FUNCTIONAL COMPOSITIONS

Abstract: The present invention provides a composition comprising an untreated or treated culture of a mixed microorganism and a functional food comprising said composition. A composition comprising an untreated or treated culture of a mixed microorganism comprising at least three lactic acid bacteria selected from the group consisting of Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillu s rhamnosus, Lactococcus lactis and Streptococcus thermophilus, mixed with Saccharomyces cerevisiae , and a functional food comprising said composition.

Description: The present invention relates to a composition comprising an untreated or treated culture of lactic acid bacteria mixed with a yeast, and to a functional food comprising said composition. BACKGROUND ART Fermented foods comprising lactic acid bacteria have been expected to prevent adult's diseases and promote health. Such foods are typified by fermented milk (yoghurt); in addition, lactic acid bacteria beverage and sour milk have been well popularized. Many reports have been made on physiological activities of lactic acid bacteria and fermented foods; therefore, they are expected to be utilized as healthy foods. In most of brewed foods including sake, bean paste (miso) and soybean sauce, it has been known that unique flavor and taste and components are generated by symbiotic or antagonistic actions between several microorganisms co-cultured in media. However, few of lactic acid bacteria or fermented foods thereof are produced by so-called cocultivation and physiological activities of the bacteria have not been known. JP-A-9 075 066 discloses mixtures of Saccharomyces cerevisiae with Lactobacillus case; ssp. case; and L. brevis, L. plantarum and L. alimentarius.) DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a composition comprising an untreated or treated culture of lactic acid bacteria mixed with a yeast, and to a functional food comprising said composition. The present inventors have made great efforts to solve the above mentioned problem and found that a culture of lactic acid bacteria mixed with a yeast or a treated material thereof exhibits various functions. Thus, the present invention has been completed. Accordingly, the present invention provides a composition comprising an untreated or treated culture of at least three lactic acid bacteria selected from the group consisting of Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis and Streptococcus thermophilus , which are mixed with Saccharomyces cerevisiae . The mixed microorganisms used in the present invention include Lactobacillus delbrueckii, Lactobacillus casei, Lactococcus lactis and Saccharomyces cerevisiae; Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactococcus lactis and Saccharomyces cerevisiae; Lactobacillus plantarum, Lactobacillus casei , Streptococcus thermophilus and Saccharomyces cerevisiae ; as well as Lactobacillus fermentum, Lactobacillus rhamnosus, Streptococcus thermophilus and Saccharomyces cerevisiae . Further, the present invention provides a functional food comprising said composition. This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. JP 98/24892, which is a priority document of the present application. Hereinafter the present invention will be described in detail. The composition of the present invention comprises a culture obtained by culturing (co-cultivating) a mixed microorganism comprising lactic acid bacteria and yeast, or a treated material thereof. Lactic acid bacteria include those belonging to the genus Lactobacillus, Lactococcus or Streptococcus, such as Lactobacillus delbrueckii , Lactobacillus acidophilus , Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis and Streptococcus thermophilus . Yeast includes Saccharomyces cerevisiae . Said microorganisms used may be generally commercially available; they are not limited to particular strains of said microorganisms provided that an untreated or treated co-culture of these microorganisms can be utilized as functional foods. For example, lactic acid bacteria belonging to the genus Lactobacillus include Lactobacillus delbrueckii strain ALAL007, Lactobacillus acidophilus strain ALAL005, Lactobacillus plantarum strain ALAL006, Lactobacillus fermentum strains ALAL001 and JCM1173, Lactobacillus casei strains ALAL002, ALAL003 and JCM1053, and Lactobacillus rhamnosus strains ALAL004, ALAL010 and JCM1136; lactic acid bacteria belonging to the genus Lactococcus