Letters in Applied Microbiology 1998, 26, 123–125

Susceptibility of bifidobacteria to nisin

V. Rada and J. DlabalCzech University of Agriculture in Prague, Kamycka, Prague, Czech Republic

1575/97: received 24 June 1997 and accepted 20 August 1997

V. RADA AND J. DLABAL. 1998. A collection of 17 bifidobacteria was tested for sensitivityor resistance to lantibiotic nisin. Minimal inhibitory concentration of the straintested was highly variable, ranging from 4·88 to 10 000 IU ml−1. In general, strainsisolated from faecal samples were more resistant than those purchased fromculture collections. These results could be useful in the production of foods containingboth bifidobacteria and nisin.

INTRODUCTION viously (Rada 1997). Bifidobacterium sp. strains 4–7 wereisolated using Rogosa agar (Oxoid, Basingstoke, UK) with

Bifidobacteria comprise one quarter of the colon flora ofcysteine hydrochloride (0·05% w/v) and identified at the

normal healthy adults (Modler et al. 1990). It was assumedgenus level by detection of fructose-6-phosphate phos-

that these organisms possessed anticancerigenic (Mitsuokaphoketolase (Scardovi 1986). Infant faeces isolates originated

1982) and anticholesterolemic (Hata et al. 1982) properties.from different, fully breast-fed individuals (two females and

The bifidobacteria also produce acetic and lactic acid whichfive males). Bifidobacterium sp. strains 8–11 were isolated

may restrict the growth of putrefactive organisms by loweringusing MRS agar (Oxoid) modified by the addition of cysteine

the pH of the intestinal contents (Rasic and Kurman 1983).hydrochloride (0·05% w/v) and with a selective supplement

Nisin is a polypeptide produced by certain strains of Lacto-containing (l−1 agar) neomycine (100mg), paromomycine

coccus lactis ssp. lactis (De Vuyst and Vandamme 1994). In(200mg), nalidixic acid (15mg) and lithium chloride (3 g)

contrast to most other bacteriocins produced by lactic acid(Teraguchi et al. 1978). The products used were purchased

bacteria, nisin displayed a relatively broad inhibitory spec-from four different manufacturers. The isolates were ident-

trum. This compound and other subtype A lantibiotics areified as described above.

characterized by a strong bactericidal action. This means thatAll strains were incubated in the MRS broth (Oxoid) with

they rapidly kill sensitive bacteria (Mattick and Hirsch 1947;cysteine hydrochloride (0·05% w/v) under anaerobic con-

Hirsch 1954; De Vuyst and Vandamme 1994). Nisin inhibitsditions, using roll-tube techniques, and stored in the same

many strains of Gram-positive bacteria including bacilli, clo-medium with glycerine (20% v/v) at – 20 °C.

stridia, corynebacteria, enterococci, lactobacilli, lactococci,leuconostocs, micrococci, pediococci, staphylococci, strepto-cocci and actinomycetes (De Vuyst and Vandamme 1994; Bacteiocins assaysLaukova 1995; Rodrıguez 1996).

The lyophylized bacteriocin preparation (Sigma, Prague,Although both bifidobacteria and nisin are used in the foodCzech Republic) was dissolved at a concentration of 1mgindustry, there is no information on susceptibility of theseml−1 in 0·02mol l−1 HCl (pH 2) and stored at – 4 °C. Anti-bacteria to nisin. Therefore, the aim of the present study wasbacterial effects were detected by the agar well diffusionto examine the possible influence of lantibiotic nisin on themethod according to Tagg et al. (1976). The bifidobacteriagrowth of 17 bifidobacteria strains.were cultivated in the modified MRS broth at 37 °C for 20 h.Modified MRS agar (20 ml) was seeded with 1ml of grown

MATERIALS AND METHODS culture and poured into Petri dishes. Plates were sub-sequently transferred to a temperature of 4 °C for 7min to

Bacterial cultures, media and growth conditions accelerate the solidification of the agar. Afterwards, portions(250ml) of nisin solution were added to wells 1 cm in diameterThe strains used are listed in Table 1. Bifidobacterium longumcut into the agar. Control wells contained 250ml of 0·02molstrains 1–3 were isolated and identified as described pre-l−1 HCl. Plates were incubated in an anaerobic jar (AnaerobicCorrespondence to: Dr V. Rada, Czech University of Agriculture in Prague,

