Detection and Enumeration of Spore-Forming Bacteria in Powdered Dairy Products

peer-reviewedWith the abolition of milk quotas in the European Union in 2015, several member states including Ireland, Luxembourg, and Belgium have seen year on year bi-monthly milk deliveries to dairies increase by up to 35%. Milk production has also increased outside of Europe in the past number of years. Unsurprisingly, there has been a corresponding increased focus on the production of dried milk products for improved shelf life. These powders are used in a wide variety of products, including confectionery, infant formula, sports dietary supplements and supplements for health recovery. To ensure quality and safety standards in the dairy sector, strict controls are in place with respect to the acceptable quantity and species of microorganisms present in these products. A particular emphasis on spore-forming bacteria is necessary due to their inherent ability to survive extreme processing conditions. Traditional microbiological detection methods used in industry have limitations in terms of time, efficiency, accuracy, and sensitivity. The following review will explore the common spore-forming bacterial contaminants of milk powders, will review the guidelines with respect to the acceptable limits of these microorganisms and will provide an insight into recent advances in methods for detecting these microbes. The various advantages and limitations with respect to the application of these diagnostics approaches for dairy food will be provided. It is anticipated that the optimization and application of these methods in appropriate ways can ensure that the enhanced pressures associated with increased production will not result in any lessening of safety and quality standards

Mesophilic sporeformers identified in whey powder by using shotgun metagenomic sequencing

peer-reviewedSpoilage and pathogenic spore-forming bacteria are a major cause of concern for producers of dairy products. Traditional agar-based detection methods employed by the dairy industry have limitations with respect to their sensitivity and specificity. The aim of this study was to identify low-abundance sporeformers in samples of a powdered dairy product, whey powder, produced monthly over 1 year, using novel culture-independent shotgun metagenomics-based approaches. Although mesophilic sporeformers were the main target of this study, in one instance thermophilic sporeformers were also targeted using this culture-independent approach. For comparative purposes, mesophilic and thermophilic sporeformers were also tested for within the same sample using culture-based approaches. Ultimately, the approaches taken highlighted differences in the taxa identified due to treatment and isolation methods. Despite this, low levels of transient, mesophilic, and in some cases potentially pathogenic sporeformers were consistently detected in powder samples. Although the specific sporeformers changed from one month to the next, it was apparent that 3 groups of mesophilic sporeformers, namely, Bacillus cereus, Bacillus licheniformis/Bacillus paralicheniformis, and a third, more heterogeneous group containing Brevibacillus brevis, dominated across the 12 samples. Total thermophilic sporeformer taxonomy was considerably different from mesophilic taxonomy, as well as from the culturable thermophilic taxonomy, in the one sample analyzed by all four approaches. Ultimately, through the application of shotgun metagenomic sequencing to dairy powders, the potential for this technology to facilitate the detection of undesirable bacteria present in these food ingredients is highlighted

Tracking the Dairy Microbiota from Farm Bulk Tank to Skimmed Milk Powder.

peer-reviewedMicroorganisms from the environment can enter the dairy supply chain at multiple stages, including production, milk collection, and processing, with potential implications for quality and safety. The ability to track these microorganisms can be greatly enhanced by the use of high-throughput DNA sequencing (HTS). Here HTS, both 16S rRNA gene amplicon and shotgun metagenomic sequencing were applied to investigate the microbiomes of fresh mid- and late-lactation milk collected from farm bulk tanks, collection tankers, milk silos, skimmed milk silos, a cream silo, and powder samples to investigate the microbial changes throughout a skim milk powder manufacturing process. 16S rRNA gene analysis established that the microbiota of raw milks from farm bulk tanks and in collection tankers were very diverse but that psychrotrophic genera associated with spoilage, Pseudomonas and Acinetobacter, were present in all samples. Upon storage within the whole-milk silo at the processing facility, the species Pseudomonas fluorescens and Acinetobacter baumannii became dominant. The skimmed milk powder generated during the mid-lactation period had a microbial composition that was very different from that of raw milk; specifically, two thermophilic genera, Thermus and Geobacillus, were enriched. In contrast, the microbiota of skimmed milk powder generated from late-lactation milk more closely resembled that of the raw milk and was dominated by spoilage-associated psychrotrophic bacteria. This study demonstrates that the dairy microbiota can differ significantly across different sampling days. More specifically, HTS can be used to trace microbial species from raw milks through processing to final powdered products.IMPORTANCE Microorganisms can enter and persist in dairy at several stages of the processing chain. Detection of microorganisms within dairy food processing is currently a time-consuming and often inaccurate process. This study provides evidence that high-throughput sequencing can be used as an effective tool to accurately identify microorganisms along the processing chain. In addition, it demonstrates that the populations of microbes change from raw milk to the end product. Routine implementation of high-throughput sequencing would elucidate the factors that influence population dynamics. This will enable a manufacturer to adopt control measures specific to each stage of processing and respond in an effective manner, which would ultimately lead to increased food safety and quality

