Post-pasteurization Contamination Research Articles

Short communication: Comparing the microbiota diversity from the core, middle part and rind of six Spanish commercial goat cheeses

The cheese core has a lower oxygen saturation and salinity and a higher acidity than the rind, but there is controversy about the incidence of such factors on the magnitude of microbial diversity. The goal of the current work was to investigate the existence of differences in α-diversity between the core, middle part, and rind of six Spanish commercial cheeses through a sequencing approach. To this end, we have collected rind, middle part, and core samples from fresh (H and M), soft semi-ripened (C and P), hard semi-ripened (B) and semi-hard aged (G) goat cheeses. After purifying deoxyribonucleic acid from these 18 samples, the V3-V4 ultravariable region of the 16S rRNA gene was sequenced. The analysis of microbial composition revealed that lactic acid bacteria from the genera Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc are predominant in all six goat cheeses. Furthermore, we identified several psychrophilic taxa often associated with the post-pasteurization contamination of refrigerated milk. Comparison of three α-diversity estimators (Chao1, Shannon and Faith's phylogenetic diversity indices) of microbiota in the core, middle part, and rind of all six goat cheeses did not reveal substantial differences, being only significant (at the nominal level) the comparison of rind vs middle part for the Shannon index (P-value = 0.031). Moreover, the construction of a dendrogram based on Aitchison distances revealed that cheese samples cluster according to their manufacturing characteristics, with a clear distinction between fresh vs semi-ripened or aged cheeses. We conclude that the magnitude of microbial α-diversity in the cheese core is similar to that in the rind despite their different physicochemical attributes. This result could be because physicochemical differences between cheese compartments are often attenuated during cheese ripening.

Analysis of Microbiological Quality and Antibiotic Resistance Patterns in Milk Supply Chain

The widespread consumption of milk for its nutritional value and health benefits brings the risk of milk-borne diseases due to the presence of various microorganisms, including antibiotic-resistant pathogens. This has led to an increased focus on ensuring the safety of milk products across the supply chain by dairy industries. The study aimed to evaluate microbiological parameters and detect multi-antibiotic-resistant pathogens at three specific supply points and to explore the association between the presence of residual antibiotics and the resistant isolates in milk samples. About 50 milk samples, including raw, soon-after-processed, packaged marketed pasteurized, and UHT milk, were subjected to microbiological analysis. This involved assessing the total bacterial count (TBC) and total coliform count (TCC), conducting antibiotic susceptibility tests through disk and well diffusion assays, detecting virulence genes in multi-antibiotic resistant isolates using gene-specific PCR, and analyzing residual antibiotics by HPLC. The study revealed that the quality of raw milk samples was unacceptable (TBC >4.5x107 CFU/mL and TCC >5.6x104 CFU/mL), while pasteurized samples from processing plants had lower counts than those from retail stores (TBC >5x105 CFU/mL and TCC >1.6x104 CFU/mL) indicating post-pasteurization contamination. About 70.37% of the isolates were Gram-negative, with Escherichia coli (21.4%) and Vibrio (18.8%) being the most prevalent. Resistance to antibiotics was substantial, particularly against ampicillin (86.3%), tetracycline (76%), and ciprofloxacin (58.9%). Gene-specific PCR analysis detected uidA, oprL, and oprI virulence genes in multi-drug-resistant Escherichia coli and Pseudomonas sp. respectively. The study also revealed a direct association between the presence of residual antibiotics and the resistant isolates, emphasizing the need for dairy industry improvements. As high bacterial counts in milk can pose health risks by fostering antibiotic-resistant pathogens, it is essential to mitigate microbiological contamination in the milk supply chain through the implementation of various precautionary measures.
 Bioresearch Commu. 10(1): 1462-1473, 2024 (January)

Phenotypic and genomic characterizations of Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata strains reveal no clear association between genetic content and ropy phenotype

