A new magnetic resonance imaging approach for discriminating Sardinian sheep milk cheese made from heat-treated or raw milk

The evaluation of milk heat treatment on dairy products via reliable analytical methods is a challenging issue that involves both industrial and fundamental research. We describe a new magnetic resonance imaging (MRI) protocol for discriminating Sardinian sheep milk cheese originating from heat-treated or raw milk. Thirty-six samples (18 pecorino cheeses manufactured from heat-treated milk and 18 Fiore Sardo cheeses made from raw milk) were investigated by means of MRI and bi-exponential signal decay analysis. The protocol is capable of discerning cheeses by virtue of the different distribution of the transversal (T₂) relaxation time constant. Cheeses from heat-treated milk showed a significantly higher area fraction (≈70–80%), corresponding to the fast relaxing water protons (T₂ ≈ 9 ms), compared with raw milk cheeses, whereas the opposite was observed for the long T₂ (T₂ ≈ 35 ms) proton population. The MRI protocol described is rapid and nondestructive, and it provides statistically significant discrimination between ewe milk cheeses made from heat-treated and raw milk.     

Temperature modulates the production and activity of a metalloprotease from Pseudomonas fluorescens 07A in milk

This work evaluated the expression and activity of a metalloprotease released by Pseudomonas fluorescens 07A in milk. Low relative expression of the protease by the strain was observed after incubation for 12 h at 25°C while the strain was in the logarithmic growth phase. After 24 h, protease production significantly increased and remained constant for up to 48 h, a time range during which the strain remained in the stationary phase. Conversely, at refrigeration temperatures, at 12 h the strain was still in the lag phase and expressed the protease at higher levels than when the logarithmic phase was reached. Casein fractions were highly degraded by P. fluorescens 07A, the purified protease, and the bacterial pellet on d 7 of incubation at 25°C and to a lesser extent at 10°C for the sample incubated with the bacterium. Heat treatment at 90°C for 5 min completely inactivated the proteolytic activity of the purified protease and the bacterial pellet. This work contributes to the knowledge about the conditions of milk storage that influence the production and activity of this extracellular metalloprotease. The results demonstrate the need to find alternative strategies to control the synthesis and activity of proteolytic enzymes in the dairy industry to ensure the quality of processed products.     

Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from north China

Lipases secreted by psychrotrophic bacteria are known to be heat resistant and can remain active even after the thermal processing of milk products. Such enzymes are able to destabilize the quality of milk products by causing a rancid flavor. Rapid detection of a small amount of heat-resistant lipase-producing psychrotrophic bacteria is crucial for reducing their adverse effects on milk quality. In this study, we established and optimized a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Pseudomonas fluorescens in raw cow milk, as the most frequently reported heat-resistant lipase-producing bacterial species. Pseudomonas fluorescens-specific DNA primers for LAMP were designed based on the lipase gene sequence. Reaction conditions of the LAMP assay were tested and optimized. The detection limit of the optimized LAMP assay was found to be lower than that of a conventional PCR-based method. In pure culture, the detection limit of the LAMP assay was found to be 4.8 × 10¹ cfu/reaction of the template DNA, whereas the detection limit of the PCR method was 4.8 × 10² cfu/reaction. Evaluation of the performance of the method in P. fluorescens-contaminated pasteurized cow milk revealed a detection limit of 7.4 × 10¹ cfu/reaction, which was 10² lower than that of the PCR-based method. If further developed, the LAMP assay could offer a favorable on-farm alternative to existing technologies for the detection of psychotrophic bacterial contamination of milk, enabling improved quality control of milk and milk products.     

Behavior of Shiga-toxin-producing Escherichia coli in ewe milk stored at different temperatures and during the manufacture and ripening of a raw milk sheep cheese (Zamorano style)

