Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/065—Microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/11—Lactobacillus

Definitions

• the present invention relates to a composition containing a culture of a mixed microorganism of lactic acid bacteria and yeast or a processed product thereof, and a functional food containing the composition.
• Fermented foods using lactic acid bacteria are expected to have the effect of preventing adult diseases and improving health.
• Fermented milk (yogurt) is a typical example of such food, and lactic acid bacteria drinks, sour milk and the like are also widely used.
• An object of the present invention is to provide a composition containing a culture of a mixed microorganism of lactic acid bacteria and yeast or a processed product thereof, and a functional food containing the composition.
• the present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a culture of a mixed microorganism of lactic acid bacteria and yeast or a processed product thereof exhibits various functions, and have completed the present invention.
• a culture of a mixed microorganism of lactic acid bacteria and yeast or a processed product thereof exhibits various functions, and have completed the present invention.
• the present invention relates to Lactobacillus' delbrueckii, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermenta.
• the mixed microorganisms include Lactobacillus delbrueckii, Lactobacillus' casei, Lactococcus lactis and Saccharomyces' Celepiche, Lactobacillus' Acidophilus, Lactobacillus rhamnosus, Lactococcus rhamnosus, Lactococcus lactis and Saccharomyces cerevisiae.
• Lactobacillus 'plantarum Including Lactobacillus 'plantarum, Lactobacillus' force, Streptococcus thermophilus and Saccharomyces cerepiche, and Lactobacillus famentumum, Lactobacillus rhamnosus, Streptococcus At least one group selected from the group consisting of Thermophilus and Saccharomyces cerevisiae.
• the present invention is a functional food containing the above composition.
• the composition of the present invention includes a culture obtained by culturing (co-cultivation) a mixed microorganism of lactic acid bacteria and yeast, or a processed product thereof.
• Lactic acid bacteria include those belonging to the genus Lactobacillus, Lactococcus or Streptococcus, such as Lactobacillus delbrueckii), Lactobacillus acidophilus, Lactobacillus acidophilus, and Lactobacillus acidophilus. Lactobacillus plantar urn, Lactobacillus ferment urn, Lac tobacillus ferment urn, Lactobacillus casei, Lactobacillus rhactobacillus rhactobacillus nosus), Lactococcus' lactois (ac tococcus lactis) and Streptococcus lactis.
• Lactobacillus plantar urn Lactobacillus ferment urn
• Lac tobacillus ferment urn Lactobacillus casei
• Lactobacillus rhactobacillus rhactobacillus nosus Lactococcus' lactois (ac tococcus lactis) and Strept
• yeast examples include Saccharomyces' Saccl romvces cerevisiae) Can be
• microorganisms As the above microorganisms, commercially available microorganisms can be used, but as long as a co-cultured culture of these microorganisms or a processed product thereof can be used as a functional food, the microorganism is limited to a specific strain of the microorganism. Not something.
• lactic acid bacteria belonging to the genus Lactobacillus include Lactobacillus delbrueckii ALAL007 strain, Lactobacillus acidophilus ALAL005 strain, Lactobacillus plantarum ALAL006 strain, Lactobacillus' Famentum ALAL001 strain, JCM1173 strain, Lactobacillus AL strain 002 , ALAL003 strain, JCM1053 strain, Lactobacillus rhamnosus ALAL004 strain, ALAL010 strain, JCM1 136 strain, etc.
• Lactococcus lactobacillus belonging to the genus Lactococcus is Lactococcus lactis subsp.hordniae ALAL008 strain, ALAL009 strain, etc.
• Examples of the lactic acid bacteria belonging to the genus Streptococcus include Streptococcus saccharomyces ALAL011 strain and ALAL012 strain, and examples of the yeasts include Saccharomyces' Celepiche JCM1499 strain, ALAY001 strain, ALAY002 strain, and the like. ALAY003 strain and ALAY004 strain are mentioned.
• Saccharomyces cerevisiae ALAY001 strain, Lactobacillus fermentum ALAL001 strain, Lactobacillus fermentum ALAL001 strain, and Lactobacillus casei ALAL003 Sus ALAL004 Lactobacillus rhamnosus ALAL004) strain was sent to the National Institute of Bioscience and Human-Technology (1-3-1 Higashi, Tsukuba-shi, Ibaraki) on November 28, 1997, and on November 28, 1997, the FERM BP- strain for Saccharomyces cerevisiae ALAY001 was 6626, Lactobacillus fermentum ALAL001 strain, FERM BP-6627, Lactobacillus casei ALAL003 strain, FERM BP-6628, and Lactobacillus rhamnosus ALAL004 strain, FERM BP-6629, have been deposited under the Budapest Treaty.
