taiwan registered number CCRC 910111, Japanese registered number FERM BP-6060, Patent Document 1). Individually selected Swiss lactic acid bacteria CP3232 strain. In addition, the Swiss lactic acid bacteria CM4 strain and the CP3232 strain are used by the applicant as a patented biological storage center of the Institute of Industrial Technology and Technology, an independent administrative agency based on the Budapest Treaty for the Collection of Patented Microorganisms (Ibaraki Prefecture, Japan) It is registered as FERM BP-6060 on August 15th, 2011 (August 15th) and August 4th (2010) in Heisei City, 1st Floor, 1st Floor, 1st Floor, Tsukuba City, 1st Floor, 305-8566) FERM BP-11271. It has been confirmed that the Swiss lactic acid bacteria CP3232 strain has all of the properties (a) to (d) described above as described in the examples. Among them, the Swiss lactic acid bacteria CM4 strain was deposited in the Food Industry Development Research Institute on September 16, 1998, the registration number is CCRC 910111; the Swiss lactic acid bacteria CP3232 strain was deposited in the Food Industry Development Institute on January 12, 2012, the registration number For BCRC 910539.

Further, the Swiss lactic acid bacteria CP3232 strain was derived from the mutant strain of the Swiss lactic acid bacterium CM4 strain as a parent strain as described above. Therefore, the mutant strains of the Swiss lactic acid bacteria CM4 strain and the Swiss lactic acid bacteria CP3232 strain are also highly likely to have any of the above properties (a) to (d). The mutant strain derived from such a Swiss lactic acid bacteria CP3232 strain or the Swiss lactic acid bacteria CM4 strain is also included in the present invention as long as it has the above properties.

In the present invention, "mutant strain" means any strain obtained from a mother strain. Specifically, it refers to a method of artificially increasing the frequency of sudden mutations by spontaneous mutation of the mother strain or by using induced mutations caused by chemical or physical mutations, or by specific sudden mutation induction techniques (eg, genetic recombination). The strain obtained. The microorganisms produced by such a method are repeatedly selected and separated, and the useful microorganism individual is cultured, whereby a mutant having a target property can be obtained.

Here, the mutant strain includes a CM4 strain, a CP3232 strain, or a strain obtained by using the mutant strain as a parent strain. That is, the mutant strain which was subjected to several variations of the CM4 strain and selected was also included in the mutant strain of the CM4 strain.

For example, the mutant strain derived from the Swiss lactic acid bacterium CM4 strain, the Swiss lactic acid bacteria CP3232 strain, and the variant thereof can be easily obtained from the molecular weight distribution of the amplified fragment of the genomic DNA of the lactic acid bacteria obtained by the polymerase chain reaction (PCR) method. The ground is distinguished from other lactic acid strains. For simplicity of explanation, a DNA reagent is prepared for a target lactic acid bacterium, and gene amplification is carried out by using a PCR method having primers having, for example, the sequences shown in SEQ ID NOs: 1 and 2, and the electrophoresis pattern of the obtained fragment is analyzed. This can be judged whether it is a mutant strain derived from the Swiss lactic acid bacteria CP3232 strain or the Swiss lactic acid bacteria CM4 strain. However, the method of confirming whether or not the mutant strain is a strain is not limited to the method, and there is also a case where the mutant strain of the lactic acid bacteria CM4 strain and the mutant strain of the Swiss lactic acid bacteria CP3232 strain are derived even when the PCR fragment is not detected. Whether or not a mutant strain can be confirmed by a method known in the art such as detection of other gene fragments or bacteriological properties.

A strain which can be used as the lactic acid bacteria of the present invention can be obtained by judging whether or not such a mutant has the properties of the above (a) to (d).

The lactic acid bacteria of the present invention can be produced by culturing under appropriate conditions using a medium which is usually used in the culture of lactic acid bacteria. The medium to be used in the culture may be any one of a natural medium and a synthetic medium, as long as it is a carbon source, a nitrogen source, an inorganic salt, or the like, and can be efficiently cultured. The strain to be used is appropriately selected from a suitable medium. As the carbon source, lactose, glucose, galactose, brown molasses or the like can be used, and as the nitrogen source, an organic nitrogen-containing substance such as a hydrolysate of casein, a whey protein hydrolyzate or a soy protein hydrolyzate can be used. Further, as the inorganic salt, phosphate, sodium, potassium, magnesium or the like can be used. Examples of the medium suitable for culturing the lactic acid bacteria include MRS liquid medium, GAM medium, BL medium, Briggs Liver Broth, animal milk, skim milk, milk whey, and the like. It is preferred to use a milk medium containing sterilized skim milk powder.

Further, the culture of the lactic acid bacteria of the present invention is carried out under anaerobic conditions at 20 ° C to 50 ° C, preferably 25 ° C to 42 ° C, more preferably 28 ° C to 37 ° C. The temperature conditions can be adjusted by a thermostatic bath, a mantle heater, a jacket, and the like. Further, under anaerobic conditions, it refers to an anoxic environment in which the lactic acid bacteria can proliferate, for example, by using an anaerobic chamber, an anaerobic tank or a closed container or bag containing a deoxidizing agent, or simply by sealing The container is cultured to produce anaerobic conditions. The culture form is static culture, shaking culture, tank culture, and the like. Also, the culture time can be set to 3 hours to 96 hours. The pH of the medium at the start of the culture is preferably maintained at 4.0 to 8.0.

A specific preparation example of the lactic acid bacteria of the present invention will be briefly described. For example, in the case of using the Swiss lactic acid bacteria CP3232 strain, the lactic acid bacteria are sterilized in a sterilized milk medium (milk medium containing 9.00% (w/w) reduced skim milk powder, etc.), and at 28%, and at 28 The culture was carried out overnight (about 18 to 28 hours) at ~37 °C. Preferably, the culturing operation is repeated.

After the culture, the obtained culture can be used as it is, or a part of purification by centrifugation or the like and/or solid-liquid separation or sterilization using filtration or the like can be carried out as needed.

Further, as long as it has a target property, the treated product of the lactic acid bacteria obtained by treating the lactic acid bacteria of the present invention may be used, and the treated product of the lactic acid bacteria may be further processed. Examples of such treatments are described below.

The fermented product can be prepared by fermenting raw milk, skim milk or soymilk using the bacteria and/or treatment of the lactic acid bacteria. For example, lactic acid bacteria or other treated lactic acid bacteria are inoculated into raw milk, skim milk, soy milk, etc., and fermentation is carried out under the conditions of lactic acid bacteria fermentation conditions (substantially the same as those of the above lactic acid bacteria culture) known in the art. The obtained fermented material can be used directly, or can be subjected to other treatments such as filtration, sterilization, dilution, concentration, and the like.

