Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced

The Food Safety Modernization Act (FSMA) includes a time-to-harvest interval following the application of non-compliant water to pre-harvest produce to allow for microbial die-off. However, additional scientific evidence is needed to support this rule. This study aimed to determine the impact of weather on the die-off rate of E. coli and Salmonella on spinach and lettuce under field conditions. Standardized, replicated field trials were conducted in California, New York, and Spain over two years. Baby spinach and lettuce were grown and inoculated with a ∼104 CFU/mL cocktail of E. coli and attenuated Salmonella. Leaf samples were collected at 7 timepoints (0-96h) following inoculation; E. coli and Salmonella were enumerated. The associations of die-off with study design factors (location, produce type, and bacteria) and weather were assessed using log-linear and biphasic segmented log-linear regression. A segmented log-linear model best fit die-off on inoculated leaves in most cases, with a greater variation in the segment 1 die-off rate across trials [-0.46 (95% confidence interval (95% CI): -0.52, -0.41) to -6.99 (95% CI: -7.38, -6.59) log10 die-off/day] compared to the segment 2 die-off rate [0.28 (95% CI: -0.20, 0.77) to -1.00 (95% CI: -1.16, -0.85) log10 die-off/day]. A lower relative humidity was associated with a faster segment 1 die-off and an earlier breakpoint (the time when segment 1 die-off rate switches to the segment 2 rate). Relative humidity was also found to be associated with whether die-off would comply with FSMA's specified die-off rate of -0.5 log10 die-off/day. Importance The log-linear die-off rate proposed by FSMA is not always appropriate, as the die-off of foodborne bacterial pathogens and specified agricultural water quality indicator organisms appear to commonly follow a biphasic die-off pattern with an initial rapid decline followed by a period of tailing. While we observed substantial variation in the net culturable population levels of Salmonella and E. coli at each time point, die-off rate and FSMA compliance (i.e., at least a 2 log10 die-off over 4 days) appear to be impacted by produce type, bacteria, and weather; die-off on lettuce tended to be faster than that on spinach, die-off of E. coli tended to be faster than that of attenuated Salmonella, and die-off tended to become faster as relative humidity decreased. As such, the use of a single die-off rate for estimating time-to-harvest intervals across different weather conditions, produce types, and bacteria should be revised.

Aims: Starter lactic acid bacteria in Cheddar cheese face physico-chemical stresses during manufacture and ageing that alter their abilities to survive and to interact with other bacterial populations. Nonstarter bacteria are derived from milk handling, cheese equipment and human contact during manufacture. Probiotic bacteria are added to foods for human health benefits that also encounter physiological stresses and microbial competition that may mitigate their survival during ageing. We added probiotic Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium animalis subsp. lactis to full-fat, reduced-fat and low-fat Cheddar cheeses, aiming to study their survival over 270 days of ageing and to determine the role of the cheese matrix in their survival. Methods and Results: Probiotic and other lactic acid bacterial populations were enumerated by quantitative PCR using primers specifically targeting the different bacterial genera or species of interest. Bifidobacteria were initially added at 10 6 CFU g À1 cheese and survived variably in the different cheeses over the 270-day ageing process. Probiotic lactobacilli that were added at 10 7 CFU g À1 cheese and incident nonstarter lactobacilli (initially at 10 8 CFU g À1 cheese) increased by 10-to 100-fold over 270 days. Viable bacterial populations were differentiated using propidium monoazide followed by species-specific qPCR assays, which demonstrated that the starter and probiotic microbes survived over ageing, independent of cheese type. Addition of probiotic bacteria, at levels 100-fold below that of starter bacteria, modified starter and nonstarter bacterial levels. Conclusions: We demonstrated that starter lactococci, nonstarter lactobacilli and probiotic bacteria are capable of surviving throughout the cheesemaking and ageing process, indicating that delivery via hard cheeses is possible. Probiotic addition at lower levels may also alter starter and nonstarter bacterial survival. Significance and Impact of the Study: We applied qPCR to study multispecies survival and viability and distinctly enumerated bacterial species in commercial-scale Cheddar cheese manufacture.

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