AU2004200137B2 - Cholesterol-lowering agents, secondary bile acid production inhibitors, and foods and drinks - Google Patents

Description

1e -~A

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Kabushiki Kaisha Yakult Honsha Actual Inventor(s): Haruji Sawada, Yasuto Yoshida, Tunekazu Watanabe Yasue Wada, Kenji Ohishi, Masahiko Itoh, Wakae Mori, Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: CHOLESTEROL-LOWERING AGENTS, SECONDARY BILE ACID PRODUCTION INHIBITORS, AND FOODS AND DRINKS Our Ref: 711645 POF Code: 1544/63311 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 1 Cholesterol-Lowering Agents, Secondary Bile Acid Production Inhibitors, and Foods and Drinks This application is a divisional of Australian patent application 60205/00 the entire content of which is herein incorporated by reference.

Technical Field The present invention relates to a cholesterol-lowering agent capable of lowering the blood or hepatic cholesterol level and improving atherogenic index; to a secondary bile acid production inhibitor useful for preventing and treating colorectal cancer, liver cancer, pancreatic cancer, cholelithiasis, etc.; and to use thereof for producing foods and drinks.

Background Art In.recent years, the mortality rate relating to cardiovascular diseases has increased year by year. If diseases caused by arteriosclerosis such as cardiac infarction and brain infarction are included, cardiovascular diseases are ranked at the top of the causes of-death in adults.

Arteriosclerosis is caused by a variety of factors.

Among them, increase in plasma lipid level, inter alia, plasma cholesterol level, is conceivably one of the most dangerous factors.

One cause of elevated plasma cholesterol level is a genetic disease. In this case, grave patients are given diet YMarvWyf NO DELETE MRWO2s0o Div.de therapy, and simultaneously, drugs such as a cholesterol synthesis inhibitor, Nicomol, Clofibrate, an ion-exchange resin, and an anabolic steroid are employed. However, these drugs cause adverse side effects such as toxicity to the liver, gastrointestinal disorders, and carcinogenicity.

Another important cause of elevated plasma cholesterol level is excessive fat consumption caused by the recent dietary custom of consuming a large quantity of eggs, butter, meat, etc., to the extent that excessive fat consumption has become the general habit for younger people. Alimentary hypercholesterolemia induced by excessive fat consumption generally does not become grave in comparison with that caused by genetic factor. But alimentary hypercholesterolemia induces a gradual accumulation of cholesterol in the vascular wall from a young age and problematically causes arteriosclerosis in adulthood, and then may cause cardiac infraction and brain infraction together with hypertriglyceridemia. In relation to these hyperlipemias, diet therapy including limitation of lipid consumption within an appropriate range is of great importance, rather than drug therapy, which raises problems such as adverse side effects. However, patients under diet limitation suffer mental pain and must abandon the joy of their regular diet. Thus, complete diet therapy is difficult to attain, and the effect thereof is usually limited.

Excessive cholesterol in plasma accumulates on the inside of the blood vessels, inducing arteriosclerosis.

However, if the cholesterol is taken up in the liver, and, then cholesterol and its metabolites, namely bile acid are excreted into the intestiral track without accumulation in the liver, followed by discharge together with feces to outside the body, the pool oize of cholesterol in the body decreases, to thereby prevent artheriosclerosis onset by the aforementioned mechanism. Thus, a cholesterol-lowering agent which exerts an effect for lowering hepatic cholesterol level as well as plasma cholesterol level has been desired.

A variety of microorganisms exerting a cholesterollowering effect with slight side effects are disclosed.

However, yeasts are disclosed in surprisingly few number.

From long ago, brewer's yeast has been known as a typical yeast, and its effect of improving lipid metabolism is disclosed in several literatures. For example, there are disclosed a case in which administration of chromium-added brewer's yeast (9 g/day) for eight weeks to elderly subjects decreased the serum cholesterol level [Ester G. Offenbacher and F. Xavier Pi-Sunyer, Beneficial effect of chromium-rich yeast on glucose tolerance and blood lipids in elderly subjects. Diabetes, 29, 919, (1980)]; a case in which administration of chromium chloride and brewer's yeast (5 g) for 10 weeks to elderly subjects caused no effect on the serum cholesterol level and triacylglycerol level [Caral J.

Rinko, and F. Xavier Pi-Sunyer, The effects of inorganic chromium and brewer's yeast on glucose tolerance, plasma lipids, and plasma chromium in elderly subjects., Am. J. Clin.

Nutr., 42, 454, (1985)]; and a case in which administration test of no-chromium-added brewer's yeast to human subjects resulted in no effect of lowering the serum cholesterol level [Arne T. Hostmark, Einar Eilertsen, and Ole Gronnerod, Plasma lipid and lipoprotein responses of rats to starch and sucrose diets with and without brewer's yeast., J. Nutri., 109, 1073, (1978)]. In addition, there is disclosed a case in which administration of a soybean-protein-added diet lowered the serum cholesterol level of rats, but a slight increase in the level was attained when 50% of the soybean protein was substituted by brewer's yeast [Jorge De Abreu and Nancy Millan, Effect of addition of brewer's yeast to soy protein and casein on plasma cholesterol levels of rabbits. Archivos Latinoamericanos de Nutricion., 44, 18, (1994)].

As described above, at present, there still remain different theories regarding the effect of brewer's yeast on improvement of lipid metabolism.

Effects of other yeasts are also disclosed in literature. For example, a methanol extract of Sporobolomyces ruberrinus and that of Saccharomyces uvarum are reported to slightly lower the serum cholesterol level and triglyceride level of choresterol-loaded rats (Shuji CHO, Hisao FUJII, and Jun Shiraishi, Effect of polysaccharide produced by Bacillus natto or alcohol extract of yeast on the lipid metabolism of rats. Bulletin of the Faculty of Home Life Science, Fukuoka Women's University, 16, 65, (1984)).

Also disclosed is a case in which the culture supernatant of Saccharomyces cerebisiae lowers the serum cholesterol level of mice [Tadaaki Kishida, On the serum Cholesterol-Lowering Effect of Saccharomyces cerebisiae Liquid Culture. Jurnal of Japanese Society of Food and Nutrition, 26, 371,(1973)].

However, there is only a few reports in which effectofyeasts other than brewer' s yeast or its constituent on improvement of lipid matabolism was demonstrated.

In the human liver, primary bile acids such as cholic acid and chenodeoxycholic acid are synthesized from cholesterol, and conjugated with glycine or taurine, to thereby yield glycine-conjugated or taurine-conjugated bile acid, which are secreted via the bile duct to the digestive tract. Bile acid causes lipid contained in foods and drinks to emulsify and disperse by its surface activation effect, and promotes digestion and absorption of lipid. The bile acid is then actively absorbed again from the ileum, returned to the liver via the portal vein, and resecreted to the digestive tract again. Thus, bile acids repeat enterohepatic circulation.

A portion of bile acid which has not been absorbed by the ileum undergoes modification by enterobacteria; e.g., deconjugation, 7a-dehydroxylation, oxidation, reduction, or epimerization, to thereby convert to secondary bile acid.

Predominant secondary bile acids produced through the process are deoxycholic acid and lithocholic acid, which are formed by 7a-dehydroxylation of primary bile acids (cholic acid and chenodeoxycholic acid, respectively. A portion of secondary bile acid is passively absorbed by the large intestine and transferred to the liver, repeating enterohepatic circulation in similar fashion to the case of primary bile acid. The remaining portion of-secondary bile acid is excreted into feces.

In recent years, secondary bile acids such as deoxycholic acid and lithocholic acid produced by enterobacteria existing in the large intestinal tract have been found to be closely related to onset of colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, etc. In general, the carcinogenesis process is conceived to include an initiation step in which a gene is mutated by a chemical carcinogen, radiation, or a virus, and a promotion step in which abnormality occurs in growth and differentiation through long-term exposure to a promoter.

Hitherto, there is disclosed that secondary bile acids such as deoxycholic acid and lithocholic acid act as a promoter in the second step, to thereby promote onset of colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, etc. (Narisawa, et al., J. Natl. Cancer Inst., 53, 1093- 1097, 1974; Tsuda, et al., Gann, 75, 871-5, 1984; and Makino, et al., J. Natl. Cancer Inst., 76, 967-75, 1986).

There is also disclosed that the carcinogenetic promoter activity of secondary bile acid is considerably stronger than that of primary bile acid (Narisawa, et al., J. Natl.

Cancer Inst., 53, 1093-1097, 1974; and Reddy, BS., et al., Cancer Res., 37, 3238-3242, 1977). Moreover, there has recently been disclosed that deoxycholic acid may be related to onset of cholelithiasis other than cancers (Marcus, SN., Heaton, KW., Gut, 29, 522-533, 1988).

Thus, since secondary bile acids such as deoxycholic acid and lithocholic acid produced by enterobacteria existing in the large intestinal tract have a strong carcinogenetic promoter activity and cause cholelithiasis, diseases such as colorectalcancer, liver cancer, pancreatic cancer, bile duct cancer, and cholelithiasis can be prevented and treated through inhibition of secondary bile acid production.

Hitherto, secondary bile acid production inhibitors have been disclosed, even though the inhibitory effect is weak. These inhibitors are ampicillin--a type of penicillin derivative---(Low-Beer, TS., Nutter, Lancet, 2(8099), 1063-1065, 1978), 3a,12p-dihydroxy-5p-cholane-24-Nmethylamine (Roda, et al., J. Pharm. Sci., 81, 237-240, 1992); lactulose--a type of oligosaccharide---(Magengast, FM., Eur. J. Clin. Invest., 18, 56-61, 1988); and wheat bran (Lampe, JW., et al., Gut, 34, 531-536, 1993).

