Project description:The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.
Project description:Beef is one of the most consumed food worldwide, and it is prone to spoilage by bacteria. This risk could be caused by resident microbiota and their alterations in fresh beef meat during processing. However, scarce information is available regarding potential spoilage factors due to resident microbiota in fresh beef meat. In this study, we analyzed the microbiota composition and their predicted functions on fresh beef meat. A total of 120 beef meat samples (60 fresh ground and 60 non-ground beef samples) were collected from three different sites in South Korea on different months, and the microbiota were analyzed by the MiSeq system. Our results showed that although the microbiota in beef meat were varied among sampling site and months, the dominant phyla were the same with shared core bacteria. Notably, psychrotrophic genera, related to spoilage, were detected in all samples, and their prevalence increased significantly in July. These genera could inhibit the growth of other microbes with using glucose by fermentation. The results of this study extend our understanding of initial microbiota in fresh beef meat and potential functions influencing spoilage and can be useful to develop the preventive measures to reduce the spoilage of beef meat products.
Project description:Meat and seafood spoilage ecosystems harbor extensive bacterial genomic diversity that is mainly found within a small number of species but within a large number of strains with different spoilage metabolic potential. To decipher the intraspecies diversity of such microbiota, traditional metagenetic analysis using the 16S rRNA gene is inadequate. We therefore assessed the potential benefit of an alternative genetic marker, gyrB, which encodes the subunit B of DNA gyrase, a type II DNA topoisomerase. A comparison between 16S rDNA-based (V3-V4) amplicon sequencing and gyrB-based amplicon sequencing was carried out in five types of meat and seafood products, with five mock communities serving as quality controls. Our results revealed that bacterial richness in these mock communities and food samples was estimated with higher accuracy using gyrB than using16S rDNA. However, for Firmicutes species, 35% of putative gyrB reads were actually identified as sequences of a gyrB paralog, parE, which encodes subunit B of topoisomerase IV; we therefore constructed a reference database of published sequences of both gyrB and pare for use in all subsequent analyses. Despite this co-amplification, the deviation between relative sequencing quantification and absolute qPCR quantification was comparable to that observed for 16S rDNA for all the tested species. This confirms that gyrB can be used successfully alongside 16S rDNA to determine the species composition (richness and evenness) of food microbiota. The major benefit of gyrB sequencing is its potential for improving taxonomic assignment and for further investigating OTU richness at the subspecies level, thus allowing more accurate discrimination of samples. Indeed, 80% of the reads of the 16S rDNA dataset were represented by thirteen 16S rDNA-based OTUs that could not be assigned at the species-level. Instead, these same clades corresponded to 44 gyrB-based OTUs, which differentiated various lineages down to the subspecies level. The increased ability of gyrB-based analyses to track and trace phylogenetically different groups of strains will generate improved resolution and more reliable results for studies of the strains implicated in food processes.
Project description:Poultry meat deterioration is caused by environmental conditions, as well as proliferation of different bacterial groups, and their interactions. It has been proposed that meat spoilage involves two bacterial groups: one group that initiates the deterioration process, known as specific spoilage organisms (SSOs), and the other known as spoilage associated organisms (SAOs) which represents all bacteria groups recovered from meat samples before, during, and after the spoilage process. Numerous studies have characterized the diversity of chicken meat SAOs; nonetheless, the identification of the SSOs remains a long-standing question. Based on recent genomic studies, it is suggested that the SSOs should possess an extensive genome size to survive and proliferate in raw meat, a cold, complex, and hostile environment. To evaluate this hypothesis, we performed comparative genomic analyses in members of the meat microbiota to identify microorganisms with extensive genome size and ability to cause chicken meat spoilage. Our studies show that members of the Pseudomonadaceae family have evolved numerous biological features such as large genomic size, slow-growing potential, low 16S rRNA copy number, psychrotrophic, and oligotrophic metabolism to initiate the spoilage of poultry meat. Moreover, inoculation experiments corroborated that these biological traits are associated with the potential to cause chicken meat deterioration. Together, these results provide new insights into the identification of SSO. Further studies are in progress to elucidate the impact of the SSO on meat quality and microbiota diversity.