Ropy defect of pasteurized fluid milk is a type of spoilage which manifests itself by an increased viscosity, slimy body, and string-like flow during pouring. This defect has, among other causes, been attributed to the growth, proliferation and exopolysaccharide production by coliform bacteria, which are most commonly introduced in milk as post-pasteurization contaminants. As we identified both Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata that were linked to a ropy defect, the goal of this study was to characterize 3 K. pneumoniae ssp. pneumoniae strains and 2 R. inusitata for (i) their ability to grow and cause ropy defect in milk at 6 and 21°C and to (ii) probe the genetic basis for observed ropy phenotype. While all K. pneumoniae ssp. pneumoniae and R. inusitata strains showed net growth of > 4 log10 over 48 h in UHT milk at 21°C, only R. inusitata strains displayed growth during 28-d incubation period at 6°C (>6 log10). Two out of 3 K. pneumoniae ssp. pneumoniae strains were capable of causing the ropy defect in milk at 21°C, as supported by an increase in the viscosity of milk and string-like flow during pouring; these 2 strains were originally isolated from raw milk. Only one R. inusitata strains was able to cause the ropy defect in milk; this strain was able to cause the defect at both 6 and 21°C, and was originally isolated from a pasteurized milk. These findings suggest that the potential of K. pneumoniae ssp. pneumoniae and R. inusitata to cause ropy defect in milk is a strain-dependent characteristic. Comparative genomics provided no definitive answer on genetic basis for the ropy phenotype. However, for K. pneumoniae ssp. pneumoniae, genes rffG, rffH, rfbD, and rfbC involved in biosynthesis and secretion of enterobacterial common antigen (ECA) could only be found in the 2 strains that produced ropy defect, and for R. inusitata a set of 2 glycosyltransferase- and flippase genes involved in nucleotide sugar biosynthesis and export could only be identified in the ropy strain. While these results provide some initial information for potential markers for strains that can cause ropy milk, the relationship between genetic content and ropiness in milk remains poorly understood and merits further investigation.

Gram-negative postpasteurization contamination patterns of single-serve fluid milk produced in 4 different processing facilities

An analysis of historic data on high temperature, short time (HTST) fluid milk quality showed higher total bacterial counts and lower sensory defect judging scores at d 14 post-processing for milk packaged in single-serve containers as compared with milk packaged in half-gallon containers from the same processing facilities. As post-pasteurization contamination with gram-negative bacteria is likely a major contributor to an increased spoilage risk associated with milk packaged in single-serve containers, we performed a comprehensive assessment of the microbial quality and shelf-life of 265 commingled single-serve HTST fluid milk samples (including white (unflavored) skim, white (unflavored) 1%, chocolate skim, and chocolate 1%) collected over 2 visits to 4 commercial fluid milk processing facilities. Over 2 initial sampling visits, the frequency of gram-negative spoilage ranged from 14 to 79% of the product collected from the 4 facilities, with significant differences of gram-negative spoilage frequency between sampling visits, facilities (sampling visit 1, sampling visit 2, and both sampling visits combined), milk types (sampling visit 2), and filler lanes (sampling visit 2). There were no significant differences in the frequency of gram-negative spoilage between sampling time points (e.g., beginning, middle, and end of production run). Across facilities, single-serve containers of milk with gram-negative contamination showed significantly higher bacterial counts on d 7 and 14 and significantly lower sensory scores as compared with those without gram-negative contamination. Follow-up investigations, based on in-facility surveys that identified carton forming mandrels as filler components that frequently failed quality assurance ATP swab checks, found that bacterial genera, including Pseudomonas and Bacillus, isolated from single-serve milk samples were also frequently isolated from mandrels. While interventions aimed at improving cleaning and sanitation of mandrels did not lead to significant reduction of gram-negative spoilage frequency in a comparison of 398 control and 400 intervention samples, our data still suggest that the unhygienic design of single-serve fillers is likely a root-cause of gram-negative contamination of single-serve milk.

When Does Milk Spoil? The Use of Rejection Threshold Methodology to Investigate the Influence of Total Microbial Numbers on the Acceptability of Fresh Chilled Pasteurised Milk

The consumer rejection threshold (RjT) method was applied to determine the total microbial numbers (TMNs) where consumers find that the quality of whole fresh chilled pasteurised milk (WFCPM) and skim milk (Trim) stored at 4.5 ± 0.5 °C is no longer acceptable. Food spoilage progression was supported by measurements of VOCs and the terms consumers used to describe the ageing fresh chilled pasteurised milk (FCPM). RjTs for TMN of 7.43 and 7.34 log10 CFU.mL−1 for WFCPM and Trim, respectively were derived using Hill’s equation from a series of paired preference tests comparing fresh and aged milks (3–26 days) assessed by consumers (WFCPM, n = 55; Trim, n = 52). A poor relationship between storage time and TMN was found, owing mainly to batch-to-batch and within-batch variation in the milk’s post-pasteurization contamination (PPC) levels. At the RjT, there was a significant change in the signal intensities for a number of spoilage-related VOCs that occurred in the FCPM headspace (p ≤ 0.05), which were measured using proton transfer reaction–mass spectrometry (PTR-MS), including m/z 33, 45, 47, 61, 63, 69, 71, 87, and 89, tentatively identified as methanol; acetaldehyde; ethanol; acetate (acetic acid and acetate esters); dimethyl sulphide (DMS); isoprene, furan, and aldehydes; 2-butanone; and pentanal and butyrates (butyric acid and butyrate esters), respectively. Consumers described the milks at TMN greater than the RjTs using terms like off, expired, sour, spoilt or rancid. This multidisciplinary study has provided data on the importance of PPC and subsequent increases in TMN on VOCs associated with FCPM and consumer’s preferences and highlighted the value of measuring a range of variables when investigating consumer’s perception of food quality and shelf-life.

