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Inhibition of Bacillus cereus by strains of Lactobacillus and Lactococcus in milk
International Journal of Food Microbiology 89 (2003) 205 – 212 www.elsevier.com/locate/ijfoodmicro
Inhibition of Bacillus cereus by strains of Lactobacillus and Lactococcus in milk Elisabeth Røssland a,*, Grethe I. Andersen Borge a,b, Thor Langsrud a, Terje Sørhaug a a
Department of Food Science, Agricultural University of Norway, P.O. Box 5036, N-1432, A˚ s, Norway b Norwegian Food Research Institute (MATFORSK), Osloveien 1, 1430, A˚ s, Norway Received 2 January 2002; received in revised form 21 February 2003; accepted 15 March 2003
Abstract The growth and death or survival of Bacillus cereus in sterile skimmed milk fermented with 18 different lactic acid bacteria (LAB) were investigated. B. cereus alone in milk reached about 107 – 108 colony-forming units (cfu)/ml. When B. cereus was cultivated together with different Lactobacillus or Lactococcus cultures at 30 or 37 jC, the B. cereus counts after 72 h of fermentation ranged between < 10 cfu/ml and about 106 cfu/ml. The inhibition patterns for the different Lactobacillus and Lactococcus cultures varied. All the Lactococcus cultures (with one exception) reduced pH to 5.3 or lower in 7 h. After 24 h, B. cereus was not detected in any of the fast Lactococcus-fermented milk samples. After 48 h, B. cereus was not detected for 4 of the 12 Lactobacillus cultures. These cultures reduced pH to below 5.0 in 24 h. The other Lactobacillus cultures also inhibited B. cereus, but the counts of B. cereus were still 104 – 106 cfu/ml after 72 h. They also reduced pH at a slower rate. Survival of B. cereus was to a variable extent linked with formation of endospores. Proteinase K did not affect the antimicrobial activity observed. Acid production with decreasing pH, particularly the initial rate of pH decrease, appears to be most important for control of B. cereus with LAB. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Bacillus cereus; Lactobacillus; Lactococcus; Inhibition; Endospore; pH
1. Introduction Bacillus cereus is an ubiquitous, spore-forming soil microorganism that is a common cause of food poisoning (Kramer and Gilbert, 1989; Andersson et al., 1995). These species produce two distinct types of food-borne illness: the diarrhoeal illness, seen occasionally following consumption of milk and other dairy products, meat products, sauces, soups and * Corresponding author. Tel.: +47-64-94-85-76; fax: +47-6494-37-89. E-mail address: [email protected] (E. Røssland).
vegetables (Granum and Lund, 1997), and the emetic (vomit-inducing) syndrome, associated with consumption of a heat-stable toxin produced in fried and cooked rice, pasta and noodles (Shinagawa, 1990; Jackson, 1991; Granum and Lund, 1997). B. cereus is a common contaminant of raw milk (Lin et al., 1998) but is also frequently isolated from different dairy products (Kramer and Gilbert, 1989). A survey conducted by Wong et al. (1988) on dairy products showed that B. cereus was found in 52% of ice creams, 35% of soft ice creams, 29% of milk powders, 17% of fermented milks and 2% of pasteurised milks and fruit flavoured milks.
0168-1605/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(03)00149-1
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Some psychrotrophic strains of B. cereus are known to grow in foods at temperatures as low as 4– 6 jC (Griffiths and Phillips, 1990; van Netten et al., 1990; Dufrenne et al., 1994; Rowan and Anderson, 1998; Borge et al., 2001). This growth represents a problem in chilled products such as milk and other dairy products, and in ‘‘long life’’ ready-to-eat or fresh-chilled foods. Griffiths and Phillips (1990) isolated endospores of psychrotrophic Bacillus spp. from 58% of farm bulk tank milks and from about 69% of pasteurised milks. In addition to B. cereus, several other species within the genus Bacillus are associated with spoilage problems in the dairy industry (Andersson et al., 1995; Larsen and Jørgensen, 1997; Mayr et al., 1999). Examples of such spoilage include bitty cream, sweet curdling of milk and off-flavours in various products leading to substantial economic losses (Barkley and Delaney, 1980). Lactic acid bacteria (LAB) used in food fermentations are able to inhibit or kill food-borne and other pathogenic microorganisms as well as simple spoilage bacteria by producing a variety of antimicrobial agents (Holzapfel et al., 1995; Adams and Nicolaides, 1997; Helander et al., 1997). Acidity is probably in most cases the primary factor in the preservation of lactic acid-fermented foods (Adams and Nicolaides, 1997; Rowan et al., 1998). However, a number of other factors may also be inhibitory, like for instance bacteriocins, diacetyl, carbon dioxide, hydrogen peroxide and ethanol (Holzapfel et al., 1995; Adams and Nicolaides, 1997; Helander et al., 1997). Inhibition of B. cereus by LAB has been reported in nonfat milk medium (Wong and Chen, 1988), in Gouda (Rukure and Bester, 2001) and in Brie (Little and Knøchel, 1994). It has been shown that foods fermented by LAB to pH 4.0 or lower inhibit the growth of B. cereus and other pathogens (Nout et al., 1987; Aryanta et al., 1991; Svanberg et al., 1992; Kingamkono et al., 1994). Lactobacillus rhamnosus LC705 has been shown to inhibit Bacillus spp. in bakery products (Suomalainen and MayraMakinen, 1999). Lactobacillus acidophilus LF221 producing two bacteriocins active against different pathogens including B. cereus was found to inhibit 1 out of 12 B. cereus strains tested in an agar-well diffusion assay (BogovicMatijasic et al., 1998).
The aim of the present work was to determine the effects of different Lactobacillus and Lactococcus cultures on B. cereus in milk.