include Lactococcus lactis subsp . hordniae strains ALAL008 and ALAL009; lactic acid bacteria belonging to the genus Streptococcus include Streptococcus thermophilus strains ALAL011 and ALAL012; and yeast includes for example Saccharomyces cerevisiae strains JCM1499, ALAY001, ALAY002, ALAY003 and ALAY004. Saccharomyces cerevisiae strain ALAY001, Lactobacillus fermentum strain ALAL001, Lactobacillus casei strain ALAL003, and Lactobacillus rhamnosus strain ALAL004 have been deposited by A.L.A. Corporation of Nishisando Yamaki under the Budapest Treaty on November 28, 1997 with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3 Higashi 1-chome, Tsukubashi, Ibaraki, 305-0046, Japan) under Accession Nos. FERM BP-6626, 6627, 6628 and 6629, respectively. According to the present invention, the mixed microorganism consists of at least three arbitrarily selected lactic acid bacteria and one yeast. For example, the combination of the microorganisms may be any of the group A to D as set forth in Table 1. group Lactic acid bacteria Yeast (1) (2) (3) A L. delbrueckii subsp. bulgaricus L. casei Lactococcus lactis subsp. hordniae S. cerevisiae B L. acidophilus L. rhamnosus Lactococcus lactis subsp. hordniae S. cerevisiae C L. plantarum L. casei Streptococcus thermophilus S. cerevisia e D L. fermentum L. rhamnosus Streptococcus thermophilus S. cerevisiae Any of the Groups A to D may be used alone (4 strains of microorganisms); or two or more groups may be combined. If microorganisms of the same species are included in two or more groups to be combined and used (for example, L. casei and S . cerevisiae are overlapped when Groups A and C are to be combined), different strains of the same species should be used. The composition of the present invention may be obtained by cultivating the mixed microorganism comprising lactic acid bacteria and yeast in a medium containing hot water extract of soybean. The medium comprises hot water extract of soybean. After mixing 1×10 5 to 1×10 6 of each of lactic acid bacteria per ml with 1×10 4 to 1×10 6 of a yeast per ml, the mixed microorganism is inoculated in the medium and cultivated at 20 to 37°C for 4 to 10 days. When a plurality of microorganisms from different Groups are to be combined, each of the Groups to be combined is cultivated under the above described conditions and then mixed with each other followed by cultivating under the above described conditions. After cultivation, the culture is boiled at 80°C to sterilize and recovered. The composition of the present invention may be obtained by freeze- or spray-drying the culture. Alternatively, the culture may be treated by filtration or centrifugation or otherwise to separate the supernatant from the cells. In this case, the supernatant and cells may also be freeze- or spray-dried to prepare each a composition of the present invention. The composition of the present invention may be in any form and can be processed into a liquid, solid, granule or the like. A granular form is preferred since it may be conveniently handled. When processed into a granule, it is included in a polysaccharide such as cyclodextrin. The composition of the present invention obtained in the above manner have various activities and, therefore, may be used as a healthy food having some function (functional food). Such activities include, for example, liver and kidney function improving activity, anti-mutagenic activity, tumor cell growth inhibiting activity, and enteric bacterial flora improving activity. When in a granular form as usual, the composition of the present invention may be used as a functional food by eating itself in the granular form or adding an appropriate amount thereof to a food. Foods include, but not limited to, for example, cakes such as jellies and candies, beverages such as juices, teas and nutritional drinks, and rice. The amount and rate of the composition of the present invention added to a food may be adjusted as appropriate depending upon preference and usually 0.1 to 1% by weight per food. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the liver function improving activity of the composition according to the present invention. Fig. 2 shows the kidney function improving activity of the composition according to the present invention. Fig. 3 shows the kidney function improving activity of the composition according to the present invention. Fig. 4 shows the kidney function improving activity of the composition according to the present invention. Fig. 5 shows the liver function improving activity of the composition according to the present invention. Fig. 6 shows the change of body weight of test mice. Fig. 7 shows the carcinogenesis inhibiting activity of the composition according to the present invention. Fig. 8 shows the anti-mutagenic activity of the composition according to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be illustrated in more detail by the following examples. However, the scope of the present invention is not limited to these examples. Example 1: Preparation of composition Eight (8) species, 12 strains of lactic acid bacteria and one species, 4 strains of yeast were divided into 4 groups (Groups A to D) as shown in Table 2. group Lactic acid bacteria Yeast (1) (2) (3) A L. delbrueckii subsp. bulgaricus ALAL007 L. casei ALAL003 Lactococcus lactis subsp. hordniae ALAL008 S. cerevisiae ALAY003 B L. acidophilus ALAL005 L. rhamnosus ALAL004 Lactococcus lactis subsp. hordniae ALAL009 S. cerevisiae ALAY004 C L. plantarum ALAL006 L. casei ALAL002 Streptococcus thermophilus ALAL011 S. cerevisiae ALAY001 D L. fermentum ALAL001 L. rhamnosus ALAL010 Streptococcus thermophilus ALAL012 S. cerevisiae ALAY002 For each Group, a preculture containing four respective strains was inoculated to hot water extract of soybean (each lactic acid bacterium: 1×10 5 to 1×10 6 /ml; yeast: 1×10 4 to 1× 10 5 /ml) and cultivated at 20 to 37°C for 5 to 10 days. Each culture was mixed into a fresh medium and further cultivated at 20 to 37°C for 2 to 5 days. After cultivation, the culture was heated to sterilize and freeze-dried to yield 90 g of a dried product per liter. On the other hand, the culture was filtered and the supernatant was freeze-dried to yield 40 g of a dried product per liter. Among the compositions thus obtained, those obtained from the culture per se and those from the supernatant were designated as RS-II and RS-I, respectively. Example 2: Liver function improving activity test (Effect on bile acid loaded hepatopathic rat) Deoxycholic acid, hereinafter referred to as DCA, is a representative secondary bile acid excreted from cholic acid by enteric bacteria. DCA is highly toxic and is known to cause cholestatic hepatopathy in experimental animals. In addition, it is also reported that DCA may be responsible for acute pancreatitis and colic cancer. The amount and composition of bile acid in the blood and bile may vary with one's eating habitude and physiological state; for example, it has been known that the amount of DCA in the bile is significantly increased in diabetic patients and experimental diabetic model animals. The effects on living bodies are important. Accordingly, to investigate the effect of the composition of the present invention on hepatopathy, DCA was used to induce hepatopathy. (1) Method Wistar male rats of 5 weeks old were purchased from Charles River and pre-bred in a well air-conditioned breeding room with a controlled room temperature 23±1°C, humidity 50±5%, photoperiod cycle (12h light/12h dark) for 1 week. At 6 weeks old, the rats were divided into two groups of 6 rats each such that the mean and variation of body weights in each group were substantially identical with each other, one group constituting a group to which the composition of the present invention was administered and the other a control group. The administration group was given MF powdery feed (Oriental Yeast Co., Ltd.) containing 0.5% DCA and 5% RS-I while the control group was given MF powdery feed containing 0.5% DCA only. These feeds were freely taken by rats of both groups together with tap water for 6 weeks. At 0, 2, 4 and 6 weeks from the start of administration, the blood was taken from the tail vein and the serum was separated. The values of Glutamic-Oxaloacetic transaminase (GOT), Glutamicpyruvic transaminase (GPT), Blood urea nitrogen (BUN), Uric acid (UA) and Cholesterol (CHL) were measured. At 5 weeks of administration, a metabolic cage was used to measure the amount of urine and the concentration of electrolytes in the urine. After administration, the rat was sacrificed and dissected. The weights of organs were measured and the serum was separated from the taken blood to analyze serum biochemical properties. (2) Results Upon load of DCA, the rat serum GOT activity rapidly increased. In the control group, the activity increased to 1366 ±467 (Karmen) at 2 weeks and 5122±1848 (Karmen) as shown