Incubation Of Milk Research Articles

Molecular Screening for Digital Dermatitis-Associated Treponemes in Bovine Ischaemic Teat Necrosis Lesions and Milk in Dairy Cattle

Bovine ischaemic teat necrosis (ITN) is a disease affecting the skin of the teats of dairy cows with an unknown aetiopathogenesis. Digital dermatitis (DD)-associated treponemes have previously been suggested as a potential aetiological agent in ITN, although the sample size was small. The current study, using established PCR techniques, aimed to examine the association with the presence of DD-associated treponemes in a large number of ITN samples from a wider geographical area, and surveyed the potential of milk as an infection reservoir. From 95 ITN lesions, 35.8% (n = 34) were positive for at least one DD-associated treponeme compared with only 5.6% (n = 1) of 18 non-lesioned teats from cows with ITN lesions on a different teat using a nested PCR approach. All 10 age- and production-matched control cows were negative for DD-associated treponemes via PCR. No DD-associated treponemes could be detected from foremilk of cows with (n = 19) and without (n = 31) a DD lesion on the hind feet. DD-associated treponemes could be detected via PCR after incubation in milk for up to 2 h. Therefore, milk does not appear to be a competent reservoir for transmission of DD-associated treponemes. Moreover, in the current study DD-associated treponemes were only detected in a subset of ITN samples, so it is unlikely these opportunistic skin-associated pathogens are the major or sole agent of ITN.

Control of Listeria monocytogenes in milk by using phage cocktail

Biocontrol applications such as using phages against the contamination of Listeria monocytogenes are promising trends in terms of reducing the use of chemical additives in food industry. The aim of this study was to determine the effectiveness of Listex P100 phage (phage P100) on different Listeria monocytogenes strains (PL2, PL3, PL9 and PL10) in pasteurized milk and broth. Survival data of L. monocytogenes were successfully described by Weibull model. Time parameter of the Weibull model was used to evaluate the phage-resistances of L. monocytogenes strains. The reduction of L. monocytogenes was greater in broth than in milk regardless of the temperature level and it was significantly higher at 30 °C than at 4 °C in both media. The reductions of L. monocytogenes strains by the phage treatment were between 2.7 to 3.4 log10 units at 30 °C and 1.4 to 2.1 log10 units at 4 °C after 4 days of incubation in broth whereas 1.9 to 2.9 log10 units and 1.0 to1.6 log10 units were observed after 4 days of incubation in milk at 30 °C and 4 °C, respectively. It was found that L. monocytogenes PL2 is the most phage-resistant strain in broth at 30 °C and at 4 °C, and in milk at 30 °C, while L. monocytogenes PL9 is the most phage-resistant L. monocytogenes strain in milk at 4 °C. This study demonstrated P100 phage could be used to control L. monocytogenes counts in milk.

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S–23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated.The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6.Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers’ health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.

Impedance microbiology to speed up the screening of lactic acid bacteria exopolysaccharide production

Bacterial production of exopolysaccharides (EPS) is of increasing interest near food manufacturers, biotechnology industries and nutritionists because of their different roles. Several analytical methods are available for recovery, quantification and characterization of EPS from lactic acid bacteria (LAB) in food. However, direct screening method for production of EPS is still based on the visual observation of filamentous texture of the colonies developed on supplemented solid growth media. To overcome weaknesses of many currently used screening methods, we propose adopting impedance microbiology to evaluate the EPS production from LAB in milk. In this work we have proven that the peculiar shape of capacitance curve of Lactobacillus delbrueckii subsp. bulgaricus 2214, measured in milk by means of a BacTrac 4300® system, is due to production of EPS. Besides the pH measurement, the amounts of EPS evaluated after 0, 8, 13 and 55 h of incubation in milk, were in agreement with the evaluation of gene expression and confirmed by the observations by confocal laser scanning microscopy and by transmission electron microscopy.With the aim to verify the applicability of the proposed method, the drop entity of the capacitance curve (ΔE%) of 22 EPS-producing (EPS+) LAB strains and one negative (EPS-) control was evaluated both in broth medium and in milk. The positive ΔE% value found for all of the strains cultivated in the clear broth medium allowed to confirm the EPS production, simply observing a strain-dependent amount of EPS on surface of the measurement electrodes of the device. When the same EPS+ strains were cultivated in milk, the obtained ΔE% values showed that only a few of them were able to produce EPS in this environment, supporting their diversified ability to utilize lactose for this purpose.Results obtained by this multidisciplinary study demonstrate that impedance microbiology represents a suitable method to overcome the limits of the most commonly used methods to screen LAB for EPS production in milk. Moreover, these results also open a door to the application to other food and beverages, in which the EPS produced in situ could be of great interest for food industry.