2. Materials and methods 2.1. Bacterial strains and growth conditions An overview of the lactic acid bacteria (LAB) and B. cereus strains used in the experiments is presented in Table 1. The stock cultures were kept at 80 jC in media containing 15% (w/v) glycerol (Merck, Darmstadt, Germany) as follows: B. cereus in brain heart infusion broth (BHI, Difco, Becton Dickinson, Sparks, MD, USA), Lactobacillus reuteri SD2112 in sterile 10% (w/v) reconstituted skimmed milk (RSM, TINE Norske Meierier, Foll Voll, Norway), other lactobacilli in de Man Rogosa Sharpe broth (MRS broth, Oxoid, Hampshire, England) and the Lactococci in M17 broth (Oxoid). The B. cereus strains were subcultured three times in 10 ml BHI broth at 30 jC for 11 –12 h before the cells were used as inocula in the different experiments. Lactic acid bacteria were subcultured three times in 10 ml MRS broth (Lactobacillus) or 10 ml M17 broth (Lactococcus) at 30 or 37 jC (Tables 2 and 3) for 11– 12 h before the cells were used as inocula. 2.2. Preparation and inoculation of milk samples Sample flasks (250 ml screw-cap flasks) containing 200 ml of 10% (w/v) RSM were prepared and autoclaved (121 jC, 15 min). The sterile milk was inoculated with B. cereus to obtain 102 colony-forming units (cfu)/ml at zero time. Simultaneously, Lactobacillus or Lactococcus cultures were inoculated at population levels of about 106 – 107 cfu/ml milk. Bottles inoculated with B. cereus alone served as controls. 2.3. Growth conditions and enumeration methods Inoculated milk samples were incubated at 30 jC for 3 days except for Lb. acidophilus NCFB 1748, Lb. acidophilus LF221 and Lb. reuteri SD2112 that were
E. Røssland et al. / International Journal of Food Microbiology 89 (2003) 205–212 Table 1 An overview of the Lactobacillus (Lb.), Lactococcus (L.) and B. cereus cultures used in the experiments Strains a
Lb. casei 2756 , Lb. casei 2752a, Lb. curvatus 2042a, Lb. plantarum 2741a Lb. paracasei subsp. paracasei INF-15Da
Source
Obtained from
isolated from cheese
Department of Food Microbiology, University College, Cork, Ireland Department of Food Science, Agricultural University of Norway National Collection of Food Bacteria, Reading, England Collection of Industrial Microorganisms, Ljubljana, Slovenia Biogaia Biologics, Stockholm, Sweden
isolated from Norvegia cheese
Lb. acidophilus NCFB 1748b Lb. acidophilus LF221b
isolated from infant faeces
Lb. reuteri SD2112b
isolated from human breast milk
Lb. rhamnosus LC705b Lb. brevis 226, Lb. plantarum 8, Lb. fermentum 99
L. lactis subsp. lactis ML8, L. lactis subsp. cremoris DRC3, L. lactis subsp. cremoris P2 L. lactis subsp. lactis biovar. diacetylactis NCDO 176, L. lactis subsp. lactis biovar. diacetylactis NCDO 184L L. lactis subsp. lactis lac 952 B. cereus NVH 38, 45, 68 and 74c
a
isolated from Bushera (fermented sorghum beverage)
Valio culture collection, Helsinki, Finland Department of Food Science, Agricultural University of Norway
Dr. Tim Cogan, Dairy Products Research Centre, Fermoy, County of Cork, Ireland National Collection of Food Bacteria, Reading, England
isolated from dairy products
Christian Hansen, Hørsholm, Denmark Culture collection of The Norwegian School of Veterinary Science (NVH)
Nonstarter lactic acid bacteria (NSLAB). Probiotic bacteria. c All strains contained the non-haemolytic enterotoxin gene (nhe) and the enterotoxin T gene. All strains, except B. cereus NVH 38, contained in addition the haemolytic enterotoxin gene (hbl) (Borge et al., 2001; Stenfors and Granum, 2001). b
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incubated at 37jC. All the experiments were carried out in triplicate. After shaking the flasks by hand, samples (0.1 ml) were serially diluted in sterile peptone water/saline (0.1%, w/v, Peptone (Oxoid), containing 0.85%, w/v, NaCl (Merck)) and surface plated (0.1 ml) in duplicate. BHI agar plates used to enumerate B. cereus were incubated at 30 jC for 24 h. Lactobacillus was enumerated on Lactobacillus-selective medium (LBS, Becton Dickinson) and the plates were incubated at 30jC for 72 h for all the strains except for Lb. acidophilus NCFB 1748 and Lb. reuteri SD2112, which were incubated at 37 jC for 72 h. Lb. acidophilus LF221 was enumerated on MRS-agar due to poor growth on LBS agar, and the plates were incubated at 37 jC for 48 h. Lactococcus was enumerated on M17 agar and the plates were incubated at 30 jC for 48 h. Plate counts of B. cereus NVH 45 were made in duplicate after different time intervals, as indicated in Tables 2 and 3, and plate counts of Lactobacillus or Lactococcus were made in duplicate after incubation for 0 and 24 h. Lactobacillus casei 2756 or Lb. acidophilus NCFB 1748 were co-inoculated with B. cereus NVH 38, NVH 45, NVH 68 or NVH 74 in 10% RSM, and plate counts of B. cereus and Lactobacillus were made in duplicates after incubation for 0, 2, 4, 7, 12, 24, 30, 48 and 72 h. The pH of each sample was also measured. 2.4. pH measurement The pH was measured at different times with a pH electrode (PHM 210, Radiometer, Copenhagen, Denmark) in 10 ml aliquots taken from each of the flasks. The pH electrode was calibrated with buffers (Merck) at pH 4.0 and 7.0. 2.5. Endospore content Quantitative determination of the number of endospores in the milk samples was made by surface plating after heat treatment (80 jC, 5 min) of 1 ml milk with serial dilutions in peptone water/saline. In addition, 1 ml of milk sample (from the same bottle) was adjusted to pH 6.5 by adding NaOH (Merck, 0.5% or 1.0%, w/v) before heat treatment. The pH adjustment prior to heating of the samples did not
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Table 2 Growth of B. cereus NVH 45 together with different Lactobacillus cultures in 10% reconstituted skimmed milk at 30 or 37 jC and the development of pH during fermentation Strains
B. cereus log10 cfu/ml 0h
24 h
B. cereus NVH 451 B. cereus NVH 45 together with: Lb. casei 27561 Lb. casei 27521 Lb. paracasei INF-15D1 Lb. plantarum 27411 Lb. curvatus 20421 Lb. acidophilus NCFB 17482 Lb. rhamnosus LC7051 Lb. acidophilus LF2212 Lb. reuteri SD21122 Lb. brevis 2261 Lb. plantarum 81 Lb. fermentum 991
2.06
7.93
2.24a 2.19a 2.19a 2.22a 2.19a 2.93b 1.97a 2.15a 2.20a 2.20a 2.20a 2.27a
4.53a 5.63a 5.37a 5.73a 7.21bc 7.19bc 6.25ab 7.88e 6.65abc 7.2bc 7.25c 7.68d
pH of the milk
48 h
72 h
8.12
8.09
< 1a < 1a < 1a < 1a 6.13bc 5.39bc 4.58b 7.2bc 6.78bc 6.16bc 7.73cd 7.86d
< 1a < 1a < 1a < 1a 5.43b 5.29b 4.45b 6.51b 6.37b 5.97b 6.44b 6.68b
0h
4h
24 h
30 h
48 h
72 h
6.5
6.5
6.0
5.7
5.3
5.0
6.4ab 6.4ab 6.4ab 6.5ac 6.4ab 6.5ac 6.5c 6.5ac 6.4b 6.4ab 6.4ab 6.5ac
6.2ab 6.2abc 6.2ab 6.2ab 6.3ad 6.4cd 6.4d 6.4cd 6.2b 6.3abd 6.2abc 6.4d
4.8b 4.8b 4.8b 5.0b 5.6cd 4.9b 4.4a 5.5c 5.6cd 5.4c 5.9de 6.0e
4.6b 4.6b 4.6b 4.7b 5.3de 4.7b 4.1a 5.1cd 5.5e 5.1c 5.6ef 5.8f
4.2b 4.2ab 4.2ab 4.2b 4.8cd 4.1ab 3.8a 4.8cd 5.3ef 4.5bc 5.0de 5.4f
4.0ab 4.0ab 4.0ab 4.0abd 4.5d 3.8ac 3.7a 4.4bd 5.1e 4.2bcd 4.4bd 5.3e
Different letters in the same column indicate significant differences between Lactobacillus cultures. Significant differences were reported when p < 0.05. 1 30 jC. 2 37 jC.
increase the endospore number of B. cereus with the exception of the B. cereus NVH 38 samples, where few spores were detected with the pH adjustment. Therefore, only results of endospore counts without a pH adjustment are presented in Fig. 1B, D. The endospore content was determined after 0, 7, 12, 24, 30, 48 and 72 h of incubation. The BHI agar plates were incubated at 30 jC for 24 h.
