JPH07155103A - Method for producing lactic bacteria fermentation solution - Google Patents

Abstract

PURPOSE:To obtain a lactic bacteria fermentation solution not having side effects and having anticancer, antibacterial and antimutagenic effects by producing a fermented milk with lactic acid bacteria, and subsequently maturing the fermented milk to produce the peptides of physiologically active substances useful for human bodies, low molecular weight organic acids such as fatty acids, etc. CONSTITUTION:Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris, Streptococcus lactis subsp cliacetylactis, Leuconostoc cremoris, etc., are individually inoculated on milk and cultured at the optimal growth temperatures for the respective bacteria. Each cultured bacterium solution is inoculated on a milk in a proper amount, and subsequently cultured at a different temperature. The starter obtained at each temperature is added to a milk in a prescribed ratio. The mixture is cultured under a constant temperature environment, and subsequently the fermentation solution is extracted from the obtained fermented curd.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a fermented lactic acid bacterium which is useful for the human body and which has an anticancer action, an immunostimulating action, an intestinal regulating action, an antibacterial action and an antimutagenic action using milk, which is said to be a complete diet. More specifically, the present invention relates to a method for producing a lactic acid bacterium fermented liquid rich in physiologically active substances, which are metabolites, by co-culturing a plurality of lactic acid bacteria and yeast.

[0002]

2. Description of the Related Art In recent years, human cancer mortality has been increasing rapidly. A chemical carcinogen that causes cancer is a mutagen that modifies DNA by itself or its metabolically active substance.
In recent years, it has become clear that foods contain an antimutagenic substance that suppresses the action of this mutagen, and research and development of substances having an anticancer activity have become active.

[0003] Conventionally, it relates to an inkmarine derivative which is an analog of the antibiotic MI43-37F11, which is known as an inkmarin compound having an antitumor effect as a substance having a therapeutic effect on cancer (for example, JP-A-5-9).
No. 7841), and irinotecan hydrochloride, which uses a plant extract called Kiki, which has an anticancer action, as a raw material.

[0004]

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

[0005] Therefore, the present invention is to produce a lactic acid bacterium fermented liquid having anti-cancer, anti-bacterial and anti-mutagenic effects that bring out useful physiologically active substances by utilizing the functions of foods using microorganisms and have no side effects on the body. The purpose is to provide a method.

[0006]

Means for Solving the Problems The present inventors have found that milk, which is said to be a complete diet, contains milk proteins such as casein and lactalbumin, and the amino acid sequences of the milk proteins include peptides having various physiological activities. Some of them have the same or very similar molecular sequences, and the present invention was devised by paying attention to the fact that a physiologically active substance is released when a milk protein is decomposed by a specific proteolytic enzyme. That is, under the idea that the extracellular enzyme secreted by lactic acid bacteria is suitable as the specific enzyme for releasing physiologically active substances such as peptides from milk protein, fermented milk by lactic acid bacteria is produced and the product is further aged. During this ripening process, proteins, lipids and the like in milk are decomposed to obtain peptides, low molecular weight organic acids such as fatty acids, which are useful physiologically active substances for the human body.

As a means therefor, the method for producing a lactic acid bacterium fermented liquid of the present invention is roughly divided into three steps of pre-culture, main culture and extraction of the lactic acid bacterium fermented liquid. As the preculture, a plurality of types of lactic acid bacteria and, if necessary, bifidobacteria are individually inoculated into milk as a single bacterium, and the inoculated ones are individually cultivated under an optimal growth temperature environment for each bacterium. On the other hand, yeast is inoculated into milk and cultured in an optimal temperature environment for an appropriate time. This yeast may be added to a nutrient solution containing vitamins, amino acids, etc. without culturing it in milk.

Next, each culture bacterium solution obtained by the above-mentioned pre-culture is individually collected, and the inoculated milk is collectively inoculated into milk, which is then cultivated for a proper time under different temperature environments to carry out main culture.

The starter obtained at each temperature by the main culture is added to milk at a predetermined ratio, and further cultured for a suitable time in a constant temperature environment. The fermentation liquid is extracted from the fermentation card obtained by this culture.