This study was conducted to assess the survival of 2 wild Shiga toxin-producing Escherichia coli strains (one serotype O157:H7 and one non-O157:H7) in ewe milk stored at different conditions and to examine the fate of the O157 strain during the manufacture and ripening of a Spanish sheep hard variety of raw milk cheese (Zamorano). The strains were selected among a population of 50 isolates, which we obtained from ewe milk, because of their high resistance to 0.3% lactic acid. Both strains were inoculated (approximately 2 log₁₀ cfu/mL) in raw and heat-treated (low-temperature holding, LTH; 63°C/30 min) ewe milk and stored for 5 d at 6, 8, and 10°C and also according to a simulation approach for assessing the effects of failures in the cold chain. The minimum growth temperature for the O157:H7 strain in LTH and raw ewe milk was 8°C. For the non-O157:H7 strain, the lowest temperature showing bacterial growth in LTH ewe milk was 6°C, but it did not grow at any of the tested conditions in raw milk. It appears that the O157 strain was more susceptible to cold stress but was likely a better competitor than the non-O157 strain against the milk autochthonous microbiota. For manufacture of Zamorano cheese, raw milk was inoculated with approximately 3 log₁₀ cfu/mL, and after 2 mo of ripening at 10 to 12°C, the cheeses showed the expected general characteristics for this variety. The O157:H7 strain increased 0.9 log₁₀ cfu/g after whey drainage and during ripening and storage decreased by 2.9 log₁₀ cfu/g. Nevertheless, its detectable level (estimated at 6.2 cfu/g) after 2 mo of ripening suggests that Zamorano cheese manufactured from raw ewe milk contaminated with E. coli O157:H7 could represent a public health concern.     

Synthesis of extracellular lipase by a strain ofPseudomonas fluorescensisolated from raw camel milk

Ten strains of psychrotrophic bacteria were isolated from raw camel milk and were arranged according to their lipase production. Lipolytic activities ranged between 0.26 to 3.43 meq of palmetic acid 100 g^-;1of bovine milk fat h^-;1.Pseudomonas fluorescensRM₄was the most active strain. This bacterium could grow and secrete lipase over a wide range of temperatures. The optimum temperature for growth was 37°C, and the maximum lipase production was at 25°C. Growth was maximal after 96 h of incubation at 25°C, and lipase activity was maximal at 72 h post-inoculation at 25°C (during the late logarithmic phase). Shaking of the cultures (100 rpm) led to an increase in both growth and enzyme activities. Pseudomonas fluorescensRM₄was able to grow and secrete lipase over a pH range of 5.5-;8.50. Synthesis of the enzyme appeared to be inducible because no enzyme was detected in the absence of organic nitrogen. Supplementation of the basal medium with milk proteins enhanced lipase production. Tryptophan and lysine induced enzyme synthesis most effectively. Addition of 4 g l^-;1of glucose to broth stimulated both growth and production of lipase. Beyond this level of supplementation, lipase activity was considerably depressed.     

Rapid detection of bovine milk in yak milk using a polymerase chain reaction technique

Yak milk contains a greater percentage of protein and has better quality than bovine milk. There has been an increasing focus on yak milk and milk products during the last few years. In the present study, a PCR-based assay was developed for the specific identification of bovine milk in yak milk by designing 3 primers targeting the mitochondrial ND1 gene. The use of 3 primers in a single PCR reaction set yielded 2 amplification fragments of 293 and 190 bp from bovine milk DNA, whereas only 1 amplification fragment of 293 bp was obtained in yak milk DNA. The technique was applied to raw and heat-treated binary mixtures of yak and bovine milks and enabled the specific detection of bovine milk with a detection limit of 0.1%. The assay developed is sensitive, fast, and straightforward, and it might be useful in the quality control of yak milk and milk products.     

A century of gray: A genomic locus found in 2 distinct Pseudomonas spp. is associated with historical and contemporary color defects in dairy products worldwide