Ampicillin and 3a,12p-dihydroxy-5p-cholane-24-Nmethylamine inhibit secondary bile acid production by removing bacteria which catalyze 7a-dehydroxylation in the large intestinal tract. However, when these inhibitors are employed for a long period of time, there arise problems such as generation of resistant bacteria and occurrence of adverse side effects. In addition, selective exclusion of bacteria which relates to secondary bile acid production by use of antibiotics is impossible, and use of antibiotics simultaneously excludes enterobacteria which play an important role in maintaining the human health; Lactobacillus and Bifidobacterium, and this is greatly problematic.

Lactulose and wheat bran are thought to suppress activity of 7a-dehydroxylase which has optional pH of a neutral region by lowering pH in the large intestinal track and thereby lower the production of secondary bile acid. However, these substances exert poor effectiveness, which does not reach the level for contributing to prevention and treatment of the diseases The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

In view of the foregoing, it would be desirable to provide a cholesterol-lowering agent capable of effectively lowering the blood or hepatic cholesterol level by use of a safe yeast causing fewer and less severe side effects.

It would also be desirable to provide a secondary bile acid production inhibitor of high effectiveness and safety, useful for preventing and treating diseases such as colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, and cholelithiasis.

Disclosure of the Invention The present inventors have carried out extensive studies, and have found that the yeasts belonging to the following specific groups exert an effect of effectively Y:MzaNK]Y NO DELETE MRW2O00 Div.dx lowering the blood or hepatic cholesterol and provide fewer and less severe side effects, and that the yeasts exert an effect of inhibiting transformation of primary bile acid to secondary bile acid. The present invention has been accomplished on the basis of these findings.

Accordingly, the present invention provides a cholesterol-lowering agent containing, as an active ingredient, at least one yeast belonging to Candida, Issatchenkia, Hanseniaspora, Kloeckera, Kluyveromyces, Pichia, or Torulaspora, as well as foods and drinks containing the yeast.

The present invention also provides use of these specific yeasts for producing a cholesterol-lowering agent, as well as foods and drinks for lowering cholesterol.

Further, the present invention provides a treatment method for lowering cholesterol through administration of these specific yeasts.

Still further, the present invention provides a secondary bile acid production inhibitor containing, as an active ingredient, a yeast, as well as foods and drinks containing the yeast.

Yet further, the present invention provides use of the yeast for producing a secondary bile acid production inhibitor, as well as foods and drinks for inhibiting secondary bile acid production.

Still further, the present invention provides a treatment method for inhibiting secondary bile acid production through administration of a yeast.

Brief Description of the Drawing Fig. 1 is a graph showing plasma cholesterol levels of a cholesterol-added diet administration group and a nocholesterol-added diet administration group.

Best Modes for Carrying out the Invention The yeast to be used in the cholesterol-lowering agent or foods and drinks according to the present invention is a yeast belonging to Candida, Issatchenkia, Hanseniaspora, Kloeckera, Kluyveromyces, Pichia, or Torulaspora. These yeast may be used singly or in combination of two or more species. The above yeast may be live cells, heated cells, lyophilized products, milled products thereof, or contents thereof.

Examples of preferred yeasts belonging to the above groups include Candida kefyr, Issatchenkia orientalis, Hanseniaspora uvarum, Kloeckera africana, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia farinosa, and Torulaspora delbrueckii. Of these, most preferred yeasts are the following: Candida kefyr YIT 8237 (FERM BP-7214, original deposit date July 16, 1999; depository organization, Ministry of International Trade and Industry, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-8566 JAPAN (the same applies hereafter)); Hanseniaspora uvarum YIT 8164 (FERM BP-7212, original deposit date July 16, 1999); Issatchenkia orientalis YIT 8266 (FERM BP-7215, original deposit date July 16, 1999); Kloeckera africana YIT 8165 (FERM BP-7213, original deposit date July 16, 1999), Kluyveromyces marxianus YIT 8292 (FERM BP-7217, original deposit date July 16, 1999), Kluyveromyces lactis YIT 8263 (FERM BP-7216, original deposit date July 16, 1999), Pichia farinosa YIT 8058 (FERM BP-7210, original deposit date July 16, 1999), and Torulaspora delbrueckii YIT 8114 (FERM BP-7211, original deposit date July 16, 1999).

From ancient times, these microorganisms have been known to be yeasts for producing foods (wine, milk liquor, cheese), and are strains that are considerably safe to the human body.

The above strains have characteristics as shown in Tables 1 to 5. The properties are nearly the same as those of similar microorganisms disclosed in "The yeast, 3rd edition," J. W. Kreger-Van Rij, Elsevier Science Publishers B. Amsterdam, 1984).

Table 1 characteristicso Stan

I

Strains D-Glucose 0-Galactose Maltose Me-a--D-qlucoside Sucrose (x,ac-Trehalose Melibiose Lactose Cellobiose Ca ndi da kefyr YIT 8237 Hanseniaspo-ra uvarum YIT 8164 Issa tchenkia orien talis YE? 8266 Kloechera africana YIT 8165 K! uyverowces marxianus YIT 8292 K! uyrveronyces lactis YIT 8263 P1 ch ia farinosa YTT 8058 Torulaspor a deibruecki Fer me nta tion Melezitose Rat finose r

I

1" i. I 1" 4 1 I nul in1 1 t f m C F n m m II I "1 1 -I D-Xylose I t I I I ID-Glucose I 1t 1 7 1 7- Growth.

0-Gelactose f D-Sorbose+-+- D-GJlucosamine__ fl% D 4 1 L/ N )O=Ja

.U

1. L I Table 2 Strains CaLdlida keffyr ~YIT 8237 H-ansenaaspora uva ru YIT 8164 Issatchenkia cr1 entalis YIT 8266 Kloech era africana YTT 8165 1-r I I D-Xylose L-Arabinose !-Arabinose L-Rhamnose Sucrose Maltose a, a-Trehalose Me-a--D-qlucoside Cal lobiose Salicin Arbutin Melibiose Lactose 7 .4

I

1(1uyverayces inarxianus YTT B292 Klyq cmce lactis YET 8263 Pichia fa riflesa YIT 8058 Torula spora clbruecki i YIT 8114 4-- Growth II I t I Rat finose Melezitose±- Inulin Starch Glycerol +4- Erythritol Ribitol---+

I

£Y _I 1 Table 3 Strains Candida kefyr Hanseniaspxra uvarwn~ Issa tchenkia orientalis Kloechera africana 1(1 uyveromyces marxianus YIT 237 YIT 8164 VT R2 vmQC i_ 1 :14oc 1 L-hrabinitol D-Glucitol 0-Mannitol-4- Gal act ito 1 myo- Ifosi tol D-Glucono-l, 2 -Keto-D-gluconate D-Gluconate '.rrm o'~rn 1 4

I

-4- -4- K! uyveromyces lactis YIT 8263 P1 oh ia fa ri nose YIT 8058 To-rulaspora delbrueckii YET 8114 4- I I r

I

Growth D-Glucronate D-Galacturonate DL-Lactate Succinate -4 4 Citrate Methanol Ethanol Propane-i, 2-diol- Butane-?, 3-diol Nitrate Nitrite Ethylamine LL tysi1ne tj I Table 4 Ca ndi da Hanseniaspora Issa tchenikia KJ aech era Kluyveramyces Xl uyveromyces P1 ab ia Tori 1a spa ra Strains kefyr ravarwn ozientais aifricana raarxianus lactis farinosa deibrueckii YIT 8237 YIT 8161 YIT 8266 YIT 8165 YIlT 8292 YET 8263 YIT 8058 YIT 8114 Cadaverine Creatine Creatinine Glucosamine Imidazole W/a Vitamins -4 W/O mya-Inasital W/O Pantothenic acid

+I

W/O Biotin Go-W/0 Thiamine Th W/O Biotin Thiamine N/O Pyridoxine N/a Pyridoxine&Thiamine N/a Niacin W/O PABA 2 StC 0 C 4 370C 4 O 0 C4 .01% Gycloheximide Table Candida Hanseniaspera Issatchenkia Kioechera Kiuyveromyces Kluyv.eronyces Pichia Torulaspora Strains kefyr uvanx orientais africana marxianus lactis farinosa deibrueckii YIT 8237 YIT 8164 YIT 8266 YIT 8165 TET 8292 YIT 8263 YIT 8058 tflT 8114 0.1% Cycloheximide Growth 1% Acetic f-Glucose f-Glucose Starch production Additionlal Acetic acid production Char acteri- Urea stics hydrolysis Diazonium Bluel B reaction The yeasts which are used in the secondary bile acid production inhibitor of the present invention will next be described.

No particular limitation is imposed on the yeasts which are used in the secondary bile acid production inhibitor.

The yeasts include live cells produced by culturing the yeasts, heated cells, lyophilized products, milled products thereof, and contents thereof.

