Several lactic acid bacteria (LAB) of the Lactococcus, Lactobacillus, Leuconostoc and Pediococcus genera were screened for inhibition of food-borne pathogens and spoilage microorganisms in raw milk and dairy products. Listeria monocytogenes was killed by Lactococcus lactis subsp. lactis and Pediococcus pentosaceus due to their production of bacteriocin-type inhibitors. Staphylococcus aureus was not able to grow in raw milk at temperatures below 5°C even without LAB being present. Gram negative Salmonella enteritidis. Salmonella typhimurium and Escherichia coli, along with spoilage bacteria of the genus Pseudomonas were dramatically inhibited by a Lactobacillus species, designated AS-1, in raw and pasteurized milk as well as in cottage cheese. However, other LAB were not able to inhibit these organisms. Lactobacillus AS-1, did not produce hydrogen peroxide but carbon dioxide was produced. The AS-1 strain was a gram positive coccobacillus, catalase and oxidase negative and produced DL-lactic acid. It deaminated arginine and grew over a temperature range of 5°C to 45°C. It was also able to ferment glucose, galactose, fructose and lactose in addition to 17 other carbohydrates. High numbers (107 CFU/ml) of AS-1 were required to obtain complete inhibition of gram negative bacteria. A selective medium (ASLM) for Listeria monocytogenes was developed to follow the fate of this particular pathogen in association with LAB in raw milk; other selective media were not able to inhibit the growth of background flora of raw milk. ASLM was superior to four other media in allowing only the growth of the target pathogen. For the Lactococcus genus, a selective and differential agar medium (Alsan) was formulated to selectively allow growth of Lactococcus spp. and to differentiate between Lactococcus lactis subsp. lactis and the biovariety diacetylactis, based on citrate utilization. / Graduation date: 1993

Graduation date: 1994

Thesis (PhD (Food Science))--University of Stellenbosch, 2007. / Thermal pasteurisation failures in the dairy industry have often been found to cause end-products of poor quality and short shelf-life. Therefore, alternative methods to eliminate microbial contaminants in raw milk are being studied. Ultrasonication is one such non-thermal technology that could offer the dairy industry an alternative to traditional pasteurisation. The main objective of this dissertation was to evaluate the use of high-power lowfrequency ultrasound (20 kHz, 750 W, 124 μm) applied in batch mode to eliminate a selection of spoilage and potentially pathogenic microbes, commonly associated with milk. These included Gram-positive and negative microbes, comprising of rods and cocci, an endospore-former, and a yeast (Escherichia coli, Bacillus cereus, Chryseobacterium meningosepticum, Lactobacillus acidophilus, Lactococcus lactis, Listeria monocytogenes, Micrococcus luteus, Pseudomonas fluorescens and Saccharomyces cerevisiae). Three strains of E. coli (1 x 106 cfu.ml-1) tested, viz. ATCC 11775, a wild strain from raw milk, and an O157:H7 strain from milk were sensitive to ultrasonication. Complete elimination of viable cells occurred within 10 min. Viable counts of P. fluorescens were reduced by 100% within 6 min of ultrasonication and L. monocytogenes was reduced by 99.0% within 10 min. Lactococcus lactis was reduced by 97.0% and M. luteus, B. cereus and C. meningosepticum by 88.0%, 87.0% and 85.0% respectively. Lactobacillus acidophilus showed the most resistance to ultrasound with only 78.0% of viable cells being eliminated. Under similar conditions, S. cerevisiae was reduced by 99.7%. Microbial cell morphology, size and Gram status did not necessarily influence the efficacy of ultrasonication. Sterile saline solution and UHT milk were used as the suspension media, and the reputed protective effect of milk fat was not observed under the parameters used in this study. A higher wave amplitude (100%; 124 μm) was found to be more efficient in eliminating microbes than a lower wave amplitude (50%; 62 μm). Pulsed-ultrasonication did not enhance the efficiency of ultrasonication indicating that standing waves were absent. Limited success was achieved by ultrasonication itself, and the long batch treatment time (10 min or more) was found to be unrealistic for industrial implementation. Hence the simultaneous application of ultrasound and heat (thermoultrasonication) was examined. Thermo-ultrasonication proved to be more effective than either an ultrasonic or heat treatment with all viable M. luteus cells being eliminated within 4 min (100% amplitude at 72°C). Similarly, to eliminate E. coli and Lb. acidophilus from milk, only 2 min and 4 min thermo-ultrasonication was required, respectively. Bacillus cereus endospores remained resistant and after a 10 min thermo-ultrasonic treatment only 78.04% were eliminated. During this investigation both extensive surface (SEM) and internal (TEM) cell damage caused by ultrasonication were observed in E. coli, Lb. acidophilus and S. cerevisiae. Hence ultrasonication physically/mechanically damages these microbial cells causing cell death/injury. Microbial proteins and DNA released from cells into the environment after an ultrasonic treatment was measured and an increase in released microbial proteins and DNA was found to be indicative of a decrease in the number of viable cells, providing that the initial cell concentration was high enough. It was, however, not possible to correlate the concentration of released microbial proteins and DNA with the exact number of viable cells eliminated, rendering it an ineffective quality indicator for the industry. Ultrasonication had no statistically significant influence on the protein, fat and lactose content of both raw and pasteurised milk. The somatic cell count of raw and pasteurised milk was found to decrease after ultrasonication. Unlike with heating, activity of alkaline phosphatase and lactoperoxidase were not reduced by ultrasonication. Hence neither enzyme can be used to indicate a successful ultrasonic treatment of milk. This study has demonstrated that ultrasonication offers a viable alternative to pasteurisation as it is effective in eliminating microbes, and does not alter native milk components. However, to attain a more effective killing, thermo-ultrasonication is recommended for the treatment of milk to be used for the production of different dairy products.