Prediction, Synthesis and Evaluation of a Synthetic Peptide as an Enzyme-Linked Immunosorbent Assay (ELISA) Candidate for Screening of Bovine Antibodies against Theileria annulata.

Tick-borne diseases (TBDs) of livestock are endemic across various parts of tropical countries. Theileriosis is one such economically important TBD, caused by the Theileriidae family of organisms, which is transmitted by ticks. Theileria annulata, the causative agent of tropical theileriosis, contributes a significant loss to the dairy sector by causing anorexia, high fever, anemia, inflammatory changes in vital organs and icterus, thus, a loss in milk yield. Though vaccines are available, their protective efficacy is not absolute, and treatment is limited to early diagnosis of the causative agent. Routinely, microscopic identification of piroplasms in the erythrocytes (Giemsa-stained) of infected animals or schizonts in lymph node biopsies are practiced for diagnosis. PCR-based techniques (multiplex, uniplex, nested and real-time) have been reported to perform well in diagnosing active infection. Several attempts have been made using serological assays like Dot blot, ELISA and ICT, but the results were of variable sensitivity and specificity. Recombinant proteins like the Theileria annulata merozoite surface antigen (Tams1) and Theileria annulata surface protein (TaSP) have been explored as antigenic candidates for these assays. In the present study, we predicted an immunogenic peptide, i.e., TaSP-34, from the TaSP using various computational tools. The predicted peptide was custom synthesized. The diagnostic potential of the peptide was assessed by indirect plate ELISA to detect the bovine-IgM against Theileria annulata. Alongside, a recombinant truncated TaSP (rTaSP(tr)) was expressed and purified, which was used to compare the performance of the peptide as a diagnostic candidate. The IgM-based peptide ELISA was 100% sensitive and 92.77% specific as compared to PCR (Tams1 targeting), while 98.04% sensitivity and 97.44% specificity were observed in comparison with rTaSP(tr) ELISA. Almost perfect agreement between peptide ELISA and Tams1 PCR was observed with a Cohen's kappa coefficient (κ-value) of 0.901 and agreement of 95.31%. Further, the κ-value between the peptide ELISA and rTaSP(tr) ELISA was found to be 0.95, and the agreement was 97.65%, which shows a good correlation between the two tests. The findings suggest that the TaSP-34 peptide can be an efficient and new-generation diagnostic candidate for the diagnosis of T. annulata. Furthermore, the peptide can be synthesized commercially at a larger scale and can be a cost-effective alternative for the protein-based diagnostic candidates for T. annulata.

Effect of Bamboo Essential Oil on the Oxidative Stability, Microbial Attributes and Sensory Quality of Chicken Meatballs.

This study explores the efficacy of bamboo essential oil (BEO) incorporated at 15 ppm (T1, BEO-I) and 30 ppm (T2, BEO-II) on the overall physicochemical and oxidative stability, microbial deterioration, and sensory acceptability of meatballs stored for 20 days under refrigerated conditions. Analysis of various parameters, including physicochemical quality, color (CIE L*, CIE a* and CIE b*), generation of oxidative products (TBARS), microbial growth, and sensory acceptability of meatballs were evaluated at 5-day intervals. In addition, the total phenolics and flavonoid content of BEO were estimated, and fatty acids were determined by Gas chromatography (GC.) To gain insights into the biological activities of the BEO, antioxidant assays were determined in vitro using various methods. The antibacterial activity of BEO was also evaluated against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Vibrio cholera, Salmonella Typhimurium, Shigella flexneri, Proteus vulgaris, Escherichia coli and Klebsiella pneumoniae) bacterial strains. The BEO contained a good quantity of total phenolics and flavonoids. In addition, the oil exhibited very potent antioxidant activity scavenging reactive oxygen and other such species, effectively showing IC50 at a very minimal concentration. Further, the BEO exhibited a strong antibacterial effect with MICs within 2 µL and MBCs from 5 to 7 µL for Gram-positive as well as Gram-negative bacteria, respectively. At both the concentrations used, BEO did not show any negative effect on the color of cooked meatballs but rather increased the microbiological and oxidative stability during the overall storage period. Meatballs treated with BEO had considerably reduced oxidative changes in terms of TBARS levels compared to the control. The total viable microbial count was lowest in BEO-treated meatballs and the highest in control. Both control and treated meatballs had a desirable flavor and good acceptability. The sensory attributes and aroma of treated meatballs were better and acceptable during the storage study, whereas the control samples were disliked by the panelists on 15th day. From this study, it can be concluded that bamboo essential oil could be used as a benign and non-toxic preservative to improve the quality and shelf life of cooked meatballs stored under refrigerated conditions.