[0010]

[Function] When milk is inoculated with lactic acid bacteria as a single bacterium and cultured at an optimum temperature, the lactic acid bacteria grow and fermented milk rich in metabolites of lactic acid bacteria is produced.

When co-culturing a plurality of lactic acid bacteria and yeast,
A complex symbiotic relationship occurs between the constituent bacterial species. Due to its self-propagating property, the bacterium produces a metabolite outside the microbial cell. As extracellular enzymes, proteolytic enzymes, lipolytic enzymes, carbohydrate degrading enzymes and the like are produced to form an optimal environment for lactic acid bacteria.
Therefore, the lactic acid bacterium is further activated, and a substance more beneficial to the bacterium is further produced. Under such an optimal environment for lactic acid bacteria, the lactic acid bacteria fully exert their functions and produce products advantageous for the bacteria. The products are determined by the constituent bacteria such as aroma components such as diacetyl, acetoin and citric acid, antibacterial components such as nisin, low molecular weight amino acids and organic acids. When this co-cultivation is carried out under different temperature environments, the bacteria whose temperatures are optimal are proliferatively active, and metabolites are produced in larger amounts. Thus, the purpose of the present invention to utilize the extracellular enzyme secreted by lactic acid bacteria is to obtain a well-balanced mixture of physiologically active substances, and for an exogenous enzyme, a well-balanced physiological activity as in the present invention. This is because it is not possible to obtain a mixture of substances.

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

[0013]

Embodiment 1 A preferred embodiment of the present invention will be described below. Lactococcus lactis subsposis
Lactis (Lc. Lactis subsp lact)
is), Lactococcus lactis subspices cremoris (Lc.la-ctis subsp c
remoris), Streptococcus lactis subsposis diacetilactis (Str. lact)
is subsp diacylactis), Leuconostoc cremoris (Leu. cr-emor)
is) is individually inoculated into animal milk at a concentration of about 25% (W / W) or less, and cultured under a constant temperature environment of the optimum temperature of each of the above bacteria for up to 48 hours. When these lactic acid bacteria are cultivated in the optimum temperature environment, these lactic acid bacteria grow individually in animal milk, and the acidity of about 0.9% is produced by the lactic acid produced.
Fermented milk of weak (W / W) is produced separately. Streptococcus salivarius subsp. Thermophilus (Str. Salivarus. Subs
p. thermophil-us), Lactobacillus
Del Bricky Sub-Spiesis Bulgarix (L
b. delbrückii subsp. bulga
ricus), Lactobacillus delbruecki subsposis lactis (Lb. delbrue-cki)
i subsp. lactis) and Lactobacillus helveticus (Lb. helveticus) are individually inoculated into animal milk at a concentration of 25% (W / W) or less, and the maximum temperature of these bacteria is 48 hours in a constant temperature environment. Culture as. 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), Lactobacillus casei (Lb. casei) Bifidobacterium (Bifidobacterium. Longum) each individually containing about 25% (W / W) soybean peptide of about 25% ( W / W) or less is inoculated into animal milk at a concentration of not more than, and cultivated in a constant temperature environment, which is the optimum temperature of these bacteria, for up to 48 hours. This culture enhances the activation of the strain. Saccharomyces cerevisiae (Sacchar
omyces. cerevisiae) is inoculated into animal milk containing yeast extract and glucose and cultivated at about 30 to 32 ° C for about 48 hours. Saccharomyces cerevisiae may be a yeast solution added to an aqueous solution containing vitamins and amino acids without culturing in milk. The yeast is not limited to Saccharomyces cerevisiae, but may be Saccharomyces delbricii (Sacc.d).
Elbrueckii), Torulopsis kefir, dried yeast and the like can also be used.