The suspension or dilution can be prepared by suspending or diluting the cells and/or treatments of the lactic acid bacteria in a suitable solvent. Examples of the solvent that can be used include water, physiological saline, and PBS (Phosphate buffered saline).

The sterilized product can be prepared by sterilizing the cells and/or the treated material of the lactic acid bacteria. When the bacteria and/or the treated material of the lactic acid bacteria are sterilized, for example, a known sterilization treatment such as filtration sterilization, radioactive sterilization, superheat sterilization, or pressurized sterilization can be performed.

Further, the heat-treated product can be prepared by heat-treating the cells and/or the treated material of the lactic acid bacteria. When the heat-treated product is prepared, the cells and/or the treated material of the lactic acid bacteria are subjected to high-temperature treatment (for example, 80 to 150 ° C) for a fixed period of time, for example, for about 10 minutes to 1 hour (for example, about 10 to 20 minutes).

The crushed material and the cell-free extract can be prepared by crushing, pulverizing or grinding the cells and/or the treated material of the lactic acid bacteria. For example, such crushing can be carried out by physical crushing (stirring, filter filtration, etc.), enzyme dissolution treatment, chemical treatment, or autolysis treatment.

The extract can be obtained by extracting the cells and/or treatments of the lactic acid bacteria using a suitable aqueous solvent or organic solvent. The extraction method is not particularly limited as long as it is an extraction method using an aqueous solvent or an organic solvent as an extraction solvent, and examples thereof include the above-mentioned lactic acid bacteria or a treatment for treating lactic acid bacteria in an aqueous or organic solvent (for example, water, methanol, or the like). A known method such as a method of immersing, stirring, or refluxing in ethanol or the like.

Further, the cells and/or the treated material of the lactic acid bacteria can be dried to obtain a powder (powder) or a granular material. The specific drying method is not particularly limited, and examples thereof include spray drying, drum drying, vacuum drying, freeze drying, and the like, and these may be used singly or in combination. At this time, an excipient which is usually used may be added as needed.

Furthermore, the lactic acid bacteria of the present invention may be wet cells or dry cells.

The above processing may be performed in a single processing, or a plurality of processing may be appropriately combined. In the present invention, such a treatment product can also be used in the same manner as the lactic acid bacteria.

The lactic acid bacteria and/or treatments obtained above may be used alone or in combination with other ingredients to form a composition for the production of amino acids and/or peptides from animal milk. That is, the present invention provides a composition comprising at least one lactic acid bacterium belonging to the lactic acid bacteria of Switzerland and used to form an amino acid and/or a peptide.

The composition of the present invention can produce a free amino acid of 25 μmol/ml or more, preferably 30 μmol/ml or more, by fermenting the mammal. Preferably, 15% or more of the free amino acid produced is a branched amino acid. Alternatively, the composition of the present invention can produce a peptide of 8.5 mg/ml or more, preferably 10 mg/ml or more, by fermenting the mammal. Alternatively, the composition of the present invention can produce a total of 260 μg/ml or more, preferably 280 μg/ml, more preferably 300 μg/ml or more of XP peptide and/or by fermenting the mammal. XPP peptide. Alternatively, the composition of the present invention is such that the amount of the IPP peptide and the VPP peptide per unit acid of the fermented milk obtained by fermenting the animal milk at 30 ° C or more and 34 ° C or less is 6.0 mg/g or more in terms of VPP. By.

The composition of the present invention may contain one type of lactic acid bacteria and/or a processed substance, and may contain a plurality of different lactic acid bacteria and/or processed materials, and may also contain a combination of the above-mentioned lactic acid bacteria and/or processed materials. A plurality of lactic acid bacteria treatments treated differently. Further, the composition of the present invention is preferably a bacterial cell containing 1 × 10 ⁷ /ml or more of lactic acid bacteria.

Further, in the composition of the present invention, in addition to the active ingredient lactic acid bacteria, additives and excipients known in the art may be added singly or in combination of plural kinds as long as the target action is not inhibited. Further, the composition of the present invention may further contain an additive (for example, saccharide or glucose such as glucose) which promotes fermentation of lactic acid bacteria. The form of the composition of the present invention is not particularly limited, and it can be prepared into a suspension, a granule, a powder, a capsule or the like. The content of the active ingredient (lactic acid bacteria) in the composition of the present invention varies depending on the form thereof, and the amount of the lactic acid bacteria is usually 0.0001 to 99% by mass, preferably 0.001 to 80% by mass, more preferably 0.001 to 75% by mass. range. Further, the lactic acid bacteria contained in the composition of the present invention is from about 10 ⁷ /g to about 10 ¹² /g.

By fermenting the animal milk using the lactic acid bacteria or composition of the present invention, a fermented milk composition containing an amino acid and/or a peptide at a high concentration can be obtained. Accordingly, the present invention provides a fermented milk obtained by fermenting a mammal with at least one lactic acid bacterium belonging to the Swiss lactic acid bacterium, and containing at least one of the following (a) to (d):

(a) 25 μmol/ml or more, preferably 30 μmol/ml of free amino acid (preferably 15% or more of which is a branched amino acid);

(b) a peptide of 8.5 mg/ml or more, preferably 10 mg/ml or more;

(c) XP peptide and/or XPP peptide in a total amount of 260 μg/ml or more, preferably 280 μg/ml or more, more preferably 300 μg/ml or more;

(d) VPP conversion amount per unit acidity of IPP peptide and VPP peptide of 6.0 mg/g or more.

The fermented milk of the present invention can be prepared by subjecting the lactic acid bacteria or composition of the present invention to a fermentation treatment according to a method known in the art. The fermentation treatment can be carried out by adding at least one lactic acid bacteria to the animal milk and culturing under appropriate conditions. As the animal milk, milk derived from a mammal such as cow's milk, goat's milk or horse's milk, or processed milk such as skim milk, reduced milk or condensed milk may be used, and one type of milk may be used, or a plurality of types of milk may be used in combination or Processing milk. The concentration of the solid content of the milk is not particularly limited. For example, when the skim milk is used, the concentration of the non-fat milk solid content is about 3 to 15% by mass, preferably 6 to 15% by mass. The animal milk can also be sterilized before the fermentation. Additives that promote lactic acid bacteria fermentation (such as sugars such as glutamic acid or glucose) may also be added to the animal milk. Alternatively, the milk protein component may be separated from the animal milk, and the milk protein component may be used as a substrate for fermentation.

The lactic acid bacteria added to the animal milk preferably use a pre-cultured lactic acid bacterium as a bacterium. In addition, the amount of the lactic acid bacteria to be added is not limited, and is usually 0.005 to 10% by mass, preferably 0.05 to 5% by mass in terms of dry lactic acid bacteria. The lactic acid bacteria to be used may be directly cultured as a lactic acid bacteria, or may be isolated from the culture medium by filtration or centrifugation of the cultured lactic acid bacteria, or may be stored by freezing or freeze-drying after separation from the culture medium.