2.6. Screening for antimicrobial activity and the effect of proteinase K Colonies of various Lactobacillus and Lactococcus cultures were grown on MRS-agar or GM17-agar plates, respectively, for 24 h. A lawn of 4 ml BHI soft agar (BHI medium containing 0.7% agar) containing 70 Al of a fresh culture of B. cereus was then
Table 3 Growth of B. cereus NVH 45 together with different Lactococcus cultures in 10% reconstituted skimmed milk at 30 jC and the development of pH during fermentation Strains
B. cereus NVH 45 together with: NCDO 1761 NCDO 184L1 ML82 lac 9522 DRC33 P23
B. cereus log10 cfu/ml
pH of the milk
0h
2h
4h
7h
24 h
48 h
0h
2h
4h
7h
24 h
48 h
2.09a 2.17a 2.11a 2.19a 2.17a 2.23a
2.51a 2.63a 2.62a 2.76ab 2.87ab 3.05b
3.35ab 3.78b 2.76a 3.18ab 3.23ab 4.07c
3.42a 4.99b 2.9a 3.13a 3.38a 4.33ab
< 1a 6.64b < 1a < 1a < 1a < 1a
< 1a 2.81b < 1a < 1a < 1a < 1a
6.5a 6.5a 6.5a 6.5a 6.5a 6.5a
6.4a 6.5a 6.4a 6.4a 6.4a 6.5a
6.0b 6.4d 6.0b 5.8a 6.2bc 6.2cd
4.9ab 6.3d 4.8a 4.6a 5.3c 5.2bc
4.3a 5.1b 4.3a 4.3a 4.3a 4.3a
4.3a 4.6b 4.3a 4.3a 4.3a 4.3a
Different letters in the same column indicate significant differences between Lactococcus cultures. Significant differences were reported when p < 0.05. 1 L. lactis subsp. lactis biovar. diacetylactis. 2 L. lactis subsp. lactis. 3 L. lactis subsp. cremoris.
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Fig. 1. Inhibition of growth and sporulation of B. cereus NVH 38 ( w ), NVH 45 (5), NVH 68 (o) and NVH 74 (D) by Lb. casei 2756 at 30 jC or Lb. acidophilus NCFB 1748 at 37 jC in 10% RSM. (A) Counts of B. cereus alone (—) or in co-culture with Lb. casei 2756 (---). (B) Counts of endospores of B. cereus alone (—) or in co-culture with Lb. casei 2756 (---). (C) Counts of B. cereus alone (—) or in co-culture with Lb. acidophilus NCFB 1748 (---). (D) Counts of endospores of B. cereus alone (—) or in co-culture with Lb. acidophilus NCFB 1748 (---).
poured over the plates. After incubation for 24 h, the plates were examined for zones of growth inhibition surrounding the colonies. Proteinase K (INC Biomedicals, Cat. No. 193504, Aurora, OH, USA, 20 mg/ml) was spotted around colonies of potential bacteriocinproducing bacteria. After an incubation period of 1 h, BHI soft agar with B. cereus was poured over the colonies. Plates were examined to judge whether the inhibitory substance was sensitive to proteolysis. 2.7. Statistical analysis Statistical analysis was performed using Minitab version 13.1 (Minitab, 1999, PA, USA) by one-way analysis of variance (ANOVA). Where applicable, standard errors of means have been given as bars in Fig. 1.
3. Results and discussion In this work, B. cereus was inoculated in skimmed milk at about 102 cfu/ml to simulate the level that commonly occurs in milk (Ahmed et al., 1983; Larsen and Jørgensen, 1997, 1999; Eneroth et al., 2001). B. cereus growing alone in milk at 30 or 37 jC reached a level of 107 –108 cfu/ml (Fig. 1A,C). Similar results have been reported for B. cereus in unfermented nonfat milk medium (Wong and Chen, 1988; Mansour and Milliere, 2001) and in pasteurised milk (Wong et al., 1988). 3.1. Effects of LAB cultures on B. cereus NVH 45 The effects of the Lactobacillus or the Lactococcus cultures on the growth of B. cereus NVH 45 in
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10% RSM are presented in Tables 2 and 3, respectively. This study shows that the different Lactobacillus and Lactococcus cultures reduced the B. cereus population within 24 –48 h, although significant differences in the degree of inhibition were observed from total inhibition to a decrease of 2 –3 log10 (cfu/ml). The inhibition seemed to be correlated with reduction in pH during the first hours of fermentation. Total inhibition was particularly true with Lactococcus cultures, reducing pH to 5.3 or lower in only 7 h, and nonstarter LAB (NSLAB) that needed 24 h to reduce pH to less than 5.0. Exceptions to this were Lactococcus lactis subsp. lactis biovar. diacetylactis 184L and Lactobacillus curvatus 2042. The probiotic cultures and the cultures isolated from cereals reduced pH slower during these initial hours of fermentation, and quite high numbers of B. cereus NVH 45 (104 – 106 cfu/ ml) were present in the milk after 72 h. These findings are supported by the work with cereal gruels of Kingamkono et al. (1994), who observed a connection between a rapid acid production rate and the inhibition rate of pathogens, including B. cereus. Inhibition of B. cereus by LAB has previously been observed in fermented milk (Wong and Chen, 1988; Wong et al., 1988) and in cheese (Little and Knøchel, 1994; Rukure and Bester, 2001). According to Goepfert et al. (1972) and Nout et al. (1987), the minimum pH for growth of B. cereus
is about 4.90 in laboratory media and in soya bean tempeh. Wong and Chen (1988) reported that pH 5.0 was the critical point for B. cereus cell growth. Aryanta et al. (1991) stated that rapid growth of LAB to 108 –109 cfu/g with a concomitant reduction of pH to below 5.0 was a critical factor in controlling growth of pathogenic bacteria in red and poultry meat sausages. These results pointed out that pH 5.0 or lower was a critical factor in controlling growth of B. cereus, however the present results indicated that the crucial point was the rate of the initial pH reduction at the start of fermentation. For instance, Lb. acidophilus NCFB 1748 and Lb. rhamnosus LC705 reduced pH to < 5.0 in 24 h, and after 72 h the pH was 3.8 and 3.9, respectively, which was the lowest pH produced by any strains of Lactobacillus. However, high numbers (104 – 105 cfu/ml) of B. cereus were still detected after 72 h. The initial pH reduction rate was slower than for the more inhibitory strains of Lactobacillus and Lactococcus during the first hours of fermentation, as shown for Lb. casei 2756 and Lb. acidophilus NCFB 1748 in Table 4. B. cereus was detected as endospores when grown together with Lb. rhamnosus LC705 (data not shown) or Lb. acidophilus NCFB 1748 (Fig. 1D). Less than 10 endospores/ml were observed after co-cultivation with rapid acid producers (Fig. 1B). These results show that a slower acid production rate gives B. cereus the opportunity to sporulate.
