16S rDNA high-throughput sequencing and MALDI-TOF MS are complementary when studying psychrotrophic bacterial diversity of raw cows' milk

Refrigeration of raw milk favours the growth of psychrotrophic bacteria (psychrotrophs) that can produce spoilage enzymes, which may deteriorate final dairy products. The psychrotrophs from raw milk across four seasons were identified using culture-dependent and culture-independent approaches. Both 16S rDNA high-throughput sequencing (HTS) and MALDI-TOF MS showed the Pseudomonas genus to predominate; however, predominant species differed between the two identification methods. There was no geographical trend in microbiota, but a seasonal variation was evident. The use of HTS and MALDI-TOF MS was complementary in describing the psychrotrophic bacterial diversity of raw milk and provides an understanding of the raw milk microflora that may influence milk quality. This is the first report to compare data obtained from 16S rDNA high-throughput sequencing and MALDI-TOF analysis to assess the psychrotrophic microbial quality of refrigerated raw milk in New Zealand.     

Short communication: Effect of bactofugation of raw milk on counts and microbial diversity of psychrotrophs

Bactofugation is a centrifugal process for removing spores of microorganisms from milk, especially when it is destined for cheese making. Other microorganisms may be removed in bactofugation. This study aimed to verify the effect of milk bactofugation on the counts and microbial diversity of psychrotrophs. The raw milk was preheated (≈55°C) before being bactofuged, and samples were collected from 3 batches of milk: refrigerated raw, preheated, and bactofuged, representing the immediate conditions before and after bactofugation. The mean psychrotrophic counts of the 3 batches were 3.08 (±1.69) × 10⁶, 193 (±232), and 20 (±26) cfu/mL, respectively. Preheating was sufficient to eliminate 99.99% of the raw milk psychrotrophs, but bactofugation further reduced 89.66% of psychrotrophs from preheated milk. Lysinibacillus fusiformis was the most frequently isolated species (45.7%) among the psychrotrophs of raw milk and, proportionally, were more frequent in preheated (37.5%) and bactofuged (60%) milk. Bacillus invictae (20%), Enterococcus faecalis (10%), and Kurthia gibsonii (10%) were also isolated from bactofuged milk. Albeit in small numbers, psychrotrophic, thermoduric, and spore-forming bacteria with known proteolytic and lipolytic activity remained in the milk after bactofugation, which apparently had no effect on a specific population of microorganisms but proportionally reduced the entire psychrotrophic microbiota of raw milk.     

Molecular identification of mesophilic and psychrotrophic bacteria from raw cow's milk

The aim of this study was to use molecular techniques to assess the microbiota of eight raw cow's milk samples at biotype and species level. Sixty-six isolates from raw milk samples were screened by Randomly amplified polymorphic DNA–PCR (RAPD–PCR) biotyping and representative strains of RAPD–PCR profiles were identified by 16S rRNA gene sequencing. Pseudomonas spp. were the most commonly occurring contaminants along with Enterobacteriaceae such as Hafnia alvei, Serratia marcescens and Citrobacter freundii. Moreover, Gram-positive isolates belonging to the genera Staphylococcus and Lactococcus were also found. Experiments of growth at different temperatures showed that more than 50% of the Gram-negative isolates could grow at chill temperatures and that 65% of the Pseudomonas spp. strains grew at 7 °C within 5 days. Only 13 Gram-negative isolates displayed proteolytic activity on milk agar, suggesting that not all the biotypes of milk contaminating species are able to perform this spoilage-associated activity. Among the Gram negative, the proteolytic strains were mainly Peudomonas spp. that displayed the activity at both 7 °C and 20 °C. A reliable molecular identification of raw milk microbiota is important for the study of the microbiological quality of raw milks and for the assessment of the ecology at species level in order to develop improved systems, preventing contamination and having the best conditions for the storage of milk.     