Some gram-negative bacteria, including Pseudomonas spp., can grow at refrigeration temperatures and cause flavor, odor, and texture defects in fluid milk. Historical and modern cases exist of gray and blue color defects in fluid milk due to Pseudomonas, and several recent reports have detailed fresh cheese spoilage associated with blue-pigment-forming Pseudomonas. Our goal was to investigate the genomes of pigmented Pseudomonas isolates responsible for historical and modern pigmented spoilage of dairy products in the United States to determine the genetic basis of pigment-forming phenotypes. We performed whole genome sequencing of 9 Pseudomonas isolates: 3 from recent incidents of gray-pigmented fluid milk (Pseudomonas fluorescens group), 1 from blue-pigmented cheese (P. fluorescens group), 2 from a historical blue milk spoilage incident (Pseudomonas putida group), and 3 with no evidence for blue or gray pigment formation (2 from P. fluorescens group and 1 from Pseudomonas chlororaphis group). All 6 isolates collected from products with a gray or blue pigment defect were confirmed to produce pigment using potato dextrose agar or pasteurized milk. A subset of 2 isolates was selected for inoculation into milk and onto the surface of a model cheese for subsequent color measurement. These isolates produced different colors on potato dextrose agar, but produced nearly identical color defects in milk and on model cheese. For the same subset of 2 isolates, the gray color defect in milk was produced only in containers with ample headspace and not in full containers, suggesting that oxygen is vital for pigment formation. This work also demonstrated that a Pseudomonas isolate from cheese can produce a pigment defect in milk, and vice versa. Comparative genomics identified an accessory locus encoding tryptophan biosynthesis genes that was present in all isolates that produced gray or blue pigment under laboratory conditions and was only previously reported in 2 P. fluorescens isolates responsible for blue mozzarella in Italy. Because this locus was found in genetically distant isolates belonging to different Pseudomonas species groups, it may have been acquired via horizontal gene transfer. These data suggest that several past and present gray- or blue-pigmented dairy spoilage events share a common genetic etiology that transcends species-level identification and merits further investigation to determine mechanistic details and modes of prevention.     

Microbiota characterization of a Belgian protected designation of origin cheese,Herve cheese,using metagenomic analysis

Herve cheese is a Belgian soft cheese with a washed rind, and is made from raw or pasteurized milk. The specific microbiota of this cheese has never previously been fully explored and the use of raw or pasteurized milk in addition to starters is assumed to affect the microbiota of the rind and the heart. The aim of the study was to analyze the bacterial microbiota of Herve cheese using classical microbiology and a metagenomic approach based on 16S ribosomal DNA pyrosequencing. Using classical microbiology, the total counts of bacteria were comparable for the 11 samples of tested raw and pasteurized milk cheeses, reaching almost 8 log cfu/g. Using the metagenomic approach, 207 different phylotypes were identified. The rind of both the raw and pasteurized milk cheeses was found to be highly diversified. However, 96.3 and 97.9% of the total microbiota of the raw milk and pasteurized cheese rind, respectively, were composed of species present in both types of cheese, such as Corynebacterium casei, Psychrobacter spp., Lactococcus lactis ssp. cremoris, Staphylococcus equorum, Vagococcus salmoninarum, and other species present at levels below 5%. Brevibacterium linens were present at low levels (0.5 and 1.6%, respectively) on the rind of both the raw and the pasteurized milk cheeses, even though this bacterium had been inoculated during the manufacturing process. Interestingly, Psychroflexus casei, also described as giving a red smear to Raclette-type cheese, was identified in small proportions in the composition of the rind of both the raw and pasteurized milk cheeses (0.17 and 0.5%, respectively). In the heart of the cheeses, the common species of bacteria reached more than 99%. The main species identified were Lactococcus lactis ssp. cremoris, Psychrobacter spp., and Staphylococcus equorum ssp. equorum. Interestingly, 93 phylotypes were present only in the raw milk cheeses and 29 only in the pasteurized milk cheeses, showing the high diversity of the microbiota. Corynebacterium casei and Enterococcus faecalis were more prevalent in the raw milk cheeses, whereas Psychrobacter celer was present in the pasteurized milk cheeses. However, this specific microbiota represented a low proportion of the cheese microbiota. This study demonstrated that Herve cheese microbiota is rich and that pasteurized milk cheeses are microbiologically very close to raw milk cheeses, probably due to the similar manufacturing process. The characterization of the microbiota of this particular protected designation of origin cheese was useful in enabling us to gain a better knowledge of the bacteria responsible for the character of this cheese.     