Table 4 The development of pH during fermentation with Lb. casei 2756 or Lb. acidophilus NCFB 1748 alone and together with B. cereus NVH 38, 45, 68 or 74 at 30 or 37 jC in 10% reconstituted skimmed milk Strains
0h 1
Lb. casei 2756 Lb. acidophilus NCFB 17482 NVH 38 + 27561 NVH 38 + NCFB 17482 NVH 45 + 27561 NVH 45 + NCFB 17482 NVH 68 + 27561 NVH 68 + NCFB 17482 NVH 74 + 27561 NVH 74 + NCFB 17482
a
6.4 6.5a 6.5a 6.5a 6.4a 6.5a 6.5a 6.5a 6.5a 6.5a
2h a
6.4 6.4a 6.4a 6.5a 6.4a 6.5a 6.4a 6.5a 6.4a 6.5a
4h a
6.2 6.3ab 6.2a 6.4ab 6.2a 6.4ab 6.3ab 6.4ab 6.2a 6.4b
7h a
5.9 6.2b 5.9a 6.3b 5.9a 6.2b 6.0a 6.2b 5.9a 6.3b
12 h
24 h
ac
ab
5.6 5.9d 5.5ab 5.9d 5.5a 5.9cd 5.5ab 5.9cd 5.5ab 5.8bcd
5.0 5.3c 5.0ab 5.2bc 4.8a 4.9a 4.9a 4.9ab 4.9a 4.9a
30 h abc
4.8 5.0c 4.8ab 4.9bc 4.6a 4.7ab 4.6a 4.7ab 4.7ab 4.7ab
48 h
72 h
cd
4.1d 4.2d 4.1cd 4.1cd 4.0bc 3.8a 4.1cd 3.9a 4.1cd 3.9ab
4.4 4.5d 4.4bcd 4.4bcd 4.2a 4.1a 4.3ac 4.2a 4.2ab 4.2a
Different letters in the same column indicate significant differences between the two Lactobacillus cultures. Significant differences were reported when p < 0.05. 1 30 jC. 2 37 jC.
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3.2. Effects of Lb. casei 2756 and Lb. acidophilus NCFB 1748 on four B. cereus strains Lb. casei 2756 inhibited B. cereus NVH 38, NVH 45, NVH 68 and NVH 74 to not detectable levels (< 10 cfu/ml) after 48 h of incubation, while Lb. acidophilus NCFB 1748 inhibited only B. cereus NVH 38 to that extent during the same period (Fig. 1A,C). B. cereus NVH 45, NVH 68 or NVH 74 had B. cereus counts of about 105 cfu/ml after 48 h. During the initial incubation (7 and 12 h) of each of these two Lactobacillus cultures, together with one of the four B. cereus strains, the pH reduction was significantly slower with Lb. acidophilus NCFB 1748 than with Lb. casei 2756 (Table 4). After 24 h, the pH-values were comparable. The slow acid production by Lb. acidophilus NCFB 1748 gave B. cereus the opportunity to sporulate and exist as endospores, with the exception of B. cereus NVH 38 (Fig. 1D). Similar results were obtained for B. cereus NVH 45 when grown with Lb. rhamnosus LC705 or Lb. acidophilus LF221 (data not shown). When B. cereus was grown together with Lb. casei 2756, only low numbers of endospores were formed by B. cereus NVH 38, NVH 45, NVH 68 and NVH 74 (Fig. 1B). B. cereus NVH 38 seemed to be more pH-sensitive than the other strains and did not sporulate when grown together with Lb. casei 2756 or Lb. acidophilus NCFB 1748, and this may be an explanation for the strong inhibition exerted by both Lactobacillus cultures. Zones of growth inhibition were observed when B. cereus was tested in a bacteriocin assay, but the inhibitory substance produced by the different Lactobacillus or Lactococcus cultures was not sensitive to proteinase K (results not shown). Lb. acidophilus LF221 is known to produce bacteriocins active against some strains of B. cereus (BogovicMatijasic et al., 1998) with highest bacteriocin titres after 10 –12 h incubation at 37 jC (BogovicMatijasic and Rogelj, 1998). In the present study, no inhibition of B. cereus was observed after 24 h in milk with Lb. acidophilus LF221, indicating that B. cereus NVH 45 is not sensitive to the bacteriocins. This study demonstrates that a rapid decrease in pH during early log phase of fermentation is strongly connected with growth inhibition of B. cereus. The pH values obtained in the product/sample during the first
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hours of fermentation determine whether B. cereus is able to survive or not. When pH is reduced at a slower rate in early log phase, B. cereus is given the opportunity to sporulate and exist as endospores.
Acknowledgements We thank Elida Sehic for technical assistance. This work was funded by a grant from The Research Council of Norway.
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Griffiths, M.W., Phillips, J.D., 1990. Incidence, source and some properties of psychrotrophic Bacillus spp. found in raw and pasteurized milk. Journal of the Society of Dairy Technology 43, 62 – 66. Helander, I.M., vonWright, A., MattilaSandholm, T.M., 1997. Potential of lactic acid bacteria and novel antimicrobials against gram-negative bacteria. Trends in Food Science and Technology 8, 146 – 150. Holzapfel, W.H., Geisen, R., Schillinger, U., 1995. Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes. International Journal of Food Microbiology 24, 343 – 362. Jackson, S.G., 1991. Bacillus cereus. Journal of the Association of Official Analytical Chemist 74, 704 – 706. Kingamkono, R., Sjo¨gren, E., Svanberg, U., Kaijser, B., 1994. pH and acidity in lactic-fermenting cereal gruels—effects on viability of enteropathogenic microorganisms. World Journal of Microbiology and Biotechnology 10, 664 – 669. Kramer, J.M., Gilbert, R.J., 1989. Bacillus cereus and other Bacillus species. In: Doyle, M.P. (Ed.), Foodborne Bacterial Pathogens. Marcel Dekker, New York, pp. 21 – 70. Larsen, H.D., Jørgensen, K., 1997. The occurrence of Bacillus cereus in Danish pasteurized milk. International Journal of Food Microbiology 34, 179 – 186. Larsen, H.D., Jørgensen, K., 1999. Growth of Bacillus cereus in pasteurized milk products. International Journal of Food Microbiology 46, 173 – 176. Lin, S., Schraft, H., Odumeru, J.A., Griffiths, M.W., 1998. Identification of contamination sources of Bacillus cereus in pasteurized milk. International Journal of Food Microbiology 43, 159 – 171. Little, C.L., Knøchel, S., 1994. Growth and survival of Yersinia enterocolitica, Salmonella and Bacillus cereus in Brie stored at 4, 8 and 20 degrees C. International Journal of Food Microbiology 24, 137 – 145. Mansour, M., Milliere, J.B., 2001. An inhibitory synergistic effect of a nisin – monolaurin combination on Bacillus sp. vegetative cells in milk. Food Microbiology 18, 87 – 94. Mayr, R., Eppert, I., Scherer, S., 1999. Incidence and identification
