954 Journal of Food Protection, Vol. 56, No. 11, Pages 954-957 (November 1993) Copyright©. International Association of Milk, Food and Environmental Sanitarians Selective Enumeration of Bifidobacterium bifidum, Enterococcus faecium, and Streptomycin-Resistant Lactobacillus acidophilus from a Mixed Probiotic Product MELISSA L. CALICCHIA*, CONNIE I. E. WANG, TAKEHIRO NOMURA1, FUMIO YOTSUZUKA2, and DARYL W. OSATO Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/11/954/1664591/0362-028x-56_11_954.pdf by guest on 27 September 2021 Silliker Laboratories of California, Inc., 1139 E. Dominguez Street, Carson, California 90746 (Received for publication October 2, 1992) ABSTRACT Shimada et al. (17) and Teraguchi et al. (20) reported methods to determine Bifidobacterium selectively from Modified VF-Bouillon agar with 0.5 mg/ml lithium chloride, 20 dairy products by utilizing susceptibility differences to ug/ml sodium lauryl sulfate, 5 mg/ml sodium propionate, and 10 ug/ml antimicrobials. These methods, however, were only appli­ neomycin sulfate was used with a triple-layer diffusion technique to selectively enumerate Bifidobacterium bifidum. Modified Brigg's agar cable to wet probiotic products where the bacteria had not was used to enumerate Enterococcus faecium. Modified Brigg's agar been injured by drying. Cell injury caused by the drying with 1,200 Ug/ml streptomycin sulfate was used in a double-layer process is likely to have caused alterations in cellular diffusion technique to selectively enumerate a streptomycin-resistant chemical susceptibility (13). Subsequently, Teraguchi et al. strain of Lactobacillus acidophilus. Selective enumeration of the indi­ (21) developed an applicable method but could not differ­ vidual bacterial components was compared to the mixture with an entiate Enterococcus from Bifidobacterium. Mitsuoka et al. average 99% recovery of each component. (8) then suggested selective biochemicals for enumeration of bifidobacteria in liquid culture. This method was suc­ Claims have been made that various intestinal microor­ cessful in separating Enterococcus and Bifidobacterium but ganisms including bifidobacteria, Lactobacillus acidophi­ was not applicable to dry cells. lus, and some strains of Enterococcus faecium are impor­ In our studies, percent recoveries were determined for tant factors for human health. Clinical and other studies are all three test microorganisms in the presence of varying currently providing more information concerning the role concentrations of LiCl. The recovery of B. bifidum and E. of probiotics for use in vivo (1,2,6,15,16,18). faecium was studied in the presence of varying concentra­ The production and use of cultured yogurt dates back tions of neomycin sulfate. Combinations of neomycin sul­ centuries. Lactic acid bacteria contained in these products fate and sodium lauryl sulfate (SLS) were also tested for can implant in the human intestine and competitively in­ their combined effects on B. bifidum and E. faecium. The hibit less desirable bacteria. effect of neomycin sulfate on B. bifidum was also compared Precise determination of viable levels of several of when applied in a multiple-layer diffusion technique in­ these bacteria, when dried and blended into one product, stead of by direct addition to the whole medium. The use can be difficult. The purpose of this procedure was to of sodium propionate in addition to SLS and neomycin provide an accurate and direct plating method for enumera­ sulfate was also studied for the improved recovery of B. tion of Bifidobacterium bifidum, E. faecium, and streptomy­ bifidum. Selective recovery of E. faecium and streptomy­ cin-resistant L. acidophilus. cin-resistant L. acidophilus in the presence of streptomycin Previous methodologies (3) for enumeration of bacte­ sulfate was also studied. ria in mixed commercial dried probiotic products are inad­ equate for selective enumeration of these three genes. A MATERIALS AND METHODS number of reports (9,10,14,19) have suggested methods to determine bifidobacteria cell counts contained in matrices VF-Bouillon agar (12) plus LiCl (Sigma Chemical Co., St. consisting of mixed bacteria such as animal feces, water, Louis, MO), SLS (Sigma), neomycin sulfate (Sigma), and sodium and human feces. Tanaka and Mutai (79) used a most propionate (Sigma) was used as a selective medium for B. bifidum. probable number procedure to statistically estimate Modified Brigg's agar (4) with streptomycin sulfate (Sigma) was bifidobacterial levels. Resnick and Levin (14) and Munoa used to selectively enumerate L. acidophilus. Modified Brigg's and Pares (10) studied selective methods for application to agar was used to enumerate E. faecium. aquatic samples only. Bacterial cultures 'Biofermin Pharmaceutical, Co., Ltd. Kobe 653, Japan. Both Bifidobacterium