Australian consumers' attitudes to packaged fresh meat products with added microbial bioprotective cultures for shelf-life extension.

The application of food-grade microbial cultures to fresh meat products is a promising natural approach for meat shelf-life extension. However, before its adoption into commercial practice, it is essential to understand consumers' attitudes to this approach and the resulting marketed products. This study investigated Australian consumers' willingness to purchase and consume packaged fresh meat products with added microbial cultures for shelf-life extension. A national online survey of over 800 respondents was conducted. Results indicated that most Australian consumers would be willing to buy and eat such products, with 17.8% of respondents less likely to buy and 11.1% unwilling to eat these products. Respondents' purchasing and consumption decisions were influenced by demographic factors, their food and meat shopping and consumption behaviors, and the value, taste, and type of the meat product. Consumer acceptance may be improved by increasing their awareness of the potential use of microbial cultures as natural antimicrobials for food shelf-life extension.

Microbial biopreservatives for controlling the spoilage of beef and lamb meat: their application and effects on meat quality.

Biopreservation is a recognized natural method for controlling the growth of undesirable bacteria on fresh meat. It offers the potential to inhibit spoilage bacteria and extend meat shelf-life, but this aspect has been much less studied compared to using the approach to target pathogenic bacteria. This review provides comprehensive information on the application of biopreservatives of microbial origin, mainly bacteriocins and protective cultures, in relation to bacterial spoilage of beef and lamb meat. The sensory effect of these biopreservatives, an aspect that often receives less attention in microbiological studies, is also reviewed. Microbial biopreservatives were found to be able to retard the growth of the major meat spoilage bacteria, Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae. Their addition did not have any discernible negative impact on the sensory properties of meat, whether assessed by human sensory panels or instrumental and chemical analyses. Although results are promising, the concept of biopreservation for controlling spoilage bacteria on fresh meat is still in its infancy. Studies in this area are still lacking, especially for lamb. Biopreservatives need more testing under conditions representative of commercial meat production, along with studies of any possible sensory effects, in order to validate their potential for large-scale industrial applications.

Evaluation of the potential of protective cultures to extend the microbial shelf-life of chilled lamb meat.

The use of protective cultures to inhibit spoilage bacteria is a promising natural preservation technique to extend the shelf-life of fresh meat. This study evaluated the effectiveness of six food-grade protective cultures (containing different combinations of Lactobacillus sakei, Pediococcus pentosaceus, Staphylococcus xylosus, and Staphylococcus carnosus) on naturally contaminated chill-stored (4 °C) lamb meat in different packaging systems. Only slight reductions of common meat spoilage bacteria Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae were observed in culture-treated samples stored in modified atmosphere packaging (80% O2:20% CO2). Greater inhibitory effects were found in vacuum-packed lamb, with mixed cultures containing either L. sakei, S. carnosus, and S. xylosus or S. carnosus and L. sakei causing the most significant reductions. Protective cultures did not adversely affect meat color or pH. This study demonstrated the potential of protective cultures comprising lactic acid bacteria and coagulase-negative staphylococci in controlling microbial spoilage of lamb and, by inference, other types of meat as a natural solution for shelf-life extension.

Assessment of the potential use of MALDI-TOF MS for the identification of bacteria associated with chilled vacuum-packaged lamb meat.

The focus of this study was to compare the effectiveness of MALDI-TOF MS and partial 16S rRNA gene sequencing for the identification of bacteria isolated from VP lamb meat stored chilled at 5 °C for 21 days, at the same time gaining insights into bacterial changes over time. The identity of bacterial isolates on non-selective and selective agars was determined by both methods and results compared. Results showed that total bacterial numbers increased over the 21 days (as expected) with Staphylococcus and Pseudomonas (day 0) being replaced by Carnobacterium, Brochothrix and members of the Enterobacteriaceae family by day 21. A high level of agreement (86-100%) for bacterial isolates' identity at genus level was observed between MALDI-TOF MS and partial 16S rRNA gene-based sequencing for isolates where identification was possible. With its cheaper cost and faster turnaround time, once optimized, MALDI-TOF MS could become a useful alternative to 16S rRNA gene-sequencing for the rapid identification of red meat bacterial isolates.

Microbiota and Metabolite Profiling Combined With Integrative Analysis for Differentiating Cheeses of Varying Ripening Ages.

Cheese maturation and flavor development results from complex interactions between milk substrates, cheese microbiota and their metabolites. In this study, bacterial 16S rRNA-gene sequencing, untargeted metabolomics (gas chromatography-mass spectrometry) and data integration analyses were used to characterize and differentiate commercial Cheddar cheeses of varying maturity made by the same and different manufacturers. Microbiota and metabolite compositions varied between cheeses of different ages and brands, and could be used to distinguish the cheeses. Individual amino acids and carboxylic acids were positively correlated with the ripening age for some brands. Integration and Random Forest analyses revealed numerous associations between specific bacteria and metabolites including a previously undescribed positive correlation between Thermus and phenylalanine and a negative correlation between Streptococcus and cholesterol. Together these results suggest that multi-omics analyses has the potential to be used for better understanding the relationships between cheese microbiota and metabolites during ripening and for discovering biomarkers for validating cheese age and brand authenticity.

New insights into cheddar cheese microbiota-metabolome relationships revealed by integrative analysis of multi-omics data.