The conditions of the fermentation treatment are substantially the same as those of the above lactic acid bacteria, and are carried out under anaerobic conditions at 20 ° C to 50 ° C, preferably 25 ° C to 42 ° C, more preferably 28 ° C to 37 ° C. Further, in the following examples, the lactic acid bacteria of the present invention particularly produced branched-chain amino acids and functional peptides at 30 to 34 ° C (about 32 ° C). Therefore, it is especially preferable to carry out the fermentation treatment at 30 to 34 °C. The temperature conditions can be adjusted by a thermostatic bath, a mantle heater, a jacket, and the like. The fermentation treatment can be carried out by means of static culture, shaking culture, tank culture, and the like. Further, the fermentation time can be set to 3 hours to 96 hours, preferably 12 to 36 hours. Preferably, the fermentation is carried out while measuring the pH and acidity of the fermentation tank, and the pH is preferably maintained at 4.0 to 8.0.

The fermented milk obtained as described above may be treated in the fermented milk as long as it contains the desired ingredients, or may be further processed in the treatment of the fermented milk. Examples of such treatments are described below.

Suspensions or dilutions can be prepared by suspending or diluting the fermented milk and/or fermented milk treatment in a suitable solvent. Examples of the solvent that can be used include water, physiological saline, and phosphate buffered saline (PBS).

The sterilization treatment can be prepared by sterilizing the fermented milk and/or the fermented milk treatment. When sterilizing the fermented milk and/or the fermented milk processed product, for example, a known sterilization treatment such as filter sterilization, radioactive sterilization, superheat sterilization, or pressurized sterilization can be performed.

The heat treatment can be prepared by heat-treating the fermented milk and/or the fermented milk treatment. The heat treatment material is prepared for a fixed period of time, for example, for about 10 minutes to 1 hour (for example, about 10 to 20 minutes), and subjected to high temperature treatment (for example, 80 to 150 ° C).

Further, the supernatant (whey) can be prepared by filtering or centrifuging the fermented milk and/or the fermented milk treated product.

Further, the fermented milk and/or the fermented milk processed product can be dried to prepare a powder (powder) or a granule. The specific drying method is not particularly limited, and examples thereof include spray drying, drum drying, vacuum drying, freeze drying, and the like, and these may be used singly or in combination. At this time, an excipient which is usually used may be added as needed.

The above treatment may be carried out in a single treatment, or a plurality of treatments may be appropriately combined. Such a fermented milk processed product is also included in the present invention.

Since the fermented milk or the treated material of the present invention contains various amino acids and/or peptides at a high concentration, it can be used in applications in which the properties of the amino acids and/or peptides are utilized. For example, the amino acid is related to flavor, energy source, mental stabilizing action, growth promoting action, and the like, and thus the fermented milk of the present invention can be used as a flavoring agent, a nutritional adjuvant, and an energy supplement. Further, a branched amino acid (BCAA) containing lysine, leucine and isoleucine is an essential amino acid which can be used to strengthen muscles or prevent physical fatigue. Further, it is considered that the decomposition reaction in the liver is promoted by the presence of alanine (Ala). Further, it is also known that Ala can be tasted in terms of taste, and has a masking effect such as salting, vinegar pickling or irritating. It is considered that the amino acid which is most easily deficient in the amino acid (Lys) essential amino acid must be ingested as a raw material such as an antibody or a hormone or an enzyme. Also, it is reported that Lys is also involved in calcium absorption, collagen formation, and bone tissue production. Serine is important as a precursor to various important substances in living organisms such as phospholipid serine or other amino acids. Further, it is known that it lowers blood cholesterol, prevents brain aging by inhibiting brain cell death by activating neurons in the brain, or forms a moisturizing component of the skin. Therefore, the fermented milk or the treatment of the present invention can be used as a flavoring agent, a nutritional adjuvant, an energy supplement, a brain function improving agent, an autonomic nervous regulator, a parasympathetic regulating agent, a sympathetic regulating agent, a muscle decomposition damage inhibitor, Muscle enhancer, fatigue reliever, endurance improver, alcohol metabolism agent, and the like. Furthermore, VPP peptide and IPP peptide have ACE inhibitory activity and relaxation effect, LPP peptide has ACE inhibitory activity, YP peptide has hypotensive effect and anxiolytic effect, VP, IP, AP, EP, RP, QP, TP, MP and GP peptide It is non-decomposable in vivo and has ACE inhibitory activity. Therefore, the fermented milk or the treatment of the present invention can be used as a blood pressure lowering agent and a stress relieving agent.

Further, various amino acid and/or peptide components or fractions containing the components may be purified from the obtained fermented milk or a processed product thereof by a known separation and purification method.

The fermented milk of the present invention or the treated product thereof may contain at least one component (for example, an amino acid or a peptide) of the above components (a) to (d), or may contain a plurality of components in combination. Further, the lactic acid bacteria used in the fermentation treatment may be contained in the fermented milk or the processed material thereof.

Further, in the fermented milk of the present invention, as long as the target action is not inhibited, the following additives, other known blood pressure lowering agents, pressure alleviators, nutritional adjuvants, energy supplements, and the like may be added singly or in combination of plural kinds. Muscle enhancers, flavoring agents, etc.

The form of the fermented milk of the present invention is not particularly limited, and for example, a suspension form of the lyophilized milk obtained after the fermentation treatment, and a tablet obtained by treating by a method known in the art, Capsules, granules, powders, powders, syrups, dry syrups and the like. The fermented milk is preferably in a form that is easily administered or ingested orally. Further, the liquid such as the suspension may be in the form of being suspended in water or other suitable medium immediately before administration or ingestion, and in the case of tablets or granules, the surface may be coated by a well-known method. .

The fermented milk of the above form can be formulated with the excipients, disintegrants, binders, wetting agents, stabilizers, buffers, lubricants, preservatives, interfacial activities commonly used in the fermented milk obtained by the fermentation treatment. Additives such as a sweetener, a flavoring agent, a flavoring agent, a flavoring agent, a coloring agent, and the like, and are manufactured according to a usual method. For example, when the fermented milk is used for medical or health-promoting purposes, a pharmaceutically acceptable carrier or additive may be added. Examples of such pharmaceutically acceptable carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, and water-soluble. Dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, tricyanidin, acacia, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, hard An ester acid, human serum albumin, mannitol, sorbitol, lactose, a surfactant which is allowed as a pharmaceutical additive, and the like.

