To culture or not to culture: careful assessment of metabarcoding data is necessary when evaluating the microbiota of a modified-atmosphere-packaged vegetarian meat alternative throughout its shelf-life period.

BACKGROUND: As the increased consumption of ready-to-eat meat alternatives is a fairly recent trend, little is known about the composition and dynamics of the microbiota present on such products. Such information is nonetheless valuable in view of spoilage and food safety prevention. Even though refrigeration and modified-atmosphere-packaging (MAP) can extend the shelf-life period, microbial spoilage can still occur in these products. In the present study, the microbiota of a vegetarian alternative to poultry-based charcuterie was investigated during storage, contrasting the use of a culture-dependent method to a culture-independent metagenetic method. RESULTS: The former revealed that lactic acid bacteria (LAB) were the most abundant microbial group, specifically at the end of the shelf-life period, whereby Latilactobacillus sakei was the most abundant species. Metabarcoding analysis, in contrast, revealed that DNA of Xanthomonas was most prominently present, which likely was an artifact due to the presence of xanthan gum as an ingredient, followed by Streptococcus and Weissella. CONCLUSIONS: Taken together, these results indicated that Lb. sakei was likely the most prominent specific spoilage organisms (SSO) and, additionally, that the use of metagenetic analysis needs to be interpreted with care in this specific type of product. In order to improve the performance of metagenetics in food samples with a high DNA matrix but a low bacterial DNA load, selective depletion techniques for matrix DNA could be explored.

1H NMR-based metabolomics profiling and taste of boneless dry-cured hams during processing.

Boneless dry-cured hams were processed by raw material treatment (deboned and tumbled), salting (vacuum packed; 4 °C and 60-70% RH for 3.5 months), resting (vacuum packed; 15 °C and 60-70% RH for 1.5 months) and ripening (vacuum packed; 15 °C and 60-70% RH for 1.5 months). To explore the mechanisms that contributed to the taste of boneless dry-cured hams, 1H NMR-based metabolomics combined with multivariate data analysis was applied to investigate metabolite changes during processing. A total of 28 metabolites, including amino acids, peptide, organic acids, nucleic acids and their derivatives, sugars and others were identified. PC1 and PC2 explained a total of 77.6% and 18.0% of variables, and thus they could well reflect the main characteristics of all samples. The contents of most metabolites had an upward trend during the processing. Amino acids (isoleucine, valine, alanine, glutamate and histidine), organic acids (lactate, acetate, succinate, citrate and formate) and nucleotide derivative (hypoxanthine) were the major contributors to the taste in our final product. We concluded that the processing time (within 6.5 months) had a positive effect on the development of taste of boneless dry-cured hams.

Suggestion for a subdivision of processed meat products on the Danish market based on their content of carcinogenic compounds.

Carcinogenic effects in humans are ascribed to processed meat by organisations such as International Agency for Research on Cancer, World Cancer Research Fund and American Institute for Cancer Research. However, the term 'processed meat' covers a heterogenic group of products whose content of potential hazards differ considerably. To improve estimates of associations between processed meat intake and cancer risk we investigated ways to divide processed meat into subgroups that more precisely reflects its carcinogenic characteristics. We collected ingredient lists and declarations of salt content for >1000 processed meat products on the Danish market and combined the information with knowledge related to processing parameters. Some compounds that could affect the products' carcinogenic characteristics, alone or in combination, were evaluated and compared for 12 types of processed meat products, and we suggest subgrouping of processed meat with similar level of carcinogenic potential, which could improve the understanding of the cancer risk associated with processed meat intake in scientific human studies.

Insight into Rapid DNA-Specific Identification of Animal Origin Based on FTIR Analysis: A Case Study.

