Authentication of traditional meat products (Haleem) sold in India: a first report confirming species mislabelling.

Traditional meat products like Haleem play a pivotal role in the culinary landscapes of Indian consumers, along with high economic value and business potential. Due to anticipated gains associated with adulterating 'Haleem' and constant evasion from regulatory oversight, the susceptibility to adulteration has substantially increased. Furthermore, no reports/surveillance regarding their labelling compliance has been reported. Hence, we conducted a 2-year surveillance using 100 samples collected from Hyderabad, India, using the Chipron™ DNA macroarray analysis technique. The method was validated for sensitivity (1%), specificity, and with proficiency test samples. Following this, the surveillance samples (beef, chicken, and mutton Haleem) were tested. The surveillance revealed an alarming adulteration of 46% of the samples, with different proportions of adulterant species. Adulteration of unconventional meat like camel meat was also found. These concerning results necessitate the requirement of stricter and constant regulatory surveillance to safeguard consumer trust and preserve the authenticity of traditional meat products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05947-9.

Duplex real-time PCR assay with high-resolution melt analysis for the detection and quantification of Listeria species and Listeria monocytogenes in meat products.

Listeria contamination in foods of animal origin is one of the most concerning food safety issues. A duplex, SYBR green-based, real-time PCR assay was developed with high-resolution melting analysis-based differentiation of the genus Listeria and Listeria monocytogenes. The primers were designed and tested against other related foodborne pathogens. The assay was optimized for standard parameters in a non-orthogonal fashion and validated following international standards. The LODabs and LOQ of the assay were calculated to be 0.78 and 1.56 ng of the target DNA. The LODrel of the assay was found to be 1% Listeria DNA in background DNA. The assay was evaluated for applicability in artificially spiked samples, providing a 120 CFU/ml detection. The assay was validated with proficiency test samples and also with samples collected for surveillance analysis. This well-established and validated assay can be utilized as a qualitative and quantitative tool for addressing the Listeria contamination in the food safety contexts. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05695-2.

Development and comparative evaluation of droplet digital PCR and quantitative PCR for the detection and quantification of Chlamydia psittaci.

Chlamydia psittaci is a zoonotic pathogen mainly transmitted by psittacine birds and poultry. The low shedding rate of the pathogen in the apparently healthy birds and human clinical cases may result in false-negative results. In the present study, a droplet digital PCR (ddPCR) assay was developed and compared with optimized quantitative PCR (qPCR) for the detection of C. psittaci from the clinical samples. The ddPCR assay was found to be comparatively more sensitive than the qPCR, wherein the limit of detection (LOD) of ddPCR was upto 2.4 copies of the DNA template, whereas, the qPCR could detect upto 38 copies of the DNA template in the reaction mixture. Overall, the developed ddPCR assay was found to be robust, specific, and could reliably quantify up to 17.8 copies of the DNA template. Finally, the applicability of the developed ddPCR assay was tested by screening the field samples (n = 124), comprising lung tissues from dead poultry and feral birds; pooled faecal samples from the free-living birds, commercial and backyard poultry farms; pharyngeal and cloacal swabs collected from the duck farms. Of these, a total of seven samples were found to be positive by the ddPCR, whereas, three samples could be detected as positive using the qPCR. The developed ddPCR could serve as a reliable screening tool, particularly in those clinical samples wherein the shedding of C. psittaci is substantially very low.

Authentication of camel meat using species-specific PCR and PCR-RFLP.

