JP2791375B2 - Production method of lactic acid bacteria fermented liquid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention is useful for a human body having an anticancer effect, a blood pressure lowering effect, an immunostimulatory effect, an intestinal regulating effect, an antibacterial effect, and an antimutagenic effect utilizing milk, which is called a complete diet. More specifically, the present invention relates to a method for producing a fermented lactic acid bacterium solution rich in a physiologically active substance, which is a metabolite of a lactic acid bacterium, by co-culturing a plurality of types of lactic acid bacteria and yeast.

[0002]

2. Description of the Related Art In recent years, human cancer mortality has been rapidly increasing. Chemical carcinogens that cause cancer are mutagens that themselves or their metabolically active substances modify DNA.
In recent years, it has been revealed that food contains an anti-mutagenic substance that suppresses the action of this mutagen, and research and development of substances having an anti-cancer effect have been active.

[0003] Ink marine derivatives which are analogs of the antibiotic MI43-37F11, which is conventionally known as an ink marine compound having a carcinostatic action as a substance having a therapeutic effect on cancer (for example, see JP-A-5-9)
No. 7841) and irinotecan hydrochloride which uses a plant extract called Kiki having an anticancer effect as a raw material.

[0004]

However, regardless of whether or not the raw material is a natural product, all existing anticancer drugs destroy not only cancer cells but also normal cells, and cause side effects such as leukopenia. There is a problem of being invited.

Accordingly, the present invention provides a lactic acid bacterium which has a function of food to extract useful physiologically active substances by utilizing microorganisms and has an antihypertensive action, anticancer, antibacterial and antimutagenic effect which does not cause side effects on the body. It is an object to provide a method for producing a fermentation liquor.

[0006] By utilizing the phenomenon of symbiotic action, a mother starter obtained by culturing low-acid type strains and high-acid type strains individually in milk is mixed and cultured in a milk medium at the same ratio to produce a fermented milk product. A method for producing a starter for use has been proposed (for example, see Japanese Patent Publication No. 45-35221). This is a method for producing a starter for producing fermented milk such as yogurt, which has a different raw acid rate so that acidity does not occur during cooling and preservation of a product such as yogurt. The invention is a completely different invention having a different purpose, configuration, and effect.

[0007]

Means for Solving the Problems The present inventors have found that milk, which is called complete diet, contains milk proteins such as casein and lactalbumin, and the amino acid sequence of the milk protein includes peptides having various physiological activities. Some of them have the same or very similar molecular sequences, and the inventors have focused on releasing a physiologically active substance when milk protein is degraded by a specific protease, and have come up with the present invention. That is, a fermented milk by lactic acid bacteria is produced under the idea that a specific enzyme that releases a physiologically active substance such as a peptide from milk protein is preferably an extracellular enzyme secreted by lactic acid bacteria, and the product is further aged. Then, in this thermogenesis process, proteins, lipids and the like in milk are decomposed to obtain low molecular organic acids such as peptides and fatty acids which are useful physiologically active substances for the human body.

As a means thereof, the method for producing a fermented lactic acid bacterium of the present invention is roughly divided into three steps: preculture, main culture, and extraction of a lactic acid bacterium fermented liquor. As the preculture, a plurality of types of lactic acid bacteria and bifidobacteria are individually inoculated into milk individually, and the inoculated products are individually cultured under an optimal temperature environment for the growth of each microorganism. On the other hand, yeast is inoculated into milk and cultured under an optimal temperature environment for an appropriate time. This yeast may be added to a nutrient solution containing vitamins, amino acids and the like without being cultured in milk.

[0009] Next, the respective cultures obtained by the pre-culture are individually collected, and the main culture is performed in which the inoculated milk is collectively inoculated in a different temperature environment for a suitable time.

[0010] The starter obtained at each temperature by the above main culture is added to milk at a predetermined ratio, and further cultured under a constant temperature environment for an appropriate time. The fermentation liquid is extracted from the fermentation curd obtained by this culture.