Project description:Brochothrix thermosphacta is one of the main spoilers in food, responsible for meat and seafood spoilage through the production of malodorous volatile organic compounds. The molecules produced by this bacterium depend on the substrate (meat or seafood) and the storage conditions such as gas mixtures used in the packaging. It seems also that the spoilage potential is strain dependent as production of diacetyl and acetoin, two molecules responsible for seafood spoilage, varies with strains. Therefore, this suggests the involvement of different metabolic functions depending on both food substrate and strain capacities. In this study, we selected two strains with different abilities to produce diacetyl and acetoin and compared their behavior after grown in beef or cooked peeled shrimp juices. We determined the genes upregulated by both strains depending on the growth substrate and those that were specifically upregulated in only one strain. The genes upregulated by both strains in meat or in shrimp juice revealed the importance of the substrate for inducing specific metabolic pathways. The examination of genes that were specifically upregulated in only one of the two strains revealed strain features associated to specific substrates and also strain-specific regulations of metabolic pathways putatively leading to different levels of spoilage molecule production. This shows that the spoilage potential of B. thermosphacta depends on nutrients provided by food substrate and on metabolic activity potential that each strain possesses.
Project description:The aim of this study was to identify the share of meat, meat products and seafood in the contribution of energy and 22 nutrients to the average Polish diet. Data from the nationally representative sample of Polish population (2016 Household Budget Survey) on meat and seafood product consumption from 38,886 households (n = 99,230) were calculated into one person per month. The analyses were conducted for seven food groups (e.g., red meat, poultry) and 16 products (e.g., beef, chicken). Approximately 18.5% of energy is delivered from the sources such as meat, meat products and seafood, providing a higher percentage of 18 nutrients to the diet (e.g., 56.0% of vitamin B12, 52.3% of niacin, 44.9% of cholesterol, 41.5% of protein, 41.4%of vitamin D, 37.6% of monounsaturated fatty acids (MUFA), 37.4% of thiamin, 33.8% of zinc, 32.0% of total fats, 30.3% of saturated fatty acids (SFA), 29.6% of vitamin B6, 25.3% of riboflavin, 24.9% of phosphorus, 24.8% of iron, 22.5% of vitamin A, 21.6% of polyunsaturated fatty acids (PUFA) and 20.3% of sodium). For the contribution of 18 nutrients and energy, processed meat products were ranked first. These results should be taken into consideration in order to compose diets with adequate energy and nutrient contribution and also to analyze benefits and risk resulting from the current level of consumption of red and processed meat, fish and other seafood.
Project description:Quorum-sensing (QS) signals (N-acyl homoserine lactones [AHLs]) were extracted and detected from five commercially produced vacuum-packed meat samples. Ninety-six AHL-producing bacteria were isolated, and 92 were identified as Enterobacteriaceae. Hafnia alvei was the most commonly identified AHL-producing bacterium. Thin-layer chromatographic profiles of supernatants from six H. alvei isolates and of extracts from spoiling meat revealed that the major AHL species had an R(f) value and shape similar to N-3-oxo-hexanoyl homoserine lactone (OHHL). Liquid chromatography-mass spectrometry (MS) (high-resolution MS) analysis confirmed the presence of OHHL in pure cultures of H. alvei. Vacuum-packed meat spoiled at the same rate when inoculated with the H. alvei wild type compared to a corresponding AHL-lacking mutant. Addition of specific QS inhibitors to the AHL-producing H. alvei inoculated in meat or to naturally contaminated meat did not influence the spoilage of vacuum-packed meat. An extracellular protein of approximately 20 kDa produced by the H. alvei wild-type was not produced by the AHL-negative mutant but was restored in the mutant when complemented by OHHL, thus indicating that AHLs do have a regulatory role in H. alvei. Coinoculation of H. alvei wild-type with an AHL-deficient Serratia proteamaculans B5a, in which protease secretion is QS regulated, caused spoilage of liquid milk. By contrast, coinoculation of AHL-negative strains of H. alvei and S. proteamaculans B5a did not cause spoilage. In conclusion, AHL and AHL-producing bacteria are present in vacuum-packed meat during storage and spoilage, but AHL does not appear to influence the spoilage of this particular type of conserved meat. Our data indicate that AHL-producing H. alvei may induce food quality-relevant phenotypes in other bacterial species in the same environment. H. alvei may thus influence spoilage of food products in which Enterobacteriaceae participate in the spoilage process.
Project description:A literature search was performed on spoilage of fresh meat products by combining keyword query, text mining and expert elicitation. From the 258 collected studies, a quantitative analysis was first performed to identify the methods which are the most used to evaluate spoilage beside the preservation strategies suggested. In a second step focusing on a subset of 24 publications providing quantitative data on spoilage occurrence time, associations between spoilage occurrence time of meat products and specific spoilage indicators were investigated. The analysis especially focused on factors well represented in the 24 publications, i.e., gas packaging (O2 and CO2) and storage temperature. Relationships between spoilage occurrence and several microbiological indicators were also sought. The results point out possible advantages of removing dioxygen in packaging to delay spoilage occurrence, whereas, in the presence of dioxygen, the carbon dioxide proportion in the gas mixtures was shown to influence spoilage occurrence. The collected data clearly reveal a potentially protective role of lactic acid bacteria. Besides, while a spoilage role could be attributed to Pseudomonas spp., the growth of mesophilic aerobic microbes, Brochothrix spp. and Enterobacteriaceae seemed independent of spoilage occurrence time.