A survey of foodborne pathogens in bulk tank milk and raw milk consumption among farm families in pennsylvania

A 2-part study was conducted to determine the risk of exposure to human pathogens from raw milk. The first part of the study focused on determining raw milk consumption habits of dairy producers. A total of 248 dairy producers from 16 counties in Pennsylvania were surveyed. Overall, 105 (42.3%) of the 248 dairy producers consumed raw milk and 170 (68.5%) of the 248 dairy producers were aware of foodborne pathogens in raw milk. Dairy producers who were not aware of foodborne pathogens in raw milk were 2-fold more likely to consume raw milk compared with dairy producers who were aware of foodborne pathogens. The majority of dairy producers who consumed raw milk indicated that taste (72%) and convenience (60%) were the primary factors for consuming raw milk. Dairy producers who resided on the dairy farm were nearly 3-fold more likely to consume raw milk compared with those who lived elsewhere. In the second part of the study, bulk tank milk from the 248 participating dairy herds was examined for foodborne pathogens. Campylobacter jejuni (2%), Shiga toxin-producing Escherichia coli (2.4%), Listeria monocytogenes (2.8%), Salmonella (6%), and Yersinia enterocolitica (1.2%) were detected in the milk samples. Salmonella isolates were identified as S. enterica serotype Typhimurium (n = 10) and S. enterica serotype Newport (n = 5). Of the 248 bulk tank milk samples, 32 (13%) contained ≥1 species of bacterial pathogens. The findings of the study could assist in developing farm community-based educational programs on the risks of consuming raw milk.     

Survival of Lipase during Manufacture of Nonfat Dry Milk

Extracellular lipases from several strains of Pseudomonas fluorescens were active against milk fat. The activity of P. fluorescens strain B52 lipase in reconstituted NDM using β-naphthyl caprylate as substrate was 70% of that found with skim milk; however, hydration of the NDM at 4°C for 12 h resulted in 85% recovery of activity. As little as .0025% (vol/vol) of P. fluorescens spent media was detected in reconstituted skim milk powder using β-naphthyl caprylate. The process of freeze-drying or spray-drying without heating had little effect on either native or P. fluorescens B52 lipase. The bacterial lipase activity was reduced by 12 to 21% and 55 to 59% in NDM treated with low heat (72°C/16 s) and high heat (110°C/2 min), respectively, while the native lipases were completely inhibited by these treatments. Both native and bacterial enzymes were stable when stored in NDM for extended periods at 20°C. In a limited study of commercial NDM samples, microbial lipase was not detected, suggesting that the quality of the raw milk was sufficiently high to restrict the presence of heat-stable lipolytic enzymes.     

Continuous flow nonthermal CO2 processing: the lethal effects of subcritical and supercritical CO2 on total microbial populations and bacterial spores in raw milk

The effect of pressurized (<50 MPa) CO₂ as a nonthermal process for bacterial reduction in raw skim milk was examined using a unique pressurized continuous flow system. The lethal effects of subcritical and super-critical CO₂ applied at different temperatures and pressures toward total native psychrotrophic microbial populations, total inoculated Pseudomonas fluorescens, and total inoculated spore populations were studied and compared. Pressures between 10.3 and 48.3 MPa; temperatures of 15, 30, 35, and 40°C; and CO₂ concentrations of 0, 3, 66, and 132 g/kg of milk were studied. For both native populations and inoculated P. fluorescens, greater total microbial lethality was observed under supercritical CO₂ conditions than under subcritical CO₂ conditions. At 30°C, there was no effect on total microbial lethality of increasing pressure up to 20.7 MPa with either 66 or 132 g/kg of CO₂; at 35°C, there was a positive relationship between pressure and lethality at CO₂ levels of 132 g/kg, but no relationship at 66 g/kg of CO₂. For total microbial populations and P. fluorescens, CO₂ applied at 132 g/kg at 30°C and pressures of 10.3 to 20.7 MPa resulted in an average standard plate count reduction of 3.81 and 2.93 log, respectively; at 35°C and 20.7 MPa, maximum reductions achieved were 5.36 and 5.02 log, respectively. For both total microbial populations and inoculated P. fluorescens, CO₂ exhibited a greater overall lethal effect at 132 g/kg than at 66 g/kg and a greater effect at 35°C than at 30°C. At 24.1 and 48.3 MPa and 40°C, microbial lethality in raw aged milk treated with 3 g/kg of CO₂ was not significantly different than that observed for uncarbonated milk; lethality achieved in milk treated with 132 g/kg of CO₂ was significantly higher than that achieved in these 2 low-level CO₂ treatments. No treatment studied had any significant impact on spore populations. Our work shows that, using the studied system, pressurized CO₂ results in greater microbial lethality in milk above critical temperatures than below and suggests that a critical concentration threshold level of CO₂ is required for lethal effects. Our work also suggests that supercritical CO₂ processing in a continuous flow system can achieve reductions in some microbial populations equal to or better than that typically achieved during high-temperature, short-time pasteurization.     