The yeast to be used in the present invention has never been reported to inhibit elimination of the 7a-hydroxyl group of cholic acid or similar substances, to thereby inhibit formation of secondary bile acid such as deoxycholic acid. Since the present invention employs the effect of yeast on suppression and inhibition of transformation of primary bile acid to secondary bile acid, yeasts which have excellent absorption power to primary bile acids, such as, particularly, cholic acid, taurocholic acid, glycocholic acid, and chenodeoxycholic acid, as well as excellent power to elevate the intestinal concentration level of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid are preferred, from the viewpoint of excellent effect of inhibiting 7a-dehydroxylation.

Examples of yeasts preferably used in the secondary bile acid production inhibitor include Issatchenkia, Kluyveromyces, Hanseniaspora, Saccharomyces, Hyphopichia, Candida, Torulaspora, Pichia, and Zygosaccharomyces. These yeasts may be used singly or in combination of two or more species.

Specific examples include Issatchenkia orientalis, Kluyveromyces marxianus, Kluyvercyces lactis, Kluyveroyces thermotolerans, Hanseniaspora uvarum, Saccharomyces cerevisiae, Saccharomyces dairensis, Saccharomyces exiguus, Saccharomyces unisporus, Saccharomyces bayanus, Hyphopichia burtonii, Candida kefyr, Candida etchelisil, Candida zeylanoides, Candida solani, Candida maltosa, Candida tropicalis, Candida cylindracea, Candida utilis, Torulaspora delbrueckii, Pichia anomala, Pichia hoistil, and Zygosaccharomyces rouxii. Of these, most preferred yeasts are the following: Issatchenkia orentalis YIT 8266 (FERN BP-7215); Kluyveroryces marxianus YIT 8292 (FERN BP-7217); Kluyveromyces lactis YTT 8263 (FERM BP-7216); Kiuyveromyces thermotolerans YIT 8294 (ATCC 20309); Hanseniaspora uvarum YIT 8164 (FERN BP-7212); Saccharomyces cerevisiae YIT 8116 (ATCC 48554); Saccharomyces dairensis YIT 8191 (CBS 3007); Saccharomyces exiguus YIT 8109 (CBS 3019); Saccharomyces unisporus YIT 8226 (FERN BP-7209, original deposit date; March 25, 1999, depository organization, Ministry of International Trade and Industry, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-8566 JAPAN); Saccharomyces bayanus YIT 8128 (CBS 380); Iyphopichia burtonil YIT 8299 (CBS 2352); Candida kefyr YIT 8237 (FERN BP-7214); Candida etchelisii YIT 8278 (ATCC 60119); Candida zeylanoides YIT 8018 (IFO 0719); Candida solani YIT 8023 (CBS 1908); Candida maltosa YIT 8283 (ATCC 28140); Candida tropicalis YIT 8286 (CBS 94); Candida cylindracea YIT 8276 (ATCC 14830); Candida utilis YIT 8204 (ATCC 9950); Torulaspora delbrueckii YIT 8313 (JCM 2204) and YIT 8133 (IFO 1172); Pichia anomala YIT 8298 (JCM 3587) and YIT 8297 (JCM 3583); Pichia holstii YIT 8038 (ATCC 58048); and Zygosaccharomyces rouxii YIT 8129 (CBS 732).

From ancient times, these yeasts have been used to produce foods (wine, cheese), and are considerably safe microorganisms to the human body. The cells thereof, which are particles having a particle size of some microns, are much easily taken as compared with particles having a size of jum or more, which provide unfavorable sensation when taken.

The above yeasts; e Issatchenkia orentalis YIT 8266 (FERM BP-7215), have specific characteristics as shown in Table 6.

The characteristics are nearly the same as those of similar yeasts disclosed in "The yeast, 3rd edition," J. W.

Kreger-Van Rij, Elsevier Science Publishers B. Amsterdam, 1984).

Table 6 Characteristics of Issatchenkia orentalis YIT 8266 Fermentation D-Glucose D-Galactose Maltose Me-a-Dglucose Sucrose a,a-Trehalose Melibiose Lactose Cellobiose Melezitose Raffinose Inulin Starch D-Xylose Growth D-Glucose D-Galactose L-Sorbose D-Glucosamine D-Ribose D-Xylose L-Arabinose D-Arabinose L-Rhamnose Sucrose Maltose a,a-Trehalose Me-a-Dglucoside Cellobiose Salcin Arbutin Melibiose Lactose Raffinose Melezitose Inulin Starch Glycerol Erythritol Ribitol Xylitol L-Arabinitol D-Glucitol D-Mannitol Galactitol myo-Inositol D-Glucono-1,5lactone 2-Keto-Dgluconate D-Gluconate D-Glucuronate D-Galacturonate DL-Lactate Succinate Citrate Methanol Ethanol Propane-1,2-diol Butane-2,3-diol Nitrate Nitrite Ethylamine L-Lysine Cadaverine Creatine Creatinine Glucosamine Imidazole W/O Vitamin W/O myo-Inositol W/O Pantothenic acid W/O Biotin W/O Thiamin W/O Biotin thiamin W/O Pyridoxine W/O Pyridoxine thiamin W/O Niacin W/O PABA 25 0

C

300C 37 0

C

40 0

C

0.01% Cycloheximide 0.1% Cycloheximide 1% Acetic acid 50% D-Glucose 60% D-Glucose Additional characteristics Starch production Acetic acid production Urea hydrolysis Diazonium Blue B reaction The yeast of the present invention can be produced through a routine method; culturing in a complex medium containing yeast extract and polypeptone or in a synthetic medium predominantly containing an inorganic salt.

The thus-produced yeast can be employed, in the forms of live cells, lyophilized products thereof, killed cells obtained through heating or similar treatment, milled products thereof, or contents thereof, in a pharmaceutical composition or foods and drinks. In addition, commercially available yeast may also be used.

The aforementioned yeast may be formed into a pharmaceutical composition of a variety of drug foams, through a routine method together with pharmaceutically acceptable carriers. When a solid drug for peroral administration is prepared, the aforementioned yeast is mixed with a vehicle and optional additives such as a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, and an aroma, and the resultant mixture is formed, through a customary method, into tablets, coated tablets, granules, powder, or capsules. Additives generally employed in the art may be used as the above additives. Examples include vehicles such as lactose, white sugar, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, calcium phosphate, and poly(vinylpyrrolidone); disintegrants such as dry starch, sodium alginate, powdered agar, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, stearic monoglyceride, and lactose; lubricants such as purified talc, stearate salts, borax, and polyethylene glycol; and flavoring agents such as white sugar, orange peel, citric acid, and tartaric acid.

When a liquid drug for peroral administration is prepared, the aforementioned yeast is mixed with additives such as a flavoring agent, a buffer, a stabilizer, and an aroma, and the resultant mixture is formed, through a customary method, into peroral liquid, syrups, and elixirs.

The aforementioned flavoring agents may also be used.

Examples of the buffer include sodium citrate, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.

The foods and drinks of the present invention can be produced by adding the aforementioned yeast to a variety of foods and drinks. Examples of preferred foods and drinks include fermented milk, fruit-juice-blended drinks, soup, rice cakes, and cookies. Animals feeds are also included in the foods and drinks.

The amount of yeast which is to be added to the aforementioned drug varies in accordance with the drug form and the condition of the patient to which the drug is to be administered. Generally, an amount of 1-100 wt.% in the drug is preferred. The daily dose of the aforementioned drug or foods and drinks varies in accordance with the condition, body weight, age, sex, etc. of the patient, and cannot be determined as a fixed value. Generally, the daily dose per adult is approximately 10 mg to 30 g based on yeast, preferably approximately 1-5 g. The yeast is administered preferably once per day or 2-4 times per day in a divided manner.

EXAMPLES

The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto. As used herein, refers to Example 1 Preparation of yeasts Each of a variety of yeasts, which had been stored in a potato dextrose agar slant medium, was inoculated for one platinum loop into a medium (100 mL) shown in Table 7 placed in a Sakaguchi flask (500 mL), and the medium was subjected to shaking culture (120 spm) at 30 0

C.

Two days after initiation of culture, the culture (amount corresponding to two Sakaguchi flasks) was inoculated into a 10-liter fermentation tank (effective volume and aerobic agitation culture was carried out at 30 0 C for two days, under the following conditions: aeration rate of vvm, rotation rate of 250 rpm, and pH of 6.0 (automatically controlled by 5N sodium hydroxide).

After completion of culturing, the supernatant and cells were separated by means of a cooling centrifugator, and the separated cells were washed twice by distilled water.

The washed cells were placed in a 2-L Erlenmeyer flask, and distilled water (1 L) was added thereto. The mixture was heated at 115 0 C for 10 minutes in an autoclave. The thusheated cells as such were lyophilized.

Table 7 Composition of Medium for Pre-Culture and Culture Glucose (Kanto Chemical) 30 g Polypeptone (Daigo Eiyo) 10 g Yeast extract (Daigo Eiyo) 5 g Monopotassium phosphate (Wako Pure Chemical) Dipotassium phosphate (Wako Pure Chemical) g Magnesium sulfate (Wako Pure 0.5 g Chemical) Tap water 1 L pH Preparation of test diets By use of the yeast produced in ingredients were mixed in a routine manner, to thereby prepare diets having a composition shown in Table 8. Each diet contains cholesterol; cholesterol and sodium cholate In order to prepare a yeast-containing diet, each type of lyophilized yeast cells was added in an amount of or 10 Table 8 Diet Composition 10% Yeast 5% Yeast Control Ingredients administration administration group Ige n group group Casein 22.30 17.35 19.83 Soybean oil 1.0 1.0 Lard 10.0 9.45 9.72 Inorganic salt 4.0 3.54 3.77 Vitamins 1.0 1.0 Choline Ch o l i n e 0.15 0.15 0.15 bitartrate Cholesterol 0.5 0.5 Sodium cholate 0.25 0.25 0.25 Filter paper 10.0 7.46 8.73 powder Sucrose 50.80 50.12 50.46 Yeast cells 0.0 10.0 Test method Cholesterol-lowering effect Test diet Test diets prepared in Example 1 were employed.