in Fig. 1A. On the contrary, it was 406±88 (Karmen) at 2 weeks in the RS-I administered group; thus, the increase of GOT activity was inhibited significantly (p<0.05). Even at 4 weeks, it was 1636± 630 (Karmen), indicating that the increase of GOT activity tended to be inhibited (Fig. 1A). It was also observed, on the other hand, that the increase of serum GPT activity was inhibited as in the GOT activity (Fig. 1B). For the BUN values, the RS-I administered group showed significantly (p<0.01) lower values at 4 and 6 weeks as compared with the control group (Fig. 2). No effect of RS-I administration on UA and CHL values was observed. The RS-I administered group tended to show larger amounts of urine excreted at 5 weeks (Fig. 3A) and also larger proportions of excretion to the amount of water taken (Fig. 3B). There was no difference in the concentration of electrolytes in the urine between the RS-I administered and control group; in the control group, the amount of urine was larger and therefore the amount of electrolytes excreted was larger (Fig. 4). According to the biochemical analysis of the sera after administration of DCA, no change due to the administration of RS-I was observed in the values of Total protein (TP), Alkaline phosphatase (ALP), γ-Glutamyl transpeptidase (γ-GTP), Leucine aminopeptidase (LAP), Glucose (GLU), Total cholesterol (T-CHL), Lipid Peroxide (LPO), β-Lipoprotein (β-LP) and bilirubin. However, the concentration of total bile acid in the sera was 81 ±36 (nmol/ml) in the control group and 46±34 (nmol/ml) in the RS-I administered group; thus, the RS-I administered group tended to show lower concentration of total bile acid in sera as compared with the control group. The RS-I administration showed a tendency to inhibit the increase of serum GOT and GPT activities caused by DCA loading and, therefore, RS-I was shown to be useful for improving damage on liver function. Further, RS-I administration lowered the serum BUN values and increased the amount of urine excreted; accordingly, RS-I was found to have an activity to improve the damage on kidney function. Example 3: Effect on galactosamine hepatopathlc rat In this example, the effect of RS-I administration on hepatopathy which has a different mechanism from the DCA induced model of Example 1 was investigated. (1) Method Wistar male rats of 5 weeks old were purchased from Charles River and pre-bred in a well air-conditioned breeding room with a controlled room temperature 23±1°C, humidity 50±5%, photoperiod cycle (12h light/12h dark) for 1 week. At 6 weeks old, the rats were divided into two groups of 6 rats each such that the mean and variation of body weights in each group were substantially identical with each other, one group constituting a group to which the composition of the present invention was administered and the other a control group. The administration group was given MF powdery feed containing 5% RS-I while the control group was given MF powdery feed only. These feeds were freely taken by rats of both groups together with tap water for 3 weeks. At 3 weeks from the RS-I feed loading, about 1 ml of an aqueous solution of D-galactosamine hydrochloride was intraperitoneally injected, said solution having been prepared so that 500 mg of D-galactosamine hydrochloride was administered per kg of body weight of rat. At 1, 2, 3 and 6 days from the galactosamine administration, the blood was taken from the tail vein and serum GOT, CHL, GLU and BUN values were measured. (2) Results After administration of galactosamine, rat serum GOT rapidly increased. At 1 day, the serum GOT value increased 5148±1711 (Karmen) in the control group (Fig. 5) while it was 2244±1241 (Karment) in the RS-I administration group. Thus, the administration of RS-I significantly (p<0.05) inhibited the increase of GOT activity due to galactosamine (Fig. 5). There was not observed any effects of RS-I administration on the serum CHL, GLU and BUN values. Example 4: Effect on DMH colic cancerized mice In this example, dimethylhydrazine (DMH) induced colic cancerized mice were used to carry out the following experiments concerning the carcinogenesis inhibiting effect of the composition of the present invention. (I) Method CF#1 mice, the line of which was maintained in Institute of Physical and Chemical Research, were mated and 60 male mice were bred. The mice of this line are highly sensitive to DMH and colic polyps are specifically induced. The breeding was