In India and some of the African countries, one of the unconventional meats receiving the latest attention in meat adulteration is camel meat. So, the objective of this study was to develop a species-specific PCR based on mitochondrial cytochrome b (CYTB) gene and a PCR-RFLP assay of mitochondrial 12S rRNA to identify camel meat in suspected samples. Known sample of camel meat, samples suspected to be from illegally slaughtered camel and known samples of cattle, buffalo, sheep, goat, pork and chicken were used in the study. DNA were extracted from all samples following spin column method and PCR amplification were carried out using both CYTB and 12S rRNA gene primers. The CYTB gene amplification produced amplicon with a size of 435 bp without any non-specific spurious amplification towards other species studied. Further, the 12S rRNA PCR products were analysed both by sequencing and by RFLP using enzyme AluI. On BLAST analysis the 448 bp sequence obtained from suspected samples showed > 99% sequence homology to previously reported Camelus dromedaries (accession no: AM 9369251.1). On AluI digestion of the 448 bp product from both known and suspected camel samples, a specific RFLP pattern with three distinct products of 90, 148 and 210 bp size were evident, which were significantly different from the pattern of cattle, sheep, goat, chicken and buffalo. Further, after in-house validation, this cost effective and rapid method of camel meat identification is placed into practice for regular screening of vetero-legal samples in the lab.

OFFGEL electrophoresis and tandem mass spectrometry approach compared with DNA-based PCR method for authentication of meat species from raw and cooked ground meat mixtures containing cattle meat, water buffalo meat and sheep meat.

The present study compared the accuracy of an OFFGEL electrophoresis and tandem mass spectrometry-based proteomic approach with a DNA-based method for meat species identification from raw and cooked ground meat mixes containing cattle, water buffalo and sheep meat. The proteomic approach involved the separation of myofibrillar proteins using OFFGEL electrophoresis, SDS-PAGE and protein identification by MALDI-TOF MS. Species-specific peptides derived from myosin light chain-1 and 2 were identified for authenticating buffalo meat spiked at a minimum 0.5% level in sheep meat with high confidence. Relative quantification of buffalo meat mixed with sheep meat was done by quantitative label-free mass spectrometry using UPLC-QTOF and PLGS search engine to substantiate the confidence level of the data. In the DNA-based method, PCR amplification of mitochondrial D loop gene using species specific primers found 226bp and 126bp product amplicons for buffalo and cattle meat, respectively. The method was efficient in detecting a minimum of 0.5% and 1.0% when buffalo meat was spiked with cattle meat in raw and cooked meat mixes.

Species identification of cattle and buffalo fat through PCR assay.

A method was standardized to isolate quality DNA from cattle and buffalo fat for species identification using QIAamp DNA stool mini kit. The quality of the DNA was sufficient enough to amplify universal primers viz., mt 12S rRNA and mt 16S rRNA, and species specific D loop primers for cattle and buffalo. The sensitivity of the PCR assay in the species specific D loop primer amplification was with a detection level of 0. 47 ng cattle DNA and 0.23 ng buffalo DNA in simplex and, 0. 47 ng cattle DNA and 0.12 ng buffalo DNA in duplex PCR. It is a potentially reliable method for DNA detection to authenticate animal fat.

Colour, myoglobin denaturation and storage stability of raw and cooked mutton chops at different end point cooking temperature.

In our study effect of different end point temperature (51 °C, 65 °C, 71 °C and 79 °C) on physicochemical and storage stability of mutton chops were evaluated. The L* (lightness) value and b* (yellowness) increased (P < 0.05) in cooked mutton chops than the raw mutton. The a* value (redness) decreased (P < 0.05) as end point temperature increased. As internal cooking temperature increased soluble myoglobin content decreased with a corresponding increase in percent myoglobin denatured. Raw mutton chops (uncooked) had lower level of oxidation (less TBA values) than cooked mutton irrespective of storage length. Initial APC of raw and cooked mutton chops ranged from log 1.75 to log 3.73 and was lower in higher end point cooking temperature. It can be concluded that as end point temperature increased, mutton chops appear less red and raw mutton had lower level of oxidation than cooked mutton chops.

Effect of incorporation of Moringa oleifera leaves extract on quality of ground pork patties.