[0011]

When lactic acid bacteria are inoculated into milk as a single bacterium and cultured at an optimum temperature, lactic acid bacteria grow and produce fermented milk rich in metabolites of lactic acid bacteria.

[0012] When co-cultivating a plurality of types of lactic acid bacteria and yeast,
A complicated symbiotic relationship occurs between the constituent bacterial species. Bacteria produce metabolites extracellularly due to their self-propagation. As extracellular enzymes, they produce proteolytic enzymes, lipolytic enzymes, carbohydrate degrading enzymes, etc., and form an optimal environment for lactic acid bacteria.
As a result, the lactic acid bacteria are more activated, and produce more advantageous substances for the bacteria. Under such an optimal environment for lactic acid bacteria, lactic acid bacteria exert their functions sufficiently and produce products that are advantageous to the bacteria. The product is determined by a constituent bacterium such as an aroma component such as diacetyl, acetoin, or citric acid, an antibacterial component such as nisin, a low-molecular-weight amino acid or an organic acid. If this co-cultivation is performed under different temperature environments, the bacteria at the optimum temperature will proliferate and produce more metabolites. The reason why the present invention utilizes the extracellular enzyme secreted by the lactic acid bacterium is to obtain a balanced mixture of physiologically active substances. This is because a mixture of substances cannot be obtained.

When a starter obtained at each temperature is added to milk at a predetermined ratio and cultured, the pH of the fermented liquid is reduced by the generated lactic acid, and a fermented curd is formed near the isoelectric point. That is, the proteolytic enzyme secreted by the lactic acid bacteria produces casein to produce a fermentation curd containing casein. The fermentation liquid extracted from the fermentation curd contains a physiologically active substance such as a peptide produced by co-cultivation of the bacteria.

[0014]

Embodiment 1 Hereinafter, a preferred embodiment of the present invention will be described. Lactococcus lactis subsp.
Lactis (Lc. Lactis subsp lact)
is-), Lactococcus lactis subsp. cremoris (Lc. la-ctis subsp c.
remoris), Streptococcus lactis subsp. diaacetylactis (Str. lac)
is subsp diacetylactis), Leuconostoc cremoris (Leu. cr-emor)
is) is individually inoculated into animal milk having a concentration of about 25% (W / W) or less, and cultured for up to 48 hours in a constant temperature environment at an optimum temperature of each of the bacteria. When these lactic acid bacteria are cultured in an optimal temperature environment, these lactic acid bacteria grow individually in animal milk, and the lactic acid produced produces an acidity of about 0.9%.
(W / W) weak fermented milk is produced separately. Streptococcus salivarius subsp. Thermophilus (Str. Salivarius. Subs)
p. thermophil-us), Lactobacillus
Del Bricky Subsbysis. Bulgarix (L
b. delbrueckii subsp. bulga
ricus), Lactobacillus delbricii subsp. lactis (Lb. delblue-cki)
i subsp. lactis), Lactobacillus. Hervetics (Lb. helveticus) are individually inoculated into animal milk at a concentration of 25% (W / W) or less, and cultured for up to 48 hours in a constant temperature environment at an optimum temperature of these bacteria. When cultured under the above conditions, these lactic acid bacteria grow in animal milk, and fermented milk having an acidity of about 1.0% (W / W) is individually produced. Lactobacillus acidophilus (Lb. acidophilus) and Lactobacillus casei (Lb. casei) Bifidobacterium (Bifidobacterium. Longum) are each individually about 0.5% (W / W) containing about 25% (W / W) soy peptide (about 25%). (W / W) Inoculate animal milk at a concentration of not more than (W / W), and culture for 48 hours in a constant temperature environment, which is the optimum temperature for these bacteria. This culture enhances the activation of the strain. Saccharomyces cerevisiae (Sacchar
omyces. cerevisiae) is inoculated into animal milk containing yeast extract and glucose and cultured at about 30-32 ° C for up to about 48 hours. Saccharomyces cerevisiae may be a yeast solution added to an aqueous solution containing vitamins and amino acids without being cultured in milk. Yeasts are not limited to Saccharomyces cerevisiae, but include Saccharomyces del Bricky (Sacc.d.
elbrueckii), Torulopsis kefir, dried yeast and the like can also be used.