In this study, a methodology has been proposed to identify the origin of animal DNA, employing high throughput extension accessory Fourier transform infrared (HT-FTIR) spectroscopy coupled with chemometrics. Important discriminatory characteristics were identified in the FTIR spectral peaks of 51 standard DNA samples (25 from bovine and 26 from fish origins), including 1710, 1659, 1608, 1531, 1404, 1375, 1248, 1091, 1060, and 966 cm-1. In particular, the bands at 1708 and 1668 cm-1 were higher in fish DNA than in bovine DNA, while the reverse was true for the band at 1530 cm-1 was shown the opposite result. It was also found that the PO2- Vas/Vs ratio (1238/1094 cm-1) was significantly higher (p < 0.05) in bovine DNA than in fish DNA. These discriminatory characteristics were further revealed to be closely related to the base content and base sequences of different samples. Multivariate analyses, such as principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were conducted, and both the sensitivity and specificity values of PLS-DA model were one. This methodology has been further validated by 20 meat tissue samples (4 from bovine, 5 from ovine, 5 from porcine, and 6 from fish origins), and these were successfully differentiated. This case study demonstrated that FTIR spectroscopy coupled with PLS-DA discriminant model could provide a rapid, sensitive, and reliable approach for the identification of DNA of animal origin. This methodology could be widely applied in food, feed, forensic science, and archaeology studies.

Near-Infrared Spectroscopy and Geostatistical Analysis for Modeling Spatial Distribution of Analytical Constituents in Bulk Animal By-Product Protein Meals.

Control and inspection operations within the context of safety and quality assessment of bulk foods and feeds are not only of particular importance, they are also demanding challenges, given the complexity of food/feed production systems and the variability of product properties. Existing methodologies have a variety of limitations, such as high costs of implementation per sample or shortcomings in early detection of potential threats for human/animal health or quality deviations. Therefore, new proposals are required for the analysis of raw materials in situ in a more efficient and cost-effective manner. For this purpose, a pilot laboratory study was performed on a set of bulk lots of animal by-product protein meals to introduce and test an approach based on near-infrared (NIR) spectroscopy and geostatistical analysis. Spectral data, provided by a fiber optic probe connected to a Fourier transform (FT) NIR spectrometer, were used to predict moisture and crude protein content at each sampling point. Variographic analysis was carried out for spatial structure characterization, while ordinary Kriging achieved continuous maps for those parameters. The results indicated that the methodology could be a first approximation to an approach that, properly complemented with the Theory of Sampling and supported by experimental validation in real-life conditions, would enhance efficiency and the decision-making process regarding safety and adulteration issues.

Classification of traditional Chinese pork bacon based on physicochemical properties and chemometric techniques.

Sixty-seven pork bacon samples from Hunan, Sichuan Guangdong, Jiangxi, and Yunnan Provinces in China were analyzed to understand their geographical properties. Classification was performed by determining their physicochemical properties through chemometric techniques, including variance analysis, principal component analysis (PCA), and discriminant analysis (DA). Results showed that certain differences existed in terms of nine physicochemical determinations in traditional Chinese pork bacon. PCA revealed the distinction among Hunan, Sichuan, and Guangdong style bacon. Meanwhile, seven key physicochemical determination criteria were identified in line with DA and could be reasonably applied to the classification of traditional Chinese pork bacon. Furthermore, the ratio of overall correct classification was 97.76% and that of cross-validation was 91.76%. These findings indicated that chemometric techniques, together with several physicochemical determination, were effective for the classification of traditional Chinese pork bacon with geographical features. Our study provided a theoretical reference for the classification of traditional Chinese pork bacon.

Classification of dry-cured hams according to the maturation time using near infrared spectra and artificial neural networks.

An attempt to classify dry-cured hams according to the maturation time on the basis of near infrared (NIR) spectra was studied. The study comprised 128 samples of biceps femoris (BF) muscle from dry-cured hams matured for 10 (n=32), 12 (n=32), 14 (n=32) or 16 months (n=32). Samples were minced and scanned in the wavelength range from 400 to 2500 nm using spectrometer NIR System model 6500 (Silver Spring, MD, USA). Spectral data were used for i) splitting of samples into the training and test set using 2D Kohonen artificial neural networks (ANN) and for ii) construction of classification models using counter-propagation ANN (CP-ANN). Different models were tested, and the one selected was based on the lowest percentage of misclassified test samples (external validation). Overall correctness of the classification was 79.7%, which demonstrates practical relevance of using NIR spectroscopy and ANN for dry-cured ham processing control.