Effects of Temperature Abuse on the Growth and Staphylococcal Enterotoxin A Gene (sea) Expression of Staphylococcus aureus in Milk.

The aim of this study was to determine the effects of different temperatures and storage time on Staphylococcus aureus growth, sea gene expression, and synthesis of staphylococcal enterotoxin A (SEA) in the pasteurized and UHT-pasteurized milk. Pasteurized and UHT-pasteurized milk were inoculated with 3.98 log10 CFU/mL of S. aureus (ATCC 13565). Inoculated milk samples were stored at 8°C, 15°C, and 22°C for 24, 48, and 72 h, respectively. SEA synthesis was detected with a fully automated miniVIDAS instrument using the Enzyme-Linked Fluorescent Assay (ELFA) technology. The patterns of gene regulation were detected by quantitative reverse transcriptase PCR. The 2-ΔΔCT method has been used as a relative quantification strategy for gene expression responses data analysis. The results indicated that growth rate, sea gene expression, and SEA synthesis were influenced by type of milk, storage time, and temperature. Incubation of milk at different temperatures (15°C and 22°C) and times was used to simulate inadequate transport and storage conditions. Storage of pasteurized milk at 22°C for 24 h significantly upregulated the expression of sea gene compared with milk stored at 8°C, which coincides with the achieved S. aureus number of 105 CFU/mL and detected amount of SEA. In addition, storage of UHT-pasteurized milk at 22°C for 24 h and at 15°C for 48 h significantly upregulated the sea gene expression compared with milk stored at 8°C, which coincides with the detected amount of SEA and the dynamics of S. aureus number change. It can, therefore, be concluded that implementing good hygiene practices to avoid pre- and post-heat treatment milk contamination and maintaining the cold chain at temperature <8°C throughout the entire dairy production chain are of paramount importance to decrease the risk of staphylococcal food poisoning.

Growth inhibition of foodborne pathogens in camel milk: Staphylococcus aureus, Listeria monocytogenes, Salmonella spp. and E. coli O157:H7

The growth behaviour of foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, E. coli O157:H7 and Salmonella spp.) was investigated in pasteurised camel milk and compared with pasteurised bovine milk at different incubation temperatures. This study also aimed to compare the growth patterns of these four foodborne pathogens in pasteurised and raw camel milk. Pasteurised or raw camel milk and pasteurised bovine milk were separately inoculated with a cocktail of three strains of each foodborne pathogen. The inoculated milk samples were incubated at 10, 25, and 37°C. The total bacterial count (TBC) in raw milk and the total thermoduric bacteria count (TDB) in pasteurised milk samples were monitored. Greater growth inhibition rates of four pathogens were obtained for the pasteurised camel milk compared to the pasteurised bovine milk. Raw and pasteurised camel milk exerted bacteriostatic effect against all tested pathogens, particularly for the first 8 h of incubation in milk at the different temperatures. Pasteurised camel milk exerted an inhibitory activity that was equivalent to that of raw camel milk.

Cellular and soluble components decrease the viable pathogen counts in milk from dairy cows with subclinical mastitis.

The present study was undertaken to clarify the factors that reduce the viable pathogen count in milk collected from the udders of subclinical mastitic cows during preservation. Milk was centrifuged to divide somatic cells (cellular components, precipitates) and antimicrobial peptides (soluble components, supernatants without fat layer); each fraction was cultured with bacteria, and the number of viable bacteria was assessed prior to and after culture. In 28.8% of milk samples, we noted no viable bacteria immediately after collection; this value increased significantly after a 5-hr incubation of milk with cellular components but not with soluble components (48.1 and 28.8%, respectively). After culture with cellular components, the numbers of bacteria (excluding Staphylococcus aureus and Streptococcus uberis) and yeast decreased dramatically, although the differences were not statistically significant. After cultivation with soluble components, only yeasts showed a tendency toward decreased mean viability, whereas the mean bacterial counts of S. uberis and T. pyogenes tended to increase after 5-hr preservation with soluble components. These results suggest that most pathogens in high somatic cell count (SCC) milk decreased during preservation at 15 to 25°C, due to both the cellular components and antimicrobial components in the milk. Particularly, the cellular components more potently reduced bacterial counts during preservation.

