Discipline: yogurt; Keywords: Listeria monocytogenes, Staphylococcus aureus, probiotics, antibacterial activity, antibiotic resistance, yogurt.

Food contamination is a problem that the food industry still struggles, especially contamination of the food product after production. There are two pathogens in particular are of concern as the chance of the occurrence of these two are relatively higher, namely Listeria monocytogenes and Staphylococcus aureus. A possible solution which can be considered is to introduce probiotics to the product which may have an inhibitory effect on the pathogens. To determine if this is viable the antibiotic resistance, antimicrobial activity as well as the effect on the pathogens in fermented milk was investigated at the University of Pretoria (see citation below). The Lactobacillus rhamnosus strain with probiotic characteristics was found to be naturally susceptible and resistant to 10 antibiotics and showed strong antibacterial activity against L. monocytogenes and S. aureus. The antibacterial activity of L. rhamnosus was further investigated in fermented milk samples inoculated with L. monocytogenes and S. aureus during refrigerated storage. Lactobacillus rhamnosus strongly inhibited the pathogens as they were completely eliminated by day 21 of refrigerated storage. The L. rhamnosus strain can therefore be utilized in the food products to combat gut infections and post-production contamination.

It was also found in vitro that Lactobacillus acidophilus can inhibit pathogens such as E. coli and Salmonella Typhimurium. Lactobacillus acidophilus strongly prevented proliferation and considerably reduced the counts of E. coli and Salmonella Typhimurium in fermented milk samples during refrigeration, even when the pathogen inoculum was at an unusual higher level of possible contamination.  Interestingly, even though yoghurt culture contributed to the suppression of E. coli in fermented milk, S. Typhimurium was not observed to be inhibited by yoghurt culture in the fermented milk during refrigeration. This underlined the need to add the L. acidophilus strain to fermented milk products to keep-in check possible foodborne pathogens and infections.  The L. acidophilus strain tested did cause a gradual pH decline in the fermented milk during refrigeration but the acid production may arguably not be strong enough to negatively increase product sourness. Hence the L. acidophilus with acceptable susceptibility to antibiotics and considerable antimicrobial activity should be tested for its suitability to process acceptable probiotic functional dairy beverages.   

The study in progress focused on characterising probiotic lactic acid bacteria from dairy sources for application in yoghurt. Lactobacillus acidophilus (strain D) and L. rhamnosus (strain V) were from dairy origin, whereas L. plantarum (strain VLL1), and L. pentosus (strain LIP) and L. acidophilus ATCC 4536 were from the American Type Culture Collection. The first phase of the study aimed at identifying and characterising the lactic acid bacteria. The identification was done using phenotypic methods which included gram staining, catalase test, oxidase test and carbon dioxide gas production from glucose. The isolates were gram-positive, catalase negative and oxidase negative, which proved them to be lactic acid bacteria. They were also able to produce carbon dioxide gas from glucose. The production of carbon dioxide gas classified the bacteria as heterofermentative, and ethanol and lactic acid were produced as byproducts of glucose metabolism.

The second phase included a safety assessment and determination of probiotic properties. The safety assessment included haemolytic activity, lipase production, gelatinase production and antibiotic resistance. The probiotic properties which were determined included acid and bile resistance, bile salt hydrolase activity and antimicrobial activity. The antimicrobial activity was against entero-invasive E. coli (EIEC), entero-haemorrhagic E. coli (EHEC), entero-aggregative E. coli (EAEC) and entero-toxigenic E. coli (ETEC), and also Candida albicans ATCC 10231 (C1), C. albicans 1051255 (C4), and C. albicans M0826 (C7). On exposure to the acidic environment, the lactic acid bacteria significantly reduced the viability of these isolates at pH 2. However, they were able to survive at pH 5 and pH 3 and  a 0.3% bile salt concentration by releasing the bile salt hydrolase enzymes. The lactic acid bacteria were able to inhibit both diarrheagenic E.coli and pathogenic Candida spp. On antibiotic susceptibility testing, the strains were resistant to vancomycin and kanamycin and susceptible to gentamicin, clindamycin, erythromycin, tetracycline, ampicillin, and chloramphenicol. The isolates from bovine origin were moderately sensitive to tetracycline. The isolates were gelatinase and lipase negative, but they showed α- hemolysis which is the partial hydrolysis of red blood cells.

The third phase was to determine the fate of the bovine and dairy probiotic strains when used with regular fermenting bacteria during fermentation as well as storage. Lactobacillus plantarum, L. rhamnosus, and L. acidophilus were selected for yoghurt processing. The yoghurt was stored for 28 days under refrigeration at 4 °C. The probiotic cells were selectively enumerated using MRS- bile agar. The cell population levels of the probiotics were not affected negatively up to day 28 of storage at 4 °C.

These results indicate the progress thus far. The next objective to be investigated early 2021 is to include the effective strains e.g. a strain of L. rhamnosus and a strain of Bifidobacterium longum or bifidum at levels of 109 cfu per mL into “ripe” commercial yoghurt that has been laboratory pasteurized and to determine the viability of the strains at refrigeration temperatures during shelf-life storage.