A Selective Enumeration Medium for Bifidobacteria in Fermented Dairy Products

A Selective Enumeration Medium for Bifidobacteria in Fermented Dairy Products

A Selective Enumeration Medium for Blfldobacterla In Fermented Dairy Products K. S. LIM, C. S. HUH, and Y. J. BAEK Research and Development Center Han...

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A Selective Enumeration Medium for Blfldobacterla In Fermented Dairy Products K. S. LIM, C. S. HUH, and Y. J. BAEK Research and Development Center Hankuk Yakult Milk Products Co., Ltd. 418-12, Kamae-ri, Kiheung-eup, Yongin-kun, Kyunggi-do, Korea

H. U. KIM College of AgriCUlture and Life Science Seoul National University 103 Seodun-dong, Kwonsun-ku Suwon, Korea ABSTRACT

A selective medium, blood-glucoseliver agar containing oxgall (.2 mglml) and gentamicin (30 JLglml), was formulated for the selective enumeration of bifidobacteria in fermented dairy products containing both lactobacilli and streptococci. Recovery rates of bifidobacteria on this selective medium were around 90% of recovery on bloodglucose-liver agar. Strains of lactobacilli and streptococci were mostly inhibited with higher dilutions on this selective medium. (Key words: bifidobacteria, fermented dairy products, selective enumeration)

=

Abbreviation key: BL blood-glucose-liver, OG = oxgall-gentamicin, NPNL = neomycinparomomycin-nalidixic acid-lithium chloride. INTRODUCTION

Bifidobacteria have been reported (3, 4, 5, 6, 9, 11, 22) to have important functions in the intestines of breast-fed infants. Potential health benefits of bifidobacteria to the host have led to their wide application in fermented dairy products. It is important that bifidobacteria live in those fermented dairy products until consumption, and the viability of bifidobacteria in products depends on the degree of acidification and on the bacterial strains. The most commonly employed bifidobacterial species are Bi-

Received October 31, 1994. Accepted May 18, 1995. 1995 J Dairy Sci 78:2108-2112

fidobacterium bifidum, Bifidobaeterium in/antis, and Bifidobacterium longum (5, 9, II, 12). For the enumeration of bifidobacteria in fermented milk products, a number of procedures have been developed (I, 2, 7, 14, 17, 19, 20), and, when only bifidobacteria are used in fermented dairy products, the enumeration should not give many problems. However, bifidobacteria are usually used in combination with other desirable lactic acid bacteria, such as lActobacillus acidophilus, lActobacillus delbrueckii ssp. bulgaricus, and Streptococcus themwphilus for health benefits (16) and ease of manufacture (13). Selective media for enumeration of bifidobacteria in fermented dairy products have been reported to contain antibiotics (14, 18, 19) or a single carbon source (17, 21) as selective agents to inhibit the growth of other lactic acid bacteria. Few media, however, are truly selective for bifidobacteria, and some of them are time-consuming to prepare. This study was conducted to develop a medium for selective enumeration for bifidobacteria in fermented dairy products containing other lactic acid bacteria. MATERIALS AND METHODS Microorganisms

The bacterial strains used in this study are listed in Table 1. Isolated bifidobacterial strains were identified on the basis of their carbohydrate fermentation by the procedure of Mitsuoka (10). Isolated lactobacilli and streptococci strains were characterized using the API 50 CH system (API BioMerieux, Vercieu, France).

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SELECTIVE ENUMERATION OF BIFIDOBACTERIA

Bifidobacteria were subcultured in bloodglucose-liver (BL) broth without blood, overlaid with liquid paraffin, at 37·C for 18 h. Lactobacilli and streptococci were subcultured aerobically in MRS broth (Difco Laboratories, Detroit, MI) at 37·C for 18 h and in M17 broth (Difco Laboratories) at 42"C for 8 h, respectively. All isolates were subcultured at least twice before use.

TABLE I. List of the microorganisms. Species and strain no.

Source l

BiJidobacterium biJidum I 2

Chr. Hansen's Laboratories Yogurt

Bifidoba.cteriurn longum

3 4 5 6 7 8

Marschall RhOne-Poulenc Laboratorium Wiesby Marschall RhOne-Poulenc Yogurt Yogurt Yogurt

BiJidobacterium infantis 9 10

Laboratorium Wiesby Marschall Rhone-Poulenc

Lactobacillus acidophilus 11 12 13 14

Chr. Hansen's Laboratories Laboratorium Wiesby Marschall RhOne-Poulenc Yogurt

Lactobacillus delbrueckii ssp. bulgaricus 15 16 17

Chr. Hansen's Laboratories Chr. Hansen's Laboratories Yogurt

Lactobacillus casei 18 19 20 21

Yakult Honsha Chr. Hansen's Laboratories Marschall RhOne-Poulenc Marschall RhOne-Poulenc

Streptococcus thermophilus 22 Chr. Hansen's Laboratories 23 Chr. Hansen's Laboratories 24 Marschall RhOne-Poulenc 25 Marschall Rhone-Poulenc 26 Chr. Hansen's Laboratories 27 Laboratorium Wiesby 28 Yogurt 29 Yogurt 30 Chr. Hansen's Laboratories 31 Yogurt lChr. Hansen's Laboratories AlS, Horsholm, Denmark; Marschall RhOne-Poulenc, Madison, WI; Laboratorium Wiesby, Niebull, Germany; and Yakult Honsha, Co., Ud., Tolcyo, lapan.

