Graduate Thesis Or Dissertation
 

Studies on the inhibition of food-borne pathogens and spoilage bacteria by lactic acid starter cultures

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  • The ability of Streptococcus diacetilactis to inhibit a variety of food-borne pathogens and spoilage organisms in milk and broth cultures was demonstrated. Test organisms inhibited included Pseudomonas and Alcaligenes species, Eschericia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens and Vibrio parahaemolyticus. In general, approximately 99.0% and 99.9% inhibition was observed in milk and broth cultures, respectively. Streptococcus lactis, Streptococcus cremoris, Pediococcus cerevisiae and Lactobacillus plantarum also inhibited S. aureus in lactic broth. Possible practical applications of the observed inhibition were examined. S. diacetilactis prevented proteolysis in milk at 7. 5C by Pseudomonas fluorescens. S. aureus was inhibited greater than 99% in vanilla cream filling, ham sandwich spread, chicken gravy, soy milk and ground beef stored at 25C for 24 hr. Development of the Gram negative flora of ground beef was also inhibited greater than 99% after storage at 7.5C for 7 days. The mechanism of inhibition of S. aureus in lactic broth was examined with emphasis on the role of pH changes and acid production by S. diacetilactis. S. aureus did not grow in cell-free culture supernatants of S. diacetilactis grown in modified lactic broth (final pH 4.3 - 4.7). However, good growth was evident when the pH was adjusted towards neutrality. When the amounts of formic, acetic and lactic acids produced by S. diacetilactis were quantitatively determined and added to lactic broth, a similar pH dependent inhibition was observed. This inhibition was not as marked as that observed with cultures or cell-free supernatants of S. diacetilactis, suggesting that factors other than acid production were involved. Nutrient depletion and hydrogen peroxide did not contribute to the inhibition in this system. Enterotoxin B synthesis by S. aureus S6 and S. aureus ATCC 14458 was studied in Brain Heart Infusion broth (BHI) and N-Z Amine NAK broth (NAK). Toxin yields by S. aureus S6 were approximately 180 μg /ml and 100 μg/m1 in NAK and BHI, respectively at 30C and 150 rpm. Yields for strain 14458 were significantly lower. When the initial inoculum of S. aureus S6 was 4 x 10⁵ cells/ml, the addition of 0.5% glucose or 0.5% maltose initially repressed both toxin synthesis and the pH rise associated with toxin synthesis; however, after 72 hr of incubation, the pH rise and toxin produced were the same as occurred without added carbohydrate. Furthermore, addition of maltose, but not glucose, reduced the toxin yield of S. aureus ATCC 14458 by about 80% at 72 hr. When S. diacetilactis (initial inoculum 1 x 10⁷ cells/ml) was added to BHI containing 0. 5% glucose or 0. 5% maltose, no toxin synthesis or pH rise was observed when the initial inoculum of S. aureus was 4 x 10⁵ cells/ml. In plain BHI, no toxin was observed under these associative growth conditions and the pH remained constant at 6.6. Inhibition of toxin production in this system could be partially reversed by the addition of 0. 5% sodium pyruvate, sodium acetate or sodium succinate. When the initial inoculum of S. aureus was 4 x 10⁸ cells/ml, only partial or no inhibition of toxin production in the presence of S. diacetilactis (inoc ulum 1 x 10⁷ c ell s /m1) was observed. Substantial inhibition by S. diacetilactis was observed at this concentration when the medium was BHI + 0. 5% maltose but not when BHI + 0. 5% glucose was used. Inhibition of toxin production but not growth was evident in NAK + 1% glucose. Sodium citrate addition inhibited both growth and toxin production by S. aureus S6. The toxin yields of the S. aureus strains were greatly reduced when static incubation was used. Inhibition by S. diacetilactis was readily demonstrated under these conditions. Three commercially available starter cultures, Lactacel, Lactacel MC and Lactacel DS, were examined for their ability to control the growth of S. aureus during a simulated beaker sausage fermentation at 21C, 30C and 37C. Although differences between cultures were evident, all three gave greater than 99% inhibition after 50 hr of incubation at 30C and 37C. Inhibition was somewhat reduced (average of 97% at 50 hr) at 21C. Chemical acidulation with gluconodelta- lactone (0.75%) plus citric acid (0. 1 %) yielded good initial control in a similar process. However, on extended incubation up to 50 hr good growth of the pathogen occurred and the inhibition was only slight or none at this time for samples incubated at 30C and 37C; at 21C inhibition was approximately 97%. A combination of starter cultures and chemical acidulation gave approximately 99.99% inhibition of S. aureus at the three temperatures after 50 hr of incubation. Possible applications of these findings to the food industry are discussed. A greater role for the lactic acid bacteria in the area of food-safety is suggested.
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