Lactobacillus caseiLow-Temperature, Dairy-Associated Proteome Promotes Persistence in the Mammalian Digestive Tract

We found that incubation of probiotic Lactobacillus casei BL23 in milk at 4 °C prior to ingestion increased its survival in the mammalian digestive tract. To investigate the specific molecular adaptations of L. casei to milk, we used tandem mass spectrometry to compare proteins produced by L. casei BL23 at 4 °C in milk to those in exponential and stationary phase cells in laboratory culture medium at either 37 or 4 °C. These comparisons revealed a core of expressed L. casei proteins as well as proteins produced in either a growth-phase or temperature-specific manner. In total, 205 L. casei proteins were uniquely expressed or detected in higher abundance specifically as a result of incubation in milk and included an over-representation of proteins for cell surface modification, fatty acid metabolism, amino acid transport and metabolism, and inorganic ion transport. Genes for DltD (d-alanine transfer protein), FabH (3-oxoacyl-ACP synthase), RecA (recombinase A), and Sod (superoxide dismutase) were targeted for inactivation. The competitive fitness of the mutants was altered in the mouse intestine compared with wild-type cells. These results show that the food matrix can have a profound influence on dietary (probiotic) bacteria and their functional significance in the mammalian gut.

Influence of microbial transglutaminase cross-linking on gelation kinetics and texture of acid gels made from whole goats and cows milk

The influence of microbial transglutaminase (mTGase) in modifying the functionality of proteins in cows milk has been extensively studied but to a lesser degree for goats milk. On a comparative basis, the effect of cross-linking by mTGase on the physical properties of chemically acidified UHT goats milk and UHT cows milk has been performed. Transglutaminase treatment (3 U mTGase/g protein) of cows and goats milk was conducted at 40 °C for 60 min and terminated by heat treatment (85 °C/10 min) for enzyme inactivation. Gelation was induced by acidification with 40 g.L−1d-glucono-δ-lactone. Monitoring gel formation by oscillatory rheometry revealed that the incubation of milk with mTGase accelerated gelation, which occurred at a higher pH for cows milk in comparison to goats milk. Electrophoretic analysis revealed that under the same conditions, nearly 31% of the cows milk proteins were cross-linked by the action of mTGase compared to only 9.6% of the goats milk proteins. This resulted in increased gel stiffness and reduced syneresis of the acid gels of both systems, but the extent of the effect depended on the milk source. In general, even with the mTGase treatment, goats milk gels were much weaker than cows milk gels. Despite this fact, the information gained in this study indicates that mTGase might be successfully applied in the production of acidified goat milk products.

Bacteriophage performance against Staphylococcus aureus in milk is improved by high hydrostatic pressure treatments

The combined effect of bacteriophages, vB_SauS-phi-IPLA35 (phiIPLA35) and vB_SauS-phi-IPLA88 (phiIPLA88), and high hydrostatic pressure (HHP) on Staphylococcus aureus Sa9 was evaluated in pasteurized whole milk under a simulated cold chain break, which was simulated by incubation of milk at 25°C for 48h. Four-hundred MPa was found to be the most suitable pressure to be used in combination with these phages. Two different levels of staphylococcal initial contamination (1×104 and 1×106CFU/mL) were tested. A synergistic effect between HHP and phages was observed in both cases. Compared to each single treatment, the combined treatment was able to reduce the initial S. aureus contamination below the detection limit (<10CFU/mL). Bacteriophage performance in pressurize milk against S. aureus enabled milder hydrostatic pressure treatments, therefore phages can be regarded as a valuable hurdle on minimally processed food.