Animal and breeding method a) Group of animals administered 10% yeast-containing diet Male, 5-week-old Wistar rats (obtained from Clea Japan) had been pre-bred for 7 days with diet powder F-2 (product of Oriental Yeast). The rats were divided into groups, each group containing 8 rats. To each group, the 10% yeastcontaining diet shown in Table 8 was administered for 7 days.

The rats were bred in group in a metal-made cage, and allowed to consume the administered diet and water ad libitum.

b) Group of animals administered 5% yeast-containing diet Male, 5-week-old Wistar rats (obtained from Nihon Kurea) had been pre-bred for 7 days with diet powder F-2 (product of Oriental Yeast). The rats were divided into groups, each group containing 8 rats. To each group, the yeast-containing diet shown in Table 8 was administered for 14 days. The rats were bred individually in a metal-made cage, and the amount of administered diet was limited to g/day, but water was provided ad libitum.

Measurement of plasma lipids a) Group of animals administered 10% yeast-containing diet Seven days after initiation of administration, blood was collected, under anesthetization with Nembutal (without fasting), from the abdominal aorta of each rat by use of a cannula, and the plasma cholesterol concentration and the plasma HDL concentration were measured. The total cholesterol concentration was measured by means of a biochemical automatic analyzer (Model 7170, Hitachi).

The HDL cholesterol concentration was measured by means of a biochemical automatic analyzer (Model 7170, Hitachi), after lipoprotein components other than HDL had been precipitated by use of Determiner HDL (product of Kyowa Medics).

b) Group of animals administered 5% yeast-containing diet Seven days after initiation of administration, blood was collected from the tail vein of each rat, and the plasma cholesterol concentration and the plasma HDL concentration were measured. The total cholesterol concentration was measured by means of a biochemical automatic analyzer (Model 7170, Hitachi).

The HDL cholesterol concentration was measured by means of a biochemical automatic analyzer (Model 7170, Hitachi), after lipoprotein components other than HDL had been precipitated by use of Determiner HDL (product of Kyowa Medics).

Measurement of intrahepatic lipids After perfusion of the liver by use of physiological saline, the liver was collected and lyophilized. Lipid extraction was performed by use of chloroform methanol (2 1) in accordance with the method of Folch et al. The resultant chloroform lower layer was concentrated to solid, and the solid was diluted again by use of ethanol. The diluted sample was subjected to measurement of lipid components contained in the liver. Among hepatic lipids, the amount of cholesterol was measured by use of Determiner TC 555.

Method for evaluating animal test results Statistical method Upon statistical analysis of the animal test results, homogeneity of variance was tested by the Bartlett method.

When the variance, was found to be homoscedastic, analysis of variance was performed in a one-way layout manner, followed by Dunnett's multiple comparison test, whereas when the variance was found to be heteroscedastic (test objects from which. significant difference was obtained through Bartlett test at a level of significance under 5 analysis of variance was performed through the Kruskal-Wallis test, followed by Dunnett's multiple comparison test. In Dunnett's multiple comparison test, the level of significance was set at 5% and and the test was performed at each level of significance.

Method for obtaining percent reduction of lipid components <Percent reduction in plasma cholesterol> The expression "100% reduction in plasma cholesterol" refers to a plasma cholesterol level lowered to a plasma cholesterol level of a rat belonging to a group administered an ordinary diet (no-cholesterol-added). The express "0% reduction in plasma cholesterol" refers to a plasma cholesterol level elevated to a plasma cholesterol level of a rat belonging to a group administered a cholesterol-added diet (control).

Atherogenic index Atherogenic index (AI) is represented by the following formula: AI (VLDL cholesterol LDL cholesterol)/HDL cholesterol.

AI was calculated from the total plasma cholesterol level and the plasma HDL cholesterol; actual plasma lipid measurements, according to the following formula: AI ((total plasma cholesterol) (plasma HDL cholesterol))/(plasma HDL cholesterol).

The percent improvement of atherogenic index was calculated from the AI value of each yeast-containing-diet administration group and the AI value of cholesterol-addeddiet administration group (control group). The "expression 100% improvement of atherosclerotic index" refers to an AI value identical to the AI value of the no-cholesterol-addeddiet administration group. The expression improvement of atherosclerotic index" refers to an AI value identical to the AI value of the cholesterol-added-diet administration group (control group).

Body weight measurement The body weight of each rat was measured upon purchase thereof. The rats were separated into groups such that no significant difference in body weight was present among the groups, and pre-breeding was started. The body weight was measured before starting the test and after completion of the test.

The aforementioned test method was adopted to the following pre-tests and Examples.

Preliminary test: Blood was collected from the tail vein of rats after administration of the cholesterol-added diet; on day 0, day 5, and day 8. On day 14, blood was collected, under anesthetization with Nembutal, from the abdominal aorta.

Plasma was separated from each collected blood sample, and subjected to lipid component measurement through an enzyme method.

As shown in Fig. 1, the plasma cholesterol level of the cholesterol-added diet administration group increased to approximately the maximum at day 5. Thereafter, a slight increase was observed until day 14, but no significant change was observed. The plasma cholesterol level of the nocholesterol-added diet administration group (diet prepared by removing cholesterol and sodium cholate from the cholesteroladded diet) did not increase during the test period, and a tendency of slight decrease in level was observed.

Table 9 shows the plasma lipid concentration on day 14 after initiation of administration of cholesterol-added diet.

The cholesterol level of the no-cholesterol-added diet administration group is 72.5 mg/dL, whereas that of the cholesterol-added diet administration group is 325.3 mg/dL, indicating an approximately 4.5 fold increase in level. In contrast, the plasma HDL cholesterol level of the cholesterol-added diet administration group is 45.9 mg/dL, significantly lower than that of the no-cholesterol-added diet administration group (31.4 mg/dL).

Table 9 Plasma Lipid Concentration (day 14) Diet administration grou Cholesterol HDL cholesterol ps (mg/dL) (mg/dL) Cholesterol free diet 72.5 6.1 45.9 4.2 (Control group) Cholesterol added diet (high-fat diet 325.3 48.6" 31.4 4.00** administration group) **p<0.01 Table 10 shows the hepatic lipid content on day 14 after initiation of administration of the cholesterol-added diet. The cholesterol level of the cholesterol-added diet administration group is about 20 times the cholesterol level of the no-cholesterol-added diet administration group.

Table Intrahepatic Lipid Content (day 14) Total cholesterol Diet administration groups (mg/liver) Cholesterol free diet 32.9 8.4 (Control group) 39 Cholesterol added diet 637.9 60.70** (high-fat diet administration group) **p<0.01 Example 2 Jar culture was performed in accordance with the method of Example 1, to thereby prepare 200 g of lyophilized cells (heated at 110 0 C for 10 minutes) of each yeast. Each cell sample was mixed into a cholesterol-added diet at 10% yeast group in Table The resultant diet was administered to each group consisting of eight rats, and consumed ad libitum for seven days.

Tables 11 to 13 show the percent reduction of cholesterol level and the percent improvement of atherosclerotic index, based on the plasma cholesterol level of the high-cholesterol-diet administration control group.

No significant change in weight was observed among the tested groups.

Table 11 YIT No.

8164 8301 8263 8292 8266 8299 8058 8237 8165 8313 8114 8298 8280 8104 8278 8026 8018 8007 8223 Percent Reduction of Plasma Cholesterol and Percent Improvement of AI Yeast taxonomic genus and Plasma lipid Av. body species Percent reduction Dunnett's test Percent improveme weight of cholesterol nt of AI Hanseniaspora uvarum 76.5 0.01 78.0 224.0 Pichia quilliermondii 74.7 0.01 60.1 216.4 Kluvveromyces lactis 72.8 0.01 68.8 220.4 Kluvveromyces marxianus 65.5 -0.01 55.8 219.6 Issatchenkia crientalis 64.2 -0.01 55.3 225.0 Hyphopichia burtonii 62.7 0.01 54.0 219.0 Pichia farinosa 60.3 0.05 51.8 212.1 Candida kefyr 60.0 0.01 59.3 216.4 Kloeckera africana 54.9 n.s. 54.1 216.8 Torulaspora delbrueckii 52.7 0.01 51.0 221.4 Torulaspora delbrueckii 52.5 0.05 46.2 217.4 Pichia anomala 50.7 0.05 38.0 223.2 Candida qlabrata 48.1 0.05 42.9 215.0 Kluyveromyces marxianus 48.0 0.05 44.9 221.0 Candida etchellsii 46.1 0.01 41.5 224.2 Debaryomyces hansenii 44.5 0.05 30.8 217.0 Candida zelanoides 43.5 0.05 48.4 218.4 Candida krusei 38.3 0.05 43.8 225.6 Candida qropenqiesseri 38.1 n.s. 37.2 220.0 Table 12 Percent Reduction of Plasma Cholesterol and Percent Improvement of AI Plasma lipid Yeast taxonomic genus v bd YIT No. Yeast taxonomic genus Percent reductin Dunnett's te Percent improveme Av body and species of cholesterol st nt of AI weight (g) we gh 8247 Pichia capsulata 38.1 n.s. 22.6 220.5 8294 Kluvveromyces thermotolerans 37.7 n.s. 63.4 220.4 8159 Candida maqnoliae 37.1 n.s. 51.8 220.6 8281 Candida qropenqiesseri 36.5 0.05 34.6 224.0 8006 Candida parapsilosis 36.4 n.s. 33.8 218.0 8314 Yarrowia lipolytica 35.8 n.s. 24.5 208.2 8110 Torulaspora delbrueckii 34.5 n.s. 23.5 217.0 8133 Torulaspora delbrueckii 33.7 n.s. 32.3 209.8 8300 Pichia etchellsii 33.3 n.s. 52.0 223 2 8302 Pichia membranaefaciens 31.5 n.s. 46.6 220.4 8241 Candida valida 30.4 n.s. 34.0 223.2 8303 Pichia subpelliculosa 27.7 n.s. 48.2 221.4 8285 Candida stellata 26.8 n.s. 21.3 219.0 8277 Candida diversa 26.0 n. s. 28.6 226.4 8291 Issatchenkia terricola 25.2 n.s. 30.1 212.2 8107 Torulaspora delbrueckii 24.8 n.s. 20.2 207.8 8034 Williopsis californica 23.2 n.s. 10.2 214.8 8023 Candida solani 22.9 n.s. 25.0 226.6 8288 Candida vini 21.8 n.s. 20.7 223.8 8033 Pichia anomala 20.4 n.s. 23.0 220.6 Table 13 YIT No.

