John A. Renye

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, 18.6s); 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 log(10) 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 log(10) BA spores/mL of milk and the 1.4-μm membrane removed 4.50±0.35 log(10) 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 log(10) 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 log(10) 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.

Short communication: Characterization of microflora in Mexican Chihuahua cheese

This work was performed to identify the bacterial species present in 10 Chihuahua cheeses obtained from commercial producers in Mexico using 16S rRNA gene analysis. As expected, some of the agar media initially used for isolation were not very selective, supporting the growth of several unrelated bacterial species. Sequence analysis identified potential pathogens, including Escherichia coli and Staphylococcus aureus, in all raw milk samples and 2 pasteurized milk samples. Streptococcus thermophilus and Lactococcus lactis ssp. lactis were identified in 9 and 6 samples, respectively, and would serve as acidifying agents during cheese production. Lactobacilli were identified in all cheeses, with the most prevalent being Lactobacillus plantarum identified in 7 raw milk and 1 pasteurized milk cheeses. Leuconostoc mesenteroides and Streptococcus macedonicus were identified in 4 raw milk cheeses and both were present in all pasteurized milk samples, suggesting that they may play a role in the development of traditional Chihuahua cheese attributes.

BlpC-regulated bacteriocin production in Streptococcus thermophilus

Streptococcus thermophilus B59671 produces a bacteriocin with anti-pediococcal activity, but genes required for its production are not characterized. Genome sequencing of S. thermophilus has identified a genetic locus encoding a quorum sensing (QS) system that regulates production of class II bacteriocins. However, in strains possessing this gene cluster, production of bacteriocin like peptides (Blp) was only observed when excess pheromone was provided. PCR analysis revealed this strain possessed blpC, which encodes the 30-mer QS pheromone. To investigate if BlpC regulates bacteriocin production in S. thermophilus B59671, an integrative vector was used to replace blpC with a gene encoding for kanamycin resistance and the resulting mutant did not inhibit the growth of Pediococcus acidilactici. Constitutive expression of blpC from a shuttle vector restored the bacteriocin production, confirming the blp gene cluster is essential for bacteriocin activity in S. thermophilus B59671.

Characterization of the indigenous microflora in raw and pasteurized buffalo milk during storage at refrigeration temperature by high-throughput sequencing

The effect of refrigeration on bacterial communities within raw and pasteurized buffalo milk was studied using high-throughput sequencing. High-quality samples of raw buffalo milk were obtained from 3 dairy farms in the Guangxi province in southern China. Five liters of each milk sample were pasteurized (72°C; 15 s); and both raw and pasteurized milks were stored at refrigeration temperature (1-4°C) for various times with their microbial communities characterized using the Illumina Miseq platform (Novogene, Beijing, China). Results showed that both raw and pasteurized milks contained a diverse microbial population and that the populations changed over time during storage. In raw buffalo milk, Lactococcus and Streptococcus dominated the population within the first 24h; however, when stored for up to 72h the dominant bacteria were members of the Pseudomonas and Acinetobacter genera, totaling more than 60% of the community. In pasteurized buffalo milk, the microbial population shifted from a Lactococcus-dominated community (7d), to one containing more than 84% Paenibacillus by 21d of storage. To increase the shelf-life of buffalo milk and its products, raw milk needs to be refrigerated immediately after milking and throughout transport, and should be monitored for the presence of Paenibacillus. Results from this study suggest pasteurization should be performed within 24h of raw milk collection, when the number of psychrotrophic bacteria are low; however, as Paenibacillus spores are resistant to pasteurization, additional antimicrobial treatments may be required to extend shelf-life. The findings from this study are expected to aid in improving the quality and safety of raw and pasteurized buffalo milk.

Insertion of a heterologous gene construct into a non-functional ORF of the Streptococcus thermophilus chromosome

An integrative vector was constructed for inserting heterologous genes within a non-functional open reading frame (ORF) on the chromosome of Streptococcus thermophilus. The vector, pINTRS, contained a temperature sensitive origin of replication and an erythromycin resistance gene for initial selection in S. thermophilus. The region of the vector containing unique cloning sites, for insertion of recombinant genes, was flanked by homologous DNA sequences corresponding to a pseudogene in S. thermophilus to facilitate chromosomal integration. The gene encoding green fluorescent protein, regulated by a plasmid borne hsp promoter of S. thermophilus, was cloned into pINTRS to demonstrate proper functioning of the vector.