Prague 6 – Suchdol, Kamycka 126, 165 21, Czech Republic. Plus System, Oxoid) at 37 °C for 30 h. Thereafter, nisin

© 1998 The Society for Applied Microbiology

124 V. RADA AND J. DLABAL

Table 1 The susceptibility of bifidobacteria to nisin—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Strain Source Zone* (mm) MIC (IU ml−1)—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Bifidobacterium bifidum ATCC 29521 18 4·88B. bifidum Collection of Dairy Cultures MILCOM, Prague, Czech Republic 9 19·53B. breve ATCC 15700 8 156·25B. infantis ATCC 17930 9 78·13B. infantis 94 Collection of Dairy Cultures MILCOM, Prague, Czech Republic 8 19.53B. longum ATCC 15707 9 156·25B. longum 1 Infant faeces 6 2500B. longum 2 Infant faeces 3 10 000B. longum 3 Infant faeces 3 2500Bifidobacterium sp. 4 Infant faeces 9 156·25Bifidobacterium sp. 5 Infant faeces 6 2500Bifidobacterium sp. 6 Infant faeces 8 625Bifidobacterium sp. 7 Infant faeces 8 312·50Bifidobacterium sp. 8 Commercial fermented milk products 8 156·25Bifidobacterium sp. 9 Commercial fermented milk products 4 312·50Bifidobacterium sp. 10 Commercial fermented milk products 9 39·06Bifidobacterium sp. 11 Commercial fermented milk products 7 156·25—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

*Clear zones surrounding wells 250 ml of nisin solution at a concentration of 1 mg ml−1.

sensitivity was observed as clear zones surrounding wells Nisin is the first bacteriocin legally permitted as a foodpreservative. The recommended concentration of nisin incontaining nisin. Each isolate was examined in triplicate.

For minimal inhibitory concentration (MIC) assessment, milk and milk products ranges from 10 to 500 IU ml−1 (DeVuyst and Vandamme 1994; Muriana and Kanach 1995). Thethe nisin solution described above was serially diluted by a

factor of two to obtain the following concentrations of nisin: results of the present study suggest that some of the strainstested were susceptible to commonly used nisin con-40 000, 20 000, 10 000, 5000, 2500, 1250, 625, 312·5, 156·25,

78·13, 39·06, 19·53, 9·77, 4·88 and 2.44 IU ml−1. All strains centrations but some were highly resistant. There is a widerange of milk products containing bifidobacteria on theof bifidobacteria were tested for susceptibility to every nisin

concentration as described above. The MIC was identified as market. These products include yoghurts, other fermentedmilks, ice cream, cheese and sweet milk (Tamine et al. 1995).the highest dilution of nisin which produced clear zones

surrounding wells. It may be possible to improve the shelf-life and safety of theseproducts by the addition of nisin, provided of course thatcarefully selected nisin-resistant bifidobacterial strains are

RESULTS AND DISCUSSIONused.

In fermented milks, bifidobacteria are in co-culture withThe results are shown in Table 1. All strains examined weresensitive to nisin at a concentration of 1mgml−1 with zones of other lactic acid bacteria (Shah 1997). Optimal fermentation

conditions for L. lactis results in maximal nisin titres of 1000–inhibition from 3 to 18mm. Minimal inhibitory concentrationdata gave more accurate information on the susceptibility or 5000 IU ml−1 (Van’t Hull and Gibbons 1996; Qiao et al.

1997). Our results show that four out of the 17 bifidobacteriaresistance of bifidobacteria to nisin. It was revealed that thestrains examined were highly variable in this parameter. strains tested exhibit MICs equal to or higher than 2500 IU

ml−1. Therefore, the combination of nisin-producing strainsBifidobacterium bifidum ATCC 29521 was the most sus-ceptible (MIC 4·88 IU ml−1). On the other hand, all B. and nisin-resistant bifidobacteria may be feasible.longum strains isolated from infant faeces exhibited highMICs ranging from 2500 to 10 000 IU ml−1. In general, the

ACKNOWLEDGEMENTstrains isolated from the faecal samples were more resistantthan those purchased from culture collections. Four strains The study was supported by grant no. 238/10/12596/0 of

the Internal Grant Agency of Czech University of Agricultureisolated from dairy products were susceptible to nisin, withMICs ranging from 39·06 to 312·50 IU ml−1. in Prague.

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 123–125

SUSCEPTIBILITY OF BIFIDOBACTERIA TO NISIN 125

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© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 123–125