Microbial diversity of naturally fermented milk produced by smallholder milk producers in South Africa
The microbial ecology of Sethemi is complex and determines its quality and safety. Raw milk and Sethemi (a naturally fermented milk from South Africa) samples were collected in the vicinity of Bloemfontein, South Africa, to determine the development and diversity of all relevant microorganisms. Simulated milk fermentations were executed in the laboratory to study environmental influences on the microbial development. A total of 116 yeasts were isolated and identified using conventional methods and sequence analysis of the D1/D2 domain. The isolates belonged to 12 different genera and 22 distinct species. Despite the high diversity during the initial stages of fermentation, only seven species proved to be dominant at all stages being present at high numbers in the final product. The predominant species were identified as C. albicans, Cryptococcus curvatus, Debaryomyces hansenii, Kluyveromyces marxianus, Clavispora lusitaniae, Saccharomyces cerevisiae and Yarrowia lipolytica. Aerobic mesophilic bacteria, lactobacilli-leuconostocs and lactococci being present at numbers in excess of 9 log10 CFU/ml, however, predominated during processing and in the final product. Sethemi was also produced in the laboratory in four different containers, namely clay pot, gourd, nickel jar and plastic jar, using the traditional methods of milk fermentation. The development of bacteria and yeasts was similar in all containers but differed in the time to reach maximum numbers. Optimum growth of LAB was obtained sooner in nickel containers. The highest yeast diversity was, however, obtained in the clay pot dominated by the yeasts Deb. hansenii, Cr. humicola and K. marxianus, which were commonly found in the raw and naturally fermented milks. Considering their industrial or health importance, four dominant yeast species, namely K. marxianus, S. cerevisiae, C. albicans, and Deb. hansenii were selected for environmental studies. S. cerevisiae showed the highest stimulatory effect on the growth of LAB and promised further study whereas the good growth of C. albicans, an opportunistic yeast pathogen, was alarming. The best Sethemi was produced at 25oC and must be consumed within two to four days. Fermentation at higher temperatures, however, resulted in early spoilage. The effect of residual levels of oxytetracycline and penicillin G on the growth of LAB and yeasts in the presence or absence of the four selected yeast species in naturally fermented milks was studied. The results showed that oxytetracycline and penicillin G at the lowest values significantly inhibited the growth of lactobacilli-leuconostocs and lactococci. In both antibiotic treatments, lactococci were more inhibited than lactobacillileuconostocs. In oxytetracycline-treated fermented milks, the addition of S. cerevisiae and D. hansenii significantly reduced the inhibition while the addition of C. albicans and K. marxianus contributed to the inhibition. In contrast, in penicillin-treated milk, all four yeast species added to the inhibition of the growth of LAB. The yeast growth was generally favored by the presence of both antibiotics and even out-competed the LAB in some instances indicating that it might pose a serious health problem amongst the immunocompromised if it is a potentially pathogenic one.