The same amount of each culture solution obtained by the above preculture is inoculated into animal milk having a concentration of about 25% (W / W) or less. The inoculation amount is 0.01 to 4.00% (V /
V), but it is preferably 2.00% (V / V) or less. After inoculation, high temperature in the range of 22-40 ℃,
Select each of medium temperature and low temperature, and about 2 in each constant temperature environment
It was cultured for 4 to 60 hours. What was cultivated in a high temperature environment is called a starter 1, what was cultivated in a medium temperature environment was a starter 2, and what was cultivated in a low temperature environment was a starter 3.
As a result of microscopic observation of Starter 1, Starter 2 and Starter 3 at 400 magnifications, in Starter 1, one yeast was present in one field of view, lactobacilli and bacilli and cocci were present in a ratio of 8: 2, and as a whole, Many long rods were observed. In Starter 2, three yeasts per field of view, lactobacillus and cocci were present in the same ratio in lactic acid bacteria, and it was found that long-coccus, short-coccus, monococcal, dicoccal, and streptococcus are all growing in a well-balanced manner. did. It was found that in the starter 3, yeast was one in 10 fields of view, and lactobacilli were 3 to 7 in the ratio of lactobacilli and cocci in total, and it was found that there were a large number of cocci, especially dicoccus. The following Table 1 shows the starter 1, the starter 2 and the starter 3.
Was added to animal milk according to the above composition 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 physiologically active substances due to the symbiosis of the lactic acid bacterium and the enzyme.

[0015]

[Table 1]

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

[0017]

[Table 2]

The fermented curds thus obtained are individually stirred while heating to about 70.degree. By this stirring and heating treatment, the fermentation curd is separated into a solid matter and a liquid. After completion of stirring, the mixture is left to cool to room temperature and then solid matters are removed. Any method may be used for removal, but in the present embodiment, the resulting filtrate is filtered through Melita filter paper (trade name of Melita Japan Co., Ltd.), and the resulting filtrate is heat-sterilized at about 80 ° C. and then cooled to ferment lactic acid bacteria. Get the liquid. This heat sterilization treatment may not be performed. This is because the lactic acid bacterium fermentation liquid itself removed from the fermentation card has an extremely strong antibacterial action, as shown in a test example described later.

[0019]

[Example 2] As in Example 1 described above, Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies cremoris, Streptococcus lactis subspecies diacetilactis, and leuconostoc cremoris were individually separated. The animal milk is inoculated into the animal milk at a concentration of about 25% (W / W) or less, and cultivated under a constant temperature environment of the optimum temperature of each bacterium for up to 48 hours. When these lactic acid bacteria are cultivated in an optimal temperature environment, these lactic acid bacteria are individually activated and proliferate in animal milk. Streptococcus salivarius subspecies thermophilus, Lactobacillus, delbricky subspecies bulgaricus, Lactobacillus delbricky subspecies lactis,
Individual Lactobacillus helveticus concentration of about 25
% (W / W) or less of animal milk is inoculated and cultivated under a constant temperature environment of the optimum temperature of each of these bacteria for up to 48 hours.
Lactobacillus acidophilus and Lactobacillus casei were individually inoculated into animal milk containing 0.5% (W / W) soybean peptide at a concentration of about 25% (W / W) or less. Incubate for up to about 48 hours in the optimal temperature environment. Saccharomyces cerevisiae was inoculated into milk containing yeast extract and glucose, and the temperature was about 30 to 32 ° C.
Incubate for about 20 to 25 hours. This yeast Saccharomyces cerevisiae may be a yeast solution dissolved in an aqueous solution containing vitamins and amino acids without culturing in milk. Dry yeast may be used as in Example 1 above.

The same amount of each culture solution obtained by the above preculture is inoculated into animal milk having a concentration of about 25% (W / W) or less. The inoculation amount is 0.01 to 4.00% (V /
V) is particularly preferably 2.00% (V / V) or less. After inoculation, select high temperature, medium temperature and low temperature within the range of 22 to 40 ° C, and about 24 to 60 in each constant temperature environment.
Incubated for hours. What was cultivated in a high temperature environment is called a starter 1, what was cultivated in a medium temperature environment was a starter 2, and what was cultivated in a low temperature environment was a starter 3. The starter 1, the starter 2 and the starter 3 were mixed at a final concentration of 0.1
% Was added to the animal milk. The fermentation curd was obtained by statically culturing at about 31 ° C. for 120 hours. The pH of the obtained fermentation card is 3.5, the number of lactic acid bacteria is 1.3 × 10 ⁹ / ml, and the number of yeasts is 8.8 × 10 ⁶ cells / ml, and the survival rate of lactic acid bacteria is high even after 120 hours of culture. There was found. This fermented curd is heated to about 70 ° C. with stirring, the solid matter and its leachate are separated, allowed to stand and cooled to room temperature, and a filtrate is obtained by a means such as filtration as in Example 1 described above. The obtained filtrate is heat-sterilized at about 80 ° C. and then cooled to obtain a lactic acid bacterium fermentation liquid. The sterilization treatment of the filtrate is not always necessary due to its strong antibacterial action as in the above-mentioned Example 1.
The lactic acid bacterium fermentation broth obtained in this example had a pH of 3.5, a lactic acid content of 1.8% (W / W) {sugar content of 6.7% (W / W),
The nitrogen (N) content was 0.07% (W / W), and when the nitrogen content was converted to milk protein, it was 0.46% (W / W).