Effect of milk centrifugation and incorporation of high heat-treated centrifugate on the microbial composition and levels of volatile organic compounds of Maasdam cheese

peer-reviewedCentrifugation is a common milk pretreatment method for removal of Clostridium spores which, on germination, can produce high levels of butyric acid and gas, resulting in rancid, gassy cheese. The aim of this study was to determine the effect of centrifugation of milk, as well as incorporation of high heat-treated centrifugate into cheese milk, on the microbial and volatile profile of Maasdam cheese. To facilitate this, 16S rRNA amplicon sequencing in combination with a selective media-based approach were used to study the microbial composition of cheese during maturation, and volatile organic compounds within the cheese matrix were analyzed by HPLC and solid-phase microextraction coupled with gas chromatography–mass spectrometry. Both culture-based and molecular approaches revealed major differences in microbial populations within the cheese matrix before and after warm room ripening. During warm room ripening, an increase in counts of propionic acid bacteria (by ∼101.5 cfu) and nonstarter lactic acid bacteria (by ∼108 cfu) and a decrease in the counts of Lactobacillus helveticus (by ∼102.5 cfu) were observed. Lactococcus species dominated the curd population throughout ripening, followed by Lactobacillus, Propionibacterium, and Leuconostoc, and the relative abundance of these accounted for more than 99% of the total genera, as revealed by high-throughput sequencing. Among subdominant microflora, the overall relative abundance of Clostridium sensu stricto was lower in cheeses made from centrifuged milk than control cheeses, which coincided with lower levels of butyric acid. Centrifugation as well as incorporation of high heat-treated centrifugate into cheese milk seemed to have little effect on the volatile profile of Maasdam cheese, except for butyric acid levels. Overall, this study suggests that centrifugation of milk before cheesemaking is a suitable method for controlling undesirable butyric acid fermentation without significantly altering the levels of other volatile organic compounds of Maasdam cheese

Microbiome-based environmental monitoring of a dairy processing facility highlights the challenges associated with low microbial-load samples

peer-reviewedEfficient and accurate identification of microorganisms throughout the food chain can potentially allow the identification of sources of contamination and the timely implementation of control measures. High throughput DNA sequencing represents a potential means through which microbial monitoring can be enhanced. While Illumina sequencing platforms are most typically used, newer portable platforms, such as the Oxford Nanopore Technologies (ONT) MinION, offer the potential for rapid analysis of food chain microbiomes. Initial assessment of the ability of rapid MinION-based sequencing to identify microbes within a simple mock metagenomic mixture is performed. Subsequently, we compare the performance of both ONT and Illumina sequencing for environmental monitoring of an active food processing facility. Overall, ONT MinION sequencing provides accurate classification to species level, comparable to Illumina-derived outputs. However, while the MinION-based approach provides a means of easy library preparations and portability, the high concentrations of DNA needed is a limiting factor.Department of Agriculture, Food and the Marin

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus

peer-reviewNisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus

peer-reviewNisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains

Evaluation of methods for the reduction of contaminating host reads when performing shotgun metagenomic sequencing of the milk microbiome

peer reviewedShotgun metagenomic sequencing is a valuable tool for the taxonomic and functional profiling of microbial communities. However, this approach is challenging in samples, such as milk, where a low microbial abundance, combined with high levels of host DNA, result in inefficient and uneconomical sequencing. Here we evaluate approaches to deplete host DNA or enrich microbial DNA prior to sequencing using three commercially available kits. We compared the percentage of microbial reads obtained from each kit after shotgun metagenomic sequencing. Using bovine and human milk samples, we determined that host depletion with the MolYsis complete5 kit significantly improved microbial sequencing depth compared to other approaches tested. Importantly, no biases were introduced. Additionally, the increased microbial sequencing depth allowed for further characterization of the microbiome through the generation of metagenome-assembled genomes (MAGs). Furthermore, with the use of a mock community, we compared three common classifiers and determined that Kraken2 was the optimal classifier for these samples. This evaluation shows that microbiome analysis can be performed on both bovine and human milk samples at a much greater resolution without the need for more expensive deep-sequencing approaches.Irish Dairy Lev