Effect of muscle type and frozen storage on the quality parameters of Iberian restructured meat preparations.

The main objective of this study was to evaluate the effect of muscle type and frozen storage on the quality of restructured meat preparations from undervalued Iberian muscle to make use of meat from a high-quality and natural pig production system. The effect of two muscle types (i.e. white-glycolytic (W) and red-oxidative (R)) and frozen storage (lasting 0, 30, 60 and 90 days) on quality characteristics were assessed. Significant differences were found between the W and R Iberian restructured preparations in most physicochemical and some colour, texture and sensory traits, and in the fatty acid profile and oxidative measurements, suggesting that the R muscles are more suitable; however, the microbial contamination should be reduced. Frozen storage reduced but did not eliminate the initial microbial contamination, and it enhanced some quality traits in the Iberian restructured preparations, i.e. increased a* values, cohesiveness and juiciness and decreased adhesiveness and pastiness, without negatively affecting any parameter. Thus, frozen Iberian restructured preparations are recommended to be commercialized. In addition, the implementation or revision of Hazard Analysis and Critical Control Point is recommended to reduce microbial contamination.

Feasibility of use of fatty acid and triacylglycerol profiles for the authentication of commercial labelling in Iberian dry-cured sausages.

In the present study, fatty acid and triacylglycerol profiles were used to evaluate the possibility of authenticating Iberian dry-cured sausages according to their label specifications. 42 Commercial brand 'chorizo' and 39 commercial brand 'salchichón' sausages from Iberian pigs were purchased. 36 Samples were labelled Bellota and 45 bore the generic Ibérico label. In the market, Bellota is considered to be a better class than the generic Ibérico since products with the Bellota label are manufactured with high quality fat obtained from extensively reared pigs fed on acorns and pasture. Analyses of fatty acids and triacylglycerols were carried out by gas chromatography and a flame ion detector. A CP-SIL 88 column (highly substituted cyanopropyl phase; 50 m × 0.25 mm i.d., 0.2 µm film thickness) (Varian, Palo Alto, USA) was used for fatty acid analysis and a fused silica capillary DB-17HT column (50% phenyl-50% methylpolysiloxane; 30 m × 0.25 mm i.d., 0.15 µm film thickness) was used for triacylglycerols. Twelve fatty acids and 16 triacylglycerols were identified. Various discriminant models (linear quadratic discriminant analyses, logistic regression and support vector machines) were trained to predict the sample class (Bellota or Ibérico). These models included fatty acids and triacylglycerols separately and combined fatty acid and triacylglycerol profiles. The number of correctly classified samples according to discriminant analyses can be considered low (lower than 65%). The greatest discriminant rate was obtained when triacylglycerol profiles were included in the model, whilst using a combination of fatty acid and triacylglycerol profiles did not improve the rate of correct assignation. The values that represent the reliability of prediction of the samples according to the label specification were higher for the Ibérico class than for the Bellota class. In fact, quadratic and Support Vector Machine discriminate analyses were not able to assign the Bellota class (0%) when combined fatty acids and triacylglycerols were included in the model. The use of fatty acid and triacylglycerol profiles to discriminate Iberian dry-cured sausages in the market according to their labelling information is unclear. In order to ensure the genuineness of Iberian dry-cured sausages in the market, identification of fatty acid and triacylglycerol profiles should be combined with the application of quality standard traceability techniques.

Identification of mtDNA lineages of Sus scrofa by multiplex single base extension for the authentication of processed food products.