Cheese microbiota and metabolites and their inter-relationships that underpin specific cheese quality attributes remain poorly understood. Here we report that multi-omics and integrative data analysis (multiple co-inertia analysis, MCIA) can be used to gain deeper insights into these relationships and identify microbiota and metabolite fingerprints that could be used to monitor product quality and authenticity. Our study into different brands of artisanal and industrial cheddar cheeses showed that Streptococcus, Lactococcus and Lactobacillus were the dominant taxa with overall microbial community structures differing not only between industrial and artisanal cheeses but also among different cheese brands. Metabolome analysis also revealed qualitative and semi-quantitative differences in metabolites between different cheeses. This also included the presence of two compounds (3-hydroxy propanoic acid and O-methoxycatechol-O-sulphate) in artisanal cheese that have not been previously reported in any type of cheese. Integrative analysis of multi-omics datasets revealed that highly similar cheeses, identical in age and appearance, could be distinctively clustered according to cheese type and brand. Furthermore, the analysis detected strong relationships, some previously unknown, which existed between the cheese microbiota and metabolome, and uncovered specific taxa and metabolites that contributed to these relationships. These results highlight the potential of this approach for identifying product specific microbe/metabolite signatures that could be used to monitor and control cheese quality and product authenticity.

Cheesomics: the future pathway to understanding cheese flavour and quality.

Cheese is a fermented dairy product, harboring diverse microbial communities (microbiota) that change over time and vary depending on the type of cheese and their respective starter and adjunct cultures. These microorganisms play a crucial role in determining the flavor, quality and safety of the final product. Exploring the composition of cheese microbiota and the underlying molecular mechanisms involved in cheese ripening has been the subject of many studies. Recent advances in next generation sequencing (NGS) methods and the development of sophisticated bioinformatics tools have provided deeper insights into the composition and potential functionality of cheese microbiota far beyond the information provided by culture-dependent methods. These advances, which include rRNA gene amplicon sequencing and metagenomics, have been complemented and expanded in recent years by the application of metatranscriptomics, metaproteomics and metabolomics. This paper reviews studies in which application of these meta-omics technologies has led to a better understanding of the microbial composition and functionality of cheese and highlights opportunities by which the integration of outputs from diverse multi-omics analytical platforms (cheesomics) could be used in the future to advance our knowledge of the cheese ripening process and identify biomarkers for predicting cheese flavor, quality, texture and safety, and bioactive metabolites with potential to influence human health.

Rapid identification of dairy mesophilic and thermophilic sporeforming bacteria using DNA high resolution melt analysis of variable 16S rDNA regions.

Due to their ubiquity in the environment and ability to survive heating processes, sporeforming bacteria are commonly found in foods. This can lead to product spoilage if spores are present in sufficient numbers and where storage conditions favour spore germination and growth. A rapid method to identify the major aerobic sporeforming groups in dairy products, including Bacillus licheniformis group, Bacillus subtilis group, Bacillus pumilus group, Bacillus megaterium, Bacillus cereus group, Geobacillus species and Anoxybacillus flavithermus was devised. This method involves real-time PCR and high resolution melt analysis (HRMA) of V3 (~70 bp) and V6 (~100 bp) variable regions in the 16S rDNA. Comparisons of HRMA curves from 194 isolates of the above listed sporeforming bacteria obtained from dairy products which were identified using partial 16S rDNA sequencing, allowed the establishment of criteria for differentiating them from each other and several non-sporeforming bacteria found in samples. A blinded validation trial on 28 bacterial isolates demonstrated complete accuracy in unambiguous identification of the 7 different aerobic sporeformers. The reliability of HRMA method was also verified using boiled extractions of crude DNA, thereby shortening the time needed for identification. The HRMA method described in this study provides a new and rapid approach to identify the dominant mesophilic and thermophilic aerobic sporeforming bacteria found in a wide variety of dairy products.

Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci.

In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes.

Genotyping of present-day and historical Geobacillus species isolates from milk powders by high-resolution melt analysis of multiple variable-number tandem-repeat loci.

Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.

Characterization and genomic analysis of phage asccphi28, a phage of the family Podoviridae infecting Lactococcus lactis.

Bacteriophage asccphi28 infects dairy fermentation strains of Lactococcus lactis. This report describes characterization of asccphi28 and its full genome sequence. Phage asccphi28 has a prolate head, whiskers, and a short tail (C2 morphotype). This morphology and DNA hybridization to L. lactis phage P369 DNA showed that asccphi28 belongs to the P034 phage species, a group rarely encountered in the dairy industry. The burst size of asccphi28 was found to be 121 +/- 18 PFU per infected bacterial cell after a latent period of 44 min. The linear genome (18,762 bp) contains 28 possible open reading frames (ORFs) comprising 90% of the total genome. The ORFs are arranged bidirectionally in recognizable functional modules. The genome contains 577 bp inverted terminal repeats (ITRs) and putatively eight promoters and four terminators. The presence of ITRs, a phage-encoded DNA polymerase, and a terminal protein that binds to the DNA, along with BLAST and morphology data, show that asccphi28 more closely resembles streptococcal phage Cp-1 and the phi29-like phages that infect Bacillus subtilis than it resembles common lactococcal phages. The sequence of this phage is the first published sequence of a P034 species phage genome.