Donor human milk is pasteurized to prevent the potential risk of the transmission of pathogens to preterm infants. Currently, Holder pasteurization (human milk held at 62·5°C for 30 min) is used in most human milk banks, but has the disadvantage that it results in excessive inactivation of important bioactive components. Power-ultrasound (20–100 kHz) is an emerging technology for the preservation of foods and could be an alternative method for the treatment of human milk. The aim of this study was to investigate the effect of different ultrasound settings on the elimination of Escherichia coli and the retention of bile salt stimulated lipase (BSSL) activity. Ultrasonication with a constant power decreased Esch. coli viability exponentially over time until the processing temperature increased to sub-pasteurization level to between 51·4 and 58·5°C, then a log10 1·3 decrease was observed (P<0·05). BSSL activity decreased to 91% until a temperature of 51·4°C and then it decreased to 8% between 51·4 and 64·9°C. Ultrasonication with a constant energy and various power and exposure times showed the highest temperature (53·7°C) when treated with the longest exposure time and lowest ultrasound-power (276 s at 3·62 W) compared with 37·6°C for 39 s at 25·64 W. The findings predict that the viability of Esch. coli could be reduced by log10 5 with a minimal loss of activity of BSSL by applying 13·8 kJ of energy in 12 ml of human milk using high ultrasound power over a short exposure time to ensure that the temperature remains below the critical level for protein denaturation. Alternatively, the use of lower power settings such as the 26 W used in the present studies would require a cooling system to ensure the human milk BSSL was protected against temperature denaturation.

A multiplex PCR (mPCR) assay using previously known genetic markers of Shigella, Escherichia coli and Shiga-toxic Esch. coli was standardized. uidA gene was targeted for the common detection of Esch. coli and Shigella, whereas ipaH and stx1 genes were used as markers for the detection of Shigella and shiga-toxin producing strains, respectively. The standardized assays detected the target organism specifically and selectively. The mPCR developed by combining all the three reactions generated specific products. The inclusivity and exclusivity tests depicted the precise specificity of the mPCR assay. Results were interpreted on the basis of the pattern of amplicons generated: amplifications of the ipaH and uidA gene fragments indicated the presence of Shigella spp., amplification of uidA alone revealed the presence of Esch. coli and additional presence of verotoxin gene amplicon indicated verotoxinogenic nature of the strain. Specific patterns of bands were obtained when different strains of Esch. coli and Shigella spp. were subjected to this assay. The reactions, individually as well as in the mPCR, could detect approximately 1 cell per 20-μl PCR assay. The protocols were validated by analyzing the coded samples of full fat milk spiked with different pathogens. In naturally contaminated raw milk samples (n=100), Esch. coli were detected in all samples and verotoxinogenic Esch. coli in 15 samples. Shigella, however, was not detected in any of the samples. When DNA purified from the samples found positive for Shiga-toxic Esch. coli was directly used as template for the mPCR, the results showed agreement with the enrichment based detection. The mPCR assay, standardized in this study, may be used for rapid microbiological evaluation of milk samples. Further, the study emphasizes the need for better hygienic conditions in dairies.