Shelf-life storage temperature has a considerably larger effect than high-temperature, short-time pasteurization temperature on the growth of spore-forming bacteria in fluid milk

In the absence of postpasteurization contamination, psychrotolerant, aerobic spore-forming bacteria that survive high-temperature, short-time (HTST) pasteurization, limit the ability to achieve HTST extended shelf-life milk. Therefore, the goal of the current study was to evaluate bacterial outgrowth in milk pasteurized at different temperatures (75, 85, or 90°C, each for 20 s) and subsequently stored at 3, 6.5, or 10°C. An initial ANOVA of bacterial concentrations over 14 d of storage revealed a highly significant effect of storage temperatures, but no significant effect of HTST. At d 14, average bacterial counts for milk stored at 3, 6.5, and 10°C were 1.82, 3.55, and 6.86 log10 cfu/mL, respectively. Time to reach 1,000,000 cfu/mL (a bacterial concentration where consumers begin to notice microbially induced sensory defects in fluid milk) was estimated to be 68, 27, and 10 d for milk stored at 3, 6.5, and 10°C, respectively. Out of 95 isolates characterized with rpoB allelic typing, 6 unique genera, 15 unique species, and 44 unique rpoB allelic types were represented. The most common genera identified were Paenibacillus, Bacillus, and Lysinibacillus. Nonmetric multidimensional scaling identified that Bacillus was significantly associated with 3 and 10°C, whereas Paenibacillus was consistently found across all storage temperatures. Overall, our data show that storage temperature has a substantially larger effect on fluid milk shelf life than HTST and suggests that abuse temperatures (e.g., storage at 10°C) allow for growth of Bacillus species (including Bacillus cereus genomospecies) that do not grow at lower temperatures. This indicates that stringent control of storage and distribution temperatures is critical for producing extended shelf-life HTST milk, particularly concerning new distribution pathways for HTST pasteurized milk (e.g., electronic commerce), and when enhanced control of spores in raw milk is not feasible.

Pasteurized milk quality in Brazil: a cross-sectional study over five years.

This research communication delineates the quality of pasteurized cow milk sold in Brazil from 2015 to 2020. A cross-sectional study was performed gathering 1749 samples, which were evaluated for microbiological and physicochemical parameters, including Salmonella spp., total and thermotolerant coliforms, freezing point, alkaline phosphatase and lactoperoxidase. The proportion of compliant and non-compliant samples was compared through the years and jurisdiction of the inspection services. Interactions between the design and response variables were assessed by log-linear analysis. Overall, a considerable non-conformity rate (12%) was found for at least one microbiological or physicochemical parameter. Post-pasteurization contamination by coliforms was the major challenge for dairy industries. Notably, the non-compliance rate for freezing point increased during the SARS-CoV-2 pandemic. In addition, the ability to comply was linked to the type of inspection service. Thus, it is suggested that the SARS-CoV-2 pandemic is affecting the dairy industries in Brazil, and we strengthen the need for more studies monitoring the quality of milk over the years, which could assist industries and regulatory agencies to ensure the compliance of pasteurized milk.

BACTERIOLOGICAL QUALITY OF UNPACKAGED POWERED MILK SOLD WITHIN KADUNA

Milk is a nutritious food and a excellent source of protein needed for growth and body building. It’s costly nature however makes it unaffordable by most people especially in a developing country like Nigeria. The aim of this study is to evaluate the bacteriological quality of unpackaged powdered milk sold within Kaduna south local government. The objectives are to determine the bacteriological load of three brands of powdered milk and to isolate and identify bacteria from the samples analyzed. This was done using standard microbiological methods. Thirty milk samples from three different brands (A, B and C) were collected from markets within Kaduna south local government. The bacterial load of brand A ranged from 1.26x 105_ 7.0x105 with a mean viable count of 4.0x105 ± 2.9 that of brand B ranged from 1.38x105-2.44x105 with a meancount of 2.0x105 ± 0.5 while that of brand C ranged from 1.00x105-2.0x105 with a mean count of 1.3x105±0.5.Three genera of bacteria (Bacillus spp Staphylococcus aureus, and Streptococcus spp) with percentage occurance of 60, 23.3 nd 16.7% respectively were isolated from all the milk samples analysed. The study establish evidence of contamination of all the milk samples with the total viable count in all the samples(>104 CFU/ g). Proper hygienic practices should be enforced and maintained during handling to reduce post pasteurization contamination and spoilage of powdered milk.