Each of the cultures obtained by the above pre-culture is inoculated in the same amount into animal milk having a concentration of about 25% (W / W) or less. The inoculation amount was 0.01 to 4.00% (V /
V), but preferably 2.00% (V / V) or less. After inoculation, high temperature within the range of 22 to 40 ° C,
Select each of the medium temperature and low temperature, about 2 each in each constant temperature environment
The culture was performed for 4 to 60 hours. The one cultured in a high temperature environment is referred to as a starter 1, the one cultured in a medium temperature environment is referred to as a starter 2, and the one cultured in a low temperature environment is referred to as a starter 3.
As a result of microscopic observation of the starter 1, the starter 2, and the starter 3 at a magnification of 400, the starter 1 contained one yeast in one visual field, and lactic acid bacteria contained bacilli and cocci at a ratio of 8: 2. Many long rods were observed. In the starter 2, three yeasts are present in one field of view, and lactobacilli and bacilli are present in the same ratio, and it is found that long bacilli, short bacilli, monococci, diplococci, and streptococci grow in a well-balanced manner. did. In the starter 3, it was found that one yeast per 10 fields of view, and lactobacilli were mostly abundant in bacillus and cocci in a ratio of 3 to 7, and particularly abundant in diplococci. The starter 1, starter 2, and starter 3 are shown in Table 1 below.
Was added to animal milk according to the composition described above and cultured at about 22 to 40 ° C. for each hour. The reason why the culture time is long is to obtain a metabolite rich in more physiologically active substances by symbiosis of the lactic acid bacteria and the enzyme.

[0016]

[Table 1]

The pH, the number of lactic acid bacteria and the number of yeasts of the obtained fermented curd were as shown in Table 2 below.

[0018]

[Table 2]

The obtained fermented curds are individually stirred while being heated to about 70 ° C. By this stirring and heating treatment, the fermented curd is separated into a solid and a liquid. After completion of the stirring, the mixture is allowed to cool to room temperature, and then, solids are removed. Although any elimination method may be used, in this embodiment, the solution is filtered with Melita filter paper (trade name of Melita Japan Co., Ltd.), and the obtained filtrate is heated and sterilized at about 80 ° C., then cooled, and lactic acid bacteria fermented. Obtain a liquid. This heat sterilization need not be performed. The obtained fermentation liquor has a clear yellow-brown color. This is because the lactic acid bacteria fermented liquid itself removed from the fermentation curd itself has an extremely strong antibacterial action, as shown in the test examples described below.

[0020]

Example 2 In the same manner as in Example 1 described above, Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies cremoris, Streptococcus lactis subspecies diacetillac, and Leuconostoc cremoris were each individually isolated. Specifically, animal milk having a concentration of about 25% (W / W) or less is inoculated, and cultured under a constant temperature environment at an optimum temperature of each of the bacteria for a maximum of 48 hours. When these lactic acid bacteria are cultured in an optimal temperature environment, these lactic acid bacteria are individually activated and propagated in animal milk. Streptococcus saliparius subspecies Thermophilus, Lactobacillus, Delbrikie subspecies Bulgarix, Lactobacillus delbricii subspecies Lactis,
Lactobacillus hervetics individually at a concentration of about 25
% (W / W) or less, and cultured for up to 48 hours in a constant temperature environment at the optimum temperature of each of these bacteria.
Lactobacillus acidophilus and Lactobacillus casei were individually inoculated into animal milk containing not more than 0.5% (W / W) soy peptide and having a concentration of about 25% (W / W) or less. Incubate for up to 48 hours under optimal temperature conditions. Saccharomyces cerevisiae is inoculated into milk containing yeast extract and glucose, at about 30-32 ° C.
For about 20-25 hours. This yeast Saccharomyces cerevisiae may be a yeast solution dissolved in an aqueous solution containing vitamins and amino acids without culturing it in milk. Dry yeast may be used as in Example 1 described above.