Prevalence,antimicrobial resistance,and molecular characterization of Campylobacter spp. in bulk tank milk and milk filters from US dairies

Campylobacter spp. are frequently isolated from dairy cows as commensal organisms. Sporadic Campylobacter infections in humans in the United States are generally attributed to poultry, but outbreaks are also commonly associated with dairy products, particularly unpasteurized or raw milk. Bulk tank milk samples and milk filters from US dairy operations were collected during the National Animal Health Monitoring System Dairy 2014 study and analyzed using real-time PCR and traditional culture techniques for the presence of thermophilic Campylobacter species. The weighted prevalence of operations from which we detected Campylobacter spp. in either bulk tank milk or milk filters was 24.9%. We detected Campylobacter spp. in a higher percentage of operations with 100–499 cows (42.8%) and 500 or more cows (47.5%) than in operations with 30–99 cows (6.5%). Campylobacter spp. were also more frequently detected in operations in the west than the east (45.9 and 22.6%, respectively). We isolated Campylobacter spp. from approximately half of PCR-positive samples, representing 12.5% (weighted prevalence) of operations. The majority (91.8%) of isolates were C. jejuni, but C. lari and C. coli were also isolated. We detected resistance to tetracycline in 68.4% of C. jejuni isolates, and resistance to ciprofloxacin and nalidixic acid in 13.2% of C. jejuni isolates. Based on pulsed-field gel electrophoresis, we found that dairy-associated C. jejuni were genotypically diverse, although clonal strains were isolated from different geographic regions. These results suggest that bulk tank milk can be contaminated with pathogenic Campylobacter spp., and that the consumption of unpasteurized or raw milk presents a potential human health risk.     

Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model

Psychrotolerant spore-forming bacteria represent a major challenge regarding microbial spoilage of fluid milk. These organisms can survive most conventional pasteurization regimens and subsequently germinate and grow to spoilage levels during refrigerated storage. To improve predictions of fluid milk shelf life and assess different approaches to control psychrotolerant spore-forming bacteria in the fluid milk production and processing continuum, we developed a predictive model of spoilage of fluid milk due to germination and growth of psychrotolerant spore-forming bacteria. We characterized 14 psychrotolerant spore-formers, representing the most common Bacillales subtypes isolated from raw and pasteurized milk, for ability to germinate from spores and grow in skim milk broth at 6°C. Complete growth curves were obtained by determining total bacterial count and spore count every 24 h for 30 d. Based on growth curves at 6°C, probability distributions of initial spore counts in bulk tank raw milk, and subtype frequency in bulk tank raw milk, a Monte Carlo simulation model was created to predict spoilage patterns in high temperature, short time-pasteurized fluid milk. Monte Carlo simulations predicted that 66% of half-gallons (1,900 mL) of high temperature, short time fluid milk would reach a cell density greater than 20,000 cfu/mL after 21 d of storage at 6°C, consistent with current spoilage patterns observed in commercial products. Our model also predicted that an intervention that reduces initial spore loads by 2.2 Log₁₀ most probable number/mL (e.g., microfiltration) can extend fluid milk shelf life by 4 d (end of shelf life was defined here as the first day when the mean total bacterial count exceeded 20,000 cfu/mL). This study not only provides a baseline understanding of the growth rates of psychrotolerant spore-formers in fluid milk, it also provides a stochastic model of spoilage by these organisms over the shelf life of fluid milk, which will ultimately allow for the assessment of different approaches to reduce fluid milk spoilage.     

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.     