Safety aspects, genetic diversity and technological characterisation of wild-type Streptococcus thermophilus strains isolated from north Italian traditional cheeses

Antibiotic susceptibility, antimicrobial activity, genotypic and technological properties of 52 Streptococcus thermophilus isolates, collected from four north Italian traditional cheeses, was investigated. RAPD-PCR, was used to study genetic variability and distinguish closely related strains; the results showed a high degree of heterogeneity among isolates. With regard to technological properties, after 6 h of incubation in milk 25% of the streptococcal strains could be defined as fast acid producers, while after 24 h the majority of isolates (79%) displayed only weak acidification activity. Reduction activity was generally low; in fact, none of these S. thermophilus strains showed a Eh < −102 mV). All the studied S. thermophilus were susceptible to ciprofloxacin, levofloxacin, penicillin G, ampicillin, mupirocin, nitrofurantoin, quinupristin/dalfopristin and rifampicin. Nine isolates were classified as resistant to tetracycline, 6 to streptomycin, 5 to oxacillin, 3 to erytromycin, 3 to vancomycin and only one to chloramphenicol. PCR-based detection did not identify any of the common genetic determinants for vancomycin (vanA, vanB, vanC1, vanC2, vanC3, vanD, vanE, vanG) or erythromycin (ermB and ermC). The genetic basis of the tetracycline resistance phenotype in these strains was linked to tetS-tetL genes (8 isolates) or the tetM gene (1 isolate), and the integrase element int of the Tn916/Tn1545 family of transposons was negative. Four strains were able to produce antimicrobial compounds against Clostridium tyrobutyricum. The study provides new evidence of the resistance of S. thermophilus to antimicrobial agents, confirming the importance of including an accurate safety assessment of phenotypic/biotechnological data when carrying out strain selection for dairy applications.

HPLC profile and dynamic surface properties of the proteose–peptone fraction from bovine milk and from whey protein concentrate

Extraction of proteose–peptones (PPs) was carried out from fresh milk, from milk after prolonged incubation (12 days at 37 °C) and from two different whey protein concentrates (WPCs). The high performance liquid chromatography profiles of these extracts were compared. PPs eluted as three chromatographic peaks, which increased during milk incubation. The PP extracts from WPCs showed a number of peaks that are attributable to small peptides belonging to the proteose–peptone fraction and probably derive from proteolysis during storage of WPC. The dynamic properties at the air–water interface of the PP extracts were investigated by the drop-volume method. The PPs extracted from fresh milk showed the lowest values and a more rapid reduction in surface tension. PPs from WPCs were found to be effective as surfactants, even though a less marked reduction in surface tension compared with PPs from milk samples was shown.

Patterns of survival and volatile metabolites of selected Lactobacillus strains during long-term incubation in milk

The focus of this study was to monitor the survival of populations and the volatile compound profiles of selected Lactobacillus strains during long-term incubation in milk. The enumeration of cells was determined by both the Direct Epifluorescent Filter Technique using carboxyfluorescein diacetate (CFDA) staining and the plate method. Volatile compounds were analysed by the gas-chromatography technique. All strains exhibited good survival in cultured milks, but Lactobacillus crispatus L800 was the only strain with comparable growth and viability in milk, assessed by plate and epifluorescence methods. The significant differences in cell numbers between plate and microscopic counts were obtained for L. acidophilus strains. The investigated strains exhibited different metabolic profiles. Depending on the strain used, 3 to 8 compounds were produced. The strains produced significantly higher concentrations of acetic acid, compared to other volatiles. Lactobacillus strains differed from one another in number and contents of the volatile compounds.

Growth and activity of Bulgarian yogurt starter culture in iron-fortified milk

Bulgarian yogurts were manufactured and fortified with 8, 15 and 27 mg of iron kg(-1) of yogurt. The growth and acidifying activity of the starter culture bacteria Streptococcus thermophilus 13a and Lactobacillus delbrueckii subsp. bulgaricus 2-11 were monitored during milk fermentation and over 15 days of yogurt storage at 4 degrees C. Fortifying milk with iron did not affect significantly the growth of the starter culture during manufacture and storage of yogurt. Counts of yogurt bacteria at the end of fermentation of iron-fortified milks were between 2.1 x 10(10) and 4.6 x 10(10) CFU ml(-1), which were not significantly different from numbers in unfortified yogurts. In all batches of yogurt, the viable cell counts of S. thermophilus 13a were approximately three times higher than those of L. delbrueckii subsp. bulgaricus 2-11. Greater decrease in viable cell count over 15 days of storage was observed for S. thermophilus 13a compared to L. delbrueckii subsp. bulgaricus 2-11. Intensive accumulation of lactic acid was observed during incubation of milk and all batches reached pH 4.5 +/- 0.1 after 3.0 h. At the end of fermentation process, lactic acid concentrations in iron-fortified yogurts were between 6.9 +/- 0.4 and 7.3 +/- 0.5 g l(-1). The acidifying activity of starter culture bacteria in the control and iron-fortified milks was similar. There was no increase in oxidized, metallic and bitter off-flavors in iron-fortified yogurts compared to the control. Iron-fortified yogurts did not differ significantly in their sensorial, chemical and microbiological characteristics with unfortified yogurt, suggesting that yogurt is a suitable vehicle for iron fortification and that the ferrous lactate is an appropriate iron source for yogurt fortification.