Each culture solution obtained by the above preculture is inoculated in the same amount to animal milk having a concentration of about 25% (W / W) or less. The inoculation amount was 0.01 to 4.00% (V /
V), particularly preferably 2.00% (V / V) or less. After inoculation, each of high temperature, medium temperature and low temperature is selected within the range of 22 to 40 ° C., and each temperature is about 24 to 60 under each constant temperature environment.
Cultured for hours. The one cultured in a high temperature environment is referred to as a starter 1, the one cultured in a medium temperature environment is referred to as a starter 2, and the one cultured in a low temperature environment is referred to as a starter 3. The starter 1, starter 2, and starter 3 were added at a final concentration of 0.1.
% To animal milk. The culture was allowed to stand still at about 31 ° C. for 120 hours to obtain a fermentation card. The pH of the obtained fermentation curd was 3.5, the number of lactic acid bacteria was 1.3 × 10 ⁹ cells / ml, and the number of yeast cells was 8.8 × 10 ⁵ cells / ml. The survival rate of lactic acid bacteria was high even after culturing for 120 hours. It has been found. The fermentation curd is heated to about 70 ° C. with stirring to separate the solid matter and the leachate, and then left to cool to room temperature. A filtrate is obtained by means of filtration or the like as in Example 1 described above.
The obtained filtrate is heat-sterilized at about 80 ° C. and then cooled to obtain a clear yellow-brown lactic acid bacteria fermented liquid. Sterilization treatment of the filtrate is not always necessary, as in Example 1 described above, because of its strong antibacterial action. The lactic acid bacteria fermentation liquor obtained according to this example is:
pH 3.5, lactic acid content 1.8% (W / W), sugar content 6.7
% (W / W), content of nitrogen (N) 0.07% (W / W)
It is 0.46% when the nitrogen content is converted into milk protein.
(W / W).

[0022]

Example 3 Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Cremoris, Streptococcus lactis subsp. Cerevisiae diacetylactis, and Leuconostoc cremoris each at a concentration of 12% (W /
The reduced skim milk of W) was inoculated and allowed to stand still for 21 hours under a constant temperature environment of 25 ° C. Streptococcus salivarius subspecies thermophilus, Lactobacillus delbricii subspecies bulgaricus, Lactobacillus delbricii subspecies subsp. The reduced skim milk was inoculated and allowed to stand still for 16 hours under a constant temperature environment of 37 ° C.
Lactobacillus acidophilus and Lactobacillus casei were individually inoculated into 12% (W / W) reduced skim milk containing 0.5% (W / W) soybean peptide, and 3%.
Each was cultured for 16 hours in a constant temperature environment of 7 ° C. for 16 hours. Saccharomyces cerevisiae was inoculated into a 12% (W / W) reduced skim milk containing 0.5% (W / W) soy peptide and 0.5% (W / W) glucose, at 31 ° C. The culture was allowed to stand in a constant temperature environment for 23 hours.

Each of the cultures obtained by the above pre-culture was subjected to a concentration of 1
0.25% (V / W) in 2% (W / W) reduced skim milk
V) Inoculate each. Inoculate for 3 minutes, 37 ° C, 3
The culture was allowed to stand still for 42 hours under a constant temperature environment of 1 ° C. and 25 ° C.
The one cultured in a constant temperature environment is referred to as a starter 1, the one cultured in a constant temperature environment of 31 ° C. is referred to as a starter 2, and the one cultured in a constant temperature environment of 25 ° C. is referred to as a starter 3. Starter 1, Starter 2 and Starter 3 were added to partially skim milk so that the final concentration was 0.1%. A fermentation curd was obtained by stationary culture for 120 hours under a constant temperature environment of 31 ° C. The pH of the obtained fermentation curd was 3.5, the number of lactic acid bacteria was 1.3 × 10 ⁹ cells / ml, and the number of yeast cells was 8.8 × 10 ⁵ cells / ml.
ml. As in the case of Example 1 or 2, the fermentation curd was heated to 70 ° C. while stirring, and then left to cool in a room temperature environment. Next, the mixture was filtered to obtain a filtrate.
And then cooled to obtain a lactic acid bacteria fermented liquid. The obtained fermented lactic acid bacterium has a clear yellow-brown color.