• lactic acid bacteria are arbitrarily selected and mixed with one type of yeast to obtain a mixed microorganism.
• a combination of the mixed microorganisms for example, those described in Groups A to D in Table 1 can be used. Table 1
• microorganisms of the above groups A to D may be used as a single group (4 microorganisms), respectively, or two or more groups may be used in any combination. However, when two or more groups are combined and microorganisms of the same species overlap (for example, L casei and 5. care s ae when group A and group C are combined), separate strains are used. Shall be used.
• composition of the present invention can be obtained by fermenting a mixed microorganism of lactic acid bacteria and yeast in a medium containing a hot water extract of soybean.
• the medium used is a hot water extract of soybeans. Then, 1 X. 10 to 1 X 10 6 cells ml per one lactic acid bacteria, yeast one per 1 X 10 4 ⁇ l X 10 6 cells Zml was inoculated into the culture medium were mixed, respectively, 4 20 to 37 ° C Incubate for ⁇ 10 days.
• composition of the present invention can be obtained by freeze-drying or spray-drying the culture itself after the culture.
• the culture supernatant and the cells may be separated by performing a treatment such as filtration or centrifugation.
• the culture supernatant and the cells can be freeze-dried or spray-dried to prepare the composition of the present invention.
• composition of the present invention can be arbitrarily set, and can be added to liquids, solids, granules, etc., but granules are preferred because they are convenient for handling. When processed into granules, it should be included with polysaccharides such as cyclodextrin. Since the composition of the present invention obtained as described above has various actions, it can be used as a functional health food (functional food). Its effects include, for example, a liver and kidney function improving effect, an anti-mutagenic effect, a tumor cell growth suppressing effect, and an intestinal flora improving effect.
• composition of the present invention is usually processed into granules, when it is used as a functional food, it may be eaten as it is, or an appropriate amount may be added to the food.
• Foods include, for example, confectionery such as jelly and candy, juice, tea, beverages such as nutritional drinks, and cooked rice, but are not limited thereto.
• the mixing amount and mixing ratio of the composition of the present invention to food are 0.1 to 1% by weight per food, but can be appropriately adjusted according to preference.
• FIG. 1 is a view showing the liver function improving effect of the composition of the present invention.
• FIG. 2 is a graph showing the renal function improving effect of the composition of the present invention.
• FIG. 3 is a graph showing the renal function improving effect of the composition of the present invention.
• FIG. 4 is a graph showing the renal function improving effect of the composition of the present invention.
• FIG. 5 is a view showing the liver function improving effect of the composition of the present invention.
• FIG. 6 is a diagram showing changes in body weight of test mice.
• FIG. 7 is a view showing the carcinogenesis-suppressing effect of the composition of the present invention.
• FIG. 8 is a view showing the anti-mutagenic effect of the composition of the present invention.
• microorganisms of 12 strains of lactic acid bacteria and 12 strains of yeast were used for a total of 16 strains.
• the combination of three strains of lactic acid bacteria and one strain of yeast was divided into four groups A to D as one group.
• Table 2
• the culture solution of each group was mixed in a new culture medium, and the mixture was further cultured at 20 to 37 ° C for 2 to 5 days. After cultivation, the mixture was sterilized by heating and freeze-dried to obtain a dried product of 90 dalam per liter. The supernatant was obtained by filtering the culture solution, and lyophilized to obtain a dried product of 40 g per liter.
• compositions thus obtained those obtained from the culture itself were designated as RS-II, and those obtained from the culture supernatant were designated as RS-1.
• Example 2 Liver function improving effect test (Effect on bile acid-loaded liver injury rat)
• DCA Deoxycholic acid
• the amount and composition of bile acids in the blood and bile change with eating habits and physiological conditions, For example, it is known that the amount of DCA in bile is significantly increased in diabetic patients and experimental diabetic model animals, and its effect on the living body is important.
• One group was divided into two groups, each group consisting of 6 animals, so that the average and the variation in body weight were almost equal at the age of 6 weeks.