Further, in the fermented milk of the present invention, various additives or various other substances used in the manufacture of medicines, foods, and feeds may be coexistent. Examples of such a substance or an additive include various oils and fats (for example, vegetable oils such as soybean oil, corn oil, safflower oil, and olive oil, animal fats such as butter and salmon oil), natural medicines (for example, royal jelly, human cockroaches, etc.). Amino acids (such as branamine, cysteine, leucine, arginine, etc.), polyols (such as ethylene glycol, polyethylene glycol, propylene glycol, glycerol, sugar alcohols, as an example of sugar alcohols) Sorbitol, erythritol, xylitol, maltitol, mannitol, etc.), natural polymers (eg gum arabic, agar, water soluble zein fiber, gelatin, sanmon gum, casein, gluten or bran) Hydrolysate, lecithin, starch, dextrin, etc.), vitamins (such as vitamin C, vitamin B group, etc.), minerals (such as calcium, magnesium, zinc, iron, etc.), dietary fiber (such as mannan, pectin, Hemicellulose, etc.), surfactant (such as glycerin fatty acid ester, sorbitan fatty acid ester, etc.), purified water, excipients (such as glucose, corn starch, lactose, dextrin, etc.), stabilizers, pH adjustment Agent, antioxidant, sweetener, flavoring ingredient, sour material, Materials and spices.

Further, in the fermented milk of the present invention, as a functional component or an additive other than the above-mentioned active ingredient, for example, taurine, glutathione, carnitine, creatine, coenzyme Q, glucuronic acid, glucose may be formulated. Aldolonic acid lactone, capsicum extract, ginger extract, cocoa extract, guarana extract, Garcinia Cambogia extract, theanine, γ-aminobutyric acid, capsaicin, capsaicin ester, various organic Acid, flavonoids, polyphenols, catechins, xanthine derivatives, oligofructose and other indigestible oligosaccharides, polyvinylpyrrolidone, and the like. The blending amount of these additives is appropriately determined depending on the kind of the additive and the required intake amount.

The fermented milk of the present invention is administered or vaccinated to a vertebrate, specifically, a mammal, such as a human, a primate (猿, chimpanzee, etc.), a livestock animal (cattle, horse, pig, sheep, bird, etc.). Animals for pets (dogs, cats, etc.), experimental animals (white mice, rats, etc.), reptiles and birds. In particular, it is preferred that the subject to take up the amino acid and/or the peptide is, for example, a hypertensive patient, a person who exhibits symptoms caused by stress, a person before and after exercise, or the like.

The dosage or intake of the fermented milk of the present invention varies depending on the age and weight of the subject, the route of administration/intake, the number of administrations/intakes, the purpose of administration, and the like, and can be achieved in accordance with the discretion of the practitioner. Make changes within a wider range. The content ratio of the active ingredient contained in the mashed milk is not particularly limited, and may be appropriately adjusted in accordance with the easiness of production or a preferred one-day dosage. Since the fermented milk of the present invention has high safety, it is possible to further increase the intake. The average intake per day can be taken once, and can be divided into several intakes. Further, the frequency of administration or ingestion is not particularly limited, and may be appropriately selected depending on various conditions of the administration/intake route, the age and weight of the subject, and the target effect.

The administration/intake route of the fermented milk of the present invention is not particularly limited, and it is preferably oral administration or oral ingestion. For example, it can be formulated into foods or foods, or made into tablets or granules, etc., orally or ingested.

The fermented milk of the present invention can also be used in combination with other medicines, treatments or prevention methods. Such other medicines may be formulated together with the fermented milk of the present invention, or may be administered as separate preparations at the same time or at intervals.

As described above, the fermented milk of the present invention can be used as a blood pressure lowering agent, a brain function improving agent, an autonomic nervous regulator, a parasympathetic regulating agent, a sympathetic adjusting agent, a nutritional adjuvant, an energy supplement, a muscle decomposition damage inhibitor, Muscle enhancer, fatigue reliever, endurance improver, flavoring agent or alcohol metabolism agent.

The lactic acid bacteria and the fermented milk of the present invention are also easy to be used for long-term continuous intake, and therefore can be used in foods and drinks and feed. The lactic acid bacteria of the present invention are those which produce a large amount of amino acids and/or peptides having various functions, and the fermented milk of the present invention contains such amino acids and/or peptides at a high concentration. Further, the amount of the functional peptide of the lactic acid bacteria of the present invention with respect to the acidity is high, and the sourness can be suppressed, and the flavor of the food or beverage itself is not hindered by being added to various foods and drinks. Therefore, the lactic acid bacteria and the fermented milk of the present invention can be continuously added to various foods and drinks, and various effects are expected.

Therefore, the food or drink of the present invention contains at least one lactic acid bacterium of the present invention and/or a treated product thereof, and/or the fermented milk of the present invention and/or a processed product thereof. In the present invention, the food and beverage also contains a beverage. In addition to the amino acid and/or peptide, the food or drink of the present invention contains healthy lactic acid foods, functional foods, and foods for specific health care, and the lactic acid bacteria and the fermented milk of the present invention. All food and drink.

As a food or drink of the present invention, a functional food or drink is particularly preferred. The "functional foods and drinks" of the present invention are foods and drinks having a fixed function for a living body, and include, for example, foods and beverages including specific health care (including conditional special care [special health foods]) and nutritional function foods and drinks. Health-care foods, foods, special-purpose foods, nutritional supplements, health-assist foods, supplements (for example, tablets, coated tablets, sugar-coated tablets, capsules, and liquids) and beauty foods and drinks ( For example, dieting foods and drinks, etc., all so-called healthy foods and drinks.

Specific examples of the food and drink include healthy foods and beverages in the form of a liquid such as a enteral nutrient, a tablet, a chewable tablet, a tablet, a powder, a powder, a capsule, a granule, and a drink. And nutritional supplement foods; tea drinks such as green tea, oolong tea and black tea, refreshing drinks, jelly drinks, sports drinks, milk drinks, carbonated drinks, vegetable drinks, fruit drinks, fermented vegetable drinks, fermented fruit drinks, fermented milk drinks, Beverages (yoghurt, solid yogurt), yoghurt beverage (sterilization), lactic acid bacteria beverage, concentrated beverage, concentrated solids, milk beverage (coffee milk, etc.), beverage powder, cocoa beverage, milk and purified water Butter, jam, pine and margarine; mayonnaise, shortening, custard, sauce, bread, rice, noodles, pasta, miso soup, tofu, yoghurt, soup or sauce Materials, pastries (for example, biscuits or cookies, chocolate, candy, cakes, ice cream, chewing gum, lozenges), etc.

In addition to the lactic acid bacteria and/or fermented milk of the present invention, other food materials, various nutrients, various vitamins, minerals, dietary fibers, and various additives used in the manufacture of the foods and drinks may be blended (for example, the food and beverage of the present invention). It is a flavoring ingredient, a sweetener, a sour material such as an organic acid, a stabilizer, a perfume, etc., and is produced according to a usual method.