Conventional and ohmic heating pasteurization of fresh and thawed sheep milk: Energy consumption and assessment of bacterial microbiota during refrigerated storage

This study aimed to assess the energy consumption of ohmic heating (OH) and conventional heating (CH) for pasteurization of fresh and thawed sheep milk and their impact on bacterial microbiota throughout refrigerated shelf-life (4 °C ± 1, 15 days). OH pasteurization using 8.33 and 5.83 V/cm electric field strength spent 72–73% less energy than CH pasteurization (515 KJ). The cultivation-dependent approach showed that at least 4.2 log cycle reductions were achieved in sheep milk submitted to CH and OH pasteurization, regardless sheep milk was fresh or thawed. Data from amplicon sequencing indicated that Staphylococcus was the most prevalent genus in raw samples at day 1 (F1_D1: 58.35%; T1_D1: 69.50%), while Pseudomonas became the most abundant after 15 days under cold storage (F1_D15: 50.15% and T1_D15: 54.50%). The relative abundance of all bacterial genera assessed remained similar on pasteurized samples by CH and OH throughout refrigerated storage.Industrial relevanceOhmic heating (OH) presents as a critical advantage rapid and uniform heating of fluid food material. No studies assessed the use of OH for pasteurization of sheep milk and to evaluate the impact of this technology on sheep milk bacterial microbiota during refrigerated storage. The findings of this study prove the feasibility of sheep milk pasteurization using OH_{8.33 V/cm}. OH also ensured the bacteriological stability of sheep milk during 2 weeks of storage under refrigeration conditions prior to dairy products manufacturing. This approach comprises a cheaper and easier way to store milk when compared to frozen storage, with potential benefits to small farmers and dairy industries.     

Effect of ultrasonication on microbiological,chemical and sensory properties of raw,thermized and pasteurized milk

The combined effect of ultrasonication and heat treatment on microbiological, chemical and sensory properties of raw, thermized and pasteurized milk was studied. Microbiological parameters monitored included total viable counts and psychrotrophs, chemical parameters included thiobarbituric acid and volatiles determination and sensory evaluation included the monitoring of odour and taste as a function of storage time. Results showed a 1–2.1 log cfu mL^?1 reduction in total viable counts and psychrotrophs for raw, thermized and pasteurized milk up to 6 days of storage. For raw and pasteurized milk ultrasonication resulted in a taste score equal to or lower than that of untreated milk. However, thermized milk, ultrasonicated for 2 min gave a higher taste score than its untreated counter part on day 4 of storage. With regard to lipid oxidation, malondialdehyde ranged between 1.20 and 1.95 mg kg^?1. Finally, volatile compounds identified in all samples were mainly products of lipid oxidation that increased in concentration with sonication and storage time.     

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 10⁴ cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.     

High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk

The grade A Pasteurized Milk Ordinance specifies minimum processing conditions of 72°C for at least 15 s for high temperature, short time (HTST) pasteurized milk products. Currently, many US milk-processing plants exceed these minimum requirements for fluid milk products. To test the effect of pasteurization temperatures on bacterial numbers in HTST pasteurized milk, 2% fat raw milk was heated to 60°C, homogenized, and treated for 25 s at 1 of 4 different temperatures (72.9, 77.2, 79.9, or 85.2°C) and then held at 6°C for 21 d. Aerobic plate counts were monitored in pasteurized milk samples at d 1, 7, 14, and 21 postprocessing. Bacterial numbers in milk processed at 72.9°C were lower than in milk processed at 85.2°C on each sampling day, indicating that HTST fluid milk-processing temperatures significantly affected bacterial numbers in fluid milk. To assess the microbial ecology of the different milk samples during refrigerated storage, a total of 490 psychrotolerant endospore-forming bacteria were identified using DNA sequence-based subtyping methods. Regardless of processing temperature, >85% of the isolates characterized at d 0, 1, and 7 postprocessing were of the genus Bacillus, whereas more than 92% of isolates characterized at d 14 and 21 postprocessing were of the genus Paenibacillus, indicating that the predominant genera present in HTST-processed milk shifted from Bacillus spp. to Paenibacillus spp. during refrigerated storage. In summary, 1) HTST processing temperatures affected bacterial numbers in refrigerated milk, with higher bacterial numbers in milk processed at higher temperatures; 2) no significant association was observed between genus isolated and pasteurization temperature, suggesting that the genera were not differentially affected by the different processing temperatures; and 3) although typically present at low numbers in raw milk, Paenibacillus spp. are capable of growing to numbers that can exceed Pasteurized Milk Ordinance limits in pasteurized, refrigerated milk.     