of psychrotrophic (7 degrees C-tolerant) Bacillus spp. in German HTST pasteurized milk. Milchwissenschaft. 54, 26 – 30. Nout, M.J.R., Beernink, G., Bonantsvanlaarhoven, T.M.G., 1987. Growth of Bacillus cereus in soyabean tempeh. International Journal of Food Microbiology 4, 293 – 301. Rowan, N.J., Anderson, J.G., 1998. Diarrhoeal enterotoxin production by psychrotrophic Bacillus cereus present in reconstituted milk-based infant formulae (MIF). Letters of Applied Microbiology 26, 161 – 165. Rowan, N.J., Anderson, J.G., Smith, J.E., 1998. Potential infective and toxic microbiological hazards associated with the consumption of fermented foods. In: Wood, B.J.B. (Ed.), Microbiology of Fermented Foods, vol. 2. Blackie Academic Professional, London, UK, pp. 812 – 837. Rukure, G., Bester, B.H., 2001. Survival and growth of Bacillus cereus during Gouda cheese manufacturing. Food Control 12, 31 – 36. Shinagawa, K., 1990. Analytical methods for Bacillus cereus and other Bacillus species. International Journal of Food Microbiology 10, 125 – 141. Stenfors, L.P., Granum, P.E., 2001. Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis. FEMS Microbiology Letters 197, 223 – 228. Suomalainen, T.H., MayraMakinen, A.M., 1999. Propionic acid bacteria as protective cultures in fermented milks and breads. Lait 79, 165 – 174. Svanberg, U., Sjo¨gren, E., Lorri, W., Svennerholm, A.M., Kaijser, B., 1992. Inhibited growth of common enteropathogenic bacteria in lactic-fermented cereal gruels. World Journal of Microbiology and Biotechnology 8, 601 – 606. van Netten, P., van De Moosdijk, A., van Hoensel, P., Mossel, D.A., Perales, I., 1990. Psychrotrophic strains of Bacillus cereus producing enterotoxin. Journal of Applied Bacteriology 69, 73 – 79. Wong, H.C., Chen, Y.L., 1988. Effects of lactic acid bacteria and organic acids on growth and germination of Bacillus cereus. Applied and Environmental Microbiology 54, 2179 – 2184. Wong, H.C., Chen, Y.L., Chen, C.L.F., 1988. Growth, germination and toxigenic activity of Bacillus cereus in milk products. Journal of Food Protection 51, 707 – 710.
Inhibition of Bacillus cereus by strains of Lactobacillus and Lactococcus in milk Elisabeth Røssland a,*, Grethe I. Andersen Borge a,b, Thor Langsrud a, Terje Sørhaug a a
Department of Food Science, Agricultural University of Norway, P.O. Box 5036, N-1432, A˚ s, Norway b Norwegian Food Research Institute (MATFORSK), Osloveien 1, 1430, A˚ s, Norway Received 2 January 2002; received in revised form 21 February 2003; accepted 15 March 2003
Abstract The growth and death or survival of Bacillus cereus in sterile skimmed milk fermented with 18 different lactic acid bacteria (LAB) were investigated. B. cereus alone in milk reached about 107 – 108 colony-forming units (cfu)/ml. When B. cereus was cultivated together with different Lactobacillus or Lactococcus cultures at 30 or 37 jC, the B. cereus counts after 72 h of fermentation ranged between < 10 cfu/ml and about 106 cfu/ml. The inhibition patterns for the different Lactobacillus and Lactococcus cultures varied. All the Lactococcus cultures (with one exception) reduced pH to 5.3 or lower in 7 h. After 24 h, B. cereus was not detected in any of the fast Lactococcus-fermented milk samples. After 48 h, B. cereus was not detected for 4 of the 12 Lactobacillus cultures. These cultures reduced pH to below 5.0 in 24 h. The other Lactobacillus cultures also inhibited B. cereus, but the counts of B. cereus were still 104 – 106 cfu/ml after 72 h. They also reduced pH at a slower rate. Survival of B. cereus was to a variable extent linked with formation of endospores. Proteinase K did not affect the antimicrobial activity observed. Acid production with decreasing pH, particularly the initial rate of pH decrease, appears to be most important for control of B. cereus with LAB. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Bacillus cereus; Lactobacillus; Lactococcus; Inhibition; Endospore; pH
1. Introduction Bacillus cereus is an ubiquitous, spore-forming soil microorganism that is a common cause of food poisoning (Kramer and Gilbert, 1989; Andersson et al., 1995). These species produce two distinct types of food-borne illness: the diarrhoeal illness, seen occasionally following consumption of milk and other dairy products, meat products, sauces, soups and * Corresponding author. Tel.: +47-64-94-85-76; fax: +47-6494-37-89. E-mail address: [email protected] (E. Røssland).
vegetables (Granum and Lund, 1997), and the emetic (vomit-inducing) syndrome, associated with consumption of a heat-stable toxin produced in fried and cooked rice, pasta and noodles (Shinagawa, 1990; Jackson, 1991; Granum and Lund, 1997). B. cereus is a common contaminant of raw milk (Lin et al., 1998) but is also frequently isolated from different dairy products (Kramer and Gilbert, 1989). A survey conducted by Wong et al. (1988) on dairy products showed that B. cereus was found in 52% of ice creams, 35% of soft ice creams, 29% of milk powders, 17% of fermented milks and 2% of pasteurised milks and fruit flavoured milks.
0168-1605/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(03)00149-1
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Some psychrotrophic strains of B. cereus are known to grow in foods at temperatures as low as 4– 6 jC (Griffiths and Phillips, 1990; van Netten et al., 1990; Dufrenne et al., 1994; Rowan and Anderson, 1998; Borge et al., 2001). This growth represents a problem in chilled products such as milk and other dairy products, and in ‘‘long life’’ ready-to-eat or fresh-chilled foods. Griffiths and Phillips (1990) isolated endospores of psychrotrophic Bacillus spp. from 58% of farm bulk tank milks and from about 69% of pasteurised milks. In addition to B. cereus, several other species within the genus Bacillus are associated with spoilage problems in the dairy industry (Andersson et al., 1995; Larsen and Jørgensen, 1997; Mayr et al., 1999). Examples of such spoilage include bitty cream, sweet curdling of milk and off-flavours in various products leading to substantial economic losses (Barkley and Delaney, 1980). Lactic acid bacteria (LAB) used in food fermentations are able to inhibit or kill food-borne and other pathogenic microorganisms as well as simple spoilage bacteria by producing a variety of antimicrobial agents (Holzapfel et al., 1995; Adams and Nicolaides, 1997; Helander et al., 1997). Acidity is probably in most cases the primary factor in the preservation of lactic acid-fermented foods (Adams and Nicolaides, 1997; Rowan et al., 1998). However, a number of other factors may also be inhibitory, like for instance bacteriocins, diacetyl, carbon dioxide, hydrogen peroxide and ethanol (Holzapfel et al., 1995; Adams and Nicolaides, 1997; Helander et al., 1997). Inhibition of B. cereus by LAB has been reported in nonfat milk medium (Wong and Chen, 1988), in Gouda (Rukure and Bester, 2001) and in Brie (Little and Knøchel, 1994). It has been shown that foods fermented by LAB to pH 4.0 or lower inhibit the growth of B. cereus and other pathogens (Nout et al., 1987; Aryanta et al., 1991; Svanberg et al., 1992; Kingamkono et al., 1994). Lactobacillus rhamnosus LC705 has been shown to inhibit Bacillus spp. in bakery products (Suomalainen and MayraMakinen, 1999). Lactobacillus acidophilus LF221 producing two bacteriocins active against different pathogens including B. cereus was found to inhibit 1 out of 12 B. cereus strains tested in an agar-well diffusion assay (BogovicMatijasic et al., 1998).