• One group was a group to which the composition of the present invention was administered, and the other group was a control group.
• the MF powder diet mixed with 0.5% DCA and 5% RS-1 (Oriental Yeast Co., Ltd.) was given to the administration group, and the MF powder diet mixed only with 0.5% DCA was given to the control group for 6 weeks. Water Free access to drinking water.
• Blood is collected from the tail vein at 2, 4 and 6 weeks after administration, serum is separated and iutamic-Oxaloacetic transaminase (GOT), Glutamic-pyruvic transaminase (GPT), Blood urea nitrogen (BUN), Uric acid (UA) and Cholesterol (CHU values were measured.
• GOT Glutamic-Oxaloacetic transaminase
• GPT Glutamic-pyruvic transaminase
• BUN Blood urea nitrogen
• Uric acid Uric acid
• CHU Cholesterol
• DCA loading resulted in a rapid increase in serum GOT activity in rats, with the control group increasing to 1366 ⁇ 467 (Karmen) at 2 weeks and 5122 ⁇ 1848 (armen) at 4 weeks ( Figure 1).
• the value was 406 ⁇ 88 (Karmen) at the second week, and the increase in GOT activity was significantly (p ⁇ 0.05) suppressed.
• it was 1636 Sat 630 (Karmen), indicating that the increase in GOT activity tended to be suppressed (Fig. 1A).
• serum GPT activity was also found to have an inhibitory effect on elevation (Fig. 1B).
• the BUN value of the RS-1 administration group was significantly (p ⁇ 0.01) lower than that of the control group at the 4th and 6th weeks (FIG. 2). UA and There was no effect of RS-1 administration on CHL levels.
• TP total protein
• ALP alkaline phosphatase
• ⁇ -GTP r-ulutamy 1 transpeptidase
• LAP leucine aminopeptidase
• G Glucose
• T-CH Total cholesterol
• LPO Lipid Peroxide
• ⁇ - ⁇ 3-Lipoprotein
• pyrilrubin did not show changes that might be due to RS-1 administration, but serum total bile acid concentration was 46 ⁇ 34 (nmol / ral) in the RS-1 administration group compared to 81 ⁇ 36 (nmol / ml) in the control group, and the serum total bile acid concentration was lower in the RS-1 administration group than in the control group There was a tendency.
• RS-I administration reduced serum BUN levels and increased urinary excretion, indicating that RS-I also has an improving effect on renal dysfunction.
• One group was divided into two groups, each group consisting of 6 animals, so that the average and the variation in body weight were almost equal at the age of 6 weeks.
• One group was a group to which the composition of the present invention was administered, and the other group was a control group.
• the MF powder diet mixed with 5% RS-I was given to the administration group, and the MF powder diet alone was given to the control group, and allowed to freely ingest with tap water for 3 weeks.
• CFiil mice (maintained at RIKEN) were bred, and only 60 males were bred. This strain of mice is highly sensitive to DMH and specifically induces colonic polyps. Rearing is performed at a temperature of 23 ⁇ 1 ° C, a humidity of 50 ⁇ 5%, and an illumination time of 8:00 am to 8:00 pm.
• special breeding feed (CF) manufactured by Oriental Yeast Co., Ltd. was given ad libitum with tap water.
• mice were used for each group so that the average and the body weight were equal at the age of 5 weeks (Fig. 6), and the three groups shown in Table 3 (RS-II-administered, RS-1-administered and control The mice were fed at the doses shown in Table 3, and the mice were intraperitoneally administered with the DMH solution at a dose of 20 mg per body weight once a week for 10 weeks. The control group received only CMF. Group name Dose (%) Number of animals
• the incidence of polyps was 94% in the control group, compared to 65% in the RS-II group, and was significantly (P ⁇ 0.05) lower in the control group than in the control group ( Figure 7A). ). However, the incidence of polyps in the RS-1 treated group was 94%, which was not different from the control group.
• the number of polyps per mouse was 4.0 ⁇ 2.7 (average soil standard deviation) in the control group, whereas it was 1.4 ⁇ 1.5 in the RS-II-administered group.
• the number was significantly (P less than 0.01) relatively small (Fig. 7B).
• the number was 2.7 ⁇ 1.9, which was significantly (P ⁇ 0.05) less than that in the control group (FIG. 7B).