A genetic method to identify the breed of origin could serve as a useful tool for inspecting the authenticity of the increasing number of monobreed foodstuffs, such as those derived from small local European pig breeds. Mitochondrial DNA (mtDNA) is practically the only reliable genomic target for PCR in processed products, and its haploid nature and strict maternal inheritance greatly facilitate genetic analysis. As a result of strategies that sought to improve the production traits of European pigs, most industrial breeds presently show a high frequency of Asian alleles, while the absence or low frequency of such Asian alleles has been observed in small rustic breeds from which highly prized dry-cured and other traditional products are derived. Therefore, the detection of Asian ancestry would indicate nonconformity in Protected Denomination of Origin products. This study presents a single base extension assay based on 15 diagnostic mtDNA single nucleotide polymorphisms to discriminate between Asian and European Sus scrofa lineages. The test was robust, sensitive and accurate in a wide range of processed foodstuffs and allowed accurate detection of pig genetic material and identification of maternal ancestry. A market survey suggested that nonconformity of products derived from Portuguese breeds is an unusual event at present, but regular surveys both in the local populations and in commercial products would be advisible. Taking into consideration the limitations presented by other methodologies, this mtDNA-based test probably attains the highest resolution for the direct genetic test for population of origin in Sus scrofa food products.

Application of Classification and Regression Tree (CART) analysis on the microflora of minced meat for classification according to Reg. (EC) 2073/2005.

In a retrospective study on the microbiology of minced meat from small food businesses supplying directly to the consumer, the relative contribution of meat supplier, meat species and outlet where meat was minced was assessed by "Classification and Regression Tree" (CART) analysis. Samples (n=888) originated from 129 outlets of a single supermarket chain. Sampling units were 4-5 packs (pork, beef, and mixed pork-beef). Total aerobic counts (TACs) were 5.3±1.0 log CFU/g. In 75.6% of samples, E. coli were <1 log CFU/g. The proportion of "unsatisfactory" sample sets [as defined in Reg. (EC) 2073/2005] were 31.3 and 4.5% for TAC and E. coli, respectively. For classification according to TACs, the outlet where meat was minced and the "meat supplier" were the most important predictors. For E. coli, "outlet" was the most important predictor, but the limit of detection of 1 log CFU/g was not discriminative enough to allow further conclusions.

Identification of important image features for pork and turkey ham classification using colour and wavelet texture features and genetic selection.

A method to discriminate between various grades of pork and turkey ham was developed using colour and wavelet texture features. Image analysis methods originally developed for predicting the palatability of beef were applied to rapidly identify the ham grade. With high quality digital images of 50-94 slices per ham it was possible to identify the greyscale that best expressed the differences between the various ham grades. The best 10 discriminating image features were then found with a genetic algorithm. Using the best 10 image features, simple linear discriminant analysis models produced 100% correct classifications for both pork and turkey on both calibration and validation sets.

High processed meat consumption is a risk factor of type 2 diabetes in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention study.

Relatively small lifestyle modifications related to weight reduction, physical activity and diet have been shown to decrease the risk of type 2 diabetes. Connected with diet, low consumption of meat has been suggested as a protective factor of diabetes. The aim of the present study was to examine the association between the consumption of total meat or the specific types of meats and the risk of type 2 diabetes. The Alpha-Tocopherol, Beta-Carotene Cancer Prevention cohort included middle-aged male smokers. Up to 12 years of follow-up, 1098 incident cases of diabetes were diagnosed from 24 845 participants through the nationwide register. Food consumption was assessed by a validated FFQ. In the age- and intervention group-adjusted model, high total meat consumption was a risk factor of type 2 diabetes (relative risk (RR) 1.50, 95 % CI 1.23, 1.82, highest v. lowest quintile). The result was similar after adjustment for environmental factors and foods related to diabetes and meat consumption. The RR of type 2 diabetes was 1.37 for processed meat (95 % CI 1.11, 1.71) in the multivariate model. The results were explained more by intakes of Na than by intakes of SFA, protein, cholesterol, haeme Fe, Mg and nitrate, and were not modified by obesity. No association was found between red meat, poultry and the risk of type 2 diabetes. In conclusion, reduction of the consumption of processed meat may help prevent the global epidemic of type 2 diabetes. It seems like Na of processed meat may explain the association.