8012 8181 8304 8312 8120 8317 8129 8315 Percent Reduction of Plasma Cholesterol and Percent Improvement of AI Plasma lipid Yeast taxonomic genus and s ie Percent reduction Dunnett's Percent and species improvement of of cholesterol test AI Yarrowia lipolytica 13.3 n.s. 22.4 Rhodotorula minuta 12.2 n.s. -15.4 Rhodotorula qlutinis 1.0 n.s. -13.4 Saccharomycopsis malanqa -2.1 n.s. -11.1 Pachytichospora transvaalensis -9.7 n.s. -24.3 Zyqosaccharomyces rouxii -10.2 n.s. 0.6 Zyqosaccharomyces rouxii -34.2 n.s. -55.9 ZyqosaccharomVces bailii -67.5 n.s. -77.9 Av. body weight (g) 216.6 224.0 221.0 215.6 207.8 216.8 199.2 209.0 Example 3: 10% Yeast-added diet administration test Test method; Jar culture was performed in accordance with the method of Example 1 to thereby prepare 200 g of lyophilized cells of each yeast. Each cell sample was mixed into a cholesterol-added diet at 10% (10% yeast group in Table 8).

The resultant diet was administered to each group consisting of eight ratsi and consumed ad libitum for seven days. Table 14 shows the results of plasma lipid measurement.

Table 14 Plasma lipid (10% mixed administration) Total chole HDL choles- Reduction of Percent Strains s-terol terol cholesterol AI improvement of (mg/dL) (mg/dL) A( Control group (high-fat-diet administration group) 226.0 14.9 21.7 1 3.4 9.62 1.75 administration group) Klueromyes marxa134.5 29.7 23.2 3.9 65.5 4.84 1.22"* 55.8 Kluyveromyces marxianus YIT 8292 ssatce a or ais YIT 826 136.2 18.7" 23.4 3.0 64.2 4.88 0.99" 55.3 Issatchenkia orientalis YIT 8266 Control group (high-fat diet administration group) 244.5 50.3 23.7 3.4 9.43 2.33 eromyes latis YI126.8 26.4" 27.2 1.8 72.8 3.67 1.06"* 68.8 Kluyveromyces lactis YIT 8263 Control group (high-fat diet 230.1 26.9 25.9 3.5 7.97 1.17 administration group) 127.5 i 19.4" 35.9 4.6* 76.5 2.58 0.54" 78.0 Hanseniaspora uvarum YIT 8164 Candida kefyr YIT 8237 133.8 19.4" 27.8 2.4 60.0 3.87 1.06" 59.3 Candida kefyr YIT 8237 Control group (high-fat diet 222.0 14.9 23.5 6.1 8.94 3.10 administration group) aruasra ru ii Y 148.1 19.9" 23.7 1.9 52.5 5.30 1.05" 46.2 Torulasa darina YIT 8054 140.6 18.7" 24.8 4.8 60.3 4.86 2.07" 51.8 PKleca africana YIT 865 143.7 37.5" 25.4 1.9 54.9 4.68 1.26" 54.1 Kloeckera africana YIT 8165 **p<0.01 Through administration of a cholesterol-added high-fat food, the control group exhibited increase in total cholesterol level and elevation of atherosclerotic index to 7.97-9.62. However, the test groups- rats to which a diet containing the yeast cells according to the present invention had been administered---were found to remarkably suppress elevation of the values in terms of all analyzed items, even though the same high-fat diet was administered.

Thus, yeasts; Kluyveromyces marxianus YIT 8292, Kluyveromyces lactis YIT 8263, Hanseniaspora uvarum YIT 8164, Issatchenkia orientalis YIT 8266, Candida kefyr YIT 8237, and Pichia farinosa YIT 8058, exhibited remarkably excellent percent reduction of plasma cholesterol and were found to improve atherogenic index.

Example 4: 5% Yeast-added diet administration test The procedure of Example 3 employing male Wistar rats was repeated, except that each lyophilized cell sample was mixed into a diet for test groups at 5% yeast group in Table the resultant diet was administered to each group consisting of eight rats; with restricted amount g/day) for 14 days.

Table 15 shows the results of plasma lipid analysis. As a result of administration of a high-fat food, the control group exhibited increase in total cholesterol level and elevation of atherogenic index. However, the test groups--rats to which a diet containing the yeast cells according to the present invention had been administered---were found to remarkably suppress elevation of the values in terms of all analyzed items, even though the same high-fat diet was administered.

Table Strains Control group (high-fat diet administration group) Kluyveromyces marxianus YIT 8292 Control group (high-fat diet administration group) Kluyveromyces lactis YIT 8263 Candida kefyr YIT 8237 Kloeckera africana YIT 8165 Control group (high-fat diet administration group) Hanseniaspora uvarum YIT 8164 Issatchenkia orientalis YIT 8266 Plasma lipid Percent cholesterol cholesterol cholesterol eent cholesterol cholesterol eent chodL (mger i m po ve( n t m g d

L

(mg/dL) of AI 339.7 34.8 205.3 30.1" 23.2 3.8 23.6 5.2 57.6 13.85 1.91 7.87 1.18** 46.8 377.8 75.3 21.9 1.9 16.33 3.83 222.5 27.2" 23.9 4.4 55.3 8.59 2.01" 50.7 228.9 69.1" 25.4 5.0 53.0 7.98 1.74" 54.7 276.9 54.6" 25.9 5.2 36.1 9.89 2.30" 42.1 360.0 44.7 237.8 34.8"* 241.5 49.6" 243.3 38.5" 21.5 5.2 24.2 2.8 20.9 2.8 24.5 3.1 46.5 45.1 44.3 18.37 6.81 8.45 2.45** 9.95 1.56"* 8.86 2.09** 57.3 48.7 55.0 Control group (high-fat diet administration group) 284.2 54.1 19.9 4.5 13.66 3.34 Torulaspora delbrueckii YIT 199.2 37.3" 21.9 5 5. .4 8.63 3.41" 40.0 p81140.01 **p<0.01 Table 16 shows the results of hepatic lipid measurement.

In all test groups, the total hepatic cholesterol level decreased significantly, as compared with the control group.

Thus, the yeasts according to the present invention have been found to serve as an excellent anti-cholesterol material which exerts an effect of lowering plasma lipid through peroral administration, as well as an effect of lowering hepatic lipid.

Table 16 Total cholesterol Strains (mg/liver) Control group 538.9 53.5 (high-fat diet administration group) Kluyveromyces marxianus YIT 8292 459.6 44.9" Control group 469.1 54.3 (high-fat diet administration group) Kluyveromyces lactis YIT 8263 370.2 51.1" Candida kefyr YIT 8237 368.5 67.1" Kloeckera africana YIT 8165 399.5 43.3" Control group 422.2 57.2 (high-fat diet administration group) Hanseniaspora uvarum YIT 8164 390.8 49.1" Issatchenkia orientalis YIT 8266 348.5 36.9" Pichia farinosa YIT 8058 331.9 40.2" Control group 463.1 66.9 (high-fat diet administration group) Torulaspora delblueckii YIT 8114 380.1 65.4" **p<0.01 Example 5 Bile acid adsorption power Test method; For each of cholic acid taurocholic acid (TCA), glycocholic acid (GCA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA), a sodium salt thereof (product of Sigma) was dissolved in a 0.1M phosphate buffer (pH 6.7) or a 0.1M phosphate buffer (pH 7.5) such that the final concentration was adjusted to 1 mM.