In the food or drink of the present invention, the blending amount of the lactic acid bacteria, the fermented milk or the processed materials can be appropriately determined by the manufacturer in consideration of the form of the food or drink or the taste or texture desired. Thus, by drinking or eating the food or drink of the present invention in a form that can manage the desired intake, blood pressure reduction, stress relief, nutritional supplementation, energy assistance, muscle enhancement, brain function improvement, and self-discipline are provided using the food or drink. Neuromodulation, parasympathetic regulation, sympathetic regulation, inhibition of muscle breakdown injury, muscle enhancement, reduction of fatigue, improvement of endurance, alcohol metabolism, etc.

The food or beverage of the present invention can be produced by any suitable method available to the manufacturer. For example, the lactic acid bacteria and/or the fermented milk (treated product) of the present invention can be prepared into a liquid form, a gel form, a solid form, a powder form or a granulated form, and then blended into a food or drink. Alternatively, the lactic acid bacteria and/or the fermented milk (treated product) of the present invention may be directly mixed or dissolved in a raw material of a food or beverage. Lactic acid bacteria and/or fermented milk (treated material) may also be coated, coated, infiltrated or attached to food or beverage. Lactic acid bacteria and/or fermented milk (treated material) can be uniformly dispersed in foods and drinks, and may be unevenly distributed. The adjustable agent is a capsule containing lactic acid bacteria and/or fermented milk (treated material). The lactic acid bacteria and/or the fermented milk (treated material) may be wrapped in an edible film or an edible film or the like. In addition, a suitable excipient or the like may be added to the lactic acid bacteria and/or fermented milk (treated product) of the present invention, and then formed into a shape such as a tablet. The food or beverage of the present invention can be further processed, and such processed products are also included in the scope of the present invention.

In the manufacture of the food or beverage of the present invention, various additives conventionally used in foods and drinks can also be used. The additive is not limited, and examples thereof include a color developer (such as sodium nitrite), a coloring material (such as scorpion dye and red 102), a fragrance (such as orange flavor), and a sweetener (stevia sinensis and aspartame). Sweet, etc.), preservatives (sodium acetate, sorbic acid, etc.), emulsifiers (sodium chondroitin sulfate, propylene glycol fatty acid esters, etc.), antioxidants (disodium EDTA, vitamin C, etc.), pH adjusters (citric acid, etc.) ), chemical seasonings (sodium inosine, etc.), tackifiers (Sanxian gum, etc.), bulking agents (calcium carbonate, etc.), defoamers (calcium phosphate), etc., adhesives (sodium polyphosphate, etc.), nutrition A strengthening agent (calcium fortifier, vitamin A, etc.), an excipient (water-soluble dextrin, etc.), and the like. Further, functional raw materials such as Asian anthraquinone extract, Acanthopanax senticosus extract, eucalyptus extract, and Eucommia tea extract may be further added.

As described above, the food or drink of the present invention has various functions caused by an amino acid and/or a peptide, and further has high safety without worrying about side effects. Further, the lactic acid bacteria and/or the fermented milk of the present invention can suppress the acidity and have a good flavor, and are added to various foods and drinks without impeding the flavor of the food or drink. The obtained foods and drinks are easy to be ingested for a long period of time, and it is expected to exhibit various functions for a long time.

Further, the lactic acid bacteria and/or the fermented milk of the present invention can be incorporated into human food and beverage products, and can be blended into feeds of animals such as livestock (bovine, pig, etc.), horse racing, pets (dogs, cats, etc.). Regarding the feed, the food is substantially the same as the food and drink, and therefore the description of the food or drink is also applicable to the feed.

[Examples]

Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to the examples.

[Example 1] Identification of L. helveticus CP3232 strain

The L. helveticus CM4 strain (Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060) was bred, and the novel L. helveticus CP3232 showing the properties shown in Examples 2 to 5 was obtained from the obtained mutant strain. Strain.

The L. helveticus CP3232 strain was anaerobic cultured on MRS agar medium at 37 ° C for 24 hours, and the morphological observation properties and physiological properties of the obtained community were studied. Further, the sequence of 16S rRNA was determined and confirmed to have 99.9% homology with other Swiss lactic acid bacteria (type strain). From this, it can be seen that the obtained strain does belong to L. helveticus and is a novel strain.

[Example 2] Preparation of fermented milk

The 9.00% (w/w) reduced skim milk powder was subjected to temperature sterilization at 95 ° C and then cooled to 15 ° C for use as a milk medium. The lactic acid bacteria-containing fermented milk was inoculated at a ratio of 5% in a milk medium, and cultured at 37 ° C for 24 hours. Further, the operation was repeated, and the obtained fermented milk was used as a fermenting agent. The culture was added with 3% fermenting agent in the milk medium, and cultured at 32 ° C or 37 ° C for 24 hours. Determine the pH and acidity of the fermented milk. The acidity was measured using a flat-automatic titrator COMTITE-450 (Pinguma Industry). As the lactic acid bacteria, L. helveticus CP3232 strain, and comparative L. helveticus CP3264 strain (separated from antihypertensive preparation), L. helveticus CM4 strain (Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060), L are used. Helveticus CP1092 strain, L. helveticus CP1100 strain, L. helveticus CP1081 strain, and L. helveticus CPN4 strain (Japanese Patent Laid-Open No. Hei 7-123977).

The results are shown in Table 1. As a result, the ACE inhibitory activity per unit weight of IPP was 1.7 times that of VPP. Therefore, the amount of IPP and the amount of VPP were expressed as VPP by the following formula:

VPP conversion amount (μg/ml) = IPP amount (μg / ml) × 1.7 + VPP amount (μg / ml)

Further, "acidity" means the concentration of the acid (lactic acid) contained in the fermented milk. As shown in Table 1, in the fermented milk obtained by fermentation of L. helveticus CP3232 strain, the amount of IPP peptide and VPP peptide was 6.2 mg in terms of VPP relative to the amount of lactic acid produced (ie, acidity). /g, which shows a higher amount of peptide in unit acidity. The fermented milk specific gravity was calculated to be 1.02.

[Example 3] Determination of free amino acid in mashed milk supernatant

10000 g of the fermented milk obtained in Example 2 was centrifuged for 10 minutes, and the supernatant was appropriately diluted with distilled water, filtered through a 0.2 μM membrane filter, and subjected to high performance liquid chromatography (HPLC, Shimadzu). The amino acid automatic analyzer (C-R7A/LC-10A) of the manufacturer was measured. The analysis conditions are as follows.

Column: Shim-pack Amino-Li (100 mmL × 6.0 mmI.D.)

Ammonia trap: Shim-pack ISC-30Li (50 mmL × 4.0 mmI.D.)

Column temperature: 38~58°C

Reaction temperature: 65 ° C

Mobile phase: mobile phase set Li-type for analysis of simian amino acid

Reaction solution: reaction solution for the analysis of oxazinamide acid OPA reagent

Flow rate: 0.4 ml/min.