In Examples 1 and 2 described above, Streptococcus, Lactobacillus (La)
ctobacillus), Leuconostoc (Le
Although u-conostoc) was used as the lactic acid bacterium, the present invention is not limited to this and the present invention also includes the case of using all kinds of lactic acid bacteria corresponding to other genera. Also, the culture time, temperature, inoculation ratio in the main culture, etc. are not limited to the values shown in Examples 1 and 2 above, and values other than these values are also included in the present invention. This is because although the obtained lactic acid bacterium solution may show changes in taste and the like, its action and effect are the same.

Further, as shown in the above-mentioned Examples, the only method for obtaining a physiologically active substance by using microorganisms to extract extracellular enzymes using the function of food is to obtain a physiologically active substance from milk and lactic acid bacteria. Instead, it can be adjusted, for example, from natto.

[0023]

[Test Example 1] Six strains of Escherichia coli (Escherichia co-li) and Vibrio parahaemolyticus
s), Salmonella typh
Imurium), Bifidobacterium bifid-u
m), Clostridium sporogenes (Clo
strideum sporogenes, Bacteroides vulgatas
Attus) was used as a test bacterium to measure antibacterial activity. The antibacterial activity was measured by the paper disc method. Place a paper disk impregnated with 50 microliters of fermentation liquid of lactic acid bacteria on 10 milliliters of agar plate medium, and
Incubated for hours. The diameter of a circular transparent blocking circle (a lysate) formed around the disc was measured and compared with that of a control disc (50 microliters of a lactic acid aqueous solution having a pH of 3.5). The results are shown in Table 3 below. With the exception of No. 5, antibacterial activity against Vibrio parahaemolyticus and Salmonella was observed. In 1 against N. coli and Clostridium sporogenes,
o. No. 3 is for E. coli. The antibacterial activity of 4 was detected against Escherichia coli and Bacteroides vulgatus. Table 3
Samples 1, 2, and 3 are lactic acid bacterium fermentation liquid 1 shown in Table 2 above.
Lactic acid bacterium fermentation liquid 2, lactic acid bacterium fermentation liquid 3, sample 4 is lactic acid bacterium fermentation liquid obtained in Example 2, 5 is Bifidobacterium Bifidoba
Lactobacillus lactis (Lac)
Lactobacillus bulgaricus (Lactobacillus bulgaricus) was obtained by culturing Tobacillus l-actis with a single bacterium, and 7 was Lactobacillus bulgaricus.
Lactobacillus acidophilus (Lactobacillus)
Lactobacillus fermented broth obtained by culturing Us. acidophilus) with a single bacterium, 9 is Streptococcus thermophilus
Lactobacillus fermented liquor 10 obtained by culturing ilatus) as a single bacterium is a control. ++, the diameter of the lytic zone is 15 mm or more, ++
Indicates the diameter of the lytic plaque is 10 mm or more, ± indicates the diameter of the lytic plaque is 1
Less than 0 mm, -indicates that no lytic plaque is observed.