Lyophylized cells (100 mg) of Issatchenkia orientalis YIT 8266 were placed in a centrifugation tube (15 mL), and each bile acid solution (3.5 mL) prepared above was added thereto. The mixture was shaken (120 spm) at 37 0 C. One hour later, yeast cells were caused to precipitate through centrifugation (12,000 rpm, 10 minutes), to thereby collect the supernatant. The bile acid contained in the supernatant was quantitated by use of Enzabile II (product of Daiichi Pure Chemicals Co., Ltd.), and percent bile acid adsorption was calculated on the basis of the following formula. As used herein, the term "percent bile acid adsorption" refers to a ratio of the amount of bile acid adsorbed by yeast to the total amount of added bile acid, and the ratio serves as an index of the adsorption strength between the yeast and the bile acid.

percent bile acid adsorption [{added bile acid (nmol) bile acid in supernatant (nmol)}/added bile acid (nmol)] x 100 Table 17 shows the percent bile acid adsorption.

Issatchenkia orientalis YIT 8266 has been found to adsorb cholic acid, taurocholic acid, glycocholic acid, or chenodeoxycholic acid, exhibiting excellent primary bile acid adsorption power. The percent adsorption of bile acid by yeast did not depend on the pH (6.7 and 7.2) of the reaction mixture. These results indicate that the yeast tightly binds to primary bile acid in the large intestinal tract and inhibits deconjugation and 7a-dehydroxylation caused by enterobacteria, to thereby inhibit production of secondary bile acid.

In addition, the yeast tightly binds to deoxycholic acid----a type of secondary bile acid. This indicates that, in addition to inhibiting of production of secondary bile acid, the yeast also adsorbs and immobilizes formed secondary bile acid, to thereby reduce the toxicity thereof.

Table 17 Percent Bile Acid Adsorption by Issatchenkia orientalis Yeast CA CA TCA GCA CDCA DCA pH6.7 pH7.5 pH7.5 pH7.5 pH7.5 Issatchenkia orientalis 40.3 40.2 29.3 43.8 68.8 73.7 YIT 8266 Example 6 In a manner similar to that employed in Example percent adsorption of bile acid by a variety of yeasts was measured. Table 18 shows the results. All of the yeasts exhibited excellent adsorption to bile acids; i.e., chenodeoxycholic acid (primary bile acid) and deoxycholic acid (secondary bile acid).

Table 18 Percent Adsorption of Bile Acid by Yeasts Percent adsorption of bile acid Strains YIT CDCA DCA No. (pH7.5) Kluyveromyces marxianus 8292 63.0 60.0 Kluyveromyces lactis 8263 54.4 41.0 Kluyveromyces thermotolerans 8294 79.5 53.6.

Hanseniaspora uvarum 8164 68.0 40.3 Saccharomyces cerevisiae 8116 66.6 54.5 Saccharomyces dairensis 8191 50.8 44.7 Saccharomyces exiguus 8109 55.5 50.4 Saccharomyces unisporus 8226 58.8 65.5 Saccharomyces bayanus 8128 61.8 64.3 Hyphopichia burtonii 8299 51.6 35.3 Candida kefyr 8237 56.2 55.3 Candida etchellsii 8278 55.8 45.1 Candida zeylanoides 8018 76.1 56.0 Candida solani 8023 52.0 36.9 Candida maltosa 8283 58.3 46.4 Candida tropicalis 8286 58.3 40.1 Candida cylindracea 8276 58.8 53.0 Candida utilis 8204 51.6 42.2 Torulaspora delbrueckii 8313 58.6 52.6 Torulaspora delbrueckii 8133 64.6 49.4 Pichia anomala 8297 56.4 43.4 Pichia anomala 8298 51.6 60.9 Pichia holstii 8038 58.8 54.6 Zygosaccharomyces rouxii 8129 48.0 39.8 Cellulose (Comparative) 4.1 3.7 Example 7 Inhibition of secondary bile acid production by lyophilized cells of Issatchenkia orientalis YIT 8266 Preliminary test Male, 5-week-old Wistar rats (obtained from Nihon Kurea) had been bred for 7 days with diet powder F-2 (product of Oriental Yeast). The rats were divided into groups such that no weight difference generates among the groups, each group containing 8 rats. To each group, the diet shown in Table 19 was administered for 14 days. The rats were bred individually in a metal-made cage, and allowed to consume diet and water ad libitum.

Table 19 Diet Composition Ordinary High-bile-acid d Ingredient diet let Casein 22.30 22.30 Sucrose 56.55 55.80 Inorganic salt 4.00 4.00 Vitamins 1.00 1.00 Soybean oil 1.00 1.00 Lard 10.00 10.00 Choline bitartrate 0.15 0.15 Cholesterol 0.00 0.50 Sodium cholate 0.00 0.25 Filter paper powder 5.00 5.00 After completion of 14 days' breeding, no significant differences in diet consumption and in body weight increase were observed between the ordinary-diet administration group and the high-bile-acid-diet administration group.

.Bile acid level Lyophylized feces (day 12 to day 14) (25 mg) were weighed and placed in a test tube (15 mL) equipped with a screw cap, and an internal standard substance 3c, 17ct, 20a-triol) (0.15 Imol) and ethanol (5 mL) were added thereto. The mixture was heated at 70 0 C for two hours by means of a block heater. The resultant insoluble matter by means "5 was pecipitated through centrifugation (3,000 rpm, minutes) The supenatant was transferred to another test tube and dried to solid under nitrogen flow. Te extract was dissolved in methanol (0.5 mL), and the resultant insoluble matter was removed by means of a filter (0.45 xm) (product of Niho Millipore Ltd.). An analysis sample (10 was Nh° l l i p o r e L t o-f an HPLC system (product subjected to separation by means of an of Jasco) under the following conditions.

Table 20 Conditions for Separating Bile Acids quAcetonitrile Methanol 30 mM Ammonium acetate Liquid

A:

(30:30:40) mixture acetate tiquid B: Acetonitrile Methanol 30 mM Ammoniumacetate (20:20:60) mixture (20:20:60)Proportional concentration gradient from 100% B Proportional coce 32 min followedby Elution 0% A to 0% B 100% A over 32 m. followed by conditions: elution for 13 min. by use of 100%

A.

1.0 mL/min Flow rate: .0'mL/mi Column 25°C t coumn product O J a s c o Bilepak-I' (4.6 mm i.d. X250 mm) (separation column, product of Jasco) The eluate provided from a separation column was mixed, at a flow rate of 1.0 m/minute, with a reaction mixture (0.3 mM p-nicotinamide adenine dinucleotide (P-NADO), 1 mM ethylenediaminetetraacetic acid, and 10 mM potassium phosphate buffer (pH 7.8) containing 0.05 ercaptoethanol) The liquid mixture was transferred to an enzyme column (Enzymepak (4.6 mm i.d. x 35 mm, product of Jasco) charged with 3a-hydroxysteroid dehydrogenase, where NADH (reduced form of P-NAD produced upon dehydroxylation of bile acid was monitored by means of a fluorescence detector (excitation wavelength 345 nm, emission wavelength 470 nm).

Each bile acid was identified on the basis of the retention time with reference to the standard. The bile acid concentration was obtained from the corresponding peak area.

In this case, percent recovery was corrected by the peak area of the internal standard.

Table 21 shows the results. The results indicate that administration of cholic acid increases the level of primary bile acid reaching the large intestinal tract, to thereby promote production of secondary bile acid. Thus, the effect of a secondary bile acid production inhibitor can be evaluated at high sensitivity.

Table 21 Level of Bile Acids Excreted in Feces [pmol/day] Ordinary diet High-bile-acid diet Primary bile acid a-Muricholic acid 1.52 0.35 8.52 3.05* P-Muricholic acid 0.20 0.09 8.48 1.55* Hyocholic acid 0.00 0.00 0.23 0.21* Cholic acid 0.00 0.00 29.20 7.44** Chenodeoxycholic acid 0.00 0.00 0.57 0.20" Total primary 1.72 0.40 47.00 10.31* bile acids Secondary bile acid Deoxycholic acid 0.72 0.26 9.13 5.92** Lithocholic acid 0.18 0.10 0.13 0.18"n.s Hyodeoxycholic acid 0.01 0.03 1.06 0.54* Total secondary 0.90 0.35 10.32 5.84* bile acids Note: Student's t-test Significance level Significance level 1% n.s. no significance Inhibition of secondary bile acid production by lyophilized cells of Issatchenkia orientalis YIT 8266 Lyophylized cells of Issatchenkia orientalis YIT 8266 were added to a high-bile-acid diet, and the resultant diet was administered for 14 days mix diet) (Table 22). The energy score of each diet was equalized by controlling the' amount of casein, saccharose, lipid, or cellulose.

Administration of yeast cells provided substantially no influence on diet consumption and substantially no influence on increase in body weight during the test period (Table 23).

Table 22 Diet Composition Yeast-added Ingredients High-bile-acid diet high-bile-acid diet Casein 22.30 20.56 Sucrose 55.80 54.15 Inorganic salt 4.00 4.00 Vitamins 1.00 1.00 Soybean oil 1.00 1.00 Lard 10.00 9.85 Choline bitart e 0.15 0.15 bitartrate Cholesterol 0.50 0.50 Sodium cholate 0.25 0.25 Filter paper Powder 5.00 3.55 Powder Heat-dried yeast cells* 0.00 5.00 *Issatchenkia orientalis YIT 8266 Table 23 Diet Consumption and Body Weight Increase during Test Period High-bile-acid diet Yeast-added high-bile-acid diet Diet consumption 236.0 [g/14 days] Body weight increase [g/14 days] 95.9 Note: Student's t-test n.s. no significance 8.6 4.0 227.8 21. 5 n s 93.6 11.9n As shown in Tables 24 and 25, through administration of Issatchenkia orientalis YIT 8266, the level of secondary bile acid excreted in feces decreased by approximately 82%, and the secondary bile acid concentration in feces decreased by approximately 84%. This tendency was observed to be particularly remarkable for deoxycholic acid and lithocholic acid. The level of primary bile acid excreted in feces and the primary bile acid concentration in feces were significantly increased through administration of the corresponding cells.