Synthesis of Conjugated Linoleic Acid by Human-Derived Bifidobacterium breve LMC 017: Utilization as a Functional Starter Culture for Milk Fermentation

This study was performed to discover bifidobacteria isolated from human intestines that optimally convert linoleic acid (LA) to conjugated linoleic acid (CLA) and to optimize the culture conditions of milk fermentation. One hundred and fifty neonatal bifidobacteria were screened for CLA-producing ability, and Bifidobacterium breve LMC 017 was selected as it showed about 90% conversion of free LA in MRS broth. The selected strain showed resistance at 0.5% LA in microaerophillic conditions. When monolinolein (LA 90%) was used as a substrate for CLA production, the conversion rate was lower compared to free LA, but the growth rate was unaffected during the milk fermentation. There was no significant difference in CLA production between aerobic and anaerobic conditions, and little decline in CLA was shown after the maximal CLA level had been reached. CLA production increased by 80% with 24 h of incubation in milk containing additional skim milk (5%), where the proteins may have facilitated the production of CLA by enhancing the interaction of substrate with the bacteria. CLA production did not decline after 9 h of fermentation and an additional 12 weeks of storage with other commercial starters. This demonstrates the possibility of using this strain as a costarter in the production of CLA-enriched yogurt.

Evaluation of Aroma Generation of Lactococcus lactis with an Electronic Nose and Sensory Analysis

There is an increased interest in exploring the potential of new Lactococcus lactis strains isolated from different natural ecosystems for the production of aroma compounds. Thus, the objective of this study was to screen the aroma generation of Lactococcus lactis strains isolated from different sources by an electronic nose and sensory evaluation for their potential use in starter cheese cultures. Twenty-three strains of Lactococcus lactis were isolated from dairy sources such as artisanal raw-milk cheeses, nondairy sources, and commercial starter cultures (industrial). All the strains were assessed for their ability to produce aromas by an electronic nose and sensory analysis after their incubation in milk. Some phenotypic characteristics of technological importance such as lactose fermentation, proteolytic activity, and citrate utilization were also evaluated. Lactococcus lactis strains showed clear phenotypic differences related to their isolation source. Strains isolated from raw-milk dairy products or commercial starter cultures presented faster lactose fermentation and proteolytic activity than those presented by strains isolated from nondairy sources. Additionally, strains isolated from dairy and nondairy sources presented better citrate utilization than strains isolated from commercial dairy starters. On the other hand, there was not a clear relationship between the source of isolation and the ability of lactococci strains to produce aroma. Principal components analysis of electronic nose data revealed 4 distinctive groups based on aroma profiles. Additionally, odor intensity scores (yogurt-like and Fresco cheese-like) for these 4 groups revealed the nature of their differences. In general, strains from dairy products presented intense yogurt-like and Fresco cheese-like aromas, with the latter being the most intense for one specific strain. On the other hand, the majority of wild strains from nondairy sources presented a stronger yogurt-like aroma, whereas industrial strains presented low intensity for both aroma descriptors. Additionally, an aroma potentiation effect was observed when mixtures of 2 lactococci strains isolated from different sources acted together.

Inhibition of Bacillus cereus in Milk Fermented with Kefir Grains

The effects of kefir-fermented milk were tested against a toxigenic strain of Bacillus cereus. The incubation of milk with B. cereus spores plus 5% kefir grains prevented spore germination and growth of vegetative forms. In contrast, when 1% kefir grains was used, no effects were observed. The presence of metabolically active kefir grains diminished titers of nonhemolytic enterotoxin A, as assessed by enzyme-linked immunosorbent assay. During fermentation, kefir microorganisms produce extra-cellular metabolites such as organic acids, which could play a role in the inhibition of spore germination and growth of B. cereus, although the effect of other factors cannot be ruled out. Results of the present study show that kefir-fermented milk is able to antagonize key mechanisms involved in the growth of B. cereus as well as interfere with the biological activity of this microorganism.