Short communication : Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide

An 8-AA (8mer) fragment (PFPEVFGK) of a known antihypertensive peptide derived from bovine αS1-casein (C12 antihypertensive peptide) was synthesized by microwave-assisted solid-phase peptide synthesis and purified by reverse phase HPLC. Its ability to inhibit angiotensin-converting enzyme (ACE) was assessed and compared with that of the parent 12mer peptide (FFVAPFPEVFGK) to determine the effect of truncating the sequence on overall hypotensive activity. The activity of the truncated 8mer peptide was found to be almost 1.5 times less active than that of the 12mer, with ACE-inhibiting IC50 (half-maximal inhibitory concentration) values of 108 and 69μM, for the 8mer and 12mer, respectively. Although the 8mer peptide is less active than the original 12mer peptide, its overall activity is comparable to activities reported for other small proteins that elicit physiological responses within humans. These results suggest that microbial degradation of the 12mer peptide would not result in a complete loss of antihypertensive activity if used to supplement fermented foods and that the stable 8mer peptide could have potential as a blood pressure-lowering agent for use in functional foods.

The effects of microfluidization on the physical, microbial, chemical, and coagulation properties of milk

This work examines the use of mild heat treatments in conjunction with 2-pass microfluidization to generate cheese milk for potential use in soft cheeses, such as Queso Fresco. Raw, thermized, and high temperature, short time pasteurized milk samples, standardized to the 3% (wt/wt) fat content used in cheesemaking, were processed at 4 inlet temperature and pressure conditions: 42°C/75 MPa, 42°C/125 MPa, 54°C/125 MPa, and 54°C/170 MPa. Processing-induced changes in the physical, chemical, and microbial properties resulting from the intense pressure, shear, and cavitation that milk experiences as it is microfluidized were compared with nonmicrofluidized controls. A pressure-dependent increase in exit temperature was observed for all microfluidized samples, with inactivation of alkaline phosphatase in raw and thermized samples at 125 and 170 MPa. Microfluidization of all samples under the 4 inlet temperature and processing pressure conditions resulted in a stable emulsion of fat droplets ranging from 0.390 to 0.501 μm, compared with 7.921 (control) and 4.127 (homogenized control) μm. Confocal imaging showed coalescence of scattered fat agglomerates 1 to 3 μm in size during the first 24 h. We found no changes in fat, lactose, ash content or pH, indicating the major components of milk remained unaffected by microfluidization. However, the apparent protein content was reduced from 3.1 to 2.2%, likely a result of near infrared spectroscopy improperly identifying the micellar fragments embedded into the fat droplets. Microbiology results indicated a decrease in mesophilic aerobic and psychrophilic milk microflora with increasing temperature and pressure, suggesting that microfluidization may eliminate bacteria. The viscosities of milk samples were similar but tended to be higher after treatment at 54°C and 125 or 170 MPa. These samples exhibited the longest coagulation times and the weakest gel firmness, indicating that formation of the casein matrix, a critical step in the production of cheese, was affected. Low temperature and pressure (42°C/75 MPa) exhibited similar coagulation properties to controls. The results suggest that microfluidization at lower pressures may be used to manufacture high-moisture cheese with altered texture whereas higher pressures may result in novel dairy ingredients.

Characterization of starter-free Queso Fresco made with sodium-potassium salt blends over 12 weeks of 4°C storage

Development of reduced-sodium cheese to meet the demands of consumers concerned about sodium levels in their diet is challenging when a high-moisture, higher pH, fresh cheese, such as Queso Fresco (QF), depends on its NaCl salt content to obtain its signature flavor and quality traits. This study evaluated the effects of different Na-K salt blends on the compositional, sensorial, microbial, functional, and rheological properties of QF stored for up to 12 wk at 4°C. Queso Fresco curd from each vat was divided into 6 portions and salted with different blends of NaCl-KCl (Na-K, %): 0.75-0.75, 1.0-0.5, 1.0-1.0, 1.0-1.3, 1.0-1.5, and 2.0-0 (control). Within this narrow salt range (1.5 to 2.5% total salt), the moisture, protein, fat, and lactose levels; water activity; pH; and the textural and rheological properties were not affected by salt treatment or aging. The total salt, sodium, potassium, and ash contents reflected the different Na-K ratios added to the QF. Total aerobic microbial count, overall proteolysis, the release of casein phosphopeptides, and the level of volatile compounds were affected by aging but not by the salt treatment. Only the 1.0-1.3 and 1.0-1.5 Na-K cheeses had sensory saltiness scores similar to that of the 2.0-0 Na-K control QF. Loss of free serum from the cheese matrix increased steadily over the 12 wk, with higher losses found in QF containing 1.5% total salt compared with the higher Na-K blends. In conclusion, KCl substitution is a viable means for reduction of sodium in QF resulting in only minor differences in the quality traits, and levels of 1.0-1.3 and 1.0-1.5 Na-K are recommended to match the saltiness intensity of the 2.0-0 Na-K control. The findings from this study will aid cheese producers in creating reduced-sodium QF for health-conscious consumers.