[0024]

Example 4 Pre-culture and the like were carried out in the same steps as in Example 3 to obtain starters 1, 2 and 3. Starter 1, Starter 2, Starter 3
Is added to the partially skimmed milk to a final concentration of 0.1%,
A fermentation curd was obtained by statically culturing in a constant temperature environment of 31 ° C. for 480 hours. The pH of the obtained fermentation curd was 3.5, the number of lactic acid bacteria was 1.1 × 10 ⁷ cells / ml, and the number of yeast cells was 3.9 × 10 ⁶ cells / ml. The fermentation curd was heated to 70 ° C. while being stirred and cooled to room temperature in the same manner as in Example 3, and then filtered to obtain a fermented lactic acid bacterium having a transparent yellowish brown color.

In the first, second, third and fourth embodiments,
Streptococcus, Lactobacillus, and Leuconostoc species were used as lactic acid bacteria, but the present invention is not limited thereto, and the present invention is not limited thereto. The use of lactic acid bacteria of the present invention is also included in the present invention. Further, the culturing time, temperature, inoculation ratio in the main culturing and the like are not limited to the values shown in Examples 1, 2, 3, and 4, but those based on other values are also included in the present invention. This is because although the obtained lactic acid bacteria solution may change in taste and the like, its action and effect are the same.

Further, as shown in the above-mentioned embodiment, the method for obtaining a physiologically active substance by extracting extracellular enzymes by using a microorganism for the function provided in food is only a production method for obtaining a physiologically active substance from milk and lactic acid bacteria. In addition, for example, it can be adjusted from natto or the like.

[0027]

[Test Example 1] Six strains of Escherichia coli (Escherichia coli) and Vibrio parahaemolyticu (Vibrio parahaemolyticus).
s), Salmonella typhi
imurium), Bifidobacterium bifidum (Bifidobacterium bifid-u)
m), Clostridium sporogenes (Clo)
strium sporogenes, Bacteroides vulgartus (Bacteroides vulg)
atus) was used as a test bacterium to measure antimicrobial activity. The antibacterial activity was measured by the paper disk method. A paper disc impregnated with 50 microliters of a fermented lactic acid bacterium solution was placed on 10 ml of an agar plate medium, and 48
Cultured for hours. The diameter of a circular transparent inhibition circle (bacteriolysis plaque) formed around the disk was measured and compared with a control disk (50 microliter of lactic acid aqueous solution of pH 3.5). The results are shown in Table 3 below, and the lactic acid bacteria fermented solution was No. 3 With the exception of Vibrio parahaemolyticus and Salmonella, antibacterial activity was observed except for No. 5. In the case of E. coli and Clostridium sporogenes,
o. No. 3 against E. coli. In No. 4, antibacterial activity was detected against Escherichia coli and Pacteroides burgatas. Table 3
Medium, samples 1, 2, and 3 were lactic acid bacteria fermented liquid 1 shown in Table 2 above.
The lactic acid bacteria fermented solution 2, the lactic acid bacteria fermented solution 3, and the sample 4 are the lactic acid bacteria fermented solution obtained in Example 2, and 5 is Bifidobacterium (Bifidob).
Lactobacillus lactis (La), which is obtained by culturing Lactobacillus acterium longum as a single bacterium.
Lactobacillus bulgaricus (Lactobacillus bulgari), a fermented solution of lactic acid bacteria obtained by culturing C. bacillus l-actis as a single cell.
lactic acid bacterium fermented broth obtained by culturing bacillus cus) with a single bacterium, 8 is Lactobacillus acidophilus (Lactoba-cil)
lactic acid bacterium obtained by culturing L. acidophilus as a single bacterium; 9 is Streptococcus thermophilus (Streptococcus thermophilus);
lactic acid bacterium fermented broth 10 obtained by culturing lac) alone. ++ indicates that the diameter of the lysis spot is 15 mm or more, + indicates that the diameter of the lysis spot is 10 mm or more, and ± indicates that the diameter of the lysis spot is 10 mm.
Less than mm,-indicates that no hot spot was observed.

