InternationalJournal o/Food Microbiology, 13 (1991) 87-94 © 1991 ElsevierScience Publishers B.V. 0168-1605/91/$03.50

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FOOD 00400

Short c o m m u n i c a t i o n

The effect of Pediococcus damnosus and Pediococcus pentosaceus on the growth of pathogens in minced meat T. M a t t i l a - S a n d h o l m , A. H a i k a r a a n d E. Skytt~ Food Research Laboratory, Technical Research Centre of Finlan~ Espoo, Finland (Received 11 October 1990, accepted 8 January 1991)

The antibacterial effects of one strain of Ped/ococcus danmosus and two strains of Ped/ococcus pento.~,_.,'__,~us12-i-st Cimtridium perfringens, Listecia monocytogenes, Salmonella in/antis and Yersinia enterocalitiea were investipted. Growth inhibition studies were conducted in juice from minced meat incubated at + 6 ° C and + I S ° C for various periods after the inoculation with pediococci. Inhibitory effects were seen for at] bacteria tested. Key words: Pedmcoccus; Pathogen; Minced meat; Natural inhibitor: Antimicrobial activity

inmm'uction The antibacterial activities of lactic acid bacteria include production of acid, hydrogen peroxide carbon dioxide, use of nutrients and formation of antibacterial components like bacteriocins (Gibbs, 1987; Klaenhammer, 1988). Pediococci have proved to be important in meat and vegetable fermentations (Fleming et al., 1975; Gibbs, 1987; Klaenhammer, 1988). Furthermore, their ability to restrict the growth of other organisms has been noticed. Most of the experimental work has been carried out with Pediococcus damnosus (former P. cerevisiae), which has shown inhibitory activity against the growth of major Gram-positive spoilage organisms like lactobacilli and streptococci and Gram-positive pathogens llke Staphylococcus aureus and Bacillus cereus (Gilliland and Speck, 1972; Klaenhammer, 1988). A corresponding antibacterial activity against Gram-negative organisms has not been reported (Daeschel, 1989; Klaenhammer, 1988). Pediococcus pentosaceus and Pediococcus acidilactici have been of great interest in recent years (Bhunia et al., 1988; Daeschel and Klaenhammer, 1985; Graham and McKay, 1985; Klaenhammer, 1988; Ray et al., 1989). This has led to the discovery of the so-called pediocin A produced by P. pentosaceus, which is believed to be a C~dence addre~: T. Mattila-Sandhoim, Food Research Laboratory, Technical Research Centre of Finland, P.O. Box 203, SF-02151 E~>o, Finland.

88 plasmid-based antibacterial component (Daeschel and Klaenhammer, 1985). The effectiveness of pediocin A against foodborne microorganisms has led to further ideas to develop new preservative systems and starter cultures for food fermentation processes (Daeschel and Klaenhammer, 1985; Graham and McKay, 1985). At present the most important field of interest is probably the intensive genetic studies cloning the pediocin A genes into cultures, which is currently used in the production of fermented foods. These studies may lead to the generation of new starter cultures which produce and excrete specific and carefully defined antibiotic factors (Daeschel and Klaenhammer, 1985; Klaenhammer, 1988). The studies reported in the literature (Bhunia et al., 1988; Daeschel, 1989; Klaenhammer, 1988) have mainly been conducted in culture media using partly purified bacteriocins and studying their effect on pure cultures of organisms. The present work was carried out in order to find out how the antibacterial activity of pediococci affects the growth potential of major pathogens in minced meat.

