Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

In vitro antioxidant and antimicrobial activities of clove extract and its effectiveness in bio-composite film on storage stability of goat meat balls.

The aim of this study was to find out clove extract's antimicrobial and antioxidant properties, as well as its efficacy as a bioactive ingredient in the development of bio-composite films to increase the storage stability of goat meat balls stored at 4 ± 1°C. The clove extracts (CLEs) were prepared in ethanol, hydroethanol (1:1), and water and evaluated for antioxidant and antimicrobial potential. In vitro assays of CLEs revealed more susceptibility for gram-positive bacteria than gram-negative bacteria. Among the different extracts, the clove ethanol extract (CLEE) had the highest antimicrobial activity against tested microorganisms as well as total phenolics (1.14 mg GAE/g), flavonoids (8.50 µg catechin/g), and DPPH assay (39.59%). Further, the concentration-dependent effect of CLEE (p < 0.05) on thickness and color values and antimicrobial properties of the bio-composite film were observed. The storage qualities of the product T1 (with film; 450 µl CLEE) such as pH (6.45 ± 0.01), TBARS (0.87 ± 0.06 mg malonaldehyde/kg) value, free fatty acid (0.193 ± 0.001% oleic acid), total mesophilic count (4.98 ± 0.05 log10 CFU/g), and sensory attributes (overall acceptability score: 5.67 on 8-point scale) were better (p < 0.05) than T0 (without film; control) on day 20 of storage. Thus, the ethanolic clove extract has a superior antioxidant and antimicrobial potential. Its inclusion in the bio-composite film prolonged the storage stability of goat meat balls by controlling lipid oxidation and microbial growth. Practical Application Today's consumers are more attracted towards meat products added with natural ingredients having preservative effects. Clove extract is a classic example of a natural preservative and has excellent antimicrobial and antioxidant potential. The present study revealed that by wrapping the ethanolic clove extract-based bio-composite film on goat meat balls extended the storage stability of the product due to controlled lipid oxidation and microbial growth. Thus, such bio-composite films can be successfully applied on goat meat balls that function as a antimicrobial packaging for providing optimum organoleptic quality and better shelf life.

In-vitro functional efficacy of extracts from Phyllanthus emblica, Eucalyptus globulus, Tinospora cordifolia as pancreatic lipase inhibitor and source of anti-oxidant in goat meat nuggets.

The present study was aimed to evaluate the functional efficacy of plant extracts as a source of pancreatic lipase inhibitor and antioxidant in goat meat nuggets to address the fat paradox issue of red meat. The PPLIA, antioxidant potential, and resistance against fat digestion were in the order ofPhyllanthus emblica > Eucalyptus globulus > Tinospora cordifolia.PPL inhibition activities of water and ethanolic extracts fromPhyllanthus emblicausing DNPB and Triolein as substrate were 63.76, 67.94 and 56.17 and 64.36 percent respectively whereas, TPC, DPPH RSA, FRPA were 40.82 and 59.52 (mgGAE/g), 54.89 and 59.84 (percent), 1.26 and 1.61 (OD) respectively. The average diameter of fat globules in digest was maximum (8.91 µm) withPhyllanthus emblicafruits extracts whereas; TBARs (0.347 mg MDA/Kg) and FFA (4.47 µg/g) values were lowest. This study showed that extracts from plants can act as a promising natural alternative in the development of healthy meat products.

Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences.