Present study was conducted to evaluate the effect of addition of different levels of Moringa oleifera leaves extract (MLE) and butylated hydroxytoluene (BHT) in raw and cooked pork patties during refrigerated storage. Five treatments evaluated include: Control (without MLE/BHT), MLE 300 (300 ppm equivalent M. oleifera leaves phenolics), MLE 450 (450 ppm equivalent M. oleifera leaves phenolics), MLE 600 (600 ppm equivalent M. oleifera leaves phenolics) and BHT 200 (200 ppm BHT). Total phenolic content ranged from 60.78 to 70.27 mg per gram. A concentration dependent increase in reducing power and 1,1-diphenyl 2-picrylhydrazyl (DPPH) radical scavenging activity of both MLE and BHT was noticed. Higher (P < 0.001) a* and lower thiobarbituric acid reactive substances values were observed in MLE 600 and BHT 200 compared to control. Addition of MLE did not affect the sensory attributes or microbial quality. These results showed that M. oleifera leaves can be used as a potential source of natural antioxidants to inhibit lipid oxidation in ground pork patties.

Relationship between the solubility, dosage and antioxidant capacity of carnosic acid in raw and cooked ground buffalo meat patties and chicken patties.

Antioxidant capacity of oil soluble and water dispersible carnosic acid (CA) extracted from dried rosemary leaves using HPLC was evaluated at two different dosages (22.5 ppm vs 130 ppm) in raw and cooked ground buffalo meat patties and chicken patties. Irrespective of total phenolic content, CA extracts reduced (p<0.05) the thiobarbituric acid reactive substances (TBARS) by 39%-47% and 37%-40% in cooked buffalo meat and chicken patties at lower dosage (22.5 ppm) relative to control samples. However, at higher dosage (130 ppm) the TBARS values were reduced (p<0.05) by 86%-96% and 78%-87% in cooked buffalo meat and chicken patties compared to controls. The CA extracts were also effective in inhibiting (p<0.05) peroxide value and free fatty acids in cooked buffalo meat and chicken patties. The CA extracts when used at higher dosage, were also effective in stabilizing raw buffalo meat color.

A rapid method for authentication of Buffalo (Bubalus bubalis) meat by Alkaline Lysis method of DNA extraction and species specific polymerase chain reaction.

Buffalo (Bubalus bubalis) meat is a major item of export from India but export of beef i.e. meat from cattle (Bos indicus) is prohibited. Also, adulteration of buffalo meat with that of beef (meat from cattle) is a common fraudulent practice because of prohibition on cow slaughter in most states of India. Food analysts require precise identification techniques to implement such regulations. In the present study, a method of DNA extraction by Alkaline lysis from meat samples and speciation of buffalo meat using species specific Polymerase Chain Reaction (PCR) has been reported. Alkaline lysis technique is a rapid method which involves triturating meat with four volumes of 0.2N NaOH, dilution of resultant liquid extract with eight volumes of 0.2N NaOH, heating the mix 75 °C for 20 min followed by neutralization with eight volumes of 0.04N Tris HCl. Entire procedure of DNA extraction takes less than 30 min and it is economical as it involves less expensive chemicals. Method was successfully applied in animal byproducts also viz., liver, heart and kidney. For authentication of buffalo meat, pair of primers was designed based on mitochondrial D loop gene nucleotide sequence. PCR amplification using the designed primers gave amplicon of size 482 bp in buffalo and no amplification was detected in closely related species viz., cattle, sheep and goat meat samples. Results of the assay were highly repetitive and reliable. An export sample referred by export regulation authorities was also analyzed by using the Alkaline lysis method of DNA extraction and species specific PCR which enabled authentication of meat within 5 h.

Effect of ammonium hydroxide on ultrastructure and tenderness of buffalo meat.