The aim of the present work was to determine the effects of different Lactobacillus and Lactococcus cultures on B. cereus in milk.
2. Materials and methods 2.1. Bacterial strains and growth conditions An overview of the lactic acid bacteria (LAB) and B. cereus strains used in the experiments is presented in Table 1. The stock cultures were kept at 80 jC in media containing 15% (w/v) glycerol (Merck, Darmstadt, Germany) as follows: B. cereus in brain heart infusion broth (BHI, Difco, Becton Dickinson, Sparks, MD, USA), Lactobacillus reuteri SD2112 in sterile 10% (w/v) reconstituted skimmed milk (RSM, TINE Norske Meierier, Foll Voll, Norway), other lactobacilli in de Man Rogosa Sharpe broth (MRS broth, Oxoid, Hampshire, England) and the Lactococci in M17 broth (Oxoid). The B. cereus strains were subcultured three times in 10 ml BHI broth at 30 jC for 11 –12 h before the cells were used as inocula in the different experiments. Lactic acid bacteria were subcultured three times in 10 ml MRS broth (Lactobacillus) or 10 ml M17 broth (Lactococcus) at 30 or 37 jC (Tables 2 and 3) for 11– 12 h before the cells were used as inocula. 2.2. Preparation and inoculation of milk samples Sample flasks (250 ml screw-cap flasks) containing 200 ml of 10% (w/v) RSM were prepared and autoclaved (121 jC, 15 min). The sterile milk was inoculated with B. cereus to obtain 102 colony-forming units (cfu)/ml at zero time. Simultaneously, Lactobacillus or Lactococcus cultures were inoculated at population levels of about 106 – 107 cfu/ml milk. Bottles inoculated with B. cereus alone served as controls. 2.3. Growth conditions and enumeration methods Inoculated milk samples were incubated at 30 jC for 3 days except for Lb. acidophilus NCFB 1748, Lb. acidophilus LF221 and Lb. reuteri SD2112 that were
E. Røssland et al. / International Journal of Food Microbiology 89 (2003) 205–212 Table 1 An overview of the Lactobacillus (Lb.), Lactococcus (L.) and B. cereus cultures used in the experiments Strains a
Lb. casei 2756 , Lb. casei 2752a, Lb. curvatus 2042a, Lb. plantarum 2741a Lb. paracasei subsp. paracasei INF-15Da
Source
Obtained from
isolated from cheese
Department of Food Microbiology, University College, Cork, Ireland Department of Food Science, Agricultural University of Norway National Collection of Food Bacteria, Reading, England Collection of Industrial Microorganisms, Ljubljana, Slovenia Biogaia Biologics, Stockholm, Sweden
isolated from Norvegia cheese
Lb. acidophilus NCFB 1748b Lb. acidophilus LF221b
isolated from infant faeces
Lb. reuteri SD2112b
isolated from human breast milk
Lb. rhamnosus LC705b Lb. brevis 226, Lb. plantarum 8, Lb. fermentum 99
L. lactis subsp. lactis ML8, L. lactis subsp. cremoris DRC3, L. lactis subsp. cremoris P2 L. lactis subsp. lactis biovar. diacetylactis NCDO 176, L. lactis subsp. lactis biovar. diacetylactis NCDO 184L L. lactis subsp. lactis lac 952 B. cereus NVH 38, 45, 68 and 74c
a
isolated from Bushera (fermented sorghum beverage)
Valio culture collection, Helsinki, Finland Department of Food Science, Agricultural University of Norway
Dr. Tim Cogan, Dairy Products Research Centre, Fermoy, County of Cork, Ireland National Collection of Food Bacteria, Reading, England
isolated from dairy products
Christian Hansen, Hørsholm, Denmark Culture collection of The Norwegian School of Veterinary Science (NVH)
Nonstarter lactic acid bacteria (NSLAB). Probiotic bacteria. c All strains contained the non-haemolytic enterotoxin gene (nhe) and the enterotoxin T gene. All strains, except B. cereus NVH 38, contained in addition the haemolytic enterotoxin gene (hbl) (Borge et al., 2001; Stenfors and Granum, 2001). b
207
incubated at 37jC. All the experiments were carried out in triplicate. After shaking the flasks by hand, samples (0.1 ml) were serially diluted in sterile peptone water/saline (0.1%, w/v, Peptone (Oxoid), containing 0.85%, w/v, NaCl (Merck)) and surface plated (0.1 ml) in duplicate. BHI agar plates used to enumerate B. cereus were incubated at 30 jC for 24 h. Lactobacillus was enumerated on Lactobacillus-selective medium (LBS, Becton Dickinson) and the plates were incubated at 30jC for 72 h for all the strains except for Lb. acidophilus NCFB 1748 and Lb. reuteri SD2112, which were incubated at 37 jC for 72 h. Lb. acidophilus LF221 was enumerated on MRS-agar due to poor growth on LBS agar, and the plates were incubated at 37 jC for 48 h. Lactococcus was enumerated on M17 agar and the plates were incubated at 30 jC for 48 h. Plate counts of B. cereus NVH 45 were made in duplicate after different time intervals, as indicated in Tables 2 and 3, and plate counts of Lactobacillus or Lactococcus were made in duplicate after incubation for 0 and 24 h. Lactobacillus casei 2756 or Lb. acidophilus NCFB 1748 were co-inoculated with B. cereus NVH 38, NVH 45, NVH 68 or NVH 74 in 10% RSM, and plate counts of B. cereus and Lactobacillus were made in duplicates after incubation for 0, 2, 4, 7, 12, 24, 30, 48 and 72 h. The pH of each sample was also measured. 2.4. pH measurement The pH was measured at different times with a pH electrode (PHM 210, Radiometer, Copenhagen, Denmark) in 10 ml aliquots taken from each of the flasks. The pH electrode was calibrated with buffers (Merck) at pH 4.0 and 7.0. 2.5. Endospore content Quantitative determination of the number of endospores in the milk samples was made by surface plating after heat treatment (80 jC, 5 min) of 1 ml milk with serial dilutions in peptone water/saline. In addition, 1 ml of milk sample (from the same bottle) was adjusted to pH 6.5 by adding NaOH (Merck, 0.5% or 1.0%, w/v) before heat treatment. The pH adjustment prior to heating of the samples did not