[0028]

[Table 3]

[0029]

[Test Example 2] Seven types of Escherichia coli (Escherich)
ia coli), Vibrio parahaemolyticus (Vibrio p.
arahaemolyticus, Salmonella (S)
a-lmonella typhimurium), Bifidobacterium bifidum (Bifidobacter)
terium bifidum, Clostridium sporodines sp.
rogenes), Bacteroides burgatas (Ba)
cteroides vulgatas, Staphylococcus aureus (Staphylococcus)
aureus) was used as a test bacterium to measure the antibacterial activity. The antibacterial activity was measured by the paper disk method. Lactic acid bacteria fermentation solution 5 on 10 ml agar plate medium
A paper disk impregnated with 0 microliter was placed on the plate and cultured for 48 hours. The diameter of a circular transparent inhibition circle (bacteriolysis plaque) formed around the disk was measured and compared with a control disk (50 microliter of lactic acid aqueous solution of pH 3.5). As a sample, 5 ml of a lactic acid bacteria fermented solution was freeze-dried, and a freeze-dried sample was dissolved in distilled water. The pH was adjusted to 3.6 with an aqueous solution of sodium hydroxide, and the final volume was adjusted to 1 ml to obtain a 5-fold concentrated lactic acid bacteria fermented solution. The results are as shown in Table 4 below. Except for Example 5, antibacterial activity was observed for all test bacteria used. In particular, NO. 1 and No.
No. 4 exhibited strong antibacterial activity against Escherichia coli and Clostridium sporogenes. Samples 1 to 10 were the same as in Test Example 1, and the symbols indicating the results also had the same meaning.

[0030]

[Table 4]

[0031]

[Test Example 3] Seven types of Escherichia coli (Escherich)
ia coli), Vibrio parahaemolyticus (Vibrio p.
arahaemolyticus, Salmonella (S)
almonella typhimurius, Bifidobacterium Bifidobacter
erium bifidum, Clostridium sporogenes
geneses), Bacteroides burgatas (Bact)
eroides vulgatas, Staphylococcus aureus (Staphylococcus a)
ureus) was used as a test bacterium, and the ascent fraction and the antibacterial activity of the fermented lactic acid bacteria solution were measured. The antibacterial activity was measured by the paper disk method. A paper disk impregnated with 50 microliters of a fermented lactic acid bacterium solution was placed on 10 ml of an agar plate medium and cultured for 48 hours. The diameter of a circular transparent blocking circle (bacteriolytic plaque) formed around the disk was measured and compared with a control disk (50 microliter of a lactic acid aqueous solution of PH 3.5). Acetone fractionation is carried out by adding 6.7 times the volume of cold acetone (-20 ° C) to 1 ml of the lactic acid bacteria fermentation broth and stretching and separating (3000 r).
(pm × 5 min), the supernatant and the precipitate were dried under reduced pressure, and dissolved in distilled water to adjust the pH to 3.5. The final dissolution volume was 1 milliliter, and 50 microliters were used for the measurement of antibacterial activity. The supernatant was concentrated under reduced pressure until the smell of acetone disappeared, and distilled water was added to dissolve the supernatant to adjust the pH to 3.5. The final volume was 1 milliliter and 50 microliters were used for measuring antimicrobial activity. As shown in Table 5 below, the results were as follows.
Samples 1, 2, 3, and 6 exhibited antibacterial activity for a). This active ingredient is considered to be a relatively high molecular weight peptide or protein. Samples 1 to 10 are the same as those in Test Examples 1 and 2, and the reference numerals indicating the results also have the same meaning.