Materials and Methods

Strains of pediococci One strain of Pediococcus danmosus VTT 76065 (Technical Research Centre of Finland), and two strains of Pediococcus pentosaceus VTT 76067 and VTI" 76068 were selected from preliminary experiments (unpublished data) showing that these strains inhibited the growth of the pathogenic bacteria used in this study. In the following only the two last digits are used for coding the different strains. Inoculatwn with pediococci and preparation of minced meat media Minced beef used for juice medium preparation was irradiated (25 kGy for 2 days at +0*C). This reduced the bacterial level to less than 102/g. Fresh cultures of pedioczocci were prepared by cultivation for 5 days at room temperature in MRS Broth (Oxoid) with subsequent inoculation into the irradiated minced meat at a level of 10~-108/g (Eamshaw et al., 1988). The control minced meat was not inoculated. The inoculated minced meat samples (each 30 g) were kept at + 6 " C and + 15°C, respectively, for 8 days. Samples were taken for juice preparation on days 1, 2, 3, 6 and 8. The juices were prepared by squeezing the meat with a hydraulic food press (HAFICO, HA Fischer Co., F.R.G.) for 3 h. The meat juice collected was diluted with sterile 0.9% saline to equal the weight of the original meat sample. The juice was centrifuged at 10 000 x g for 1 h and sterile filtered (0.45/~m, Millipore filter). Pathogenic bacteria The pathogenic bacteria used as test organisms were cultivated as follows for preparation of inocula: Clostridium perfringens (ATCC 3624) anaerobically in Bacto Fluid Thiogiycollate Medium (Difco); Listeria monocytogenes (V'I~ 4126) in Tryptose Phosphate Broth (Farber and Speirs, 1987); Salmonella infantis (VTT 38) and

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Yersinm enterocolitica (ATCC 27729) in IsoSensitest Broth (Oxoid) at 37 °C (or at 30 ° C, Y. enterocolitica) for 18 h. Measurement of bacterial growth The number of colony forming units in minced meat was determined either in Plate Count Agar (Dffco) or anaerobically on blood agar (Orion Diagnostica). The plates were incubated at 300C for 3 days. MRS Agar (Oxoid) plates used for enumeration of pediococci were incubated for 5 days at 25 *C in an atmosphere with 5% C02. Bacterial growth in the stcrile-f'dtered meat juice cultured at 37 °C for 24 h was measured using an automated turbidometer, Bioscreen (Lahsysterns, Finland) which monitors bacterial growth kinetically by a vertical pathway at 620 nm (wide band) each 10 min during the incubation period. The analyser runs 200 samples in the same batch. 270/~1 of minced meat juice was used in each well and the inoculum size of the test bacteria was 30/~1. The fresh inocula contained 10~-108/ml sterile saline suspension. Experiments were performed in parallel. The bacterial growth and growth inhibition in the juice samples from meat inoculated with pediccocci was expressed by the areas under the growth curves within 24 h of incubation. The area values were calibrated against the plate count results, separately for each strain. The plate count results were obtained by using the media given above. The following mean number of area units corresponding to the factor to be multiplied by ten to give the change in bacterial counts were found: C. perfringens, 6.9; L monocytogenes, S. in/antis and Y. enterocolitica, 1.2.

Results and Disemsion Figs. 1 to 3 indicate that during incubation all tested bacteria grew better in the early control samples (storage days 1 and 2) than in the late ones (storage days 6 and 8). This is most likely due to the effects caused by deterioration of the samples by the end of the storage period caused by the increasing number of normal flora (the bacterial count of the control samples was less than 102/g at the beginning of the experiment but increased graduatly during the incubation to 10e-10~/g). The pH of all the samples varied from pH 6 to pH 8 throughout experiments (data not shown).

Fig. 1 illustrates the growth pattern of C perfr/agens in minced meat medium at both temperatures. It can be seen that only one strain of P. pemosaceus (VTT 68) inhibited the growth of C. perfringens at + 6 ° C, whereas at + 15 ° C inhibition was evident for all the selected pediococcal strains (WIT 65, 67, 68). The growth pattern of L monocytogenes in meat samples followed thai of C. perfringens (data not shown). Figs. 2 and 3 describe the growth of $. infantis and Y. enterocolitica, respectively. Both figures follow the same trend, the strains P. pentosaceus VT'I" 67 and VTT 68 show clear inhibition at both temperatures. P. danmo.ms VTT 65, however, did not follow this pattern. P. pentosaceus and P. acidilactici have been found to control the

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Fi& 1. Growth of C. ~rfringens m jmcc from minced meat samples L,~culatcd with P. d~nnonu V'rT 65 and P. p e n t ~ ~ 67 ~nd V'TT 68. Growth expressed by means of area umts under the growth curve within 24 h. Slorage ume indicates time of incubation of inoculated meat before juice preparation.

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Fig. 3. ~ of Y. ~u,,roco~/~ in ju~e from mineecl meat samples , ~ u l a t e d with P. d , a ~ x m u V'I'T 65and P. pem___,~c~m_VTT 67 and V'IT 68. Growth cxpmned by m e a ~ of area units under the growth

curve within 24 ix. Stoca~ time indicat~ time of immbadon of inoculated me,at beJorc juice preparation.