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Table 2 Growth of B. cereus NVH 45 together with different Lactobacillus cultures in 10% reconstituted skimmed milk at 30 or 37 jC and the development of pH during fermentation Strains
B. cereus log10 cfu/ml 0h
24 h
B. cereus NVH 451 B. cereus NVH 45 together with: Lb. casei 27561 Lb. casei 27521 Lb. paracasei INF-15D1 Lb. plantarum 27411 Lb. curvatus 20421 Lb. acidophilus NCFB 17482 Lb. rhamnosus LC7051 Lb. acidophilus LF2212 Lb. reuteri SD21122 Lb. brevis 2261 Lb. plantarum 81 Lb. fermentum 991
2.06
7.93
2.24a 2.19a 2.19a 2.22a 2.19a 2.93b 1.97a 2.15a 2.20a 2.20a 2.20a 2.27a
4.53a 5.63a 5.37a 5.73a 7.21bc 7.19bc 6.25ab 7.88e 6.65abc 7.2bc 7.25c 7.68d
pH of the milk
48 h
72 h
8.12
8.09
< 1a < 1a < 1a < 1a 6.13bc 5.39bc 4.58b 7.2bc 6.78bc 6.16bc 7.73cd 7.86d
< 1a < 1a < 1a < 1a 5.43b 5.29b 4.45b 6.51b 6.37b 5.97b 6.44b 6.68b
0h
4h
24 h
30 h
48 h
72 h
6.5
6.5
6.0
5.7
5.3
5.0
6.4ab 6.4ab 6.4ab 6.5ac 6.4ab 6.5ac 6.5c 6.5ac 6.4b 6.4ab 6.4ab 6.5ac
6.2ab 6.2abc 6.2ab 6.2ab 6.3ad 6.4cd 6.4d 6.4cd 6.2b 6.3abd 6.2abc 6.4d
4.8b 4.8b 4.8b 5.0b 5.6cd 4.9b 4.4a 5.5c 5.6cd 5.4c 5.9de 6.0e
4.6b 4.6b 4.6b 4.7b 5.3de 4.7b 4.1a 5.1cd 5.5e 5.1c 5.6ef 5.8f
4.2b 4.2ab 4.2ab 4.2b 4.8cd 4.1ab 3.8a 4.8cd 5.3ef 4.5bc 5.0de 5.4f
4.0ab 4.0ab 4.0ab 4.0abd 4.5d 3.8ac 3.7a 4.4bd 5.1e 4.2bcd 4.4bd 5.3e
Different letters in the same column indicate significant differences between Lactobacillus cultures. Significant differences were reported when p < 0.05. 1 30 jC. 2 37 jC.
increase the endospore number of B. cereus with the exception of the B. cereus NVH 38 samples, where few spores were detected with the pH adjustment. Therefore, only results of endospore counts without a pH adjustment are presented in Fig. 1B, D. The endospore content was determined after 0, 7, 12, 24, 30, 48 and 72 h of incubation. The BHI agar plates were incubated at 30 jC for 24 h.
2.6. Screening for antimicrobial activity and the effect of proteinase K Colonies of various Lactobacillus and Lactococcus cultures were grown on MRS-agar or GM17-agar plates, respectively, for 24 h. A lawn of 4 ml BHI soft agar (BHI medium containing 0.7% agar) containing 70 Al of a fresh culture of B. cereus was then
Table 3 Growth of B. cereus NVH 45 together with different Lactococcus cultures in 10% reconstituted skimmed milk at 30 jC and the development of pH during fermentation Strains
B. cereus NVH 45 together with: NCDO 1761 NCDO 184L1 ML82 lac 9522 DRC33 P23
B. cereus log10 cfu/ml
pH of the milk
0h
2h
4h
7h
24 h
48 h
0h
2h
4h
7h
24 h
48 h
2.09a 2.17a 2.11a 2.19a 2.17a 2.23a
2.51a 2.63a 2.62a 2.76ab 2.87ab 3.05b
3.35ab 3.78b 2.76a 3.18ab 3.23ab 4.07c
3.42a 4.99b 2.9a 3.13a 3.38a 4.33ab
< 1a 6.64b < 1a < 1a < 1a < 1a
< 1a 2.81b < 1a < 1a < 1a < 1a
6.5a 6.5a 6.5a 6.5a 6.5a 6.5a
6.4a 6.5a 6.4a 6.4a 6.4a 6.5a
6.0b 6.4d 6.0b 5.8a 6.2bc 6.2cd
4.9ab 6.3d 4.8a 4.6a 5.3c 5.2bc
4.3a 5.1b 4.3a 4.3a 4.3a 4.3a
4.3a 4.6b 4.3a 4.3a 4.3a 4.3a
Different letters in the same column indicate significant differences between Lactococcus cultures. Significant differences were reported when p < 0.05. 1 L. lactis subsp. lactis biovar. diacetylactis. 2 L. lactis subsp. lactis. 3 L. lactis subsp. cremoris.
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Fig. 1. Inhibition of growth and sporulation of B. cereus NVH 38 ( w ), NVH 45 (5), NVH 68 (o) and NVH 74 (D) by Lb. casei 2756 at 30 jC or Lb. acidophilus NCFB 1748 at 37 jC in 10% RSM. (A) Counts of B. cereus alone (—) or in co-culture with Lb. casei 2756 (---). (B) Counts of endospores of B. cereus alone (—) or in co-culture with Lb. casei 2756 (---). (C) Counts of B. cereus alone (—) or in co-culture with Lb. acidophilus NCFB 1748 (---). (D) Counts of endospores of B. cereus alone (—) or in co-culture with Lb. acidophilus NCFB 1748 (---).
poured over the plates. After incubation for 24 h, the plates were examined for zones of growth inhibition surrounding the colonies. Proteinase K (INC Biomedicals, Cat. No. 193504, Aurora, OH, USA, 20 mg/ml) was spotted around colonies of potential bacteriocinproducing bacteria. After an incubation period of 1 h, BHI soft agar with B. cereus was poured over the colonies. Plates were examined to judge whether the inhibitory substance was sensitive to proteolysis. 2.7. Statistical analysis Statistical analysis was performed using Minitab version 13.1 (Minitab, 1999, PA, USA) by one-way analysis of variance (ANOVA). Where applicable, standard errors of means have been given as bars in Fig. 1.