To detect the proline, sodium hypochlorite was added to the reaction solution. Finally, the fluorescence coloration of the amino acid reacted with o-phthalaldehyde was measured by a spectroscopic fluorescence detector (Ex: 348 nm, Em: 450 nm). As a standard substance, the content was calculated using an amino acid mixed standard type (H) having a known concentration.

The results are shown in Figures 1 and 2. Fig. 1A shows the results of the amount of each amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C for 24 hours, and the B system shows that each lactic acid bacterium is in 37 The amount of amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermentation for 24 hours at °C. Further, A of Fig. 2 shows the total amount of free amino acid (μmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours, and the B system indicates that The amount of branched amino acid (μmol/ml) contained in the fermented milk supernatant obtained by lactic acid bacteria at 32 ° C or 37 ° C for 24 hours. According to these results, the L. helveticus CP3232 strain has a high proteolytic activity, and a free amino acid of 25 mol/ml or more is produced in the fermentation for 24 hours. In particular, 29.9 μmol/ml of free amino acid was produced in the fermentation at 37 ° C, and 33.1 μmol/ml of free amino acid was produced in the fermentation at 32 ° C ( FIG. 2A ). Further, it is shown that the amount of the branched amino acid contained in the produced free amino acid is large, and 15% or more of the free amino acid produced in the fermentation for 24 hours is a branched amino acid. Specifically, about 19.4% of the fermentations at 37 ° C were branched-chain amino acids, and about 21.5% of the fermentations at 32 ° C were branched-chain amino acids ( FIG. 2B ).

[Example 4] Peptide amount in fermented milk supernatant (OPA method)

The amount of the peptide in the fermented milk supernatant obtained in Example 2 was measured in accordance with the method of Church et al. (J. Dairy Sci. 1983. 66: 1219-1227.). For simple explanation, mix 25 ml of 100 mM sodium tetraborate solution, 2.5 ml of 20% SDS (Sodium dodecyl sulfate), 0.1 ml of 2-mercaptoethanol, and add it to the solution. The phthalic acid was dissolved in 1 ml of a 4% by weight solution in methanol, and finally 50 ml was prepared in distilled water to prepare an OPA reagent. 10000 g of the fermented milk obtained in Example 2 was taken and centrifuged for 10 minutes, and the culture supernatant was appropriately diluted to prepare an analysis sample. 200 μl of the OPA reagent was mixed with 10 μl of the analysis sample and stirred for ten minutes, after which it was allowed to stand at room temperature for 5 minutes, and the absorption at 340 nm was measured. Trypsin was used as a standard substance.

The results are shown in Fig. 3. Based on the results, it was revealed that the L. helveticus CP3232 strain has high proteolytic activity, and a large amount of peptide is produced by fermentation. Specifically, a peptide of 8.5 mg/ml or more was produced in 24 hours of fermentation, and 11.1 mg/ml of the peptide was produced in the fermentation at 32 ° C, and a peptide of 9.6 mg/ml was produced at 37 ° C ( image 3).

[Example 5] Quantification of functional peptides

10000 g of the fermented milk obtained in Example 2 was taken and centrifuged for 10 minutes, and the supernatant was appropriately diluted with distilled water, and then filtered with a 0.2 μM membrane filter, and analyzed by LC/MS. LC/MS analysis was carried out according to the method of Inoue et al. (J. Biosci. Bioeng., 2009, 108, 111-115.). That is, RP-aqueous Column was used as a separation column in the LCMS-2010A system (Shimadzu Corporation) for analysis. The content was calculated using a synthetic peptide as a standard substance. Synthetic peptides were used ( ¹³ C ₅ )Val-( ¹³ C ₅ )Pro-Pro, Ile-( ¹³ C ₅ )Pro-Pro, Glu-Pro, Gln-Pro (supplied from Scrum Corporation), Leu-Pro- Pro, Tyr-Pro, Val-Pro, Ile-Pro, Ala-Pro, Arg-Pro, Thr-Pro, Met-Pro, Gly-Pro (above purchased from Bachem).

The results are shown in Figures 4 and 5. According to the results shown in A and B of Fig. 4, the L. helveticus CP3232 strain has high proteolytic activity, and various functional peptides are produced in the fermentation for 24 hours. It is reported that among the functional peptides listed in Figure 4, VPP and IPP have ACE inhibitory activity and relaxation effect, LPP has ACE inhibitory activity, YP has hypotensive effect and anxiolytic effect, VP, IP, AP, EP, RP , QP, TP, MP and GP are non-decomposable in vivo and have ACE inhibitory activity. The L. helveticus CP3232 strain in particular produces a large amount of VPP, IPP, YP, QP and TP.

Further, according to the results shown in A and B of Fig. 5, L. helveticus CP3232 strain has a high proteolytic activity, and a functional peptide having a total amount of 260 μg/ml or more is produced in 24 hours of fermentation ( XP+XPP). In particular, a functional peptide of 318 μmol/ml was produced in the fermentation at 32 ° C (Fig. 5A), and a functional peptide of 282 μmol/ml was produced in the fermentation at 37 ° C (Fig. 5B).

[Example 6] Preparation of mashed milk and determination of peptide amount

In the same manner as in the case of the lactic acid bacteria, L. helveticus CP3232 strain, and comparative L. helveticus CNCM I-3435 strain (patent document 4), L. helveticus FERM BP-5445 strain (patent document 7), and L were used. Helveticus CM4 strain (Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060), the pH and acidity of the fermented milk after culture, and the amount of the produced IPP peptide and VPP peptide were measured.

The results are shown in Table 2. As a result, in the same manner as in the second embodiment, the amount of IPP and the amount of VPP were expressed as VPP converted amounts. Further, "acidity" means the concentration of the acid (lactic acid) contained in the fermented milk. As shown in Table 2, the amount of the IPP peptide and the VPP peptide in the fermented milk obtained by the fermentation of the L. helveticus CP3232 strain was 7.0 mg/v in terms of VPP with respect to the produced lactic acid (ie, acidity). g, showing a higher amount of peptide per unit acidity. The fermented milk specific gravity was calculated to be 1.02.

[Example 7] Determination of free amino acid in mashed milk supernatant

The free amino acid was measured in the same manner as in Example 3 using the fermented milk obtained in Example 6. The results are shown in Figures 6 and 7.

Fig. 6A shows the results of the amount of each amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C for 24 hours, and B indicates that each lactic acid bacterium was subjected to 37 ° C at 37 ° C. The amount of amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermentation for 24 hours. Further, A of Fig. 7 shows the total amount of free amino acid (μmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours, and B indicates each lactic acid bacterium. The amount of branched amino acid (μmol/ml) contained in the fermented milk supernatant obtained by fermentation at 32 ° C or 37 ° C for 24 hours. According to these results, the L. helveticus CP3232 strain has a high proteolytic activity, and a free amino acid of 25 mol/ml or more is produced in the fermentation for 24 hours. In particular, 28.1 μmol/ml of free amino acid was produced in the fermentation at 37 ° C, and 32.4 μmol/ml of free amino acid was produced in the fermentation at 32 ° C ( FIG. 7A ). Further, it was shown that the amount of the branched amino acid contained in the produced free amino acid was large, and 15% or more of the free amino acid produced in the fermentation for 24 hours was a branched amino acid. Specifically, about 20% of the fermentations at 37 ° C were branched-chain amino acids, and about 21% of the fermentations at 32 ° C were branched-chain amino acids ( FIG. 7B ).