bifidum G9-1 and Lactobacillus aci­ 2Takeda U.S.A. Inc., Orangeburg, NY 10962-2614. dophilus KS-13 were obtained from the Tokyo University culture JOURNAL OF FOOD PROTECTION, VOL. 56, NOVEMBER 1993 SELECTIVE ENUMERATION IN PROBIOTICS 955 collection. The call numbers are internal Biofermin identifica­ Sample preparation and incubation tion numbers. Enterococcus faecalis 129 BIO 3B (reclassified Five-gram samples of dried bacterial product were diluted as E. faecium) was obtained from the Kyoto. University culture 1:10 in appropriate amount of diluting fluid (8) (KH2P04 [Sigma], collection. The call numbers are also internal Biofermin identi­ 4.5 g; Na2HP04 [Sigma], 6 g; L-cystine HC1*H20 [Sigma], 0.5 g; fications. Tween 80 [Sigma], 0.5 g; agar [BBL], 0.4 g; 10% antifoam B silicone emulsion [Dow Corning No. B531-05], 1.0 ml; and DI Preparation of wet and dry cultures water, 1,000 ml). The diluting fluid was adjusted to pH 7.0 ± 0.1 B. bifidum and E. faecium were grown in modified VF- and then autoclaved at 121°C for 30 min. The initial 1:10 sample Bouillon broth at 37°C for 24 h. This was modified VF-Bouillon homogenate was preincubated at 37°C for 25 min, then diluted agar without LiCl (Sigma), agar (granulated, BBL, Cockeysville, accordingly in dilution solution. MD), SLS (Sigma), sodium propionate (Sigma), and antifoam (Dow Corning No. B531-05). L. acidophilus was grown at 37°C Selective plating procedure for 24 h in the same medium with 0.1% Tween 80 (Sigma) added. B. bifidum. Each dilution portion plus 10 ml of modified VF- The cultures were spray dried using a Niro atomizer (Denmark). Bouillon agar was poured into respective 100 x 15-mm sterile The inlet temperature was 90°C and the outlet was approximately disposable petri dishes and mixed. After solidification, a 10-ml 65°C. Dextrin was the excipient. overlay of the base media was applied to each plate. After the overlay solidified, 10 ml of the base media with 30 ug/ml Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/11/954/1664591/0362-028x-56_11_954.pdf by guest on 27 September 2021 Media for viable cell counts neomycin sulfate was overlayed as the third layer on each plate. Modified VF-Bouillon Agar for B. bifidum, and modified All plates were incubated anaerobically (C02-Steel wool method) Brigg's agar for L. acidophilus and E. faecium were the basal (9) at 37°C for 48 to 72 h (5). media used, respectively. All agar media were autoclaved for 15 E. faecium. Each dilution portion was applied on top of 8 ml min at 121°C and tempered to 45 °C prior to use or addition of of solidified base media (modified Brigg's agar), followed by a biochemicals. 12-ml overlay of the same base media. Plates were incubated Prevot (12) developed VF-Bouillon agar, which has been under aerobic conditions at 37°C for 24 h. modified for selective enumeration of B. bifidum. Our modifica­ L. acidophilus. Each dilution portion was applied on top of tion of VF-Bouillon agar consisted of lean beef liver extract, 8 ml of solidified base media (modified Brigg's agar), followed 1,000 ml; lactose (Sigma), 10 g; peptone (Difco), 10 g; L-cystine by a 12-ml overlay of base media with 2,000 ug/ml streptomycin (Sigma), 400 mg; 10% antifoam B silicone emulsion (Dow Corn­ sulfate. Plates were incubated under aerobic conditions at 37°C ing No. 531-05), 1 ml; agar (BBL), 9 g; LiCl (Sigma), 500 mg; for 48 h. SLS (Sigma), 20 mg; and sodium propionate (Sigma), 5 g. The lean beef liver extract and the L-cystine (Sigma) solu­ Identification of colonies tions were prepared prior to being combined with the other In order to validate selectivity of the media, 10% of the ingredients in the final medium. colonies within countable range (25-250) were selected at random Lean beef liver extract was made using 1 kg each finely and Gram stained. L. acidophilus were gram-positive rods with ground raw lean ground beef and beef liver. Four liters of rounded ends, while B. bifidum were club-shaped gram-positive deionized (DI) water was added and the mixture heated to 55°C rods. E. faecium were gram-positive cocci. for 3 h. The mixture was then brought to a boil, removed from the Percent recoveries for each of the three dried test microor­ flame, cooled to 30°C, and coarse filtered using gauze. The pH of ganisms in the presence of LiCl were determined. B. bifidum and the filtrate was then adjusted to between 4.0 to 4.2 and then E. faecium recoveries were compared on VF-Bouillon agar filtered using Whatman No. ] paper. The pH of the filtrate was incubated at 37°C for 72 h. Varying concentrations (0, 0.25, 0.50, then readjusted to 7.2 and filtered again using a Whatman No. 1 1.00, and 3.0 mg/ml) of LiCl were used. Brigg's agar with the filter. Volume of the final filtrate was then brought up to 4 L using same LiCl variables was used to test dry L.