carried out at a temperature of 23±1°C, a humidity of 50±5% and an photoperiod cycle (12h light/12h dark). As a breeding feed, special breeding feed (CMF) manufactured by Oriental Yeast Co., Ltd. was freely given together with tap water. At 5 weeks, the mice were divided into three groups (each group of 20 mice) as set forth in Table 3 so that the mean and variation of body weights was identical (Fig. 6). As shown in Table 3, three groups consisted of RS-II administered group, RS-I administered group and control group, which were administered at the respective amounts as shown. A solution of DMH was intraperitoneally injected into the mice at 20 mg per body weight, once a week, for 10 weeks. The control group was given only CMF. Group Amount administered (%) Number of mice Control - 20 RS-II 3 20 RS-I 5 20 Said three groups of mice were observed for colic polyps at 35 weeks after DMH administration (at 40 weeks old). (2) Results Incidence of polyp was 94% in the control group but 65% in RS-II administration group; thus, the administration group showed a significantly (p<0.05) lower rate as compared with the control group (Fig. 7A). However, RS-I administration group showed a incidence of polyp of 94%; thus, no difference from the control group was observed. The number of polyps per mouse was 4.0±2.7 (mean ± standard deviation) in the control group and 1.4±1.5 in the RS-II administration group, which was significantly (p<0.01) lower than the control group (Fig. 7B). The RS-I administration group showed 2.7±1.9 which was a significantly (p<0.05) lower value than the control group (Fig. 7B). The tumor size was 3.1±1.8 mm in the control group and 2.5± 1.3 mm in RS-II administration group; thus, RS-II administration group showed a significantly (p<0.05) smaller tumor size than the control group (Fig. 7C). From the above results, the composition of the present invention was recognized to have carcinogenesis inhibiting effects. Example 5: Anti-mutagenic test of substances produced by lactic acid bacteria It is known that most of substances having mutagenesis have carcinogenicity. Since the results of Example 4 showed that the composition of the present invention prevented colic cancer, it was suggested that the composition of the present invention also had inhibiting effects on mutagenesis. Thus, the effects of the composition of the present invention on the following mutagenic substances were investigated. (1) Experimental method (i) Mutagenic substances Each of 4-nitroquinoline-1-oxide (4NQO: 0.25 µg/plate), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG: 0.5 µg/plate), and 3-amino-1-methyl-5H-pyrido(4,3-b)indolacetate (Trp-P-2: 5µg/plate) was dissolved in DMSO. Trp-P-2 is a substance generated upon burning a protein or amino acid, such as burnt fish. MNNG is a substance causing gastric cancer. (ii)Anti-mutagenic test The anti-mutagenic test was carried out by Ames method. The test cell used was Salmonella typhmurium strain TA100 (his+). The strain TA100 was cultivated overnight in Nutrient Broth and washed with Na-K buffer. The final concentration of cells in the suspension was adjusted to about 2 x 10 9 per ml. The dried RS-I product was dissolved in sterilized water at each concentration. To a test tube, RS-I solution, 100 µl of a mutagenic substance, 0.5 ml of S9 mix or Na-K buffer, and 0.5 ml of a cell solution to be tested were sequentially added. After reaction at 37°C for 30 minutes, centrifugation was effected and the supernatant was discarded. A soft agar containing histidine (1 µM) and biotin (1 µM) was added and seeded in a minimal glucose agar medium. This was cultivated at 37°C and the number of colonies was counted after 2 or 3 days. (2) Results The mutation inhibition was 64% in 4NQO at RS-I concentration of 0.06 mg/plate, 86% in MNNG at RS-I concentration of 0.25 mg/plate, and 72% in Trp-P-2 at RS-I concentration of 0.5 mg/plate (Fig. 8). DMH, Benzopyrene (BP) and DCA were tested up to a concentration of 20 µg/plate, 20 µ g/plate and 0.5 µg/plate, respectively, but no mutagenesis of strain TA100 by these substances occurred. Thus, no inhibiting rate of RS-I was detected. From the above results, it was found that the composition of the present invention had an anti-mutagenic activity against 4NQO, MNNG and Trp-P-2. Example 6: Intestinal flora improving activity This example was done to test the intestinal bacterial flora improving activity of the composition according to the present