Characterization of crude extract prepared from Indian curd and its potential as a biopreservative.

The adverse effects of chemical preservatives used to prevent food spoilage have led to the search for various biopreservatives. Considering this, a study was undertaken to prepare crude extract (CE) from Indian curd (a fermented dairy product) and characterized it in terms of antioxidant and antimicrobial activities against some common food-borne bacteria. The CE exhibited well pronounced antimicrobial activity against Bacillus cereus and Salmonella typhimurium. The minimum inhibitory concentration (MIC) of CE was recorded for 2-fold concentrated solution prepared from a 10-fold stock. The CE exhibited a significantly higher (p < 0.05) antioxidant and antimicrobial activities compared to its fractions. The CE was found to be heat stable (up to 100 ℃ for 30 min) and exhibited a significant (p < 0.05) increase in activity at pH 2-7 and in combination with 2% citric acid solution. Trypsin treatment suggested it to be of proteinaceous in nature. The antibacterial activity of CE remained intact at 4 ℃ for seven days, whereas non-significant (p > 0.05) changes in its activity were noted during storage at -20 ℃ for 30 days. The curd sample used for preparation of CE, when tested for bacteriocin production and subsequent antimicrobial activity, did not show inhibition against S. typhimurium. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of CE and its fractions revealed multi-banding pattern. By virtue of its bioactivities observed, CE can be explored as a promising food biopreservative.

Inhibition of lipid and protein oxidation in raw ground pork by Terminalia arjuna fruit extract during refrigerated storage.

OBJECTIVE: Terminalia arjuna plant, specially its leaves, bark, and roots, are widely used in traditional herbal medicine due to presence of bioactive components and being a rich source of natural antioxidants. But its fruit has not been used for any such purposes despite its potential to retard oxidation. Hence, the antioxidant potential of Arjuna fruit extract (AFE) in retarding lipid and protein oxidation of raw ground pork was evaluated during refrigerated storage for 9 days. METHODS: The AFEs were prepared using different solvents viz. ethanol (EH), water, ethanol: water (60:40) and methanol:hot water (60:40). The AFEs were analysed for total phenolic content (TPC), 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power. Water extract (WE) and ethanol-water extract (EH-WE) were selected and incorporated at 1.0% into freshly minced pork meat and compared with a synthetic antioxidant, in retarding lipid and protein oxidation during storage. RESULTS: The TPC in AFEs using different solvents ranged from 11.04 to 16.53 mg gallic acid equivalents/g and extracts exhibited appreciable scavenging activity ranging from 50.02% to 58.62%. Arjuna extracts significantly (p<0.05) improved the colour score of meat samples by reducing the formation of metmyoglobin during storage. Both the AFEs (WE and EH-WE) significantly (p<0.05) lowered the thiobarbituric acid reactive substances value, peroxide formation and formation of protein carbonyls in raw pork than control sample during storage. Upon sensory evaluation of all samples, it was found that AFE treatment could prolong the storage period of meat samples, without influencing the colour and odour score, up to 6 days. CONCLUSION: AFEs used at 1% improved the oxidative stability, colour and odour score and prolonged the refrigerated shelf life of ground pork up 6 days. Therefore, AFE could be explored as an alternative natural antioxidant in retarding lipid and protein oxidation in meat products.