[0024]

[Table 3]

[0025]

[Test Example 2] Seven strains of Escherichia coli (Escherich
ia coli), Vibrio parahaemolyticus (Vibrio p
arahaemolyticus), Salmonella (S)
a-lmonella typhimurium), Bifidobacterium (Bifidobac)
terium bifidum), Clostridium spo
rogenes), Bacteroides burgatas (Ba)
cteroides vulgatus, Staphylococcus aureus
aureus) was used as a test bacterium to measure antibacterial activity. The antibacterial activity was measured by the paper disc method. Lactobacillus fermentation liquid 5 on 10 ml of agar plate medium
A paper disc impregnated with 0 microliter was placed and incubated for 48 hours. The diameter of a circular transparent blocking circle (a lysate) formed around the disc was measured and compared with that of a control disc (50 microliters of a lactic acid aqueous solution having a pH of 3.5). As a sample, 5 ml of the fermentation liquid of lactic acid bacteria was freeze-dried, and the freeze-dried preparation was dissolved in distilled water. The pH was adjusted to 3.6 with an aqueous sodium hydroxide solution, and the final volume was adjusted to 1 ml to prepare a 5 times concentrated lactic acid bacterium fermentation broth. As a result, as shown in Table 4 below, the antibacterial activity of the 5-fold concentrated liquid was No. With the exception of 5, antibacterial activity was observed against all the test bacteria used. Especially No. 1 and No.
4 had a strong antibacterial activity against Escherichia coli and Clostridium sporogenes. Samples 1 to 10 are the same as those in Test Example 1, and the symbols indicating the results also have the same meaning.

[0026]

[Table 4]

[0027]

[Test Example 3] Seven strains of Escherichia coli (Escherich
ia coli), Vibrio parahaemolyticus (Vibrio p
arahaemolytius, Salmonella (S
a-lmonella typhimurius), Bichidobacterium bufidam (Bifidobac)
terium bifidum), Clostridium spo
rogenes), Bacteroides burgatas (Ba)
cteroides vulgatus, Staphylococcus aureus
aureus) was used as a test bacterium and the acetone fraction and antibacterial activity of the lactic acid bacterium fermentation broth were measured. The antibacterial activity was measured by the paper disc method. A paper disk impregnated with 50 microliters of the fermentation liquid of lactic acid bacteria was placed on 10 ml of the agar plate medium and cultured for 48 hours.
Circular transparent blocking circle (lytic zone) formed around the disk
Was measured and compared with a control disk (50 microliters of a lactic acid aqueous solution having a pH of 3.5). Acetone fractionation was carried out by adding 6.7 times the volume of cold acetone (-20 ° C) to 1 ml of the fermentation liquid of lactic acid bacteria and centrifuging (300
The supernatant and the precipitate fractions were dried under reduced pressure (0 rpm × 5 min) and dissolved in distilled water to adjust the pH to 3.5.
The final dissolution volume was 1 milliliter and 50 microliters was used for the measurement of antibacterial activity. The supernatant fraction was concentrated under reduced pressure until the odor of acetone disappeared, and distilled water was added to dissolve it and the pH was adjusted to 3.5. The final volume was 1 milliliter and 50 microliter was used for the measurement of antibacterial activity. The results are shown in Table 5 below, with the antimicrobial activity Salmonella in the acetone precipitation category.
1), antibacterial activity was observed in Samples 1, 2, 3 and 6. The active ingredient is considered to be a relatively high molecular weight peptide or protein. Samples 1-10
Is the same as that of Test Examples 1 and 2, and the reference numerals indicating the results have the same meanings.

[0028]

[Table 5]

The antibacterial activity of the acetone supernatant category is shown in Table 6 below.
As shown in. As shown in Table 6, the antibacterial activity of the fermentation liquid of lactic acid bacteria was found mainly in the acetone fraction supernatant section. Further, by fractionating with acetone, No. 3 and No.
4, the antibacterial activity against Clostridium is detected. This would indicate that it was removed by fractionation because the acetone precipitation compartment contained components that neutralized its activity. Samples 1 to 10 are the same as Test Examples 1, 2 and 3, and the reference numerals indicating the results also have the same meaning.