Breaking strength and elasticity of synthetic absorbable suture materials incubated in phosphate-buffered saline solution, milk, and milk contaminated with Streptococcus agalactiae

To determine in vitro effects of PBSS, milk, and bacteria-contaminated milk (BCM; contaminated by Streptococcus agalactiae) on properties of 3 synthetic absorbable suture materials. 3 types of synthetic absorbable suture materials (poliglecaprone 25, polyglycolic acid, and polydioxanone). Suture materials were tested to determine breaking strength and elasticity before (day 0) and after incubation in 3 media (PBSS, milk, and BCM) for 7, 14, and 21 days. A loop of suture material was elongated at a rate of 60 mm/min until it reached the breaking point. Tensile properties were statistically analyzed among media and incubation times. Incubation in milk and BCM significantly decreased breaking strength and elasticity of poliglecaprone 25, compared with results for incubation in PBSS. Incubation in BCM significantly decreased tensile properties of polyglycolic acid suture, compared with results for incubation in PBSS and milk. After incubation for 21 days, tensile properties of polydioxanone did not differ significantly among the media but were significantly decreased from values on day 0. On the basis of this study, poliglecaprone 25 is an inappropriate suture material for use in teat surgery. Polyglycolic acid suture should be avoided in teats of cattle with mastitis. Of the suture materials tested, polydioxanone was best suited for use in teat surgery, as determined on the basis of material testing after incubation in milk, even when the milk was contaminated with bacteria.

Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk

Neutrophils are the first line of defense in a mammary gland infection. However, the process of neutrophil transmigration across a membrane and ingestion of fat and/or casein when incubated in milk have been shown to inhibit bacterial phagocytosis and oxidative burst functions. Recently, a killing mechanism has been described whereby stimulated neutrophils release nuclear and granule material in fibrous webs that physically trap and kill bacteria. We demonstrate that these neutrophil extracellular traps are also produced by bovine blood neutrophils stimulated with PMA/ionomycin. Importantly, neutrophil extracellular traps can be formed when neutrophils have been incubated for up to 6 h in milk prior to stimulation. This contrasts milk's rapid inhibition of bacterial phagocytosis and oxidative burst functions in the neutrophil. Furthermore, stimulation of neutrophils with bacteria common to mammary gland infections leads to neutrophil extracellular traps being formed in milk. Some bacteria tested stimulated enhanced formation of neutrophil extracellular traps in milk compared to culture media. Therefore, being unaffected by incubation in milk may indicate an important role for neutrophil extracellular traps in defense against mastitis.

Biocontrol of Psychrotrophic Enterotoxigenic Bacillus cereus in a Nonfat Hard Cheese by an Enterococcal Strain–Producing Enterocin AS-48

Bacillus cereus is a food poisoning bacterium of great concern, especially in milk products. In this study, we describe the efficient control of the psychrotrophic and toxigenic strain B. cereus LWL1 in milk and in a nonfat hard cow's cheese by the enterocin AS-48 producer strain Enterococcus faecalis A-48-32 (Bac+). No viable B. cereus cells were detected after 72 h incubation in milk coinoculated with the AS-48–producing strain and B. cereus. Diarrheic toxin production was also markedly inhibited by the Bac+ strain to eightfold lower levels compared with control cultures of B. cereus. In cheeses manufactured by inoculation with a commercial starter (about 6.8 log CFU/ml) and B. cereus (about 4 log CFU/ml), the latter reached 6.27 log CFU/g after 5 days of maturation, and approximately 8 log CFU/g after 15 days. However, in cheeses made from milk inoculated with the starter along with a mixture of E. faecalis–B. cereus (2/1 ratio), counts of B. cereus decreased by approximately 1.0, 2.0, 4.32, and 5.6 log units with respect to control cheeses after 5, 10, 15, and 30 days of ripening, respectively. Growth of E. faecalis A-48-32 was associated with enterocin AS-48 production and persistence in cheese. Interestingly, growth of starter cultures was not affected by the Bac+ strain, and neither was lactic acid production. These results clearly indicate that E. faecalis A-48-32 produced satisfactory amounts of bacteriocin in cheese and support the potential use of AS-48–producing strains as culture adjuncts to inhibit B. cereus during cheese manufacture and ripening.