Prevalence of Yersinia enterocolitica in milk and dairy products and the effects of storage temperatures on survival and virulence gene expression

Yersinia enterocolitica was isolated from raw milk and dairy products from 10% of examined samples. The highest isolation rate was 22%, from raw milk, followed by 12%, 4% and 2% from fermented milk (Rayeb), pasteurised milk and ripened salted cheese, respectively. The virulence-associated genes ail and yst were detected in 30% and 10% of the isolates, respectively, while these genes were present simultaneously in 10% of the isolates. All the isolates showed susceptibility to gentamicin, ciprofloxacin and chloramphenicol, while only two of the isolates exhibited multidrug resistance. Storage of inoculated pasteurised milk at refrigeration (4 °C), freezing (−20 °C) and room (25 °C) temperatures revealed significant differences in Y. enterocolitica counts and relative expression of the two virulence genes. The isolation of potentially pathogenic Y. enterocolitica isolates from retail dairy products indicates risk to consumers; screening of prevalence, pathogenicity potential and antibiotic resistance is essential to implement control measures.     

Molecular identification of the microbiota of peeled and unpeeled brown shrimp (Crangon crangon) during storage on ice and at 7.5 °C

The dominant microbiota of brown shrimp (Crangon crangon) were systematically identified during storage under different conditions. Freshly caught shrimp were processed on board the fishing vessel under the best possible hygienic conditions (IDEAL), unpeeled and manually (sterile) peeled, then stored on ice and at 7.5 °C until microbiologically spoiled. Results were compared with industrially processed (INDUSTRIAL) shrimp. Isolates grown on various media were identified by 16S rRNA and gyrB gene sequencing. We examined the total microbiota and microbial population shifts of shrimp under various storage conditions using denaturant gradient gel electrophoresis (DGGE). The microbiota differed somewhat during storage and among the various storage conditions; however, members of the genera Psychrobacter and Pseudoalteromonas were found to dominate the microbiota of all shrimp samples regardless of processing procedures or storage conditions. Most isolates could be identified by gyrB gene sequencing as Psychrobacter immobilis or Psychrobacter cibarius. Also Pseudoalteromonas nigrifaciens, Pseudoalteromonas elyakovii or Pseudoalteromonas paragorgicola dominated the microbiota of brown shrimp during storage. Also species from the genera Planocuccus, Exiguobacterium, Carnobacterium, Pseudomonas, Chryseobacterium and Staphylococcus were detected during storage of brown shrimp.     

Incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia

The purpose of this study was to evaluate the incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia. The distribution of Bacillus species in raw milk, pasteurised milk and UHT milk were 47.5%, 27.5% and 25%, respectively. Seven Bacillus species, including Bacillus pumilus (10%), Bacillus subtilis (12.5%), Brevibacillus brevis (10%), Bacillus cereus (22.5%), Bacillus sphaericus (7.5%), Bacillus licheniformis (12.5%) and Bacillus sporothermodurans (25%) were identified in different milk samples. Bacillus cereus was predominant in raw and pasteurised milk. Although B. sporothermodurans was the predominant sporogenous micro‐organism in UHT milk, B. cereus, B. sphaericus and B. licheniformis were also present. This study showed that there is a high degree of diversity, both phenotypic and genotypic, among Bacillus isolates from Tunisian milk and the persistence of spoilage risk in UHT milk.     

Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control

Microbial contamination of bovine raw milk often occurs at the farm. To acquire a deeper knowledge of the microbiota of farm tank milk, we studied milk from 45 farms situated in 2 geographical areas in Norway. Each farm was visited on 3 different occasions, with at least 2 wk between visits. We combined both bacterial cell counts and a sequence variant inference method of amplicon-based high-throughput sequencing to achieve a high-resolution overview of the microbiota in each sample. Compositional variation of the farm milk microbiota was shown in relation to the 2 areas, between the farms and between the sampling times. Despite the near constant level of bacteria enumerated in milk from each individual farm, the dominant microbiota differed significantly between the samplings. The predominant microbiota was dominated by spoilage genera, such as Pseudomonas and Bacillus, as well as the dairy fermentation genus Lactococcus and mastitis-causing organisms (Streptococcus). Analysis of the identified sequence variants within these genera showed that the populations of Pseudomonas and Lactococcus in milk had similar composition between the farms, but that Bacillus and, in particular, Streptococcus populations changed between collection days from the same farm and between farms and geographical areas. Furthermore, the levels and composition of Bacillus and Paenibacillus were different between the 2 geographical areas. The results presented here provide new insight into the farm milk microbiota and show that this microbiota is a dynamic community highly subject to variation.     

Molecular characterisation,genotyping and survival of Aeromonas hydrophila isolated from milk,dairy products and humans in Egypt

Two Aeromonas species, Aeromonas hydrophila and Aeromonas sobria, were isolated from raw milk (8% and 5.3% of samples tested, respectively), yoghurt (12% and 8% of samples tested, respectively) and cheese (4% and 2% of samples tested, respectively). Only A. hydrophila was isolated from human stool samples (18.8% of samples tested). Aerolysin and haemolysin associated genes were characterised in 12 and 3 isolates, respectively, while both genes were identified simultaneously in 9 isolates. Genotyping of the isolates by random amplified polymorphic DNA (RAPD) PCR revealed a high discriminatory index (D = 0.966). The storage of yoghurt samples inoculated with A. hydrophila showed the ability of the bacteria to survive for 14 days, resembling the shelf-life of yoghurt at 4 °C and 12 °C. To the best of our knowledge, this is the first study to assess the survival of A. hydrophila in yoghurt at refrigeration temperature.     

Lactic acid bacteria diversity of African raw and fermented camel milk products reveals a highly competitive,potentially health-threatening predominant microflora

The microflora of 59 East African camel milk samples of unfermented raw milk supply chains and spontaneously fermented milk (suusac) was analyzed to describe the diversity of predominant lactic acid bacteria (LAB), identify potential health risks and study bacteriocin-like inhibitory substance (BLIS) production. Bacterial isolates (n = 532) were identified using a genotypic approach incorporating rep-PCR, 16S rRNA gene sequencing, and species-specific PCR assays. The bacterial numbers on MRS and M17 agar exceeded log₁₀ 6.5 colony forming units (CFU) mL^?1 for raw milk supply chain samples and log₁₀ 8 CFU mL^?1 for suusac. Streptococcus agalactiae and Staphylococcus spp. were predominant in unfermented products, while suusac contained predominantly Streptococcus infantarius subsp. infantarius followed by Lactococcus lactis subsp. lactis, Streptococcus thermophilus, and lactobacilli. Fifty per cent of S. infantarius subsp. infantarius isolates originating from 15 out of 24 suusac samples produced a BLIS active against other LAB or Listeria and representing a potential selective advantage during fermentation. This study provides a detailed insight at the genotypic level into the LAB diversity of previously unstudied dairy products. It indicates potential health risks for consumers and the need for hygienic and manufacturing interventions and reports a potentially novel BLIS produced by S. infantarius subsp. infantarius.     