Thus, Issatchenkia orientalis YIT 8266 exerted an effect of strongly inhibiting conversion of primary bile acid to secondary bile acid.

Table 24 Level of Bile Acids Excreted in Feces [pmol/day] Yeast-added High-bile-acid high-bile-acid diet diet P uricd 13.70 3.40 15.27 3.12ns a-Muricholic acid 7.32 2.07 11.60 2.48 p-Muricholic acid 0.12 0.17 0.29 0.15n"s.

Hyocholic acid 24.37 5.86 35.30 2.55" Chenodeoxycholic acid 63.80 6.81 Total primary 46.60 7.30 63.80 6.81 Bile acids Secondary bile acid Secoile 10.10 6.51 0.22 0.46* Deoxycholic acid 0.35 0.31 0.00 0.00* Lithocholic acid 080 0.32 1.81 0.6 Hyodeoxycholic acid 0.81 Total secondary 11.25 6.59 2.03 0.81 Bile acids Note: Student's t-test significance level Significance level 1% n.s. no significance Table Composition of Bile Acids in Feces Yeast-added High-bile-acid diet high-bile-acid diet Primary bile acid a-Muricholic acid 23.58 5.32 23.11 3.47 n s P-Muricholic acid 12.59 3.07 17.50 2.51** Hyocholic acid 0.19 0.27 0.43 0.22"n s Cholic acid 42.47 11.42 53.87 3.01* Chenodeoxycholic acid 1.89 0.65 2.06 0.50 n Total primary 80.72 11.55 96.97 0.98"" bile acids Secondary bile acid Deoxycholic acid 17.30 11.46 0.30 0.67* Lithocholic acid 0.59 0.53 0.00 0.00* Hyodeoxycholic acid 1.39 0.59 2.73 0.89" Total secondary 19.28 11.55 3.03 0.98** bile acids Yeast-added High-bile-acid diet high-bile-acid diet Note: Student's t-test Significance level Significance level 1% n.s. no significance Lowering pH in the cecum by lyophilized cells of Issatchenkia orientalis YIT 8266 The contents in the decum (day 14) (0.5 g) were suspended in purified water (4.5 mL), and a 10% aqueous perchloric acid solution (0.5 mL) was added to the suspension.

The resultant suspension was stored at 4 0 C overnight. The suspension was centrifuged (9,000 rpm, 10 minutes), and the supernatant was collected and passed through a filter (0.45 pm) (product of Nihon Millipore Ltd.). The thus-prepared analysis sample was subjected to HPLC.

Short-chain fatty acids were analyzed by means of an HPLC system (product of Toa Dempa Kogyo) under the following conditions. The pH buffer was mixed with the eluent immediately before passage of the detector.

Table 26 Short-Chain Fatty Acid Analysis Conditions Column: Eluent(flow): Column temperature: pH-Regulator (flow): Detector: Cell temperature: KC-811 Shodex (Showa Denko) x 2 Perchloric acid solution containing 7% acetonitrile (I mL/min) 42 0

C

mM Perchloric acid solution containing 7% acetonitrile and mM tris(hydroxymethyl)aminomethane (1 mL/min) Conductivity detector (Toa Denpa Kogyo) 45 0

C

Table 27 pH and Short-Chain Fatty Acid Content in the Cecum High-bile-acid Yeast-added diet high-bile-acid diet pH in the cecum 6.65 0.08 6.46 0.20* Short-Chain Fatty Acid content [mM] Acetic acid 26.6 8.4 37.5 Propionic acid 7.2 2.3 17.5 Butyric acid 2.2 1.4 3.9 0.8* Lactic acid 2.5 1.0 2.6 1.2" n s Succinic acid 15.5 7.3 21.8 7.4"n..

Formic acid 1.1 0.6 1.8 1.3.s Isobutyric acid 0.7 0.6 0.4 0.4 "1- Total short-chain fatty acid55.9 15.7 85.6 5.2** fatty acids Note: Student's t-test Significance level Significance level 1% n.s. no significance High-bile-acid-diet groups exhibited a pH in the cecum of 6.65 and a total amount of short-chain fatty acid of 55.9 mM, whereas yeast (Issatchenkia orientalis YIT 8266)-added high-bile-acid-diet groups exhibited a pH in the cecum of 6.46 (significance level and a total amount of shortchain fatty acid of 85.6 mM (significance level indicating decrease in pH in the cecum. Thus, the large intestinal pH condition was shifted to more acidic, to thereby deactivate 7a-dehydroxylase of enterobacteria.

Inhibition of cholesterol metabolism by lyophilized cells of Issatchenkia orientalis YIT 8266 Feces (day 14) (25 mg) were weighed and placed in a test tube (15 mL) equipped with a screw cap, and an internal standard substance (50-cholestane) (0.1 pmol) and a ethanol solution containing IN sodium hydroxide (1 mL) were added thereto. The mixture was heated at 80°C by means of a block heater. One hour after, distilled water (0.5 mL) was added, and neutral sterols were taken through two rounds of extraction by use of petroleum ether (2.5 mL). The resultant extract was washed with distilled water (7.5 mL), dehydrated over sodium sulfate, and dried to solid under nitrogen gas flow. The recovered neutral sterols were trimethylsilylated, and analyzed through gas chromatography under the following conditions (Table 28).

Table 28 Chromatographic (GC) Analysis Conditions GC apparatus: Model 5890 (Yokogawa-Hewlett-Packard) Column: SPB-1 FS-coated capillary glass column (Supelco) 0.32 mm i.d. x 30 m 2500C, 8 min 270°C, 10 0 C/min 270°C, Column temperature: 15 min Carrier (flow): He (0.8 mL/min) Injector: Spilit, T 2300C Detector: FID, T 280°C Sample injection: 1 4L Analysis time: 25 min Through administration of Issatchenkia orientalis YIT 8266, the concentrations in feces of coprostanol and coprostanone--decreased cholesterol metabolites in the intestine significantly (Table 29). Since these cholesterol metabolites in the large intestine are related to onset of diseases such as colovectal cancer (Suzuki, et al., Cancer Lett., 33, 307-316, 1986), Issatchenkia orientalis YIT 8266 is thought to be capable of preventing or treating a variety of diseases, by means of inhibiting production of cholesterol metabolites.

Table 29 Neutral Cholesterol Composition in Feces Yeast-added High-bile-acid high-bile-acid diet diet Coprostanol 25.7 12.5 9.2 11.9* Coprostanone 1.9 2.5 0.4 0.6 n Cholesterol 72.3 14.2 90.5 12.4* Coprostanol Coo 27.7 14.2 9.5 12.4" Coprostanone Note: Student's t-test Significance level n.s. no significance Example 8 Inhibition of secondary bile acid production by cell contents of Issatchenkia orientalis YIT 8266 Preparation of cell contents Lyophilized cells of Issatchenkia orientalis YIT 8266 were suspended in a 0.1M phosphate buffer (pH and the cells were mechanically ground by means of DYNO-MILL (product of Shinmaru Enterprises). During grinding, 0.45 mm glass beads were employed as a grinding agent, and grinding operation was repeated until grinding of 95% or more cells was confirmed through microscopic observation. The milled cell liquid was centrifuged (9,000 g, 15 minutes), to thereby isolate cell contents in the supernatant. The cell contents were collected and lyophilized.

Inhibition of secondary bile acid production by cell contents of Issatchenkia orientalis YIT 8266 In a manner similar to that employed in Example 7, the effect of the cell contents prepared from Issatchenkia orientalis YIT 8266 on inhibition of secondary bile acid production was confirmed.

Table 30 shows the diet composition employed in the test. Administration of yeast cell contents exerted substantially no influence on diet consumption and substantially no influence on increase in body weight during the test period (Table 31).

Table Diet Composition S. Cell-content-added High-bile-acid Ingredients diet high-bile-acid dietdiet Casein 22.30 20.78 Sucrose 55.80 53.12 Inorganic salt 4.00 4.00 Vitamins 1.00 1.00 Soybean oil 1.00 1.00 Lard 10.00 9.77 Choline bitartrate 0.15 0.15 Cholesterol 0.50 0.50 Sodium cholate 0.25 0.25 Filter paper powder 5.00 4.43 Cell contents 0.00 5.00 Table 31 Diet Consumption and Increase in Body Weight during the Test Period Hid Cell-content-added High-bile-acid diet high-bile-acid diet Diet consumption 232.8 9.9 220.7 6.5 n [g/14 days] Body weight increase 95.9 4.0 88.4 4.5 n [g/14 days] Note: Student's t-test n.s. no significance As shown in Tables 32 and 33, as a result administration of cell contents of Issatchenkia orientalis YIT 8266, the level of secondary bile acid excreted in feces decreased by approximately 76%, and the secondary bile acid concentration in feces decreased by approximately Thus, similar to the case of lyophilized Issatchenkia orientalis YIT 8266, cell contents thereof also exerted an effect of strongly inhibiting conversion of primary bile acid to secondary bile acid.

