Cow Milk Fermentation Trials With Streptococcus Infantarius Subsp. Infantarius and Its Effect on the Growth of Selected Pathogenic Bacteria

Abstract

Streptococcus infantarius subsp. infantarius (Sii) is a lactic acid bacterium predominant in most traditional African fermented dairy products such as suusac, fene and gariss. Most lactic acid bacteria (LAB) have antagonistic activity against food spoilage microorganisms and pathogenic groups such as Listeriamonocytogenes, Staphylococcus aureus, Clostridium species (spp) and Bacillus spp. The aim of this study was to identify the optimum fermentation conditions of Sii that leads to fast pH drop to inhibit common human pathogens. A factorial experimental design was used to conduct fermentation trials of local raw and pasteurized milk. Raw/pasteurized cow milk were divided into equal aliquots of 100 mL which were inoculated with separate starter cultures of Sii CJ 18 and Sii CCUG 43820T at the following inoculation rates; 0% (control), 5%, 10% and 15% v/v. Incubations were done at 20, 30, 37 and 45oC in parallel. pH and titratable acidity were measured at 0 h, 3 h, 6 h and 9 h. The antibacterial properties Sii strains against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella enterica subsp. enterica were done using disc diffusion and competition assays methods. Disc diffusion was done as follows;culture pathogens S. aureus ATCC 25923, E. coli ATCC 25922, Salmonella ATCC 722569 and L. monocytogenes ATCC 7644 were grown aerobically on TSA (Oxiod) at 37oC for 24 h and suspended in 0.85% normal sterile water. A lawn of each indicator strain was made by spreading cell suspension over the surface of Muller Hinton agar plates (Oxiod) with sterile cotton swabs and allowed to dry.

Sterile discs were soaked with Sii strains cell-free supernatants for 5 minutes, excess carefully removed, placed on the surface of Muller Hinton agar plates and allowed to dry for 10 minutes. After incubation at 37oC for 48h, the plates were observed for a zone of inhibition around the discs. Inhibition patterns of African Sii strains isolates CJ18 and CCUG 43820T against selected pathogenic bacteria were done by competition assay using local raw and pasteurized milk. Each type of cow milk were divided into equal aliquots of 100 mL, spiked with known CFU/mL of each pathogenic bacterium, inoculated with 15% v/v Sii CJ 18 and Sii CCUG 43820T starter cultures independently and incubated at 37oC. Appropriate double serial dilutions were plated in duplicate onto appropriate selective or semi-selective growth medium after every 3 h and enumerated. Enumeration of E. coliwas done by surface plating 0.1 ml of each sample on brilliance E. coli coliform agar (Oxiod).Salmonella was enumerated by pour plating onto Violet Red Bile Agar (Merck) and plates incubated at 37oC for 24 h. Staphylococcus aureus was enumerated by surface plating onto Baird Parker Agar media (Oxoid) and plates incubated at 37 oC for 24 h. L. monocytogenes was surface plated in Listeria semi-selective agar (Oxiod). Fermentation was faster in pasteurized milk than raw milk for both Sii CJ 18 and Sii CCUG 43820T. The target pH of below 4.5 was attained at 45oC, sixth hour at 10% inoculation rate by CCUG 43820T pH(4.36±0.00) while CJ 18 attained a pH of 4.55±0.00 at 45oC, 6th h, 15% inoculation rate. The highest percent lactic acid formed by CCUG 43820T was 1.29±0.02% at 45oC, after nine hours with 15%

inoculation, while CJ 18 attained 0.82±0.02% lactic acid under same conditions. In disc diffusion assay, the growth of Staphylococcus aureus and Listeria monocytogenes were inhibited. In competition assays, Growth of pathogens increased within 9 h of fermentation in control samples whereas growth in decreased after 3 h of fermentation in samples inoculated with Sii strains within 9 h. The growth was higher in raw milk than pasteurized milk. In conclusion, African dairy Sii isolates could be potentially used as African-specific native starter cultures that will result to the fast drop in pH to inhibit human pathogens resulting in an improved and safe traditional fermented product. Safety assessment such as resistance to antibiotics, acid tolerance, viability, safety, and organoleptic properties should be carried out.