A small, anaerobic, gram-positive coccobacillus that reduces cholesterol to coprostanol was isolated from a hog sewage lagoon. This isolate, strain HLT (T = type strain) does not require cholesterol for growth, but it requires lecithin and has phospholipase activity. Much acid is produced by the fermentation of amygdalin, lactose, and salicin. Arabinose, cellobiose, fructose, glucose, mannose, and melibiose are fermented weakly. Acetic, formic, and succinic acids are produced, as is hydrogen, The isolate does not reduce nitrate, produce indole, or hydrolyze starch and gelatin. Esculin is hydrolyzed. The properties of strain HLT are most similar to those of members of the genus Eubacterium. Because strain HL (= ATCC 51222) has unique morphological and physiological properties, we propose that it should be the type strain of a new species in the genus Eubacterium, Eubacterium coprostanoligenes.To date, 13 strains of cholesterol-reducing bacteria have been isolated and characterized (2, 4, 12,14). All of these organisms were isolated from the fecal contents of rats, humans, or baboons and were classified as strains of Eubactenurn species.Because of the methods used and the sources sampled, most of the strains of cholesterol-reducing bacteria that have been characterized have similar properties. These bacteria require strict anaerobic conditions for growth, and all but two strains require a plasmalogen (plasmenylethanolamine) to reduce cholesterol to coprostanol (1). Many also require cholesterol or a related sterol for growth (4, 12,14).Coprostanol is not absorbed by the human gastrointestinal system. Therefore, the use of bacteria that can convert cholesterol to coprostanol holds promise for medical reasons and is of interest to food industries. New methods and a wide range of sample types have been tested to facilitate the isolation of new cholesterol-reducing bacteria (5, 6). In this paper, we describe characterization of a cholesterol-reducing bacterium isolated from a hog sewage lagoon. Because strain H L has unique morphological and physiological properties, including the capacity to reduce cholesterol to coprostanol, we propose that it should be the type strain of a new species, Eubactenum coprostanoligenes. MATERIALS AND METHODSMedia and culture conditions. Base cholesterol medium (BCM) contained (per liter) 2 g of Casitone (Difco Laboratories, Detroit, Mich.), 10 g of yeast extract, 2 g of cholesterol (ash-free; Sigma Chemical Co., St. Louis, Mo.), 1 g of lecithin (type IV-S; Sigma), 0.5 g of sodium thioglycolate, 1 g of CaCl, . 2H,O, and 1 mg of resazurin. Plasmenylethanolamine was not added to this medium. BCM was prepared as described by Brinkley and coworkers (l), with the following modifications: cholesterol and lecithin were homogenized in distilled, purified water in a Waring blender under a stream of 0,-free N, gas (7); other components were added; and the pH was adjusted to 7.2 with 3 N KOH. The medium was boiled under an N, atmosphere and cooled, and 9-ml aliquots were dispensed into culture tube...

Two media were developed that allowed both a total coliform count and an Escherichia coli count to be determined on the same medium after 24 h of incubation at 35°C. The new media were tested along with two standard media on 10 surface water and 7 sewage samples. The experimental media yielded equivalent or higher counts relative to the standard media and recovered more specifically the desired indicator groups as determined by colony identification. Current membrane filtration methods have been criticized for their many shortcomings (for a review, see reference 15). Several investigators have suggested that Escherichia coli be used as an indicator because it is specific for fecal pollution and provides a more accurate indicator-to-pathogen ratio than total or fecal coliform enumeration methods (4, 7, 9). Increased attention is being given to the recovery of injured indicator bacteria (3, 5, 6, 19, 20, 22, 23). One of the major factors causing reduced recoveries of injured bacteria is the selective agent(s) that is used. Monensin is an ionophore that selectively inhibits all gram-positive bacteria while it allows growth of injured gram-negative bacteria (25). Tergitol 7, a surfactant, has a similar effect (17, 19). Recently, a very sensitive and specific method for the detection of P-glucuronidase production by E. coli was developed (10, 15, 25). Over 95% of E. coli hydrolyze a nonfluorescent substrate, 4-methylumbelliferyl-p-D-glucuronide (MUG), to produce 4-methylumbelliferone, which fluoresces under long-wave UV light. Approximately 50% of Shigella spp., some Salmonella spp., and a few strains of Yersinia enterocolitica are the only other members of the families Enterobacteriaceae and Vibrionaceae that produce this enzyme (12, 14, 18, 21, 25, 29). The purpose of this study was to develop an improved membrane filtration method by using media that contain monensin, Tergitol 7, and MUG. The goal was the enumeration of total coliforms and E. coli on a single medium incubated at 35°C with improved recovery of injured cells and improved selective specificity when compared with standard membrane filtration media and methods. MATERIALS AND METHODS Media. MUG was obtained from Hach Co. (Loveland, Colo.). Monensin (90 to 95% pure) was obtained from Sigma. All media were prepared with 1.5% agar, sterilized, tempered to 47°C, and poured in approximately 3-ml volumes into petri plates (diameter, 47 mm). Peptone-Tergitol-glucuronide (PTG) agar was prepared as described previously (5). Peptone glucuronide (PG) agar was prepared exactly as PTG agar, except that the Tergitol 7 (J. T. Baker Chemical Co., Phillipsburg, N.J.) was omitted. PG agar was poured into standard petri plates (diameter, 100 mm) for the isolation and purification of colonies. Enriched

Health Benefits of Reduced Serum Cholesterol Levels 3 Cholesterol and Colon Cancer 4 Absorption of Sterols by the Human Digestive System 7 Cholesterol-reducing Bacteria 11 Mechanism of Cholesterol Reduction 22 Other Related Microbial Steroid Reactions 26 Eubacteria 38 Technologies for Reducing Cholesterol in Foods PART I. ISOLATION OF CHOLESTEROL-REDUCING BACTERIA Introduction Materials and Methods Bacteria Growth media Clear cholesterol-containing media 53 Isolation media Growth of cholesterol-reducing bacteria in clear media Isolation procedure Sterol conversion assay Results and Discussion 65 Media development 65 iii Preliminary studies on the isolation of cholesterol-reducing bacteria and on cholesterol reductase 68 Successful isolation of pure cultures of CRB 72 PART II. CHARACTERIZATION OF A CHOLESTEROL-REDUCING BACTERIUM HO

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