[Example 8] Peptide amount in fermented milk supernatant (OPA method)

The amount of the peptide in the fermented milk supernatant obtained in Example 6 was measured in the same manner as in Example 4. The results are shown in Fig. 8. According to the results, the L. helveticus CP3232 strain has a high proteolytic activity, and a large amount of peptide is produced by fermentation. Specifically, a peptide of 8.5 mg/ml or more was produced in 24 hours of fermentation, and a peptide of 10.4 mg/ml was produced in the fermentation at 32 ° C, and a peptide of 9.1 mg/ml was produced in the fermentation at 37 ° C ( Figure 8).

[Example 9] Quantification of functional peptide

The fermented milk obtained in Example 6 was analyzed by LC/MS in the same manner as in Example 5, and the amount of functional peptide was measured. The results are shown in Figures 9 and 10. According to the results shown in A and B of Fig. 9, the L. helveticus CP3232 strain has a high proteolytic activity, and various functional peptides are produced in the fermentation for 24 hours. The L. helveticus CP3232 strain in particular produces a large amount of VPP, IPP, YP, QP and TP.

Further, according to the results shown in A and B of Fig. 10, L. helveticus CP3232 strain has a high proteolytic activity, and a functional peptide having a total amount of 260 μg/ml or more is produced in 24 hours of fermentation ( XP+XPP). In particular, a functional peptide of 280 μmol/ml was produced in the fermentation at 32 ° C (Fig. 10A), and a functional peptide of 295 μmol/ml was produced in the fermentation at 37 ° C (Fig. 10B).

[Reference Example 1] L. helveticus CP3232 strain and its mutant strain identification method

The lactic acid bacteria L. helveticus CP3232 strain obtained in Example 1 and its variant strain were measured by the methods as described in Examples 2 to 9, and in addition to the identification by the polymerase chain reaction (PCR) method, thereby being easily The ground is distinguished from other lactic acid strains. Further, it is also possible to easily determine whether or not it is a mutant strain of the L. helveticus CM4 strain which is a mother strain of L. helveticus CP3232 strain by a PCR method.

When an example is briefly described, a DNA reagent is prepared for a target lactic acid bacterium, and the gene amplification is carried out by a PCR method using the following primers, and electrophoresis is carried out on a 0.9% agarose gel to analyze the migrating pattern, thereby determining whether or not L. helveticus CP3232 strain variant, and whether it is a variant of L. helveticus CM4 strain.

Introduction:

Agccacttcctccgattacag (SEQ ID NO: 1) and gctattttagcagcgattcg (SEQ ID NO: 2).

In the present example, the above PCR method was carried out using a plurality of L. helveticus strains. The result is shown in FIG. The zones in Figure 11 are as follows.

Zone 1: L. helveticus CP3232 strain

Zone 2: L. helveticus CM4 strain

Zone 3: L. helveticus CP3231 strain

Zone 4: L. helveticus CP3264 strain

Zone 5: L. helveticus CP1081 strain

Zone 6: L. helveticus CP209 strain

Zone 7: L. helveticus CP210 strain

Zone 8: L. helveticus CP293 strain

Zone 9: L. helveticus CP617 strain

Zone 10: L. helveticus CP39 strain

Zone 11: L. helveticus CP790 strain

Zone 12: L. helveticus JCM1120 strain

M: λ phage DNA restriction enzyme HindIII decomposition product.

As shown in Fig. 11, characteristic DNA fragments were confirmed in the CM4 mutant L. helveticus CP3232 strain, CP3231 strain, and parental strain CM4 strain (zones 1 to 3), and the other L. helveticus strain could not confirm the fragment. As a DNA marker, the restriction enzyme HindIII decomposition product of λ phage DNA was simultaneously subjected to electrophoresis, and the molecular weight of the characteristic DNA fragment was measured and found to be about 2.2 kb.

Further, the L. helveticus CP3232 strain, the CP3231 strain, and the parent strain CM4 strain were identified by the peptide amount analysis in the fermented milk using the OPA reagent described in Examples 4 and 8, and the L. helveticus CP3232 strain was selected. That is, if the value of the amount of the peptide in the fermented milk prepared according to Example 2 was measured by the OPA reagent, the L. helveticus CP3231 strain and the CM4 strain were 7.5 mg/ml and 4.5 mg/ml, respectively, whereas L. The helveticus CP3232 strain shows a higher value of 11.1 mg/ml and can be easily identified.

[Reference Example 2] L. helveticus CP3232 strain and its mutant strain identification method

In the same manner as in Reference Example 1, it was tested whether the lactic acid bacteria L. helveticus CP3232 strain obtained in Example 1 and its variant strain and other lactic acid strains (L. helveticus CNCM I) can be obtained by polymerase chain reaction (PCR). -3435 strain (Patent Document 4) and L. helveticus FERM BP-5445 strain (Patent Document 7)) were distinguished.

The results of gene amplification and electrophoresis by PCR using primers are shown in Fig. 12. In Figure 12, the zones are as follows:

Zone 1: L. helveticus CP3232 strain

Zone 2: L. helveticus CM4 strain

Zone 3: L. helveticus CNCM I-3435 strain

Zone 4: L. helveticus FERM BP-5445 strain

M: λ phage DNA restriction enzyme HindIII decomposition product.

As shown in Fig. 12, a characteristic DNA fragment was confirmed in L. helveticus CP3232 strain and mother strain CM4 strain (zones 1 and 2) which are CM4 mutant strains, and this fragment was not confirmed in other L. helveticus strains.

All publications, patents and patent applications cited in this specification are hereby incorporated by reference in their entirety.

[Industrial availability]

According to the present invention, there is provided a lactic acid bacterium belonging to a Swiss lactic acid bacterium which has high proteolytic activity and is excellent in peptide-forming ability and/or amino acid-forming ability. The amino acids and/or peptides produced by the present lactic acid bacteria have various functions, and peptides such as IPP, VPP, and YP have blood pressure lowering effects. Further, by using the lactic acid bacteria for fermentation, it is possible to obtain a fermented milk having a high concentration of such a plurality of amino acids and/or peptides having various functions and having a good flavor. Therefore, the present invention can be used in the fields of medicine, food and drink, and promotion of health.