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Preparation of Cultures

Overnight cultures of bifidobacteria were inoculated in 12% (wt/vol) reconstituted skim milk (Difco Laboratories) supplemented with .2% (wt/vol) yeast extract (Difco Laboratories) and incubated at 37"C for 18 to 24 h. Lactobacilli and streptococci were cultured in 12% (wt/vol) reconstituted skim milk at 37"C for 18 to 24 h and at 42"C for 6 to 8 h, respectively. All cultures were kept at 4"C before use. Yogurt Products

Fermented dairy products, which were claimed to contain bifidobacteria, were purchased from the domestic market and stored at 4"C prior to examination until the end of shelflife. Selective Media

Blood-glucose-liver agar (Difco Laboratories) was used as a basal medium. Oxgall (Difco Laboratories) and gentamicin (Sigma Chemical Co., St. Louis, MO) (OG) were added to the basal medium at the concentration of .2 mg/ml and 30 ~g/ml (BL-~ a.g~), respectively. Neomycin-paromomycm-nalldIxic acid-lithium chloride (NPNL) agar (18) was used as a reference selective medium. Recovery of the Bacteria

The number of viable bifidobacteria in each yogurt sample was determined by the pour plate methods on BL, BL-OG, and NPNL agars, respectively. The viable counts of lactobacilli were determined on MRS, BL-OG, and NPNL agars, and streptococci were counted on M17 (Difco Laboratories), BL-OG, and NPNL agars. The plates of the selective media (BL-OO and NPNL agar) and BL agar plates were incubated at 37"C for 48 ~ in an anaerobic jar with steel wool (Hrrayarna Manufacturing Co., Tokyo, Japan) and flushed with C02. The MRS and Ml7 agar plates inoculated were incubated aerobically at 37·C for 48 h and at 42·C for 24 h, respectively. Statistical Analysis

The experiments were replicated three times in a randomized block design. All data were analyzed by ANOVA using the general linear models procedure of SAS (15). Differences Journal of Dairy Science Vol. 78, No. 10. 1995

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UM ET AL

TABLE 2. Recovery of bifidobacteria on blood-glucose-liver (BL) and the selective media, blood-g1ucose-liver-oxgallgentamicin {BL-OG} and neomycin-pArOmomycin-nalidixic acid-lithium chloride (NPNL) agars. Media Species and strain no.

BL

Bifidobacterium bifidum 22 x lOS l 28 x lOS l

I 2

BL-OG: BL

NPNL

NPNL:BL

.95 .93

(cfufml)19 x lOS b 25 x lOS l

.86 .89

23x108b 28 X 108 l 14 x lOS b 16 x lOS l 8.4 X 108 b 14 x lOS lb

.88 .90 .88 .88 .76 .88

19 x lOS C 28 x lOS • 13x1OS b 10 x lOS b 7.9 x lOS b 13 x lOS b

.73 .90 .81 .56 .72 .81

26 x 108 l 21 X 108 lb

.96 .91

23 x lOS b 19x1OS b

.85 .83

BL-OG

Bifidobacterium longum 3 4 5 6 7 8

26 31 16 18 11 16

Bifidobacterium infantis 9 10

27 x lOS l 23 x lOS l

x x x x x x

lOS lOS lOS lOS lOS lOS

(cfufml) 21 x lOS lb 26 x lOS l

l l l l l l

l,b.CMeans in row without a common superscript differ (J' < .05). Means of three trials. and each trial was examined in duplicate.

among means were tested for significance (P < .05) by Duncan's multiple range test. RESULTS AND DISCUSSION

Various fermented dairy products containing bifidobacteria are on the market because of the beneficial claims of these anaerobic bacte-

ria for human health. For dietary purposes, high numbers of bifidobacteria should be present in these fermented dairy products until their consumption. A number of selective media have been developed for the enumeration of bifidobacteria, but few are truly selective for bifidobacteria and occasionally even inhibit bifidobacteria somewhat.

TABLE 3. Growth l of lactobacilli on MRS and the selective media, blood-glucose-Iiver-oxgall-gentamicin (BL-OO) and neomycin-pArOmomycin-nalidixic acid-lithium chloride (NPNL) agars. Species and strain no.