This study was conducted with an objective to improve the tenderness of tough buffalo meat using ammonium hydroxide. Buffalo meat chunks from Biceps femoris muscle were marinated with distilled water (control), 0.1%, 0.5% and 1.0% solution of ammonium hydroxide for 48 h at 4±1 °C and subjected to various physico-chemical analysis and ultrastructural studies. Ammonium hydroxide increased (P<0.05) the pH, water holding capacity (WHC), collagen solubility, total and salt soluble protein extractability and cooking yield. Reduction (P<0.05) in Warner-Bratzler shear force values were observed in all ammonium hydroxide treated samples compared to non-treated control. Electrophoretic pattern of muscle proteins exhibited reduction in the intensity and number of certain protein bands for 0.1% and 0.5% ammonium hydroxide treated samples compared to control. Scanning and transmission electron microscopy also revealed breakdown of endothelium layers surrounding muscle fibers and weakening of Z-discs respectively, in treated samples compared to controls. These results suggest that ammonium hydroxide might be used to tenderize tough buffalo meat.

Effect of dipping in pomegranate (Punica granatum) fruit juice phenolic solution on the shelf life of chicken meat under refrigerated storage (4°C).

An experiment was conducted to evaluate the effect of dipping in pomegranate fruit juice phenolics (PFJP) solution on the shelf life of chicken meat held under refrigerated storage at 4°C. Breast muscle obtained from spent hens was dipped (1:2w/v; muscle: liquid) in sterile water or in sterile water with 0.02% (v/v) PFJP, packed, stored at 4°C for 28 days and samples were analyzed on 2 days of intervals. Thiobarbituric acid reactive substance values were lower in samples treated with PFJP. Total sulfhydryl and protein bound sulfhydryl content values were higher in samples treated with PFJP. Microbial quality evaluation showed that aerobic and psychrotrophic counts were higher in samples treated without PFJP. Sensory evaluation revealed that acceptability level of samples treated without PFJP decreased on 12th day of storage. It is concluded that spent hen breast meat samples dipped in 0.02% PFJP reduced protein oxidation and inhibited microbial growth and sensorily acceptable up to 12 days of refrigerated storage at 4°C.

Selection of white-rot basidiomycetes for bioconversion of mustard (Brassica compestris) straw under solid-state fermentation into energy substrate for rumen micro-organism.

AIMS: Selection of white-rot fungi of bio-conversion of mustard straw (MS) into feed for ruminants. METHODS AND RESULTS: Mustard straw was cultured with Ganoderma applanatum, Coriolus versicolor and Phanerochaete chrysosporium for solid-state fermentation at 35 degrees C from 7 to 63 days for delignification and for 21 days to study dry matter digestibility and protein enrichment. Lignin loss in fungus cultured straw varied between 100 and 470 g kg(-1) lignin. Delignification was higher between 7 and 28 days fermentation with C. versicolor. Among the three fungi P. chrysosporium was the most effective in degrading lignin for longer fermentation. In-vitro dry matter digestibility (IVDMD) and crude protein content was higher in C. versicolor cultured straw. Large quantity of straw was cultured by C. versicolor for 21 days, for in vivo evaluation. Mean pH and metabolites of rumen fermentation were not different while, pH and volatile fatty acid increased at 6 h postfermentation on cultured straw feeding. Cultured straw fermentation increased (P = 0.001) small holotricks and reduced (P = 0.005) large holotricks population. Fungus cultures straw did not improve microbial enzyme concentration. CONCLUSIONS: Coriolus versicolor and P. chrysosporium were the promising fungus for MS bio-delignification. SIGNIFICANCE AND IMPACT OF THE STUDY: Coriolus versicolor treated MS improved dry matter digestibility and protein content.

Biochemical and physicochemical changes in spent hen breast meat during postmortem aging.

