Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk

High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6 s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log10 BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91 ± 0.05 log10 BA spores/mL of milk and the 1.4- μm membrane removed 4.50 ± 0.35 log10 BA spores/ mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log10 spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log10 BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-μm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization

Table_1_The quorum sensing peptide BlpC regulates the transcription of genes outside its associated gene cluster and impacts the growth of Streptococcus thermophilus.DOCX

Bacteriocin production in Streptococcus thermophilus is regulated by cell density-dependent signaling molecules, including BlpC, which regulates transcription from within the bacteriocin-like peptide (blp) gene cluster. In some strains, such as S. thermophilus ST106, this signaling system does not function properly, and BlpC must be supplied exogenously to induce bacteriocin production. In other strains, such as S. thermophilus B59671, bacteriocin (thermophilin 110 in strain B59671) production occurs naturally. Here, transcriptomic analyses were used to compare global gene expression within ST106 in the presence or absence of synthetic BlpC and within B59671 to determine if BlpC regulates the expression of genes outside the blp cluster. Real-time semi-quantitative PCR was used to find genes differentially expressed in the absence of chromosomal blpC in the B59671 background. Growth curve experiments and bacteriocin activity assays were performed with knockout mutants and BlpC supplementation to identify effects on growth and bacteriocin production. In addition to the genes involved in bacteriocin production, BlpC affected the expression of several transcription regulators outside the blp gene cluster, including a putative YtrA-subfamily transcriptional repressor. In strain B59671, BlpC not only regulated the expression of thermophilin 110 but also suppressed the production of another bacteriocin, thermophilin 13, and induced the same YtrA-subfamily transcriptional repressor identified in ST106. Additionally, it was shown that the broad-spectrum antimicrobial activity associated with strain B59671 was due to the production of thermophilin 110, while thermophilin 13 appears to be a redundant system for suppressing intraspecies growth. BlpC production or induction negatively affected the growth of strains B59671 and ST106, revealing selective pressure to not produce bacteriocins that may explain bacteriocin production phenotype differences between S. thermophilus strains. This study identifies additional genes regulated by BlpC and assists in defining conditions to optimize the production of bacteriocins for applications in agriculture or human and animal health.</p