Survey of naturally fermented raw milk in Bhutan for the prevalence of Bacillus cereus, Escherichia coli and Staphylococcus aureus : a thesis presented in partial fulfilment of the requirements for the degree of Master in Food Technology at Massey University, Manawatū, New Zealand

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Massey University
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In Bhutan, naturally fermented raw milk (NFRM) is an ethnic dairy product consumed as a nutritional and refreshing beverage. It is produced by spontaneously fermenting raw milk at ambient temperatures. Across the globe, raw milk and raw milk products are considered as high-risk foods that may contain human pathogens. However, no survey has been conducted to study the presence of pathogens in NFRM in Bhutan. Therefore, in the present survey, 114 NFRM samples from 19 milk outlets and stalls in Thimphu, Phuntsholing and along the Thimphu-Phuntsholing NH (NH) in Bhutan were screened for the presence of Escherichia coli, Bacillus cereus and Staphylococcus aureus using culture-independent (high throughput sequencing (HTS)) and culture-dependent methods (most probable number (MPN) and selective plating). The culture isolates were confirmed by the PCR using species-specific primers. HTS identified the Escherichia and Staphylococcus genera. The culturing methods found more than 90 % and 100 % of NFRM samples contaminated with E. coli and S. aureus respectively. 45 % of NFRM samples contained more than 3.04 log10 cfu/ml E. coli based on MPN estimates. 61 % of NFRM samples contained more than 4 log10 cfu/ml S. aureus and is an immediate food safety concern. B. cereus was suspected since HTS detected Bacillus anthracis which is closely related to B. cereus. Shigella and Vibrio genera, and Streptococcus agalactiae and Streptococcus parauberis were also identified by HTS. Ideally, fermented milk products including NFRM should be prepared from pasteurised milk and fermented using commercial starter cultures since pasteurisation is the most effective method to inactivate the vegetative pathogens and ensure the safety of the product. Hygienic practices and control measures during NFRM production should also be adopted to minimise bacterial contamination in NFRM in Bhutan.
Fermented milk, Microbiology, Bhutan, Raw milk, Milk contamination, Bacillus cereus, Escherichia coli, Staphylococcus aureus