An experiment was conducted to study the biochemical and physicochemical changes with respect to improvement in tenderness of spent hen breast meat. Breast muscle obtained from freshly slaughtered spent hens (72 wk old) was divided into 5 equal lots and dipped in 1 mM NaN(3) before being packed in low-density poly-ethylene pouches under aerobic conditions and stored at refrigeration temperature (4 degrees C). Lots were removed on 0, 7, 14, 21, and 28 d of storage and analyzed for pH, TBA reactive substances (TBARS), total sulfhydryl content, protein-bound sulfhydryl content, nonprotein-bound sulfhydryl content, perimysial fraction, collagen content, free OH-proline, N, nonprotein N, and proteolysis rate. Shear force value and penetrometer readings were also determined after making patties from the stored muscle samples. Results showed that pH values were gradually decreasing over the storage period. The TBARS values were increasing (P < 0.001), whereas the sulfhydryl content was decreasing (P < 0.001) over the storage period. The TBARS values were negatively (P < 0.05) correlated with total sulfhydryl content. This suggests that sulfhydryl content may prevent further higher oxidation of lipids. The soluble collagen content, collagen solubility, free OH-proline, and proteolysis rate were increasing (P < 0.001) during postmortem aging. These results suggest that collagen degradation into free amino acids occurs postmortem. A gradual decrease (P < 0.001) in shear force value and a gradual increase (P < 0.001) in penetrometer readings were recorded in the patties made from matured breast meat. Therefore, postmortem aging of spent hen breast meat resulted in 23% improvement in tenderness of minced patties on 14 d and 39% on 28 d as evidenced by biochemical and physicochemical changes.

Comparative efficacy of pomegranate juice, pomegranate rind powder extract and BHT as antioxidants in cooked chicken patties.

A study was carried out to evaluate the antioxidant potential of pomegranate juice (PJ), rind powder extract (RP) and butylated hydroxyl toluene (BHT) in cooked chicken patties during refrigerated storage. Freshly minced chicken meats were assigned to one of the following four treatments: control (meat treated with no antioxidants); 10mg equivalent PJ phenolics per 100g meat; 10mg equivalent RP phenolics per 100g meat; 10mg BHT per 100g meat. The patties formed from the minced meats were grilled for 20min and stored under aerobically at 4°C for 15 days. Total phenolic content (as tannic acid equivalent) significantly (P<0.05) increased from 152 in control to 195 and 224µg/g in PJ and RP patties. Addition of PJ or RP did not affect any of the sensory attributes. The TBARS values were significantly (P<0.05) reduced from 1.272 in control patties to 0.896, 0.763 and 0.203mg malonaldehyde per kg samples in BHT, PJ and RP patties, respectively. The RP treatment substantially inhibited (P<0.01) lipid oxidation in cooked chicken patties to a much greater extent than BHT treatment. The PJ or RP at a level of 10mg equivalent phenolics/100g meat would be sufficient to protect chicken patties against oxidative rancidity for periods longer than the most commonly used synthetic antioxidant like BHT.

Isolation, partial characterization and growth characteristics of a bacterium resistant to aflatoxin B1.

A bacterium was isolated from the soil of dumping ground for cattle yard waste by enrichment culture containing aflatoxin B1. This bacterium was closely related to Bacillus firmus that was found to be a non-pathogenic bacterium. The minimum inhibitory concentration of aflatoxin B1 to the bacterium was found to be 80 microg ml(-1) as measured by total viable count and soluble protein content methods. The bacterium was sensitive to all the tested antibiotics. Plasmid curing by chemical agents did not show the resistance character residing in the plasmid. Protein profiles of cell extracts of aflatoxin B1 resistant bacterium grown in the presence and absence of the toxin showed 46 and 44 protein bands respectively in SDS-PAGE. It was observed that 39 bands were common in both the extracts and the remaining bands were showing differences near the high molecular weight range.

Mechanism of removal of aflatoxin B1 by a resistant bacterium.

A bacterium resistant to aflatoxin B1 isolated from the soil was shown to remove a maximum 26% of toxin at 3 microg ml(-1). A comparison of spectrophotometric and radioisotope methods showed that a maximum of 48 hr was sufficient to remove the toxin. Radioisotope analysis showed that the radioactivity decreased in the chloroform phase while it increased in the aqueous phase during the time course of the experiment. An analysis of the supernatant of culture medium showed that the bacterium had converted aflatoxin B1 to water soluble compounds with lambda(max) of 338 and 374.