The microbiological safety of ready-to-eat specialty meats from markets and specialty food shops: a UK wide study with a focus on Salmonella and Listeria monocytogenes.

From 2359 specialty meats (continental sausages, cured/fermented, dried meats) sampled from markets and specialty food shops, 98.9% of samples were of satisfactory or acceptable microbiological quality. However, 16 (0.7%) were unsatisfactory as a result of Escherichia coli, Staphylococcus aureus or Listeria spp. contamination (>or=10(2) CFU/g), and nine (0.4%) were unacceptable due to presence of Salmonella spp. or Listeria monocytogenes (>10(2) CFU/g). Meats with unacceptable levels of L. monocytogenes were within shelf life (range: 8-143 days remaining). Nine different subtypes of L. monocytogenes were detected with sero/AFLP type 1/2c VII predominating (37%), although this subtype was not overrepresented in any particular meat type (P > 0.05). Ninety-six percent of continental sausages and cured/fermented products were stored at <8 degrees C at premises, including seven of the nine unacceptable samples. These nine meats were all pre-packed prior to supply to retail premises (OR = 0.1 P = 0.003) indicating that contamination with bacterial pathogens occurred earlier in the production chain. Most samples (72.7%, 8/11) with unsatisfactory levels of E. coli were sliced on request, suggesting cross-contamination at point of sale. This study highlights the importance of ensuring that products do not become contaminated before final packaging, that storage conditions are controlled, and that durability dates are an accurate indication of the shelf life of the product so as to minimise the potential for L. monocytogenes to be present at levels hazardous to health at the point of sale.

Postmortem metabolic rate and calpain system activities on beef longissimus tenderness classifications.

In this study, we adopted a model of tenderness classification in order to determine the factors affecting the tenderness and tenderization characteristics of beef longissimus, using cluster analysis on the basis of Warner-Bratzler shear force and myofibril fragmentation index, at 1, 7, and 14 d. The rate of tenderization was effectively differentiated by pH, R-values, mu-calpain activity, and calpastatin activity. Differences among tenderness classes were generally detected at 3 and 9 h postmortem for metabolic rate, and at 9 and 24 h for the activities of mu-calpain and calpastatin. Early postmortem metabolic rate and calpain system activities were verified as important factors with regard to longissimus tenderization.

[A comparison of N-nitrosodimethylamine contents in selected meat products]. / Porównanie zawartosci N-nitrozodimetyloaminy w wybranych produktach miesnych.

Nitrosamines are know as the most potent group of carcinogens. Approximately 300 of these compounds have been tested, and about 90% of them have been found to be carcinogenic in laboratory animals. N-nitrosodimethylamine causes liver cancer, whereas some of tobacco specific nitrosamines causes lung cancer. Volatile N-nitrosamines induce tumors in a variety of human organs, including the tongue, esophagus, lung, pancreas, liver, kidney and bladder. They are formed during reaction of secondary or tertiary amino compounds and nitrite or nitrogen oxides. Nitrosamines occurs as contaminants in many foodstuff including food and beverages: beers, cheeses, sausages, smoked and pickled foods. They are formed during frying, smoking and food preserved with pickling salt. These compounds can also be produced in man and other mammals under the acidic conditions in the stomach. The present study was carried out to determine level of N-nitrosodimethylamine in selected 13 meat products. The extraction procedure was based on Raoul's method, ie. on two consecutive extraction-concentration step using extrelut and florisil columns. The level of N-nitrosodimethylamine was varied from 0.049 mg/kg to 16.47 mg/kg. The highest level of NDMA was found in smoked sausage.