[0032]

[Table 5]

The antimicrobial activity of the acetone supernatant fraction is shown in Table 6 below.
As shown in FIG. As shown in Table 6, the antibacterial activity of the lactic acid bacteria fermented liquid was mainly observed in the acetone fraction supernatant. In addition, No. 1 was obtained by acetone fractionation. 3 and No.
In No. 4, antibacterial activity against Clostridium is detected. This would indicate that the acetone-precipitated fraction contained components that neutralized its activity and was thus removed by fractionation. Samples 1 to 10 are the same as in Test Examples 1, 2 and 3, and the reference numerals indicating the results also have the same meaning.

[0034]

[Table 6]

[0035]

[Test Example 4] Vibrio parahaemolyticus (Vibrio para)
haemolyticus) was used as a test bacterium, and the pH-dependent antibacterial activity of the fermented lactic acid bacterium obtained in Example 2 on Vibrio parahaemolyticus was measured. The antibacterial activity was measured by the paper disk method. Agar plate medium 10
A paper disk impregnated with 50 microliters of a fermented lactic acid bacterium solution was placed on the milliliter and cultured for 48 hours. The diameter of a circular transparent blocking circle (bacteriolysis spot) formed around the disk was measured and compared with the control disks (50 microliter of hydrochloric acid aqueous solution, 50 microliter of acetic acid aqueous solution, and 50 microliter of lactic acid aqueous solution). The pH of the fermented lactic acid bacterium of Example 2 was adjusted to 6.9, the precipitate was removed by centrifugation, the supernatant was recovered, and the pH was adjusted to individual pH with an aqueous hydrochloric acid solution, and the antibacterial activity was measured. As a control, hydrochloric acid, acetic acid, and lactic acid were added to distilled water, and the antibacterial activity was also measured for those adjusted to the same pH. The results are as shown in Table 7 below. The antibacterial activity of the fermented lactic acid bacterium against Vibrio parahaemolyticus was PH5.
0 or less, and particularly strong at PH 3.6 or less. An antibacterial activity is observed in an aqueous acetic acid solution having a pH of 3.0, but no antibacterial activity is observed in an aqueous hydrochloric acid solution and an aqueous lactic acid solution having the same pH. The symbols indicating the results in Table 7 are those in Table 1 above.
-The same meaning as shown in Table 6 is shown.

[0036]

[Table 7]

[0037]

Test Example 5 A test was conducted on the antimutagenic activity of the fermented lactic acid bacterium obtained in Examples 1 and 2. Many chemical carcinogens are mutagens that themselves or their metabolically active substances modify DNA. An anti-mutagen refers to a substance that inhibits the action of this mutagen and that prevents carcinogenesis. The test strains used as materials include Salmonella TA98 (Salmonella) having histidine auxotrophy, ampicillin resistance, and a frameshift mutation.
typhimurium TA98). As a mutagenic agent, 3-amino-1-methyl-5H-pyrido [4,3-b] indole (Trp to p2) is 0.5
At a concentration of μg / ml, N-methyl-N′-nitro-N-
Nitrosoguanidine (MNNG) was used at a concentration of 10 μg / ml. The method of measuring antimutagenic activity is a method applying the Salmonella mutagen test method (Ames test).
00 microliters of Trp-p2 (or MNN)
G), 100 microliters of lactic acid bacteria fermentation broth and 10
Mix 0 microliters of the S-9 mix (microsome preparation), then add 100 microliters of Salmonella culture and mix with 2 ml of an agar solution containing biotin-histidine maintained at 36 ° C. for 20 minutes. -Bonner E Medium 3
Overlay 0 ml. Incubate at 37 ° C for 48 hours,
Count the colonies. The test was performed in triplicate. The results show that for Trp-p2, as shown in Table 8 below, N
o. 4 showed the strongest anti-mutagenicity (73%).