The present study was carried out with the objective of development of species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin. The cattle-specific LAMP primer set was designed by targeting mitochondrial D-loop gene. The conditions for LAMP reaction for amplification of template DNA from cattle using designed cattle-specific primer set were optimized for the components of mixture and temperature of reaction. Amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. The developed species-specific LAMP assay was evaluated for its specificity, sensitivity and validated in laboratory on samples from known, coded, binary meat admixture with other than cattle at relative percentage of 20%, 10%, 5% and 1%, Phire tissue direct PCR master mix treated tissues of cattle and on species-specific polymerase chain reaction assay positive samples. The developed LAMP assay using self-designed primer set was highly specific, amplifying the DNA template exclusively from cattle tissue under the optimized LAMP reaction conditions. The sensitivity assay using serially diluted DNA templates revealed lowest level of detection as 0.01 ng of absolute DNA from target species. Laboratory validation substantiated the accuracy of assay in known/unknown (coded) samples and up to the 1% level of admixture in binary meat sample. DNA present in supernatant of Phire Animal tissue kit treated samples were also amplified successfully eliminating the extra step of extraction of genomic DNA. The developed assays exhibited comparable results with previously established species-specific PCR assay taken as gold standards. Thus, it was concluded that developed species-specific loop-mediated isothermal amplification assay was effective in identification of tissue of cattle origin.

Development and evaluation of isothermal amplification assay for the rapid and sensitive detection of Clostridium perfringens from chevon.

Loop-mediated isothermal amplification (LAMP) is a promising, simple, rapid and sensitive molecular detection method. In the present study, LAMP assay was developed for detecting Clostridium perfringens in chevon. Primers were designed to detect the cpa gene of C. perfringens. A panel of 19 bacterial strains, including 3 C. perfringens and 16 other strains, were included in this study to standardize and evaluate the LAMP assay. No false positive amplification was observed indicating 100% specificity of the assay. The detection limit of LAMP and conventional PCR in the DNA extracted from pure C. perfringens was 0.34 pg and 3.4 pg, respectively. This revealed that LAMP assay is 10 times more sensitive than conventional PCR. The sensitivity of the LAMP assay for the detection of C. perfringens in raw chevon was found to be 1.2 × 102 CFU/g after 6-h enrichment and 1.2 × 105 CFU/g without enrichment in artificial spiking studies. Improved C. perfringens detection of 12 CFU/g within 12 h was obtained proving that LAMP assay is significantly faster than traditional methods that take >2 d. The developed LAMP assay also detected the targeted organism in clinical and environmental samples with the sensitivity and specificity of 97% and 84%, respectively with Kappa agreement of 0.824 respects to PCR assay. This method shows immense potential for routine diagnosis and monitoring of C. perfringens in food, environment and clinical samples. This is the first report in which the LAMP assay was optimized for the detection of C. perfringens in chevon.

Effect of essential oils incorporated edible film on quality and storage stability of chicken patties at refrigeration temperature (4 ± 1 °C).

A blend of oregano and thyme essential oils (EOs) incorporated edible film was evaluated to improve the storage quality of chicken meat patties. Several preliminary trials were carried out to optimize the levels of bio-polymer to obtained desired edible film as a carrier and blend of EOs as bio preservatives. Preliminary studies indicated that 1.5% (w/v) solution of carrageenan as bio-polymer and 0.10% (v/v) oregano with 0.15% (v/v) thyme EOs in blend form as antimicrobial were suitable. Chicken meat patties wrapped with edible film incorporated with aforementioned EOs, packaged aerobically were stored at refrigeration temperature (4 ± 1 °C) for further studies. Results of refrigeration storage, showed that control samples had significantly higher pH and thiobarbituric acid reacting substances value than EOs treated products. There were significantly lower microbial counts observed in treatment samples (with EOs) and found well within permissible limit as compared to control. All the treatment samples showed lower or comparable flavour score in regard with control. It was found that shelf-life of chicken meat patties increased significantly (P < 0.05) during refrigerated storage and showed acceptable quality up to storage period of 30 days.

Development and Evaluation of Simple Dot-Blot Assays for Rapid Detection of Staphylococcal Enterotoxin-A in Food.

The present study was aimed to develop and evaluate dot-blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot-blot format, recombinant SEA was directly coated on NCM dot-blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot-blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot-blot assays exhibited a sensitivity of ~48 ng ml-1 when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot-blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot-blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.

Effect of feeding sugarcane press mud on carcass traits and meat quality characteristics of lambs.