• the control group was 3.1 ⁇ 1.8 mm, whereas the RS-II-administered group was 2.5 ⁇ 1.3 mm.
• the RS-II-administered group was significantly (P ⁇ 0.05) compared to the control group.
• Fig. 7C shows a small tumor size.
• composition of the present invention has a carcinogenesis inhibitory effect.
• Example 4 From the results of Example 4, it was confirmed that the composition of the present invention suppresses the occurrence of colorectal cancer, suggesting that the composition of the present invention also has an inhibitory effect on mutagenicity. .
• Trp-P-2 4-Nitroquinoline-1-oxide (4NQ0: 0.25 ⁇ g / plate), N-methyl-N'-nitro-N-nitrosoguanidine (M-G: 0.5 M g / plate), 3-amino- 1-Methyl-5H-pyrido (4,3-b) indole acetate (Trp-P-2: 5 ⁇ g / plate) was dissolved in DMS0. Trp-P-2 is a substance that is produced when proteins and amino acids are burned, such as when burning fish. Also, NG is a substance that causes gastric cancer.
• the Ames method was used for the anti-mutagenicity test.
• Salmonella typhmurium TA100 (his +) strain was used as a test strain. After culturing the TA100 strain in Nutrient Broth, it was washed with Na-K buffer, and the final concentration of the bacterial suspension was adjusted to about 2 ⁇ 10 9 Zml.
• the dried RS-I was dissolved in sterilized water at each concentration. Add 100 ⁇ l of the RS-1 solution at each concentration, 100 ⁇ l of the mutagen, 0.5 ml of S9 mix or Na- ⁇ ⁇ ⁇ ⁇ buffer, and 0.5 ml of the test bacterial solution to the test tube in this order, and react at 37 ° C for 30 minutes. After centrifugation, the supernatant was discarded, and soft agar containing histidine (1 ⁇ ) and biotin ( ⁇ ) was added, followed by seeding on a minimal glucose agar medium. This was cultured at 37 ° C, and the number of colonies was counted after 2-3 days.
• 4NQ0 had a mutation suppression rate of 64% at an RS-1 concentration of 0.06 mg / plate
• MNNG had an inhibition rate of 86% at 0.25 mg / plate
• Trp-P-2 had a mutation inhibition rate of 72% at 0.5 mg / plate (Fig. 8 ).
• DMH, benzopyrene (BP), and DCA were tested at concentrations of 20 / ig / plate, 20 ⁇ g / plate, and 0.5 ⁇ g / plate, respectively, but mutagenesis in the TA100 strain by these substances occurred. However, no inhibition rate of RS-I could be detected.
• composition of the present invention has anti-mutagenic activity against 4NQ0, MNNG and Trp-P-2.
• This example is intended to improve the intestinal flora of humans, that is, to increase the number of useful bacteria such as bifidobacteria and lactic acid bacteria in the intestine and suppress the growth of harmful bacteria such as Escherichia coli.
• the intestinal flora improving effect of the composition of the present invention which contributes to the promotion of human health, was tested.
• GAM medium Nesui Pharmaceutical
• GAM medium with hemi-menadione and ABCM medium 2 ml each was dispensed into small test tubes, and RS-1 dissolved in the same medium was added to a concentration of 1% (w / v).
• the bacteria solution containing a predetermined bacteria precultured (Table 4) was diluted to a final concentration of 1 10 5 Bruno 1111 was added to the medium and 18-24 hours at 37 ° C.
• the turbidity was measured by measuring the absorbance at 655 nm using a microprecipitator (Bio-Rad model 3550), and expressed as a relative value when the turbidity of the control was set to 100 as the growth rate.
• composition of the present invention showed a growth promoting effect on useful bacteria such as bifidobacteria and lactobacilli at a concentration of 1% (Table 4, upper row). Meanwhile, the effect of suppressing the growth against harmful bacteria such as Escherichia coli Ya bar Kuteroidesu was observed (Table 4 lower) c
• Bifidobac terium infant is 12 187
• a composition comprising a culture of a mixed microorganism of lactic acid bacteria and yeast or a processed product thereof and a functional food comprising the composition.
• the composition of the present invention acts to promote the growth of useful bacteria such as bifidobacteria and lactic acid bacteria in human intestine, and is expected to have a growth inhibitory effect on harmful bacteria such as Escherichia coli. It greatly contributes to the improvement of the health of people.

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