Sporulating behavior influences the population dynamics of sporeformers during raw milk holding.

Thermoduric sporeformers are predominant in raw milk and form thermoduric endospores. Our previous research showed these sporeformers to cause biofouling of dairy contact surfaces and membranes, leading to cross contamination of final products. A critical factor influencing thermal inactivation is their form as vegetative cells or endospores. It would thus be of interest to understand the population dynamics of sporeformers in raw milk during storage at low temperatures. In our previous study, a low sporulating strain of Bacillus licheniformis showed an increasing trend in vegetative cell population during 72 h of storage at 10°C or higher, while maintaining spore population relatively static. In continuation, this study investigates population dynamics of a high sporulating strain of B. licheniformis (ATCC 14580). Raw milk samples were separately spiked with an average 4.0 log vegetative cells and 2.0 log spores/mL, and stored at 4°, 6°, 8°, 10°, and 12°C for 0, 24, 48, and 72 h. Standard protocols were followed for enumerating vegetative cells and spores. Three trials were conducted, in replicates of 3, and means were compared using ANOVA. Contour plots were developed using quadratic regression models to predict the population of vegetative cells and spores. In the vegetative cell spiking study, cell population remained mainly unchanged for 72 h up to 10°C, with more than 1.0 log change observed only at 12°C. As it was a sporulating strain, the spore spiking study validated a shift toward spores during storage at 4° to 8°C, with evidence of some parallel germination at 10°C or higher. The regression models helped us to develop contour plots across the holding temperature and duration. Based on the initial cell population of the spore former, such contour plots would help predict the presence of vegetative cells and spore populations in raw milk at a given time and temperature. This information will prove useful in optimizing raw milk holding conditions to keep the sporeformer population toward vegetative cells, which can subsequently be inactivated easily with thermal treatments such as pasteurization

Influence of sampling intervals on the standard plate counts of milk samples

High accuracy in sampling is critical in determining the microbiological quality and safety of milk and dairy products. The issue arises with the lack of a standard approach to lower the variability of sampling results of low count milk (LCM) and high-count milk (HCM). It may occur in extended and multi-processing conditions that are triggered by the heterogeneous and aggregating nature of bacteria. This study focuses on the influence of sampling intervals on the microbial count variability of LCM and HCM samples. For a pilot-scale study, 380 gallons of raw whole milk (\u3c4°C) were spiked with Bacillus licheniformis vegetativecells (4 log cfu/mL) and pasteurized at 72°C/16 s at a 0.5 gpm flow rate for 12 h. Samples (100mL each) were drawn aseptically at intervals 0, 4, 8, and 12 h from each of the raw (HCM) and pasteurized (LCM) sides of the unit operation using commercially available a port-septum- based sampling device with needle insertion. Two continuous composite samples (2-L each) were also collected during the 12 h run in sterilized sampling bags at 2.5mL/min rate and held at \u3c 4°C. Samples were analyzed at each interval for standard plate counts using tryptic soy agar and incubated at 32°C for 48 h. The study was done in duplicates and ANOVA was used to compare the microbial counts. Mean counts for the individual samples at 0, 4, 8, 12 h., and the composite samples at 12 h., were found to be 4.18 ± 0.10, 4.29 ± 0.12, 4.24 ± 0.12, 4.41 ± 0.12, and 4.38 ± 0.13 log cfu/mL for the HCM, and 1.52 ± 0.13, 1.57 ± 0.13, 1.66 ± 0.12, 1.69 ± 0.12, and 1.78 ± 0.10 log cfu/mL for the LCM, respectively. Results showed an increasing trend in the counts for LCM with no significant difference between the intervals (P \u3e 0.1). The microbial counts of samples from various intervals compared with composite samples were also not significantly different (P \u3e 0.1) for both LCM and HCM. Under the conditions of analysis, a single sample of 100 mL volumes at any interval during processing is as effective as composite sampling for estimating the microbial quality of milk under prolonged dairy processing conditions of 12 h

Sporulation behavior of Bacillus licheniformis strains influences their population dynamics during raw milk holding

To understand the role of strain variability, population dynamics of 2 strains of Bacillus licheniformis, ATCC 6634 and ATCC 14580, were modeled as a function of temperature (4.0–12.0°C) and duration (0–72 h) using regression analysis. Based on the initial spiking of vegetative cells (approximately 4.0 log cfu/mL) and spores (approximately 2.0 log cfu/mL), regression equations, elucidating B. licheniformis growth behavior during raw milk holding at low temperature, were obtained. Contour plots were developed to determine the time-temperature combinations, keeping the population changes to less than 1.0 log. In vegetative cell spiking study of B. licheniformis ATCC 6634 (S1), cell population changes remained below 1.0 log up to 72 h at 8°C. For B. licheniformis ATCC 14580 (S2), 1.0 log shift was not observed only after 80 h at 8°C, indicating higher multiplication potential of S1 as compared with S2. As S2 was a readily sporulating strain, the vegetative spiking study showed spore formation at different storage temperatures. Evidence of some parallel germination was observed for this strain at 8°C or higher, when raw milk samples were spiked with spores. The experimental values obtained for sporeformers and spore counts were validated with contour plot-generated values. Overall, for raw milk samples predominated by the low sporulating strain, the contour plots suggested holding at 8°C or below for up to 72 h. In the case of the readily sporulating strain (S2), raw milk could be held at 8°C for 80 h, where little or no sporulation is observed. Sporulation behavior, germination and multiplication ability, strain variability, and temperature and duration of holding raw milk influenced the population dynamics of Bacillus species. However, in the presence of equivalent numbers of both types of sporulating strains in raw milk, despite strain variability, holding milk at 8°C for not more than 72 h would keep any cell population changes below 1.0 log. In addition, under these storage conditions, the population would remain as vegetative cells that are likely to be inactivated by pasteurization. The contour plots, so generated, would help predict the population shifts and define optimum holding conditions for raw milk before further processing