Table 32 Level of Bile Acids Excreted in Feces [pmol/day] Cell-content-added High-bile-acid i. high-bile-acid diet diet Primary bile acid a-Muricholic acid 8.56 1.85 6.80 2.04 n s P-Muricholic acid 6.80 2.18 8.70 3.04 n s Hyocholic acid 0.20 0.15 0.25 0.06 Cholic acid 28.96 11.99 29.99 4.77n.

s Chenodeoxycholic acid 0.44 0.29 0.64 0.35 n Total primary 44.96 13.17 46.37 9.40"bile acids Secondary bile acid Deoxycholic acid 9.87 5.46 2.01 2.26" Lithocholic acid 0.02 0.05 0.00 0.00 n s Hyodeoxycholic acid 0.65 0.36 0.49 0.14n" s Total secondary 10.54 5.29 2.51 2.18* bile acids Note: Student's t-test Significance level 1% n.s. no significance Table 33 Composition of Bile Acids in Feces Hd Cell-content-added High-bile-acid diet high-bile-acid diet Primary bile acid a-Muricholic acid 16.01 4.33 13.75 1.82 n s P-Muricholic acid 12.39 3.19 17.30 3.29" Hyocholic acid 0.34 0.20 0.51 0.09n.' Cholic acid 51.04 10.63 62.47 6.95** Chenodeoxycholic acid 0.76 0.34 1.27 0.56"- Total primary 79.82 8.31 94.02 3.61** bile acids Secondary bile acid Deoxycholic acid 8.29 8.90 3.64 3.76** Lithocholic acid 0.04 0.11 0.00 0.00" s Hyodeoxycholic acid 1.13 0.38 1.07 0.44 n s Total secondary 20.18 8.31 5.98 3.61* bile acids Note: Student's t-test Significance level 1% n.s. no significance In terms of the total amount of short-chain fatty acid and the pH in the cecum, high-bile-acid-diet groups exhibited a total amount of 54.0 mM and a pH of 6.65, whereas yeastcell contents-added high-bile-acid-diet groups exhibited a total amount of 68.3 mM (significance level and a pH of 6.46 (significance level Thus, the yeast cell contents shifted the large intestinal pH condition to more acidic by increasing the amount of short-chain fatty acid, to thereby deactivate 7a-dehydroxylase of enterobacteria.

Table 34 pH and Short-Chain Fatty Acid Content in the Cecum .Cell-content-added High-bile-acid ie high-bile-acid diet pH in the cecum 6.65 0.086 diet pH in the cecum 6.65 0.08 6.46 0.20' Short-Chain Fatty Acid content [mM] Acetic acid 26.2 9.0 30.2 7.1 n s 3 Propionic acid 7.2 2.5 14.3 2.2" Butyric acid 2.2 1.5 2.1 0.9n".

Lactic acid 2.4 1.0 1.9 1.2 n Succinic acid 14.3 7.1 17.8 6.4 n s Formic acid 0.9 0.3 1.8 1.7 n s Isobutyric acid 0.7 0.7 0.2 0.2n.

Total short-chain 54.0 15.9 68.3 13.9* fatty acids Note: Student's t-test Significance level Significance level 1% n.s. no significance As a result of administration of cell contents of Issatchenkia orientalis YIT 8266, the concentrations in feces of coprostanol and coprostanone---decreased cholesterol metabolites in the intestine significantly. Thus, similar to the case of the lyophilized cells, cell contents of Issatchenkia orientalis YIT 8266 are thought to be capable of preventing or treating a variety of diseases by inhibiting production of cholesterol metabolites.

Table Neutral Sterol Composition in Feces High- Cell-content-added bile-acid high-bile-acid diet diet Coprostanol 21.1 8.2 10.0 9.9 Coprostanone 2.0 1.6 0.5 Cholesterol 76.9 9.6 89.6 10.2 Coprostanol Coprostanone 23.1 9.6 12.1 10.7* Note: Student's t-test Significance level Significance level 1% Example 9 By use of a variety of yeast cells employed in the aforementioned Examples, foods and drinks of the following compositions were produced.

1) Food for maintaining and promoting health (tablets) A composition containing the following additives was pelletized, to thereby prepare tablets.

Table 36 Composition of Food for Maintaining and Promoting Health Dried yeast cells Plant extract powder Royal jelly powder Collagen powder Lactose Corn starch Hydroxypropyl celllose 4 Mg stearate 1 2) Drink for maintaining and promoting health .A drink for maintaining and promoting health was produced in accordance with the following formulation.

Table 37 Composition of Drink for Maintaining and Promoting Health Dried yeast cells Honey Citric acid 0.1 dl-Malic acid 0.1 Plant extract (cinnamon) D-Solbitol liquid Sodium benzoate 0.05 Flavors suitable amount Purified water balance 3) Fruit-juice-blended drink The drink was produced in accordance with the following formulation.

Table 38 Composition of Fruit-Juice-Blended Drink Glucose liquid sugar 33 Grapefruit juice Dried yeast cells Flavors suitable amount Sour agent suitable amount Fermented milk Fermented milk containing heated yeast cells was produced in accordance with the following formulation.

To a sterilized mixture of 10% skimmed milk powder liquid and 5% glucose, a bacterium belonging to Lactobacillus was inoculated, to thereby produce yogurt. To the yogurt, heated yeast cells obtained in Example 1 were added in an amount of 0.1-20%, to thereby produce fermented milk.

Milk liquor To a sterilized mixture of 10% skimmed milk powder liquid and 5% glucose, a bacterium belonging to Lactobacillus and heated yeast cells obtained in Example 1 were inoculated simultaneously. The mixture was subjected to stationary culturing at 37 0 C for 48 hours, to thereby produce milk liquor.

Industrial Applicability The cholesterol-lowering agent of the present invention comprising the aforementioned yeast cells or yeast cell contents exerts a remarkable effect of lowering plasma cholesterol and hepatic cholesterol, and therefore is very effective for preventing arteriosclerosis and other diseases caused by accumulation of cholesterol.

The secondary bile acid production inhibitor of the present invention strongly inhibits production of secondary bile acid through adsorption of bile acid, acidic-shift of pH condition in the large intestine, etc., and therefore is very effective for prevention and treatment of colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, cholelithiasis, etc.

In addition, the yeasts employed in the present invention having no pathogenicity, are considerably safe, as evidenced by these yeasts having been used for producing cheese, koumiss, wine, etc. from ancient times. Thus, the cholesterol-lowering agent of the present invention comprising the yeast cells or yeast cell contents raises no problem when administered for a long period of time. This is confirmed by the finding that no death case has been observed among rats to which the agent had been perorally administered at a dose of 8 g/kg.

Thus, the cholesterol-lowering agent and secondary bile acid production inhibitor of the present invention can be used as a peroral pharmaceutical, and are also very effective for maintaining health and preventing arteriosclerosis, colovectal cancer, liver cancer, pancreatic cancer, bile duct cancer, cholelithiasis, etc. by causing constant consumption of foods containing the agent or inhibitor.

Claims (7)

1. A food or drink when used for inhibiting secondary bile zacid production containing a yeast as an active ingredient.

2. A food or drink according to claim 1, wherein the yeast is at least one species Issatchenkia, Kluyveromyces, Hanseniaspora, Saccharomyces, Hyphopichia, Candida, Torulaspora, Pichia, and Zygosaccharomyces. (i

3. A food or drink according to claim 1, wherein the yeast is (iat least one species selected from among Issatchenkia orien tais, Kluyverornyces marxianus, Kluyveromyces lactis, Kluyveromyces theriotolerans, Hanseniaspora uvarum, Saccharomyces cerevisiae, Saccharomyces dairensis, Saccharomyces exiguus, Saccharomyces unisporus, Saccharomyces bayanus, Hyphopichia burtonii, Candida kefyr, Candida etcheilsil, Candida zeylanoides, Candida solani, Canclida maltosa, Candida tropi calls, Candida cylindracea, Candida utilis, Torulaspora deibrueckii, Pichia anornala, Pichia hoistii, and Zygosaccharomyces rouxii.

4. A treatment method for inhibiting secondary bile acid production, comprising administration of a yeast.

A method according to claim 4, wherein the yeast is at least one species selected from among Issatchenkia, Kluyveromyces, Hanseniaspora, Saccharomyces, Hyphopichia, Candida, Torulaspora, Pichia, and Zygosaccharomyces.

6. A method according to claim 4, wherein the yeast is at least one species selected from among issatchenkia orientalis, Kluyveromyces rnarxianus, Kluyveromyces lactis, Kluyveromyces thermotolerans, Hanseniaspora uvarum, Saccharornyces cerevisiae, Saccharomyces dai-rensis, Sacoha-romyces exiguus, Saccharomyces unisporus, Saccharornyces bayanus, Hyphopichia burtonii, Candida 66 W:Wjies\71 1645\71 1645 spwi~o tt~ keffyr, Candida etchelisii, Candida zeylanoides., Candida solani, Candida maltosa, Candida tropi calls, Candida cylindracea, Candida utilis, Torulaspora clbrueckli, Pichia anomala, Pichia zholstii, Pachiticospora transverensis, and yoacrmce rouxii.

7. A method according to claim 4 substantially as herein M described. CI 10 DATED: 3 November 2005 CIPHILLIPS ORMONDE FITZPATRICK Patent Attorneys for: KABUSHIKI KAISHA YAKULT HONSHA WA\Fil.\7l1645\71 1645 .Lido