Raw milk from vending machines: Effects of boiling,microwave treatment,and refrigeration on microbiological quality

In Italy, the sale of raw milk from vending machines has been allowed since 2004. Boiling treatment before its use is mandatory for the consumer, because the raw milk could be an important source of foodborne pathogens. This study fits into this context with the aim to evaluate the microbiological quality of 30 raw milk samples periodically collected (March 2013 to July 2013) from 3 vending machines located in Molise, a region of southern Italy. Milk samples were stored for 72 h at 4°C and then subjected to different treatments, such as boiling and microwaving, to simulate domestic handling. The results show that all the raw milk samples examined immediately after their collection were affected by high microbial loads, with values very close to or even greater than those acceptable by Italian law. The microbial populations increased during refrigeration, reaching after 72 h values of about 8.0 log cfu/mL for Pseudomonas spp., 6.5 log cfu/mL for yeasts, and up to 4.0 log cfu/mL for Enterobacteriaceae. Boiling treatment, applied after 72 h to refrigerated milk samples, caused complete decontamination, but negatively affected the nutritional quality of the milk, as demonstrated by a drastic reduction of whey proteins. The microwave treatment at 900 W for 75 s produced microbiological decontamination similar to that of boiling, preserving the content in whey proteins of milk. The microbiological characteristics of raw milk observed in this study fully justify the obligation to boil the raw milk from vending machines before consumption. However, this study also showed that domestic boiling causes a drastic reduction in the nutritional value of milk. Microwave treatment could represent a good alternative to boiling, on the condition that the process variables are standardized for safe domestic application.     

Interactions in biofilms of Lactococcus lactis ssp. cremoris and Pseudomonas fluorescens cultured in cold UHT milk

Three Lactococcus lactis ssp. cremoris isolates from refrigerated bulk raw milk were cultured separately and in association with a known psychrotrophic dairy Pseudomonas fluorescens strain, in skim UHT milk for 72 h at 7°C, to determine mutual influences in both the planktonic and biofilm phases. Two levels of inoculum of each culture partner were combined. Protocooperation and commensalism cases were found, all of them in the biofilm phase. Type and intensity of the interactions depended on Lactococcus strain and on the cell density of each partner. Maximum enhancement of attachment was observed to be approximately 100-fold for P. fluorescens and 20,000-fold for one of the L. lactis strains. Confocal scanning laser microscopy images show compact masses of Pseudomonas trapping lactococci cells in cooperative biofilms.     

Effect of dairy product environment on the growth of Bacillus cereus

pH is one of the most important parameters to manage bacterial replication in foodstuffs. In this study, the ability of 2 Bacillus cereus strains, 1 clinical human isolate (GPe2) and 1 isolate from a dairy product (D43), were investigated for in vitro growth at different pH values (from 3.5 to 7.5) at 2 temperatures (15 and 37°C), showing their ability to grow from 5.5 to 7.5 and from 5.0 to 7.5, respectively. The ability of spores of these 2 microorganisms to germinate in different typologies of dairy products (unflavored yogurt, Taleggio cheese, mascarpone cheese, and raw and pasteurized milk) was also investigated by inoculating the spores and maintaining the products at 15°C. No growth was observed in yogurt, likely due to the combined effect of low pH (<5) and the presence of natural microflora. An inhibitory action of the natural microflora on the growth of B. cereus was also hypothesized for Taleggio cheese and raw milk, as these substrates were characterized by a high natural lactic acid bacteria population and permissive pH values (5.8/6.8 in Taleggio cheese, >7 in raw milk). In pasteurized milk and mascarpone cheese, where pH was not restrictive for B. cereus growth and where no significant natural microflora was present, growth occurred rapidly up to loads close to 7 log cfu/g.     

Influence of storage and preservation on microbiological quality of silo ovine milk

