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. acidophilus. The DI water. This final solution can be stored up to 2 months frozen. plates were incubated at 37°C for 48 h. L-cystine (Sigma) was added to the modified VF-Bouillon Percent recoveries for dried individual cultures of B. bifidum agar as a solution. Four hundred milligrams L-cystine (Sigma) and E. faecium in the presence of neomycin sulfate were deter­ was dissolved in 1 ml warm dilute 1 N HC1 (Sigma). mined. Varying concentrations (200, 100, 50, 20, 10, 5, and 0 ug/ In the final medium, 1,000 ml lean beef liver extract was ml) of neomycin sulfate were tested using VF-Bouillon agar. The combined with the 1 ml L-cystine solution and the other ingredi­ plates were incubated at 37°C for 72 h. ents listed above. All ingredients were mixed, brought to a boil, Wet and dry recoveries of B. bifidum and E. faecium in the and cooled to 45°C. The pH was adjusted to 7.1 ±0.1 and the presence of a combination of neomycin sulfate and SLS were medium autoclaved. compared. VF-Bouillon agar containing 5, 10, and 20 |ig/ml Basal modified Brigg's agar was prepared by combining neomycin sulfate plus 20 ug/ml SLS was tested. B. bifidum and tomato juice solution, 200 ml; glucose (Sigma), 20 g; peptone E. faecium plates were incubated at 37°C for 72 h. (Difco), 20 g; yeast extract (Difco), 5 g; beef extract (Difco), 10 The recovery of E. faecium and L. acidophilus in the g; Tween 80 (Sigma), 2 g; L-cystine HC1*H20 (Sigma), 1 g; 10% presence of streptomycin sulfate was compared on Brigg's agar. antifoam B silicone emulsion (Dow Corning No. B531-05), 1 ml; Varying concentrations of 500, 1,000, 1,200, 1,500, and 2,000 ug/ and agar (BBL), 8 g. ml streptomycin sulfate were used. The plates were incubated at The tomato juice solution was prepared prior to combining it 37°C for 48 h. with the above ingredients. Three hundred grams of commercial tomato juice (Del Monte) was blended 1:1 with DI water. The RESULTS AND DISCUSSION tomato juice solution was then adjusted to pH 6.5 using NaOH (Sigma), boiled for 5 min, cooled to ambient temperature, and filtered using Whatman No. 1 paper. Selective media for Bifidobacterium The other ingredients in modified Brigg's agar were then Mitsuoka et al. (8) and Teraguchi et al. (20) suggested mixed with 200 ml of the tomato juice solution. The medium was that LiCl at a concentration of 3 mg/ml restricted the then adjusted to a volume of 1,000 ml using DI water, boiled, growth of L. acidophilus and had little effect on the growth cooled, adjusted to pH 7.0 ± 0.1, and autoclaved. of B. bifidum. This level had an inhibitory effect on B.

JOURNAL OF FOOD PROTECTION, VOL. 56, NOVEMBER 1993 956 CALICCHIA, WANG, NOMURA, YOTSUZUKA AND OSATO bifidum when used with dry cells. The percent recoveries with SLS are presented in Table 3. Because the recovery of for each dried test microorganism with variable levels of B. bifidum was reduced to only 55% for dry cells, the LiCl are presented in Table 1. The optimal concentration of neomycin sulfate was applied by diffusion. LiCl was 0.5 mg/ml. There was no effect on B. bifidum, while the growth of L. acidophilus was restricted. The TABLE 3. Recovery of wet and dry B. bifidum and E. faecium growth of E. faecium, however, was not affected at this cells in the presence of neomycin sulfate (NM) with sodium lauryl concentration. sulfate (SLS).

NM and SLS Recovery (%) TABLE 1. Recovery of B. bifidum, E. faecium, and L. acidophi­ concentration lus at various concentrations (mg/ml) of LiCl. (ug/ml) Wet mixed culture Dry mixed culture

Concentration Recovery (%) B. bifidum E. faecium B. bifidum E. faecium of LiCl (mg/ml) B. bifidum L. acidophilus E. faecium NM 5 + SLS 20 100 0.01 58 0.01 NM 10 + SLS 20 100 <0.01 55 <0.01