Antifungal activity of protective cultures against the yogurt drink-specific spoilage yeasts.

Fungal agents emerged as post-pasteurization contamination are responsible for the spoilage in yogurt drink. In this work, the antifungal effects of some lactic acid bacteria (LAB) on the spoilage yeasts isolated from yogurt drink (Doogh) were evaluated. First, the microbial growth in the yogurt drink samples during the storage time was investigated, and the isolated microorganisms were identified using biochemical methods and sequencing of the specific amplicons. Yeasts (3-7 log CFU ml-1 ) were found to be the most abundant microorganisms (specific spoilage organisms) in several samples. Using the amplification technique of rDNA by ITS1 and ITS4 primers, the dominant yeasts were identified as Pichia kudriavzevii, Kluyveromyces marxianus, and Candida parapsilosis. Then, the antimicrobial activity of 37 strains of LAB against the isolated yeasts was studied using broth microdilution. Eventually, the strains of Lacticplantibacillus plantarum (245, 24, P6, and P7), Lactiplantibacillus pentosus (20), and Levilactobacillus brevis (30) exhibited significant antifungal activity. In the most effective impacts, lag times of C. parapsilosis, K. marxianus, and P. kudriavzevii were increased by almost 12-19h, 12-19h, and 2-6h, respectively, while the area under the growth curve for these yeasts was reduced to lower than 40%, near 16%, and approximately 67%, in the order given. Overall, these bacteria showed high potential as the substituents for chemical preservatives in yogurt drinks. PRACTICAL APPLICATION: Spoilage yeasts were isolated from yogurt drink and identified by molecular method. Isolated yeasts belonged to Pichia, Kluyveromyces, and Candida genera. Inhibitory effects of 37 strains were evaluated against the spoilage yeasts. Cell-free supernatant was used against the isolated fungi in microdilution method. Several LAB strains showed a significant antimicrobial activity.

Serological identification, virulence factors, antibiogram and plasmid profile of Escherichia coli serotypes in raw milk and pasteurized milk products

Milk is an excellent medium that supports microbial growth with resultant spoilage of the products. Thus, this study determined the antibiogram, presumptive location of antibiotic resistance markers (ARM), and plasmid DNA molecular weight of pathogenic Escherichia coli serotypes in raw milk (n = 76) and pasteurized milk products (n = 77). Quality of raw milk and pasteurized milk products was evaluated using Methylene Blue Reduction Test (MBRT). Occurrence of E. coli in the samples was determined by Cefixime tellurite-sorbitol MacConkey agar and latex agglutination kits. The virulence factors, antibiotic susceptibility, location of ARM, and plasmid DNA molecular weight of E. coli serotypes were assayed using bacteriological media, disc diffusion, acridine orange and agarose gel electrophoresis, respectively. Based on the MBRT, 2.6% samples were graded as excellent, 9.8% samples were of good quality, while 22.2% samples were of a very poor quality as decolourization of methylene blue occurred in ˂ 20 min. E. coli 0157 and E. coli 0145 had the highest and lowest percentage of occurrence, respectively; ≤ 29 E. coli serotypes were caseinase, amylase and lecithinase producers. E. coli serotypes were highly sensitive to Ciprofloxacin and Gentamycin with percentage sensitivity ranging from 74.6 % to 81.4 %; ≤ 50.0 % E. coli 0125 were sensitive to Ofloxacin; between 33.3 % and 50.0 % E. coli 0103 were resistant to Nalidixic Acid and Ofloxacin; ≥ 53.7 % E. coli serotypes were multi-drug resistant; 19.0 % had their entire ARM located on the plasmids, while 50.0 % had their ARM residing both on the plasmids and chromosomes with plasmids sizes ranging from 0.54 kb to 43.5 kb. The occurrences of pathogenic E. coli in the milk samples indicated the need to enlighten milkers on the hygienic practices to adopt during milking and as well showed that post-pasteurization contamination of milk products should be avoided.

An Overview of Antimicrobial Activity of Lysozyme and Its Functionality in Cheese.