A microbiological perspective of raw milk preserved at room temperature using hyperbaric storage compared to refrigerated storage

The effects of hyperbaric storage (HS, 50–100 MPa) at room temperature (RT) on endogenous and inoculated pathogenic surrogate vegetative bacteria (Escherichia coli, Listeria innocua), pathogenic Salmonella enterica and bacterial spores (Bacillus subtilis) were assessed and compared with conventional refrigeration at atmospheric pressure for 60 days. Milk stored at atmospheric pressure and refrigeration quickly surpassed the acceptable microbiological limit within 7 days of storage, regarding endogenous microbiota, yet 50 MPa/RT slowed down microbial growth, resulting in raw milk spoilage after 28 days, while a significant microbial inactivation occurred under 75–100 MPa (around 4 log units), to counts below 1 log CFU/mL throughout storage, similar to what was observed for B. subtilis endospores. While inoculated microorganisms had a gradually counts reduction in all HS conditions. Results indicate that HS can not only result in the extension of milk shelf-life but is also able to enhance its safety and subsequent quality.Industrial relevanceThis new preservation methodology could be implemented in the dairy farm storage tanks, or during milk transportation for further processing, allowing a better microbial control, than refrigeration. This methodology is very promising, and can improve food products shelf-life with a considerable lower carbon foot-print than refrigeration.     

Quantification of the proteolytic and lipolytic activity of microorganisms isolated from raw milk

Heat-resistant enzymes of psychrotolerant microorganisms are associated with the spoilage of UHT-milk and milk products. In this study, we investigated the extracellular peptidase, esterase and lipase activity after submerged cultivation at 6 °C of 231 recently isolated microorganisms in milk medium. In contrast to the widely used agar diffusion tests for secretion of hydrolases, here a more realistic liquid screening approach was used. The advantages of the latter are the possibility of quantifying the enzyme activity in volumetric units and the opportunity to simulate the growth conditions of the microorganisms to the storage conditions of raw milk. The majority of enzymatically active isolates belonged to Gram-negative bacteria, especially the genus Pseudomonas. Surprisingly, among them, twelve novel Pseudomonas species were discovered. In this study, we demonstrated that numerous raw milk isolates, including bacteria and yeasts, produce extracellular enzymes that may cause spoilage problems for the dairy industry.     

Influence of Bactrian camel milk on the gut microbiota

Bactrian camel milk has become popular in the market as an important source of nutrients with diverse functional effects. In this study, the influence of Bactrian camel milk on the gut microbiota of mice was studied using metagenomic-based sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene. Bioinformatics analysis showed that Firmicutes and Bacteroidetes were the predominant phyla, accounting for more than 80% of the bacteria present. At the genus level, Allobaculum, Akkermansia, Romboutsia, Bifidobacterium, and Lactobacillus were most abundant in the gut microbiota; of these, Allobaculum and Akkermansia were the predominant genera, representing 40.42 and 7.85% of all the bacteria present, respectively. Camel milk was found to reduce relative abundance of Romboutsia, Lactobacillus, Turicibacter, and Desulfovibrio (decreased by 50.88, 34.78, 26.67, and 54.55%, respectively) in the gut microbiota compared with the control. However, some genera such as Allobaculum, Akkermansia, and Bifidobacterium in the gastrointestinal flora increased in abundance in the presence of camel milk; these genera are correlated with beneficial effects for organisms. Our research suggests that the gut microbiota should be taken into account when conducting functional studies on camel milk, and this work provides a useful foundation for further study on functions of camel milk.     

Milk with different somatic cells counts and the physicochemical,microbiological characteristics and fatty acid profile of pasteurised milk cream: is there an association?

In this study, raw cow milk containing somatic cells counts (SCC) at mean levels of 39 000 cells mL^?1 (low), 349 000 cells mL^?1 (intermediate) and 1 297 000 cells mL^?1 (high) was used for the production of pasteurised cream. Physicochemical (pH, fat and fatty acid profile) and microbiological analyses (mesophilic and psychrotrophs) were performed in the obtained creams during 30 days of refrigerated storage at 5 °C ± 2. No interactions (P > 0.05) were found between SCC, storage time and the physicochemical and microbiological variables studied. Fatty acid profile was similar among the SCC creams, except for oleic acid (C18:1), which decreased (P < 0.05) in intermediate and high SCC creams. Considering the technological aspect, our findings suggest that milk cream manufacture can be an interesting option for the use of high SCC milk.     