0.00 100 100 100 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 NM 20 + SLS 20 90 <0.01 40 <0.01 0.25 100 14 100 0.50 100 <0.01 100 1.00 93 <0.01 100 In a comparison between application of neomycin 3.00 63 <0.01 88 sulfate directly and by diffusion, recoveries of B. bifidum were higher with triple-layer diffusion. This technique Miller and Finegold (7) found that the majority of caused slow dispersion of the neomycin sulfate to a final bifidobacteria strains show resistance to neomycin and concentration of 10 ug/ml. To confirm this, 10 ug/ml suggested the use of this antibiotic in bifidobacteria selec­ neomycin sulfate was added to a single-layer medium tive media. In this case it was intended to restrict the with a suspension of B. bifidum and E. faecium from dried growth of enterococci while allowing bifidobacteria to culture. It was compared with 30 ug/ml added to the top grow. Mitsuoka et al. (8) and Teraguchi et al. (20) chose a layer of a three-layer plate where the cells were contained combination of neomycin sulfate, and paromomycin sul­ in the bottom layer. Recovery of dried B. bifidum in­ fate, and Ueno (23) reported that anaerobic bacteria show creased by 71% when the triple-layer diffusion technique strong resistance against amino-sugar antibiotics in general. was used. By utilizing this method, cell count recovery of Neomycin sulfate was used throughout the study to select dried B. bifidum in a mixture was improved to 94% as against enterococci. compared to individual dried cells. Use of the triple-layer diffusion technique caused E. faecium recovery to in­ The concentration of neomycin sulfate required to crease to 2.3% however. prevent growth of E. faecium suggested by Mitsuoka et al. (8) and Teraguchi et al. (20) ranged from 100 to 200 |ig/ml Uehara et al. (22) reported that sodium propionate for wet cultures. This level, however, was too high since it inhibits growth of E. faecalis. Mitsuoka et al. (<5) used 15 restricted the growth of dry B. bifidum as well as E. mg/ml sodium propionate in Bifidobacterium selective faecium cells. Percent recoveries of wet and dry B. bifidum media. When 5 mg/ml sodium propionate was added in and E. faecium cells in the presence of varying levels of addition to applying the triple-layer diffusion technique neomycin sulfate are presented in Table 2. Decreasing the with neomycin sulfate, the percent recovery of B. bifidum usage level of neomycin sulfate alone was not effective in increased from 94% of the total to 99% of the total. The selecting for B. bifidum because it led to the outgrowth of percent recovery of E. faecium decreased to <0.01%. E. faecium. The combined effect at or below 10 ug/ml neomycin sulfate, plus 20 ug/ml SLS as suggested by Selective media for E. faecium and L. acidophilus Ohbayashi (77), was effective in selectively recovering E. faecium colonies were easily counted when they D bifidobacteria in a wet culture. Percent recoveries of B. were grown in modified Brigg's agar at 37 C for 24 h bifidum and E. faecium in the presence of neomycin sulfate under aerobic conditions. B. bifidum is an obligate anaerobe and, thus, cannot be cultured under the above conditions. L. TABLE 2. Recovery of wet and dry B. bifidum and E. faecium acidophilus was able to grow to form visible colonies in cells on VF-Bouilion agar with neomycin sulfate. this medium if incubation time exceeded 24 h. As men­ tioned previously, L. acidophilus can be cultured in modi­ Neomycin Recovery (%) fied Brigg's agar at 37°C under aerobic conditions. A sulfate selective biochemical which inhibited the growth of E. concentration Wet culture Dry culture faecium was desired so that a direct enumeration of the L. (Ug/ml) B. bifidum E. faecium B. bifidum E. faecium acidophilus could be made. Tetracycline and streptomycin 0 100 100 100 100 were considered for inhibition of E. faecium. Tetracycline 5 100 97 63 83 could not be used because it reacted with other media 10 100 82 56 74 constituents. Streptomycin sulfate restricted growth of E. 20 92 74 45 65 faecium while having no influence on growth of L. aci­ 50 82 0.05 31 0.01 dophilus when used at 1,000 to 2,000 Ug/ml level, as 100 79 <0.01 21 <0.01 presented in Table 4. Using our procedure with Brigg's 200 57 <0.01 2.8 <0.01 agar, the final equilibrated concentration of streptomycin