Due to the concern of consumers about the presence of synthetic preservatives, researchers and food manufacturers have recently conducted extensive research on the limited use of these preservatives and the introduction and use of natural preservatives, such as herbal extracts and essential oils, bacteriocins, and antimicrobial enzymes. Lysozyme is a natural enzyme with antimicrobial activity that has attracted considerable attention to be potentially utilized in various industries. Since lysozyme is an intrinsic component of the human immune system and has low toxicity; it could be considered as a natural antimicrobial agent for use in food and pharmaceutical industries. Lysozyme exerts antimicrobial activity against microorganisms, especially Gram-positive bacteria, by hydrolyzing 1,4-beta-linkages between N-acetylmuramic acid and N-acetylglucosamine in the cell wall. In addition, increased antimicrobial activity of lysozyme against Gram-negative bacteria could be achieved by the modification of lysozyme through physical or chemical interactions. Lysozyme is presented as a natural preservative in mammalian milk and can be utilized as a bio-preservative in dairy products, such as cheese. Both bacteria and fungi can contaminate and spoil the cheese; especially the one that is made traditionally by raw milk. Furthermore, uncontrolled and improper processes and post-pasteurization contamination can participate in the cheese contamination. Therefore, besides common preservative strategies applied in cheese production, lysozyme could be utilized alone or in combination with other preservative strategies to improve the safety of cheese. Hence, this study aimed to review the antimicrobial properties of lysozyme as natural antimicrobial enzyme and its functionality in cheese.

Development of a Monte Carlo simulation model to predict pasteurized fluid milk spoilage due to post-pasteurization contamination with gram-negative bacteria

Psychrotolerant gram-negative bacteria introduced as post-pasteurization contamination (PPC) are a major cause of spoilage and reduced shelf life of high-temperature, short-time pasteurized fluid milk. To provide improved tools to (1) predict pasteurized fluid milk shelf life as influenced by PPC and (2) assess the effectiveness of different potential interventions that could reduce spoilage due to PPC, we developed a Monte Carlo simulation model that predicts fluid milk spoilage due to psychrotolerant gram-negative bacteria introduced as PPC. As a first step, 17 gram-negative bacterial isolates frequently associated with fluid milk spoilage were selected and used to generate growth data in skim milk broth at 6°C. The resulting growth parameters, frequency of isolation for the 17 different isolates, and initial concentration of bacteria in milk with PPC, were used to develop a Monte Carlo model to predict bacterial number at different days of shelf life based on storage temperature of milk. This model was then validated with data from d 7 and 10 of shelf life, collected from commercial operations. The validated model predicted that the parameters (1) maximum growth rate and (2) storage temperature had the greatest influence on the percentage of containers exceeding 20,000 cfu/mL standard plate count on d 7 and 10 (i.e., spoiling due to PPC), which indicates that accurate data on maximum growth rate and storage temperature are important for accurate predictions. In addition to allowing for prediction of fluid milk shelf life, the model allows for simulation of "what-if" scenarios, which allowed us to predict the effectiveness of different interventions to reduce overall fluid milk spoilage due to PPC through a set of proof-of-concept scenario (e.g., frequency of PPC in containers reduced from 100% to 10%; limiting distribution temperature to a maximum of 6°C). Combined with other models, such as previous models on fluid milk spoilage due to psychrotolerant spore-forming bacteria, the data and tools developed here will allow for rational, digitally enabled, fluid milk shelf life prediction and quality enhancement.

Machine Learning and Advanced Statistical Modeling Can Identify Key Quality Management Practices That Affect Postpasteurization Contamination of Fluid Milk

Spoilage of high-temperature, short-time (HTST)- and vat-pasteurized fluid milk due to the introduction of gram-negative bacteria postpasteurization remains a challenge for the dairy industry. Although processing facility-level practices (e.g., sanitation practices) are known to impact the frequency of postpasteurization contamination (PPC), the relative importance of different practices is not well defined, thereby affecting the ability of facilities to select intervention targets that reduce PPC and provide the greatest return on investment. Thus, the goal of this study was to use an existing longitudinal data set of bacterial spoilage indicators obtained for pasteurized fluid milk samples collected from 23 processing facilities between July 2015 and November 2017 (with three to five samplings per facility) and data from a survey on fluid milk quality management practices, to identify factors associated with PPC and rank their relative importance. This ranking was accomplished using two separate approaches: multimodel inference and conditional random forest. Data preprocessing for multimodel inference analysis showed (i) nearly all factors were significantly associated with PPC when assessed individually using univariable logistic regression and (ii) numerous pairs of factors were strongly associated with each other (Cramer's V ≥ 0.80). Multimodel inference and conditional random forest analyses identified similar drivers associated with PPC; factors identified as most important based on these analyses included cleaning and sanitation practices, activities related to good manufacturing practices, container type (a proxy for different filling equipment), in-house finished product testing, and designation of a quality department, indicating potential targets for reducing PPC. In addition, this study illustrates how machine learning approaches can be used with highly correlated and unbalanced data, as typical for food safety and quality, to facilitate improved data analyses and decision making.