PCR identification of beef, sheep, goat, and pork in raw and heat-treated meat mixtures.

A PCR assay has been developed for the specific and qualitative detection of pork (Sus scrofa domesticus), beef (Bos taurus), sheep (Ovis aries), and goat (Capra hircus) in raw and heat-treated meat mixtures. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear species identification. Analysis of experimental meat mixtures demonstrated that the detection limit of the assay was 1% (wt/wt) for each species analyzed. This assay can be useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in meat mixtures.

Improving meat inspection and control in resource-poor communities: the Nepal example.

Meat is an important source of protein and a valuable commodity in resource-poor communities. In many developing countries, lack of appropriate slaughtering facilities and unsatisfactory slaughtering techniques are causing unnecessary losses of meat as well as invaluable by-products from animal carcasses. Slaughtering places are frequently contaminated and may not be protected against dogs, rodents and insects. Meat products coming from such conditions are often deteriorated due to bacterial infection or contaminated, which may cause food poisoning or diseases in consumers. In many developing countries, regulations concerning meat inspection and/or control are inadequate or non-existent allowing consumers to be exposed to pathogens including zoonotic parasites. In Nepal, buffaloes contribute about 64% of the meat consumed, followed by goat meat (20%), pork (7%), poultry (6%) and mutton (2%). Goat and poultry meat is acceptable to all castes of people while buffalo meat is consumed mainly by the Newar ethnic group. Previously, pork was consumed only by people belonging to low castes, however, in recent years, the consumption of pork has increased in higher castes as the caste system has become more relaxed. Until recently, there were no official meat inspection regulations in the country, however, in 1999, the national government legislated an as-yet-to-be implemented Animal Slaughtering and Meat Inspection Act which mandates slaughterhouse construction and meat inspection and control. Due to the lack of implementation of the Meat Inspection Act and resultant absence of meat inspection, meat from sick or parasite-infected animals is serving as a source of infection to humans as well as other animals. In addition, meat quality is adversely affected by careless handling conditions in the slaughtering places as well as in the meat markets or shops. For improvement in animal slaughtering and meat inspection in both rural and urban areas of Nepal, several strategies are to be recommended. Sustainable capacity building should be introduced including training of veterinarians, meat inspectors and butchers as well as building of slaughter facilities. Government policies on slaughter procedures including ante-mortem examination, meat inspection and stamping of meat should be implemented. Programmes should be instituted with strong focus on prevention and control of meat-borne diseases to reduce infection risk of consumers and meat handlers and to avoid contamination of the environment. Lastly, emphasis should be put on improving the animal husbandry system in Nepal. These same actions can be undertaken in other developing countries to assist with improving meat inspection and control, thus helping with prevention and control of cysticercosis as well as other important meat-borne diseases.

Comparative microbial character of consumed food and drinking water.

Humans ingest large numbers of microbes daily. Food provides distinctly different physical and physiological conditions from drinking water. With high concentrations of carbohydrate, protein, and ionic strength, food is much closer to the human physiological state than drinking water, which is essentially devoid of nutrients and ionic strength. Accordingly, microbes that can multiply in humans and cause disease can grow in food, but do not multiply in drinking water. Virtually all food sources contain many thousand times more bacteria than drinking water. Therefore, based on both observed microbial content and the presence of large numbers of pathogens or their indicators in food, in this country food is more of a health risk to humans than drinking water. Compounding this disparity is the fact that much food is imported with limited control over the means of production. Naturally occurring bacteria (HPC or autochthonous flora) do not have virulence factors, making their numbers irrelevant to health risk except in the case of the most severely immunocompromised--a very defined population group. Consequently, public health regulations should not be directed to eliminating naturally occurring HPC, but should be focused toward controlling pathogens through measures such as sanitary crop systems in the steps from production (e.g., quality of irrigation and fertilization, animal feed lot sanitation) through storage to consumer preparation. Food possesses a far greater risk than drinking water, and government agencies should take this fact into account when writing regulations.