JOURNAL OF FOOD PROTECTION, VOL. 56, NOVEMBER 1993 SELECTIVE ENUMERATION IN PROBIOTICS 957

TABLE 4. Recovery of E. faecium and L. acidophilus using upon the survival of Salmonella in randomly selected human carri­ various concentrations of streptomycin sulfate on Brigg's agar. ers. Prog. Food Nutr. Sci. 7:13-17. 2. Bhattacharya, P. R.. and M. K. Majumdar. 1983. Survival of orally administered isolated intestinal Lactobacillus acidophilus Streptomycin Recovery (%) in different parts of the gastrointestinal tract of mice. J. Biol. Sci. sulfate concentration 5:97-105. E. faecium L. acidophilus 3. Brennan, M., B. Wanismail, and E. Ray. 1983. Prevalence of viable Lactobacillus acidophilus in dried commercial products. J. Food <0.01 100 Prot. 46:887-892. <0.01 100 4. Briggs, M. 1953. An improved medium for lactobacilli. J. Dairy Res. <0.01 100 20:36-40. <0.01 100 5. Busta, F. F., E. H. Peterson, D. M. Adams, and M. G. Johnson. 1984. <0.01 92 Colony count methods, pp. 62-83. In M. L. Speck (ed.), Compendium of methods for the microbiological examination of foods. American Public Health Association, Washington, DC. sulfate decreased from 2,000 to 1,200 |4g/ml throughout the 6. Clements, M. S., M. M. Levine, P. A. Ristaino, V. E. Daya, and T. plate after diffusion. P. Hughes. 1983. Exogenous lactobacilli fed to man. Their fate and ability to prevent diarrheal disease. Prog. Food Nutr. Sci. 7:29-37. 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 The recoveries for each test organism from both indi­ 7. Miller, T„ and S. M. Finegold. 1967. Antibacterial sensitivity of vidual and mixed cultures, when assayed using the final Bifidobacterium. J. Bacteriol. 93:125-130. procedure, are presented in Table 5. This method has been 8. Mitsuoka, T., T. Seqa, and S. Yamamoto. 1965. Eine verbesserte used successfully with excellent agreement between two methodik der qualitativen und quantilativen analyse der darmflora von menschen an tieren. Zentralbl. Bakteriol. Parasitenkd. Abt. I laboratories for over 2 years. As presented in Table 5, cell Orig. 195:455-469. counts for each microorganism were compared by assaying 9. Mitsuoka, T. 1974. Microbiology of lactic acid producing bacteria. dry single culture samples versus dry mixed culture. We Rinshou kensa. 18:1163-1172. concluded that our methods were effective for accurate 10. Munoa, F. J., and R. Pares. 1988. Selective medium for isolation and enumeration of Bifidobacterium spp. Appl. Environ. Microbiol. enumeration of dried B. bifidum, E. faecium, and strepto­ 54:1715-1718. mycin-resistant L. acidophilus contained in a mixed probiotic 11. Ohbayashi, A. 1961. Effects of surfactants on microorganisms. product. Nippon Nogeikagaku Kaishi. 35:61-66. 12. Prevot, A. R. 1960. Techniques pour le diagnostic des bacteries anaerobies. Editions de la Tourelle, Mande (Seine), France, pp. 23- TABLE 5. Bacterial counts in dry single culture and dry mix 24. culture. 13. Ray, B., and D. M. Adams, Jr. 1984. Repair and detection of injured microorganisms, pp. 112-123. In M. L. Speck (ed.), Compendium of B. bifidum E. faecium L. acidophilus methods for the microbiological examination of foods. American Public Health Association, Washington, DC. Dry single culture 103 + 11.3" 100 + 5.9 105 + 9.2 14. Resnick, I. G., and M. A. Levin. 1981. Quantitative procedure for (CFU x 107g) enumeration of bifidobacteria. Appl. Environ. Microbiol. 42:427- 432. 15. Shahani, K. M., and A. D. Ayebo. 1980. Role of dietary lactobacilli Dry mix culture 104 + 9.5 101 + 6,4 107 + 8.9 in gastrointestinal microbiology. Am. J. Clin. Nutr. 33:2448. (CFU x 107g) 16. Shahani, K. M., and R. C. Chandun. 1979. Nutritional and healthful aspects of cultured and culture containing dairy foods. J. Dairy Sci. " Mean + standard deviation; n = 9. 62:1685. 17. Shimada, K., M. Mada, M. Mutai, A. Suzuki, and H. Konuma. 1977. Caution must be observed when assaying for E. fae­ Techniques for viable cell counts in the fermented milk with Bifidobacterium Shokuhin Eiseigaku Zasshi. 18:537-546. cium that the incubation time not exceed 24 h. L. acidophi­ 18. Tamura, N., M. Norimoto, K. Yoshida, C. Hirayama. R. Nakai, and lus will begin to form visible colonies on modified Brigg's A. Takagi. 1983. Alteration of fecal bacterial flora following oral agar if incubated incorrectly. administration of bifidobacterial preparation. Gastroenterol. Jpn. In later studies, we found that the strong resistance 18(l):47-55. 19. Tanaka, R., and M. Mutai. 1980. Improved medium for selective against streptomycin was a unique characteristic of L. enumeration of Bifidobacterium. Appl. Environ. Microbiol. 40:866- acidophilus KS-13 and that some other strains cannot grow 869. in the presence of the above streptomycin concentration. 20. Teraguchi, S., M. Uehara, K. Osaga, and T. Mitsuoka. 1978. Enu­ Further studies are needed to find an appropriate biochemi­ meration of bifidobacteria in dairy products. Nihon Saikingaku cal to differentiate other nonstreptomycin-resistant L. aci­ Zasshi. 33:753-761. 21. Teraguchi, S., T. Kawashima, and M. Kuboyama. 1982. Test tube dophilus strains from E. faecium. method for counting bifidobacteria in commercial dairy and phar­ Further studies are currently being conducted to com­ maceutical bacteria products. Shokuhin Eiseigaku Kaishi. 23:39- pare application of some commercially available media to 44. 22. Uehara, T., H. Yabushita, and S. Fukui. 1968. Studies on pyruvate this procedure. oxidation and related metabolism in Streptococcus faecalis. Nippon REFERENCES Nogeikagaku Kaishi. 42:146-151. 23. Ueno, K. 1978. Classification and chemical susceptibility of anaero­ 1. Aim, L. 1983. The effect of Lactobacillus acidophilus administration bic bacteria. Nippon Nogeikagaku Kaishi. 35:61-66.

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