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growth of Y. enterocolitica in meat (Raccach and Hermingsen, 1984). There are also reports which indicate antibacterial effects towards Staphylococcus aureus, S. infantis and Bacillus cereus (Dubois et al., 1979, Fleming et a1., 1975; LitapoulouTzanetaki, 1987). However, these reports as well as the present study do not discriminate between the effects of pH, depletion of nutrients and antibacterial factors. Gill (1976) has stated that as the bacterial population approaches 1Og/cm 2, depletion of amino acids takes place. Furthermore, at a cell density of 10s-109/cm 2 the glucose concentration will be zero (Gill, 1976). The cell densities in meat samples with P. pentosaceus ~ 67 and 68 were so high (10S/g) that nutrient depletion may have been involved (data not shown). It shall be mentioned that the amount of lactic acid produced between P. pentosaceus strains VTT 67 and 68 varied significantly (data not shown). This study has demonstrated a model, where the characteristics of potential pediococcal strains can be tested for increasing the shelf-life and minimizing the growth risk of pathogens. The present results do not differentiate between the possible effects of acids and antibiotic-like factors. Studies are underway to elucidate this.

Relerenees Bhunia. A.IC, Johnson, M.C. and Ray, B. (1988) Purification, characterization and antimicrobial spectrum of • bacteriocin produced by Pediococcus acidilactici. J. Appl. Bacteriol. 65, 261-268. ~ l , M.A. (1989) Antimicroblal substances from lactic acid bacteria for use as food preservatives. Food Technoi. 43, 164-167. DaescheL M.A. and Klaenhammer, T.IL (1985) Association of • 13.6-mepdahon Plasmid in Pedwcoccus pentasac'eus with bacteriocin activity. Appl. Environ. Microbiol. 50, 1538-1541. Dubois, G., Beaumier, H. and Charbonneau, IL (1979) Inhibition of bacteria isolated from ground meat by Streptococcaceae and Lactobaciilae. J. Food Sci. 44, 1649-1652. ~ w , IL, Stratford, J. and Bank~ J.G. (1988) The inhibition of pathogens and spoilage bacteria in raw beef snd oottase cheese by Pediococcus pento,mceus. Techn. Memor. No 516, The Campden Food and Drmk Research Aumciation, Chipping Campden, U.K. Farbcr, J.M. and Speirg J.l. (1987) Potential use of continuous cell lines to distinguish between pathogenic and nonpathollenic l,~teria spp. J. Clin. Microbiol. 25, 1463-1466. Fleming, H.P., Etehells, J.l_ and Costilow, ILN. (1975) Microbial inhibition by an isolate of Ped/ococcua from cucumber brines. Appl. Microbiol. 30, 1040-1042. Gibbs, P.A. (1987) Novel uses for lactic acid fermentation in food preservation. J. Appl. Bacteriol, Syrup. Suppl. 51 S-58. Gill, C.O. (1976) Substrate limitation of bacterial growth at meat surfaces. J. Appl. Bacteriol. 41. 401-410. Gilliland, S.E. and Speck. M.L, (1972) Interactions of food starter cultures and food-borne pathogens:lactic streptococci versus staphylococci and salmonellae. J. Milk Food Technol. 35, 307-310. Graham, D.C. and McKay, L.L. (1985) Plasmid DNA in strains of Pedwcoccus cerevisiae and Pediococ. cus pentasacaa. Appl. Environ. Microbiol. 50, 532-534. Klaenhammer. T.R. (1988) Bacteriocins of lactic acid bacteria. Biochimie 70, 337-349. Litapoeiou-Tzanetaki, E. (1987) Interactions of Pediococcus peatosacem and some food-borne pathogens. Food Microbiol. 4, 293-302.

94 Raccach. M. and Henmngsen, E.C. (1984) Role of lactic acid bacteria, curing salts, spices and temperature m controlling the growth of Yersima enterocohtica. J. Food Protect. 47, 354-358. Ray, S.K., K.im, W.J., Johnson, M.C. and Ray, B. (1989) Conjugal transfer of a plasm/d encoding bacteriocin production and urununity in Pedzococcus acidilactici H. J. Appl. Bacteriol. 66, 393-399.