Assessing hygiene indicators in two dairies in Algeria in producing pasteurized milk.

Background and Aim:There is a worldwide controversy about the choice of microbial flora for use as process hygiene indicators. This study aimed to evaluate the pertinence of using either coliforms or Enterobacteriaceae (EB) as process hygiene indicators in the pasteurized milk production line. Two flora families and total flora were used as bacterial indicators in some stages of pasteurized milk production line to identify the origin of post-pasteurization contamination and compare the results obtained for each flora. In addition, the bacteriological profile of isolated coliforms and EB was developed.Materials and Methods:One thousand and two hundred samples of pasteurized cow milk and surfaces (pipes and tank) at various processing stages were taken from two dairies in the northern region of Algeria. The total microbial flora (TF), total coliforms (TC), thermotolerant coliforms, and EB were enumerated, following the recommendations of ISO 4833:2006, ISO 4832:2006, and ISO 21528-2:2017 methods, respectively. The bacteriological profile was determined using the API 20E and 10S tests (bioMérieux, France). Furthermore, the cleaning efficiency and disinfection protocol of surfaces were evaluated using contact agar slides 1 (Liofilchem™, Italy).Results:Enumeration of the different indicators shows that the highest contamination rate is recorded by the total flora in the two units, 3.28 and 3.78 log CFU/mL, respectively. EB (−0.60 log CFU/mL) at post-pasteurization stage in Unit 1 and coliforms (0.44 log CFU/mL) at the pasteurized packaged milk stage in Unit 2 are the least significant germ families. The lowest compliance rates of bacterial contamination were reported for total flora (82-85%) at the three sampled sites in Unit 2. In comparison, the highest was reported in Unit 1 (99.8%) and 2 (98%) by the EB indicator. Assessing the surface cleaning and disinfection protocol compliance shows that the tank records the highest non-compliance rates for EB and TF (4% and 3%) in Unit 2. EB are represented in both units by various species. Acinetobacter baumannii in Unit 1 and Enterobacter cloacae in Unit 2 are the common species of the three indicator families. Acinetobacter and Enterobacter in Unit 1, Escherichia, Citrobacter, Enterobacter, Klebsiella, and Hafnia in Unit 2 are the most time persistent bacterial genera along the production line. Stenotrophomonas, Serratia, Salmonella, Enterobacter, and Escherichia are common genera in both units.Conclusion:The results obtained show no difference in the use of EB or TC as hygiene indicators. However, if the objective is to identify the species of bacterial populations, using EBs are the most appropriate.

Identification, subtyping, and tracking of dairy spoilage-associated Pseudomonas by sequencing the ileS gene

Pseudomonas spp. are important spoilage bacteria that negatively affect the quality of refrigerated fluid milk and uncultured cheese by generating unwanted odors, flavors, and pigments. They are frequently found in dairy plant environments and enter dairy products predominantly as postpasteurization contaminants. Current subtyping and characterization methods for dairy-associated Pseudomonas are often labor-intensive and expensive or provide limited and possibly unreliable classification information (e.g., to the species level). Our goal was to identify a single-copy gene that could be analyzed in dairy spoilage-associated Pseudomonas for preliminary species-level identification, subtyping, and phenotype prediction. We tested 7 genes previously targeted in a Pseudomonas fluorescens multilocus sequence typing scheme for their individual suitability in this application using a set of 113 Pseudomonas spp. isolates representing the diversity of typical pasteurized milk contamination. For each of the 7 candidate genes, we determined the success rate of PCR and sequencing for these 113 isolates as well as the level of discrimination for species identification and subtyping that the sequence data provided. Using these metrics, we selected a single gene, isoleucyl tRNA synthetase (ileS), which had the most suitable traits for simple and affordable single-gene Pseudomonas characterization. This was based on the number of isolates successfully sequenced for ileS (113/113), the number of unique allelic types assigned (83, compared with 50 for 16S rDNA), nucleotide and sequence diversity measures (e.g., number of unique SNP and Simpson index), and tests for genetic recombination. The discriminatory ability of ileS sequencing was confirmed by separation of 99 additional dairy Pseudomonas spp. isolates, which were indistinguishable by 16S rDNA sequencing, into 28 different ileS allelic types. Further, we used whole-genome sequencing data to demonstrate the similarities in ileS-based phylogenetic clustering to whole-genome-based clustering for 27 closely related dairy-associated Pseudomonas spp. isolates and for 178 Pseudomonas type strains. We also found that dairy-associated Pseudomonas within an ileS cluster typically shared the same proteolytic and lipolytic activities. Use of ileS sequencing provides a promising strategy for affordable initial characterization of Pseudomonas isolates, which will help the dairy industry identify, characterize, and track Pseudomonas in their facilities and products.