Characterisation of the spoilage microbiota in raw salmon (Salmo salar) steaks stored under vacuum or modified atmosphere packaging combining conventional methods and PCR-TTGE

In order to characterise the spoilage related to microbiota of raw salmon, a combination of culture-dependent and -independent methods, including PCR–TTGE, was used to analyse 3 raw salmon batches stored for 3 days at chilled temperature in modified atmosphere packaging (MAP) (50% CO₂/50% N₂) or under vacuum. Sensory evaluation, microbiological enumeration and chemical analysis were performed after 3, 7 and 10 days of storage. At the onset of spoilage, 65 bacterial isolates were picked from the plates. Thus, 13 different genera or species were identified by phenotypic and molecular tests: Serratia spp., Photobacterium phosphoreum, Yersinia intermedia, Hafnia alvei, Buttiauxella gaviniae, Pseudomonas sp., Carnobacterium maltaromaticum, Carnobacterium divergens, Lactococcus piscium, Lactobacillus fuchuensis, Vagococcus carniphilus, Leuconostoc gasicomitatum and Brochothrix thermosphacta. The PCR–TTGE profiles and band identification enabled a shift of the dominant populations during the storage to be visualised for all the batches, probably due to the temperature change and the packaging. At the beginning of storage, Pseudomonas sp. dominated the raw salmon microbiota while in the following days (7 and 10), P. phosphoreum and L. piscium were identified as the main bacterial groups. This study enhances the knowledge of MAP and vacuum-packed raw salmon spoilage microbiota.     

Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States

To determine the microbial ecology of pasteurized milk within the United States, 2% fat pasteurized fluid milk samples were obtained from 18 dairy plants from 5 geographical areas representing the Northeast, Southeast, South, Midwest, and West. Of the 589 bacterial isolates identified using DNA sequence-based subtyping methods, 346 belonged to genera characterized as gram-positive endospore-forming bacteria (i.e., Bacillus and Paenibacillus). Of the 346 gram-positive endospore-forming bacteria isolated in the present study, 240 were classified into 45 allelic types identical to those previously identified from samples obtained in New York State, indicating the widespread presence of these microbes in fluid milk production and processing systems in the United States. More than 84% of the gram-positive spore-forming isolates characterized at d 1, 7, and 10 were of the genus Bacillus, whereas more than 92% of isolates characterized at d 17 of shelf life were of the genus Paenibacillus, indicating that the predominant gram-positive spoilage genera shifts from Bacillus spp. to Paenibacillus spp. during refrigerated storage.     

Quantitative and qualitative microbial analysis of raw milk reveals substantial diversity influenced by herd management practices

The quantitative (n = 260) and qualitative (n = 24) microbial compositions of raw milk samples were assessed using culture analyses and a molecular inventory based on the rDNA sequences of 1697 isolates. A statistical analysis was also performed to correlate farm practices with the microbial populations present in the milk samples. Ripening bacteria and Pseudomonas were the dominant groups. Milk samples were distinguishable by the different proportions of their microbial groups, which varied among producers. Individual practices influenced the levels of technologically important microorganisms, for example, pre-dipping of teats reduced levels of lactococci. Standard plate counts correlated with the levels of Gram-negative bacteria, Pseudomonas, lactococci and ripening bacteria, depending on the milking season. Lactic acid bacteria and yeasts, which were poorly represented, nevertheless seemed to constitute a resilient basal flora. Qualitative species diversity was considerable, with more than 100 bacterial species identified, dominated by Gram-positive bacteria, notably staphylococci and Corynebacterium.