This study was designed to analyze the effects of the storage and preservation conditions on counts of mesophilic, thermoduric, psychotrophic, coliform, Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus organisms in silo ovine milk. A total of 910 analytical determinations were conducted from aliquots of 10 silo ovine milks. The conditions tested were unpreserved and azidiol-preserved milk stored at 4°C, and unpreserved milk stored at −20°C. Milk aged 2, 24, 48, 72, and 96 h post-collection for refrigerated aliquots, and 7, 15, and 30 d post-collection for frozen aliquots. The factors silo and storage conditions significantly contributed to variation of all microbiological variables, although milk age effect within storage was only significant for mesophilic, psychrotrophic, and coliform bacteria counts. In refrigerated raw milk, mesophile, psychrotroph, and coliform counts significantly increased over 96 h post-collection, whereas the other groups and bacteria species tested maintained their initial concentration. In all cases, azidiol preservation maintained the initial bacterial concentration in raw sheep milk under refrigeration throughout 96 h. Thus, azidiol was a suitable preservative for microbiological studies in sheep milk. Smallest counts were registered for frozen samples, particularly for coliforms, E. coli, Strep. agalactiae and Staph. aureus. Estimates of mesophilic, thermoduric and psychrotrophic organisms showed similar values on both azidiol-preserved and frozen milk samples. Coliforms and E. coli counts significantly decrease over time after freezing. Consequently, freezing at −20°C could also be appropriate for analysis of mesophilic, thermoduric, and psychrotrophic bacterial groups, but not for coliforms or mammary pathogens.     

Comparative analysis of bacterial community composition in bulk tank raw milk by culture-dependent and culture-independent methods using the viability dye propidium monoazide

Microbial diversity of 3 raw milk samples after 72 h of storage at 4°C in a bulk tank was analyzed by culture-dependent and -independent methods. The culture-dependent approach was based on the isolation of bacteria on complex and selective media, chemotaxonomic differentiation of isolates, and subsequent identification by 16S rRNA gene sequencing. The culture-independent approach included the treatment of raw milk with the dye propidium monoazide before direct DNA extraction by mechanic and enzymatic cell lysis approaches, and cloning and sequencing of the 16S rRNA genes. The selective detection of viable bacteria improved the comparability between bacterial compositions of raw milk based on culture-dependent and -independent methods, which was the major objective of this study. Several bacterial species of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were detected by the culture-dependent method, whereas mainly bacteria of the phylum Proteobacteria as well as low proportions of the phyla Bacteroidetes and Actinobacteria were detected by the culture-independent method. This led to the conclusion that the phylum Firmicutes was strongly discriminated by the culture-independent approach. Generally, species richness detected by the culture-dependent method was higher than that detected by the culture-independent method for all samples. However, few taxa could be detected solely by the direct DNA-based method. In conclusion, the combination of culture-dependent and -independent methods led to the detection of the highest bacterial diversity for the raw milk samples analyzed. It was shown that DNA extraction from raw milk as the essential step in culture-independent methods causes the discrimination of taxa by incomplete cell lysis. Treatment of raw milk with the viability dye propidium monoazide was optimized for the application in raw milk without former removal of milk ingredients and proved to be a suitable tool to ensure comparability of bacterial diversity depicted by both methods.     

Low pressure CO2 storage of raw milk: microbiological effects

The effects of holding raw milk under carbon dioxide pressures of 68 to 689 kPa at temperatures of 5, 6.1, 10, and 20°C on the indigenous microbiota were investigated. These pressure-temperature combinations did not cause precipitation of proteins from the milk. Standard plate counts from treated milks demonstrated significantly lower growth rate compared with untreated controls at all temperatures, and in some cases, the treatment was microcidal. Raw milk treated with CO₂ and held at 6.1°C for 4 d exhibited reduced bacterial growth rates at pressures of 68, 172, 344, and 516 kPa; and at 689 kPa, demonstrated a significant loss of viability in standard plate count assays. The 689-kPa treatment also reduced gram-negative bacteria and total Lactobacillus spp. The time required for raw milk treated at 689 kPa and held at 4°C to reach 4.30 log₁₀ cfu/mL increased by 4 d compared with untreated controls. Total coliform counts in the treated milk were maintained at 1.95 log₁₀ cfu/mL by d 9 of treatment, whereas counts in the control significantly increased to 2.61 log₁₀ cfu/mL by d 4 and 2.89 log₁₀ cfu/mL by d 9. At d 8, Escherichia coli counts had not significantly changed in treated milk, but significantly increased in the control milk. Thermoduric bacteria counts after 8 d were 1.32 log₁₀ cfu/mL in treated milk and 1.98 log₁₀ cfu/mL in control milk. These data indicated that holding raw milk at low CO₂ pressure reduces bacterial growth rates without causing milk protein precipitation. Combining low CO₂ pressure and refrigeration would improve the microbiological quality and safety of raw milk and may be an effective strategy for shipping raw single strength or concentrated milk over long distances.