invention contributing to enhancement of human health by improving human intestinal bacterial flora, that is, by increasing the number of beneficial bacteria including Bifidobacterium and Lactobacillus in intestines while inhibiting the growth of harmful bacteria such as Escherichia coli . (1) Experimental method (i) Investigation of antibacterial properties in liquid media The media used were a medium containing Brain-Heart infusion (Difco) and ABCM medium (Eiken) for aerobic bacteria and GAM medium (Nissui Seiyaku) or Hemine-menadione added GAM medium and ABCM medium for anaerobic bacteria. The medium was distributed into small test tubes in each 2 ml and the RS-I dissolved in the same medium was added at a concentration of 1% (w/v). A cell solution containing a given pre-cultivated cell (Table 4) was diluted to a final concentration of 1×10 5 per ml, added to the above medium and cultivated at 37°C for 18 to 24 hours. Absorbance at 655 nm was measured using Microplate Reader (Bio-Rad, Model 3550) and the thus measured turbidity was expressed as a relative value to that of the control (100) and represents the growth rate. (2) Results As seen from the results in Table 4, the composition of the present invention showed growth promoting activity on the beneficial bacteria such as Bifidobacterium and Lactobacillus at a concentration of 1% (Table 4, upper part). On the other hand, the activity of inhibiting the growth of the harmful bacteria such as Escherichia coli and Bacteroides was observed (Table 4, lower part). Strain Growth ratio (%) Bifidobacterium longum E1946 206 Bifidobacterium infantis 12 187 Bifidobacterium bifidum 560 132 Lactobacillus plantarum JCM1149 360 Lactobacillus acidophilus I-65 118 Lactobacillus reuteri JCM1112 302 Escherichia coli 123 58 Staphylococcus aureus JCM2413 31 Streptococcus pyogenes JCM5674 53 Salmonella typhimurium TA98 44 Streptococcus mutans JCM5705 49 Clostridium perfringens JCM3816 72 Bacteroides coagulans ATCC 29798 38 Porphyromonas gingivalis 381 53 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety. INDUSTRIAL APPLICABILITY According to the present invention, there are provided a composition or an untreated or treated culture of lactic acid bacteria mixed with a yeast and a functional food comprising said composition. Thus, the composition of the present invention may be expected to promote the growth of beneficial bacteria such as Bifidobacterium and Lactobacillus in human intestines and to inhibit the growth of harmful bacterial such as Escherichia coli and, accordingly, may contribute to promotion of human health.

Claims: A composition comprising an untreated or treated culture of a mixed microorganism comprising: (i) at least three lactic acid bacteria from Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis or Streptococcus thermophilus ; and (ii) Saccharomyces cerevisiae . A composition according to claim 1, wherein (i) is one or more of: (a) Lactobacillus delbrueckii, Lactobacillus casei and Lactococcus lactis ; (b) Lactobacillus acidophilus, Lactobacillus rhamnosus and Lactococcus lactis ; (c) Lactobacillus plantarum, Lactobacillus casei and Streptococcus thermophilus ; or (d) Lactobacillus fermentum, Lactobacillus rhamnosus and Streptococcus thermophilus . A composition according to claim 1 or 2 in the form of a liquid, solid or granule. A functional food comprising a composition according to any one of the preceding claims. A functional food according to claim 4 comprising from 0.1 to 1% by weight of the composition. A composition according to any one of claims 1 to 3 or a functional food according to claim 4 or 5 for use in a method of improving liver and kidney function, reducing mutagenic activity, inhibiting tumor cell growth or improving enteric bacterial flora growth. A method for producing a composition according to any one of claims 1 to 3 comprising: (i) cultivating the mixed microorganism comprising lactic acid bacteria and yeast in a medium comprising hot water extract of soybean; (ii) boiling at 80°C or filtering the resulting culture; and (iii) freeze- or spray-drying the boiled culture or supernatant from the filtered culture.

Inventors: MIURA KAZUSHI, MIZUTANI TAKEO, SHIN RYOUICHI, SUZUKI MOMOYO, MIURA, KAZUSHI, MIZUTANI, TAKEO, SHIN, RYOUICHI, SUZUKI, MOMOYO
Applicants: A L A CORP, MIZUTANI TAKEO, RIKEN, A.L.A. CORPORATION, MIZUTANI, TAKEO
Application year: 1999
Main classification: C12N1
IPC codes: C12R1, A23L1, C12N1