AIM: To explore the possibilities of feeding unconventional agro-industrial byproduct for livestock production. Sugarcane press-mud (SPM), is a byproduct derived from sugarcane industry, which is rich in protein as well as minerals. The effects of dietary inclusion of SPM at different levels on the carcass characteristics of lambs were evaluated. MATERIALS AND METHODS: SPM was incorporated in concentrate mixture at different levels 0% (SP0 - concentrate mixture without SPM [Control diet]), 10% (SP10 - concentrate mixture containing 10% SPM) and 20% (SP20 - concentrate mixture containing 20% SPM). The concentrate mixtures were fed along with wheat straw for 180 days. At the end of the experimental period, six lambs per group were slaughtered to evaluate carcass and meat quality characteristics. RESULTS: No significant difference was observed in dressing percentage on pre-slaughter weight or empty body weight basis of lambs fed different levels of SPM incorporated diets. Likewise carcass weight, carcass length, and wholesale cuts appeared to have similar values among groups. The yield of visceral organs, chemical composition, and sensory attributes were not statistically affected by inclusion of SPM in the diets except juiciness of control group meat was significantly (p<0.05) higher than treatment group (SP20). CONCLUSION: The SPM can be incorporated in the diet of lambs up to the level of 20% without affecting the carcass characteristics of lambs.

Determination of sex origin of meat and meat products on the DNA basis: a review.

Sex determination of domestic animal's meat is of potential value in meat authentication and quality control studies. Methods aiming at determining the sex origin of meat may be based either on the analysis of hormone or on the analysis of nucleic acids. At the present time, sex determination of meat and meat products based on hormone analysis employ gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS), and enzyme-linked immunosorbent assay (ELISA). Most of the hormone-based methods proved to be highly specific and sensitive but were not performed on a regular basis for meat sexing due to the technical limitations or the expensive equipments required. On the other hand, the most common methodology to determine the sex of meat is unquestionably traditional polymerase chain reaction (PCR) that involves gel electrophoresis of DNA amplicons. This review is intended to provide an overview of the DNA-based methods for sex determination of meat and meat products.

Identification of species origin of meat and meat products on the DNA basis: a review.

The adulteration/substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness, and unhealthy competition in meat market. Consumer should be protected from these malicious practices of meat adulterations by quick, precise, and specific identification of meat animal species. Several analytical methodologies have been employed for meat speciation based on anatomical, histological, microscopic, organoleptic, chemical, electrophoretic, chromatographic, or immunological principles. However, by virtue of their inherent limitations, most of these techniques have been replaced by the recent DNA-based molecular techniques. In the last decades, several methods based on polymerase chain reaction have been proposed as useful means for identifying the species origin in meat and meat products, due to their high specificity and sensitivity, as well as rapid processing time and low cost. This review intends to provide an updated and extensive overview on the DNA-based methods for species identification in meat and meat products.

Comparison of quality attributes of buffalo meat curry at different storage temperature.

BACKGROUND: The product quality of curry is determined by the food animal source, raw materials and the method of processing. Moreover the scientific information on processing and quality of traditional buffalo meat curry from different groups of buffaloes is not available. This study was undertaken to develop processed curry from different buffalo groups and to compare its quality during storage at ambient and refrigeration temperature. MATERIAL AND METHODS: The meat samples were collected from the longissimus dorsi muscle of the carcasses from each group of buffaloes slaughtered according to the traditional halal method. Buffalo meat curry was prepared in a pressure cooker with the standardized formulation. This final product was subjected to evaluation of quality and shelf life. RESULTS: To evaluate the effect of different groups of meat samples on the quality of curry, product yield, pH, proximate composition, water activity (aw), thiobarbituric acid reactive substances (TBARS), calorific value, sensory attributes and microbiological assay were determined The energy of meat curry from young buffaloes was significantly lower than the meat curry from spent animal groups. The overall acceptability of curry decreased significantly during 3 days ambient storage compared to refrigeration storage. CONCLUSIONS: Scientific processing by adopting good manufacturing practices and suitable packaging helped greatly to improve the shelf life of the ambient temperature stored buffalo meat curry. Buffalo meat curry from young male group showed better product characteristics and overall acceptability scores than spent buffalo group.