Media MRS

BL-OG

NPNL

- - - - - - - - (cfufml) - - - - - - - -

Lactobacillus acidophilus 11 12 13 14 Lactobacillus delbrueckii ssp. bulgaricus 15 16 17 Lactobacillus casei 18 19 20 21

2.1 2.1 2.1 2.1

x lOS

x ]OS x ]OS x lOS

35 x lOS 28 x lOS 7.5 x lOS 6.6 9.9 4.3 4.5

x lOS x lOS x lOS x lOS

(Means of three trials, and each trial was examined in duplicate. Joumal of Dairy Science Vol. 78. No. 10. ]995

<1()3
3.4 x 10" 2.0 x 10" <10" 2.8 x 10"
1.5 x lOS <1()3

<]()3
3.4 x 10"
3.4 x 1()4
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SELECTIVE ENUMERATION OF BIFIDOBACfERIA

TABLE 4. Growth I of Streptococcus thennophilw on MI7 and the selective media, blood-gIucose-liver-oxgaIlgentamicin (BL-oG) and neomycin-paromomycin-nalidixic acid-lithium chloride (NPNL) agars. Media

Species and strain no.

BL-oG

MI7

NPNL

(cfu/m1)

S. thennophilw 22 23 24 25 26 27 28 29 30 31

12

11 9.1 10 14 20 15 5.0 7.4 3.0

x 1(Ill x l(lll x lOS x lOS x lOS xl()8 xl()8 x lOS x lOS x 1()8

<1()3 <1()3 <1()3 <1()4 <1()3 <1()3 <1()3 <1()3 <1()3 <1()3

4.5 x 1.2 x 4.5 x pp2 2.0 x 5.0 x 3.1 x <1()3 5.0 x <1()4

1()4 lOS 1()4 1()4 1()4 lOS lor

(Means of three trials, and each trial was examined in duplicate. 2Pinpoint colonies overgrown on agar plate.

Thus, the antibiotic susceptibility of bifidobacteria and lactic acid bacteria used in fermented dairy products must be studied for the right selection of antibiotics and their optimal concentration. Our previous studies on the antibiotic susceptibility of 37 strains of bifidobacteria (8) and 58 strains of lactic acid bacteria [including 9 L acidophilus, 11 L delbrueckii ssp. bulgaricus, 12 Lactobacillus casei, and 26 S. thermophilus (data not shown)] concluded that 30 Ilg of gentamicin/ml of medium achieved the optimal selectivity for bifidobacteria. We also used .2 mg of oxgalll ml of medium to inhibit other nonintestinal

lactic acid bacteria, S. thermophilus, which is the predominant flora of yogurts. The BL-OO agar was evaluated for the recovery of IO strains of bifidobacteria isolated from commercial starters and yogurts (fable 2). Recovery of bifidobacteria on BL-OG agar was around 90% or higher compared with that on BL agar, except for I strain of B. longum. Colonies of bifidobacteria on BL and BL-OG media were larger than .5 rom in diameter and slightly smaller on NPNL agar. In contrast, all tested strains of L. acidophilus, L delbrueckii ssp. bulgaricus, and L casei were inhibited in

TABLE 5. Enumeration I of bifidobacteria and lactic acid bacteria from yogurts in blood-g1ucose-liver-oxgall-gentamicin (BI..-oo), neomycin-paromomycin-nalidixic acid-lithium chloride (NPNL) agar, and in optimal growth media (MRS and Ml7 agars).

Yogurt

pH

Titratable acidity

Media MRS

BL-OG

MI7

A B C D E F G

4.25 4.13 4.20 4.35 4.25 4.36 3.98

1.10 1.15 1.10 1.05 1.12 1.04 .65

NPNL

(cfulml)

(%)

5.4 2.1 1.8 5.8 1.2 3.2 8.5

x x x x x x x

1()6 1()6 10' 1()6 l()'l 10' loa

2.1 x 1.8 x 2.2 x 1.0 x 1.8 x 2.6 x NC3

1()9 1()9 1()9 1()9 l()'l l()'l

1.6 8.7 8.0 4.7 1.2 7.1 4.9

x x x x x x x

10' 1()6 11)4 1()6 loa 10' lOS

1.7 X 10' 8.3 x 10' pp2 4.2 x 1()6 8.1 X 10'

pp 3.2 x lOS

IMean of duplicates. 2Pinpoinl colonies overgrown on agar plate. 3No colonies were detected on lhe agar plate. Journal of Dairy Science Vol. 78, No. 10, 1995

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LIM ET AL.

BL-OG and NPNL agar with higher dilution (10-3 and 10-4; Table 3). Their recovery rates were less than .1 % of that on MRS agar. Ten strains of S. thermophilus were completely inhibited on BL-OG agar (Table 4). No streptococcal colonies were detected at dilutions of 10-3 or 10-4 plated on BL-OG agar. The NPNL agar had fewer inhibitory effects on S. thermophilus strains than did BL-OG agar, and pinpoint colonies of 1 S. thermophilus strain were overgrown on NPNL agar. All of the yogurts examined contained 1()4 to 107 cfu of bifidobacterialml, and recovery on NPNL agar was relatively less than on BLOG agar, except for 1 yogurt. Furthermore, NPNL agar plates of two yogurts (C and F) were overgrown with pinpoint colonies, which were identified as S. thermophilus (Table 5). The BL-OG agar is much simpler to prepare than NPNL agar and gives higher recovery of bifidobacteria in yogurts. Therefore, BL-OG agar appears to be the most suitable medium for the isolation and selective enumeration of bifidobacteria in fermented dairy products that contain other lactic acid bacteria.

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