Epidemiological Investigation, Serotypes and Distribution of Verocytotoxigenic <i>Escherichia coli</i> (VTEC) in Raw Milk and Milk Products in Uyo, Nigeria

Food borne diseases are of great concern globally especially in the developing countries where poor sanitation is applied during collection and processing of milk from animals. The epidemiological investigation, serotypes and distribution of verocytotoxin (VTI and VT2)- producing Escherichia coli in raw milk and milk products were determined using structured questionnaire, Cefixime tellurite-sorbitol MacConkey agar, agglutination kits and VTEC-RPLA Toxin detection Kit. Out of 27 milkers, 7.4 % had primary education, 22.2 % washed the milk utensils with cold water and soap, 11.1 % washed their hands before milking, while 7.4 % milkers washed the udder of the animals before milking. All the yoghurts had the product names; 85.7 % had NAFDAC numbers; 80.0% had Batch Numbers, while 71.4 % had Manufacturer s’ Addresses. The unpasteurized milk samples had E. coli 0157 and non 0157 E. coli counts (CFU.ml-1) ranging from 4.0 x 102 to 1.7 x 103 and 6.0 x 102 to 2.0 x 103 , respectively, while E. coli 0157 and non 0157 E. coli counts of milk products were between 1.0 x 102 and 1.0 x 103 CFU.ml-1. E. coli 0157 had the highest percentage occurrence (38.3%), while E. coli 0145 had the lowest percentage occurrence (2.1%). More than 38.3% of the E. coli serotypes produced VT2, while ≥ 12.8% were VT1 producers. The occurrence of VTEC in the unpasteurized milk shows that the milkers should be enlightened on the necessary sanitary practices to adopt during milking and also post-pasteurization contamination of milk products should be avoided.
 Key Words: Verotoxigenic, Escherichia coli, Milk, Yoghurt, Nono, Serotypes.

Interventions designed to control postpasteurization contamination in high-temperature, short-time-pasteurized fluid milk processing facilities: A case study on the effect of employee training, clean-in-place chemical modification, and preventive maintenance programs

Postpasteurization contamination (PPC) with gram-negative bacteria adversely affects the quality and shelf-life of milk through the development of flavor, odor, texture, and visual defects. Through evaluation of milk quality at 4 large fluid milk processing facilities in the northeast United States, we examined the efficacy of 3 strategies designed to reduce the occurrence of PPC in fluid milk: (1) employee training (focusing on good manufacturing practices) alone and (2) with concurrent implementation of modified clean-in-place chemistry and (3) preventive maintenance (PM) focused on replacement of wearable rubber components. Despite increases in employee knowledge and self-reported behavior change, microbiological evaluation of fluid milk before and after interventions indicated that neither training alone nor training combined with modified clean-in-place interventions significantly decreased PPC. Furthermore, characterization of gram-negative bacterial isolates from milk suggested that specific bacterial taxonomic groups (notably, Pseudomonas sequence types) continued to contribute to PPC even after interventions and that no major changes in the composition of the spoilage-associated microbial populations occurred as a consequence of the interventions. More specifically, in 3 of 4 facilities, gram-negative bacteria with identical 16S rDNA sequence types were isolated on multiple occasions. Evaluation of a PM intervention showed that used rubber goods harbored PPC-associated bacteria and that PPC may have been less frequent following a PM intervention in which wearable rubber goods were replaced (reduction from 3/3 samples with PPC before to 1/3 samples after). Overall, our findings suggest that commonly used "broad stroke interventions" may have a limited effect on reducing PPC. Our case study also demonstrates the inherent complexities of identifying and successfully addressing sanitation problems in large and complex fluid milk processing facilities. For example, broad changes to sanitation practices without improvements in PM and sanitary equipment design may not always lead to reduced PPC. Our data also indicate that although short-term evaluations, such as pre- and post-tests for employee training, may suggest improvements after corrective and preventive actions, extensive microbial testing, ideally in combination with isolate characterization, may be necessary to evaluate return on investment of different interventions.