Project description:Besides intrinsic and extrinsic factors such as antagonism for organic substrates or temperature, the storage atmosphere of meat has a high influence on the development of its initial microbiota. Specific modified atmospheres (MAs) selectively suppress growth of aerobic and anaerobic bacteria, thus reshaping the initial microbiota. As some microorganisms are more tolerant to MA, they overgrow competitors and produce metabolites that cause rejection of the product. In order to elucidate responses to different MA by means of metabolic adaptation and competition for organic substrates on meat, the typical representative meat spoilage bacteria Brochothrix (B.) thermosphacta TMW2.2101 and four lactic acid bacteria Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618 and L. gelidum subsp. gasicomitatum TMW2.1619 were chosen. Bacteria were grown in sterile glass bottles filled with a meat simulation medium, which was aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth of bacteria during incubation at 25°C and stirring at 120 rpm was monitored over 48 h and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to oxygen and carbon dioxide. Both Leuconostoc subsp. were intrinsically tolerant to MA, exhibiting no proteomic regulation of enzymes, whereas the other species provide a set of metabolic adaptation mechanism, enabling higher resistance to the detrimental effects of MA. Those mechanisms comprise: enhanced oxidative stress reduction, adjustment of the pyruvate metabolism and catabolic oxygen consumption in response to oxygen and intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition in response to carbon dioxide. We further evaluated the potential of industrial used MA to inhibit specific bacterial spoilage. No bacterial inhibition is predicted for 30%_CO2/70%_O2 for the analyzed species, whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101. Furthermore, species-specific metabolic pathways enabling different and preferential carbon source utilization were identified, which enable non-competitive coexistence of respective bacteria on meat, resulting in synergistic spoilage. In conclusion, this study gives mechanistically explanations of their acknowledged status as typical spoilage organisms on MAP meats.
Project description:Background:Use of traditional methods for determining meat spoilage is quite laborious and time consuming. Therefore, alternative approaches are needed that can predict the spoilage of meat in a rapid, non-invasive and more elaborative way. In this regard, the spectroscopic techniques have shown their potential for predicting the microbial spoilage of meat-based products. Consequently, the present work was aimed to demonstrate the competence of Fourier transform infrared spectroscopy (FTIR) to detect spoilage in chicken fillets stored under aerobic refrigerated conditions. Methods:This study was conducted under controlled randomized design (CRD). Chicken samples were stored for 8 days at 4 + 0.5 °C and FTIR spectra were collected at regular intervals (after every 2 days) directly from the sample surface using attenuated total reflectance during the study period. Additionally, total plate count (TPC), Entetobacteriaceae count, pH, CTn (Color transmittance number) color analysis, TVBN (total volatile basic nitrogen) contents, and shear force values were also measured through traditional approaches. FTIR spectral data were interpreted through principal component analysis (PCA) and partial least square (PLS) regression and compared with results of traditional methods for precise estimation of spoilage. Results:Results of TPC (3.04-8.20 CFU/cm2), Entetobacteriaceae counts (2.39-6.33 CFU/cm2), pH (4.65-7.05), color (57.00-142.00 CTn), TVBN values (6.72-33.60 mg/100 g) and shear force values (8.99-39.23) were measured through traditional methods and compared with FTIR spectral data. Analysis of variance (ANOVA) was applied on data obtained through microbial and quality analyses and results revealed significant changes (P < 0.05) in the values of microbial load and quality parameters of chicken fillets during the storage. FTIR spectra were collected and PCA was applied to illuminate the wavenumbers potentially correlated to the spoilage of meat. PLS regression analysis permitted the estimates of microbial spoilage and quality parameters from the spectra with a fit of R2 = 0.66 for TPC, R2 = 0.52 for Entetobacteriaceae numbers and R2 = 0.56 for TVBN analysis of stored broiler meat. Discussion:PLS regression was applied for quantitative interpretation of spectra, which allowed estimates of microbial loads on chicken surfaces during the storage period. The results suggest that FTIR spectra retain information regarding the spoilage of poultry meat. Conclusion:The present work concluded that FTIR spectroscopy coupled with multivariate analysis can be successfully used for quantitative determination of poultry meat spoilage.