3. Results and discussion In this work, B. cereus was inoculated in skimmed milk at about 102 cfu/ml to simulate the level that commonly occurs in milk (Ahmed et al., 1983; Larsen and Jørgensen, 1997, 1999; Eneroth et al., 2001). B. cereus growing alone in milk at 30 or 37 jC reached a level of 107 –108 cfu/ml (Fig. 1A,C). Similar results have been reported for B. cereus in unfermented nonfat milk medium (Wong and Chen, 1988; Mansour and Milliere, 2001) and in pasteurised milk (Wong et al., 1988). 3.1. Effects of LAB cultures on B. cereus NVH 45 The effects of the Lactobacillus or the Lactococcus cultures on the growth of B. cereus NVH 45 in
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10% RSM are presented in Tables 2 and 3, respectively. This study shows that the different Lactobacillus and Lactococcus cultures reduced the B. cereus population within 24 –48 h, although significant differences in the degree of inhibition were observed from total inhibition to a decrease of 2 –3 log10 (cfu/ml). The inhibition seemed to be correlated with reduction in pH during the first hours of fermentation. Total inhibition was particularly true with Lactococcus cultures, reducing pH to 5.3 or lower in only 7 h, and nonstarter LAB (NSLAB) that needed 24 h to reduce pH to less than 5.0. Exceptions to this were Lactococcus lactis subsp. lactis biovar. diacetylactis 184L and Lactobacillus curvatus 2042. The probiotic cultures and the cultures isolated from cereals reduced pH slower during these initial hours of fermentation, and quite high numbers of B. cereus NVH 45 (104 – 106 cfu/ ml) were present in the milk after 72 h. These findings are supported by the work with cereal gruels of Kingamkono et al. (1994), who observed a connection between a rapid acid production rate and the inhibition rate of pathogens, including B. cereus. Inhibition of B. cereus by LAB has previously been observed in fermented milk (Wong and Chen, 1988; Wong et al., 1988) and in cheese (Little and Knøchel, 1994; Rukure and Bester, 2001). According to Goepfert et al. (1972) and Nout et al. (1987), the minimum pH for growth of B. cereus
is about 4.90 in laboratory media and in soya bean tempeh. Wong and Chen (1988) reported that pH 5.0 was the critical point for B. cereus cell growth. Aryanta et al. (1991) stated that rapid growth of LAB to 108 –109 cfu/g with a concomitant reduction of pH to below 5.0 was a critical factor in controlling growth of pathogenic bacteria in red and poultry meat sausages. These results pointed out that pH 5.0 or lower was a critical factor in controlling growth of B. cereus, however the present results indicated that the crucial point was the rate of the initial pH reduction at the start of fermentation. For instance, Lb. acidophilus NCFB 1748 and Lb. rhamnosus LC705 reduced pH to < 5.0 in 24 h, and after 72 h the pH was 3.8 and 3.9, respectively, which was the lowest pH produced by any strains of Lactobacillus. However, high numbers (104 – 105 cfu/ml) of B. cereus were still detected after 72 h. The initial pH reduction rate was slower than for the more inhibitory strains of Lactobacillus and Lactococcus during the first hours of fermentation, as shown for Lb. casei 2756 and Lb. acidophilus NCFB 1748 in Table 4. B. cereus was detected as endospores when grown together with Lb. rhamnosus LC705 (data not shown) or Lb. acidophilus NCFB 1748 (Fig. 1D). Less than 10 endospores/ml were observed after co-cultivation with rapid acid producers (Fig. 1B). These results show that a slower acid production rate gives B. cereus the opportunity to sporulate.
Table 4 The development of pH during fermentation with Lb. casei 2756 or Lb. acidophilus NCFB 1748 alone and together with B. cereus NVH 38, 45, 68 or 74 at 30 or 37 jC in 10% reconstituted skimmed milk Strains
0h 1
Lb. casei 2756 Lb. acidophilus NCFB 17482 NVH 38 + 27561 NVH 38 + NCFB 17482 NVH 45 + 27561 NVH 45 + NCFB 17482 NVH 68 + 27561 NVH 68 + NCFB 17482 NVH 74 + 27561 NVH 74 + NCFB 17482
a
6.4 6.5a 6.5a 6.5a 6.4a 6.5a 6.5a 6.5a 6.5a 6.5a
2h a
6.4 6.4a 6.4a 6.5a 6.4a 6.5a 6.4a 6.5a 6.4a 6.5a
4h a
6.2 6.3ab 6.2a 6.4ab 6.2a 6.4ab 6.3ab 6.4ab 6.2a 6.4b
7h a
5.9 6.2b 5.9a 6.3b 5.9a 6.2b 6.0a 6.2b 5.9a 6.3b
12 h
24 h
ac
ab
5.6 5.9d 5.5ab 5.9d 5.5a 5.9cd 5.5ab 5.9cd 5.5ab 5.8bcd
5.0 5.3c 5.0ab 5.2bc 4.8a 4.9a 4.9a 4.9ab 4.9a 4.9a
30 h abc
4.8 5.0c 4.8ab 4.9bc 4.6a 4.7ab 4.6a 4.7ab 4.7ab 4.7ab
48 h
72 h
cd
4.1d 4.2d 4.1cd 4.1cd 4.0bc 3.8a 4.1cd 3.9a 4.1cd 3.9ab
4.4 4.5d 4.4bcd 4.4bcd 4.2a 4.1a 4.3ac 4.2a 4.2ab 4.2a
Different letters in the same column indicate significant differences between the two Lactobacillus cultures. Significant differences were reported when p < 0.05. 1 30 jC. 2 37 jC.
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3.2. Effects of Lb. casei 2756 and Lb. acidophilus NCFB 1748 on four B. cereus strains Lb. casei 2756 inhibited B. cereus NVH 38, NVH 45, NVH 68 and NVH 74 to not detectable levels (< 10 cfu/ml) after 48 h of incubation, while Lb. acidophilus NCFB 1748 inhibited only B. cereus NVH 38 to that extent during the same period (Fig. 1A,C). B. cereus NVH 45, NVH 68 or NVH 74 had B. cereus counts of about 105 cfu/ml after 48 h. During the initial incubation (7 and 12 h) of each of these two Lactobacillus cultures, together with one of the four B. cereus strains, the pH reduction was significantly slower with Lb. acidophilus NCFB 1748 than with Lb. casei 2756 (Table 4). After 24 h, the pH-values were comparable. The slow acid production by Lb. acidophilus NCFB 1748 gave B. cereus the opportunity to sporulate and exist as endospores, with the exception of B. cereus NVH 38 (Fig. 1D). Similar results were obtained for B. cereus NVH 45 when grown with Lb. rhamnosus LC705 or Lb. acidophilus LF221 (data not shown). When B. cereus was grown together with Lb. casei 2756, only low numbers of endospores were formed by B. cereus NVH 38, NVH 45, NVH 68 and NVH 74 (Fig. 1B). B. cereus NVH 38 seemed to be more pH-sensitive than the other strains and did not sporulate when grown together with Lb. casei 2756 or Lb. acidophilus NCFB 1748, and this may be an explanation for the strong inhibition exerted by both Lactobacillus cultures. Zones of growth inhibition were observed when B. cereus was tested in a bacteriocin assay, but the inhibitory substance produced by the different Lactobacillus or Lactococcus cultures was not sensitive to proteinase K (results not shown). Lb. acidophilus LF221 is known to produce bacteriocins active against some strains of B. cereus (BogovicMatijasic et al., 1998) with highest bacteriocin titres after 10 –12 h incubation at 37 jC (BogovicMatijasic and Rogelj, 1998). In the present study, no inhibition of B. cereus was observed after 24 h in milk with Lb. acidophilus LF221, indicating that B. cereus NVH 45 is not sensitive to the bacteriocins. This study demonstrates that a rapid decrease in pH during early log phase of fermentation is strongly connected with growth inhibition of B. cereus. The pH values obtained in the product/sample during the first
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hours of fermentation determine whether B. cereus is able to survive or not. When pH is reduced at a slower rate in early log phase, B. cereus is given the opportunity to sporulate and exist as endospores.
Acknowledgements We thank Elida Sehic for technical assistance. This work was funded by a grant from The Research Council of Norway.
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