[0038]

[Table 8]

For MNNG, as shown in Table 9 below, No. 1 and No. 4 showed strong antimutagenicity (75%).

[0040]

[Table 9]

Samples 1 to 10 in Tables 8 and 9 are the same as Test Examples 1 to 4.

Higher anti-mutagenicity is observed in a culture in which various lactic acid bacteria including yeasts are symbiotic than a lactic acid bacterium fermented liquid cultivated as a whole. That is, physiologically active factors related to maintenance of human health, such as antibacterial action, anticancer action, and anticholesterol action, of the obtained lactic acid bacteria fermented liquid, which is the object of the present invention, are present in the fermented liquids (No. 1 to No. 4). This is demonstrated by Tables 8 and 9.

[0043]

Test Example 6 A test on the blood pressure lowering effect of the fermented lactic acid bacterium obtained in Example 4 was conducted. Average weight 3 at 20 weeks of age
12 male rats weighing 51.6 g were treated at a temperature of 24 ° C. and a humidity of 50%.
Were illuminated with a 12-hour cycle, and the feed was fed to the test using a solid feed for breeding (a product of Oriental Co., Ltd.) throughout the entire test period, with tap water being freely taken. After breeding for 4 weeks in this way, the rats were fasted for 14 hours, and 6 rats of Group I were orally administered with 5 ml of fermented lactic acid bacteria solution, and rats of Group II were orally administered with 5 ml of water. 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 4 hours after administration
Blood pressure was measured after 8 hours. As a condition for the measurement, the sample was left in a temperature environment of 38 ° C. for 10 minutes before the measurement.
After 12 hours from the start of the test, the animals were allowed to freely ingest the feed. Rat, mouse non-invasive automatic blood pressure monitor TK-350
Blood pressure was measured using (trade name of MMI Service Co., Ltd.), and the results are as shown in Tables 10 and 11 below.

[0044]

[Table 10]

[0045]

[Table 11]

From the above Tables 10 and 11, it can be seen that the blood pressure of the rats to which the fermented lactic acid bacterium solution was administered was lowered 6, 9, and 12 hours later. In particular, the average descent is 6
After 39 hours, it reached 39%, and after 9 hours, it reached 37%. The slight decrease in blood pressure after 3 hours and 6 hours of the rat is due to daily changes, and it is considered that the blood pressure of the rat slightly decreased because the rat entered a resting state in the daytime.

[0047]

Industrial Applicability The present invention produces a physiologically active substance rich in metabolites of lactic acid bacteria and yeast, since a co-cultivation of various lactic acid bacteria and yeast is performed with a special starter without using any chemical additives. However, as shown in the results of Test Examples 1 to 5, the lactic acid bacteria fermented solution obtained according to the present invention has an antibacterial effect, an anticancer effect, an immunostimulatory effect, an antimutagenic effect that suppresses the expression of carcinogens, and the like. Yes, and Test Examples 10 and 1
As shown in the result of No. 1, it has a blood pressure lowering effect and has a remarkable effect in maintaining human health.

In addition, the present invention is a method of using a microorganism, extracting extracellular enzymes, and extracting a physiologically active substance from foods, since it has the function inherent in foods. There is an effect that it is safe just to do. Therefore, the obtained lactic acid bacteria fermented liquid has an effect that it has a wide range of uses not only for pharmaceuticals but also for food materials and the like.

Claims (1)

(57) [Claims] 1. A method of inoculating milk individually with a plurality of types of lactic acid bacteria and bifidobacteria, culturing the inoculated products individually for an appropriate time under an optimum temperature environment for the growth of each bacterium, and then obtaining each cultured bacterium. A suitable amount of the liquid is individually collected, and after inoculating the milk together with the yeast in a batch at a different temperature environment for a suitable time, each starter obtained at each temperature is added to the milk at a predetermined ratio. A method for producing a fermented lactic acid bacterium, comprising fermenting a fermented liquor from the obtained fermentation curd after culturing for a certain time in a constant temperature environment.