Manufacturing low-spore-count skim milk powder by controlling raw milk holding conditions—A pilot-scale trial

Milk powder is one of the most traded dairy products globally, largely being used to manufacture commercially sterilized products. Skim milk powder is frequently contaminated with spores of Bacillus species and carried over from raw milk due to their ability to survive processing conditions. Our previous study proved that it is possible to use optimized raw milk holding conditions to keep their population low, which may help in making low spore count powder. This pilot scale trial was conducted to produce low spore count powder by controlling the raw milk holding conditions, to keep the spore populations low. Bacillus licheniformis being a predominant spore former in milk powders was used as an inoculant for the challenge study. Inoculated raw milk samples (batches of 1500 lbs each) were held at optimized storage conditions T1 (4°C for 24 h) and T2 (8°C for 72 h). The PMO based storage condi tions (10°C for 4 h followed by 7°C for 72 h) were kept as a control, before producing skim milk powder. Samples were drawn at different stages pre- and post-storage, pasteurization (73°C for 15 s), evaporation, and spray drying (outlet and inlet temperature of 200 and 95°C, respectively), and analyzed for sporeformers using standard culturing methods. Spore counts were done by heating the samples at 80°C for 12 min, before plating on Brain Heart Infusion agar. All samples were analyzed in the replicates of 3, and means were compared using ANOVA. Treatment T1 and T2 log spore counts (1.79 ± 0.03 and 1.82 ± 0.04, respectively), were significantly lower than control (2.59 ± 0.05), after raw milk storage step. Similarly, sporeformers log counts for T1 (3.84 ± 0.02) were significantly lower than T2 (4.07 ± 0.08) and control (4.13 ± 0.04). Skim milk powders prepared using optimized storage conditions T1 showed significantly (P \u3c 0.05) lower spore and sporeformer counts (0.58 ± 0.04 and 1.82 ± 0.05 log cfu/g), as compared with T2 (0.86 ± 0.16 and 1.90 ± 0.03) respectively, and control (1.03 ± 0.06 and 2.74 ± 0.03, respectively). This shows that skim milk powders with reduced counts can be prepared by just optimizing raw milk holding conditions

Relationship of follicle size and concentrations of estradiol among cows exhibiting or not exhibiting estrus during a fixed-time AI protocol

Cows exhibiting estrus near fixed-time artificial insemination (AI) had greater pregnancy success than cows not showing estrus. The objective of this study was to determine the relationship between follicle size and peak estradiol concentration between cows that did or did not exhibit estrus during a fixed-time AI protocol. Ovulation was synchronized in beef cows by applying the CO-Synch protocol [GnRH (100 mg) on day-9, prostaglandin F2a (PGF2α; 25 mg) on day-2, and a second injection of GnRH 48 h after PGF2α (day 0)] to both suckled (experiment 1) and nonsuckled (experiment 2) cows. Follicle size (day 0) and ovulation (day 2) was determined by ultrasonography. Blood samples were collected every 3 or 4 h beginning at the time of PGF2α injection (0 h). Estrus was detected by visual observation with the aid of estrus-detection patches, and cows that ovulated were classified as exhibited estrus (n = 46) or did not exhibit estrus (n = 63). In both suckled and nonsuckled cows, there was a positive relationship between all cows (P \u3c 0.05) and among those that exhibited estrus (P \u3c 0.05) between follicle size and peak estradiol concentration, but no linear relationship (P \u3e 0.50) between follicle size and peak estradiol concentration was observed among cows not exhibiting estrus. Cows that exhibited estrus had greater (P \u3c 0.01) peak estradiol concentrations than cows that did not exhibit estrus. Suckled cows exhibiting standing estrus had greater (P \u3c 0.001) preovulatory concentrations of estradiol beginning 6 h (replicate 1) or 4 h (replicate 2) after the injection of PGF2α on day-2 compared with cows not exhibiting standing estrus. Nonsuckled cows exhibiting standing estrus had greater (P \u3c 0.001) preovulatory concentrations of estradiol beginning at the injection of PGF2α on day-2 compared with cows not exhibiting standing estrus. Furthermore, cows that exhibited estrus had an increased (P \u3c 0.01) rate in the rise in concentrations of estradiol following the PGF2α to peak estradiol than cows not exhibiting estrus. In summary, follicle diameter had a positive relationship with peak concentrations of estradiol, but only among cows that exhibited standing estrus, and estradiol increased earlier in cows that exhibited estrus compared with cows that did not