[registration number]

Taiwan registration number BCRC 910539, Japanese registration number FERM BP-11271 (Lactobacillus Helveticus (Swiss lactic acid bacteria) CP3232 strain, Taiwan was deposited on January 12, 2012, Japan was deposited on August 4, 2010 (2010))

Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060 (Lactobacillus Helveticus CM4 strain, Taiwan was deposited on September 16, 1998, Japan was deposited on August 15, 1997 (1997))

[sequence table free content]

Sequence numbers 1 and 2: DNA (synthetic oligonucleotides)

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<120> Japanese Business Co., Ltd.

<130> PH-5013TW

<140> 100140985

<141> 2011-11-09

<150> 2010-250754

<151> 2010-11-09

<160> 2

<170> PatentIn version 3.4

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 1

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 2

Fig. 1 is a graph showing the amount of each amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours.

Figure 2 is a graph showing the total amount of free amino acid (μmol/ml) (A) and branched-chain amino acid contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours. A graph of the amount (μmol/ml) (B).

Fig. 3 is a graph showing the amount of peptide (mg/ml) in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours.

Fig. 4 is a graph showing the amount of functional peptide (μg/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours.

Fig. 5 is a graph showing the amount of XP peptide and XPP peptide (μg/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours. .

Fig. 6 is a graph showing the amount of each amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours.

Figure 7 is a graph showing the total amount of free amino acid (μmol/ml) (A) and branched-chain amino acid contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours. A graph of the amount (μmol/ml) (B).

Fig. 8 is a graph showing the amount of peptide (mg/ml) in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours.

Fig. 9 is a graph showing the amount of functional peptide (μg/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours.

Fig. 10 is a graph showing the amount of XP peptide and XPP peptide (μg/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours. .

Figure 11 is a diagram showing an electrophoresis pattern of a fragment amplified from genomic DNA of various lactic acid strains by polymerase chain reaction (PCR).

Figure 12 is a diagram showing an electrophoresis pattern of a fragment amplified from genomic DNA of various lactic acid strains by polymerase chain reaction (PCR).

(no component symbol description)

Claims (23)

A lactic acid bacterium belonging to the lactic acid bacteria of Switzerland characterized by having the following properties (a), (b) and (c) or the properties of (a), (b) and (d), and the lactic acid bacteria is a lactic acid bacteria CM4 strain (Taiwan) The variant strain of the registration number CCRC 910111, Japanese registration number FERM BP-6060, the Swiss lactic acid bacteria CP3232 strain (Taiwan registration number BCRC 910539, Japanese registration number FERM BP-11271) or its variant: (a) by making the colander Fermentation produces 25 μmol/ml or more of free amino acid; (b) produces 8.5 mg/ml or more of the peptide by fermenting the animal's milk; (c) produces a total amount of 260 μg by fermenting the animal's milk. XP peptide and/or XPP peptide of more than ml; (d) The amount of IPP peptide and VPP peptide of the acidity of the fermented milk obtained by fermenting the animal's milk at 30 ° C or more and 34 ° C or less is 6.0 mg in terms of VPP /g or more. The lactic acid bacterium according to claim 1, wherein 15% or more of the free amino acid is a branched amino acid. A lactic acid bacterium according to claim 1 or 2 which produces a YP peptide of 60 μg/ml or more. A lactic acid bacterium which is a strain of Swiss lactic acid bacteria CP3232 (Taiwan registered number BCRC 910539, Japanese registered number FERM BP-11271). A use of at least one lactic acid bacterium belonging to the lactic acid bacteria of Switzerland according to claim 1 for producing a composition derived from animal milk to produce an amino acid and/or a peptide. The use of claim 5, wherein a free amino acid of 25 μmol/ml or more is produced by fermenting the mammal. The use of claim 6, wherein more than 15% of the free amino acid is a branched amino acid. The use according to any one of claims 5 to 7, wherein the peptide of 8.5 mg/ml or more is produced by fermenting the mammal. The use according to any one of claims 5 to 7, wherein a total of 260 μg/ml or more of XP peptide and/or XPP peptide is produced by fermenting the mammal. The use according to any one of claims 5 to 7, wherein a YP peptide of 60 μg/ml or more is produced by fermenting the mammal. The use of any one of the items 5 to 7, wherein the amount of the IPP peptide and the VPP peptide of the acidity of the fermented milk obtained by fermenting the animal milk at 30 ° C or more and 34 ° C or less is 6.0 mg in terms of VPP. /g or more. A fermented milk obtained by fermenting a mammalian lactic acid bacteria by using at least one lactic acid bacteria belonging to the Swiss lactic acid bacteria, which is a Swiss lactic acid bacteria CM4 strain (Taiwan registered number CCRC 910111, Japanese registered number FERM BP-6060) The mutant strain, the Swiss lactic acid bacteria CP3232 strain (Taiwan registration number BCRC 910539, Japanese registration number FERM BP-11271) or a variant thereof, and contains the following components (a), (b) and (c) or (a), (b) And (d) components: (a) free amino acid of 25 μmol/ml or more, (b) peptide of 8.5 mg/ml or more, (c) XP peptide and/or XPP peptide of 260 μg/ml or more in total (d) A VPP conversion amount of the unit acidity of the IPP peptide and the VPP peptide of 6.0 mg/g or more. The fermented milk of claim 12, which contains at least one species belonging to Swiss lactic acid The lactic acid bacteria of bacteria. The fermented milk of claim 12 or 13 which is at least one lactic acid bacteria according to any one of claims 1 to 4. The fermented milk of claim 12 or 13, wherein more than 15% of the free amino acid is a branched amino acid. The fermented milk of claim 12 or 13 which contains a YP peptide of 60 μg/ml or more. The fermented milk of claim 12 or 13 wherein the fermentation is carried out at a temperature above 30 ° C and below 34 ° C. The fermented milk of claim 12 or 13 is used as a blood pressure lowering agent, a brain function improving agent, an autonomic nervous regulator, a parasympathetic regulating agent, a sympathetic regulating agent, a nutritional adjuvant, an energy supplement, and a muscle decomposition damage inhibition. Agent, muscle enhancer, fatigue reliever, endurance improver, flavoring agent or alcohol metabolism agent. A food or drink comprising at least one lactic acid bacterium according to any one of claims 1 to 4 and/or a treatment thereof, and/or the fermented milk of any one of claims 12 to 18 and/or Its treatment. A method for producing an amino acid and/or a peptide, characterized in that at least one lactic acid bacterium according to any one of claims 1 to 4 is used to ferment animal milk or milk protein, and an amine is extracted from the obtained mash Base acid and / or peptide. The method of claim 20, wherein the amino acid is a branched amino acid. The method of claim 20 or 21, wherein the peptide is at least one selected from the group consisting of XP peptides and/or XPP peptides. The method of claim 20 or 21, wherein the fermentation is carried out at a temperature of from 30 ° C to 34 ° C.