Short communication: Coliform Petrifilm as an alternative method for detecting total gram-negative bacteria in fluid milk

Postpasteurization contamination (PPC) of fluid milk remains a challenge for some dairy processors. Pseudomonas is the most common contaminant of fluid milk after pasteurization, and therefore methods to detect PPC should be inclusive of Pseudomonas and other gram-negative contaminants (e.g., coliform bacteria). Our objective was to compare the ability of 3M (St. Paul, MN) coliform and Enterobacteriaceae (EB) Petrifilm to detect total gram-negative bacteria with that of the standard method, crystal violet tetrazolium agar. To that end, we evaluated coliform Petrifilm, EB Petrifilm, and crystal violet tetrazolium agar to detect gram-negative bacteria in naturally contaminated samples of fluid milk. A total of 92 observations derived from shelf-life testing of 33 milk samples from 5 different processing facilities were evaluated for (1) presence of coliforms on coliform Petrifilm at both 24 and 48 h of incubation; (2) presence of any growth, regardless of gas production, on coliform Petrifilm at both 24 and 48 h of incubation; (3) presence of EB on EB Petrifilm at both 24 and 48 h of incubation; (4) presence of any growth, regardless of gas or acid production, on EB Petrifilm at both 24 and 48 h of incubation; and (5) presence of gram-negative bacteria on crystal violet tetrazolium agar after 48 h of incubation. Sensitivity and specificity analysis of results indicated that compared with the standard method (i.e., crystal violet tetrazolium agar), the method that performed the best, based on balanced accuracy (i.e., the average of sensitivity and specificity), was coliform Petrifilm evaluated for the presence of any growth after 48 h of incubation (sensitivity = 0.787; specificity = 0.839). This method can be easily adopted by the dairy industry as many processing facilities already test for coliforms using coliform Petrifilm. Improving the ability of processors to detect PPC will improve the quality of the fluid milk supply.

Gouda cheese spoilage prevention: Biodegradable coating induced by Bunium persicum essential oil and lactoperoxidase system.

This study aimed to prepare an inhibitory edible coating for Gouda cheese based on whey protein containing lactoperoxidase system (LPOS) and Bunium persicum essential oil (EO) in order to control postpasteurization contamination. Using a full factorial design, the effects of LPOS and EO on microbiological characteristics and chemical indices of manufactured Gouda cheeses were evaluated during 90 days of storage time. Listeria, lactic acid bacteria, Enterobacter, Escherichia, and Pseudomonas species were considered as potential pathogenic and spoilage indicators of produced Gouda cheese samples. Chemical properties of cheeses were assessed using the free fatty acid, peroxide value, and thiobarbituric acid experiments. The results showed that bacteria counts remained constant in cheese samples coated with EO and also EO–LPOS. However, the survival of gram‐positive lactic acid bacteria and Enterobacter spp. was more pronounced in LPOS‐based coating. The most effective treatments on oxidation stability parameters in cheese samples were EO‐ and EO–LPOS coatings. By the addition of B. persicum EO and LPOS, further inhibition of the lipid oxidation of the cheese samples was achieved. Lipolysis, as a result of lipid degradation, was more pronounced in the control, whey‐coated, and whey–LPOS‐coated samples in comparison with whey–EO‐ and whey–EO–LPOS‐coated samples during the final days of storage time. These results indicate that antibacterial, lipid oxidation, and oxygen barrier properties of the coatings were developed by the addition of B. persicum EO and LPOS.

Listeria monocytogenes in Milk: Occurrence and Recent Advances in Methods for Inactivation

Milk is one of the most important food items consumed by humans worldwide. In addition to its nutritional importance, milk is an excellent culture medium for microorganisms, which may include pathogens such as Listeria monocytogenes (L. monocytogenes). Traditional processing of milk for direct consumption is based on thermal treatments that efficiently eliminate pathogens, including pasteurization or sterilization. However, the occurrence of L. monocytogenes in milk as a consequence of failures in the pasteurization process or postpasteurization contamination is still a matter of concern. In recent years, consumer demand for minimally processed milk has increased due to the perception of better sensory and nutritional qualities of the products. This review deals with the occurrence of L. monocytogenes in milk in the last 10 years, including regulatory aspects, and recent advances in technologies for the inactivation of this pathogen in milk. The results from studies on nonthermal technologies, such as high hydrostatic pressure, pulsed electric fields, ultrasounds, and ultraviolet irradiation, are discussed, considering their potential application in milk processing plants.