[0030]

[Table 6]

[0031]

[Test Example 4] Vibrio para
Haemolyticus) was used as a test bacterium, and the pH dependence of the antibacterial activity of the lactic acid bacterium fermentation liquid obtained in Example 2 against Vibrio parahaemolyticus was measured. The antibacterial activity was measured by the paper disc method. Agar plate medium 1
A paper disk impregnated with 50 microliters of the lactic acid bacterium fermentation liquid was placed on 0 milliliter and cultured for 48 hours. The diameter of the circular transparent inhibition circle (bacteriolytic plaque) formed around the disk was measured and compared with the control disk 50 microliter hydrochloric acid aqueous solution, acetic acid aqueous solution 50 microliter and lactic acid aqueous solution 50 microliter. 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 with an aqueous hydrochloric acid solution to measure the antibacterial activity. As a control, antibacterial activity was also measured for a product prepared by adding hydrochloric acid, acetic acid and lactic acid to distilled water and adjusting the same pH. The results are shown in Table 7 below. The antibacterial activity of the fermentation liquid of lactic acid bacteria against Vibrio parahaemolyticus is pH 5.
It is recognized at 0 or less, and is extremely strong especially at pH 3.6 or less. Further, the antibacterial activity is recognized in the acetic acid aqueous solution of pH 3.0, but the antibacterial activity is not recognized in the hydrochloric acid aqueous solution and the lactic acid aqueous solution of the same pH. The symbols indicating the results in Table 7 are shown in Table 1 above.
~ Shows the same meaning as shown in Table 6.

[0032]

[Table 7]

[0033]

[Test Example 5] The lactic acid bacterium fermentation broth obtained in Examples 1 and 2 was tested for antimutagenic activity. Many chemical carcinogens are mutagens that modify DNA by themselves or by their metabolically active substances. An antimutagenic substance is a substance that inhibits the action of this mutagen and means a substance that prevents carcinogenesis. As the material, the test strains were Salmonella TA98 (Salmonella) having histidine auxotrophy, ampicillin resistance and frameshift mutation.
typhimurium TA98). As a mutagen, 3-amino-1-methyl-5H-pyrido [4,3-b] indole (Trp to P2) was 0.5.
N-methyl-N'-nitro-N- at a concentration of μg / ml
Nitrosoguanidine (MNNG) was used at a concentration of 10 μg / ml. The antimutagenic activity was measured by applying the Salmonella mutagen test method (Ames test).
00 microliters of Trp-p2 (or MNN
G), 100 microliters fermentation liquid of lactic acid bacteria and 10
0 microliters of S-9 mix (microsome preparation) was mixed, then 100 microliters of Salmonella culture was added and mixed with 2 milliliters of biotin-histidine agar solution which was kept at 36 ° C for 20 minutes,
Overlay 30 ml of Vogle-Bonner E medium. Incubate at 37 ° C for 48 hours and count colonies. The test was performed in triplicate. The results are shown in Table 8 below for Trp-p2. 4 showed the strongest antimutagenicity (73%).

[0034]

[Table 8]

With respect to MNNG, as shown in Table 9 below, No1 and No4 showed strong antimutagenicity (75%).

[0036]

[Table 9]

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

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

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, a coexisting culture of various lactic acid bacteria and yeast is carried out with a special starter without using any chemical additives, so that a physiologically active substance rich in metabolites of lactic acid bacteria and yeast is produced. However, as shown by the results of Test Examples 1 to 5, the lactic acid bacterium fermentation liquid obtained by the present invention has antibacterial action, anticancer action, immunostimulatory action, antimutagenic effect for suppressing the expression of carcinogens, intestinal action, etc. It has a remarkable effect on maintaining human health.

Further, the present invention is a method in which the function originally possessed by food is a method in which microorganisms are used, extracellular enzymes are extracted, and physiologically active substances are extracted, so that it acts on the body without causing side effects. So it has the effect of being safe. Therefore, the obtained lactic acid bacterium fermentation liquid has an effect that it has a wide range of applications such as food materials as well as pharmaceuticals.

Claims (1)

[Claims] 1. A milk is inoculated with a plurality of types of lactic acid bacteria and, if necessary, bifidobacteria individually, and the inoculated ones are individually cultivated under an optimum temperature environment for growth of each bacterium, and then obtained. After collecting an appropriate amount of each culture broth individually and inoculating milk together with the yeast liquid as needed, incubate the milk for a suitable time under different temperature environments, and then starter obtained at each temperature A method for producing a lactic acid bacterium fermented liquor, comprising adding the lactic acid bacterium fermented liquor to a milk at a predetermined ratio and culturing for a certain period of time in a constant temperature environment, and then extracting the fermented liquor from the obtained fermentation curd.