Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis.

This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

Are online meatball restaurants in Indonesia committed to their declared Halal label?

Background and Aim: Halal restaurants participating in online food delivery services do not require halal certification. The Halal status of products through the Halal logo provides the consumer with information on the basis of which he decides to buy. Online transactions involve potential risks related to online processes, payment methods, and product quality. The aim of this study was to determine whether a declared Halal label is in accordance with the business processes implemented. Materials and Methods: Halal authentication of Gofood's meatball partner products in Yogyakarta and Solo Raya determined the incompatibility of meatball ingredients. Sixty meatball samples were collected from Yogyakarta and 30 samples from Solo Raya. Halal certification test was carried out using the thermal cycle polymerase chain reaction method at Universitas Gadjah Mada Animal Husbandry Laboratory and the results were used to identify pork contamination in meatballs. The addition of pork or pork meatballs was used as a control. Results: Eight meatball restaurants in the Solo Raya and Yogyakarta areas were found to be contaminated with pig DNA. The results of the tracing materials and processes, i.e., the grinding stage, are critical because all samples were supposed to be made from beef. It is known from interviews that contamination with pig DNA at the milling stage was accidental. Conclusion: Restaurants that sell meatballs are committed to adhering to product labels that are 91.1% safe from pork contamination. The Halal and original beef labels were in accordance with their statements. This study highlights the concept of Halal authentication with traceability to overcome pork contamination in meat products.

Analysis of dog meat adulteration in beef meatballs using non-targeted UHPLC-Orbitrap HRMS metabolomics and chemometrics for halal authentication study.

Due to the different price and high quality, halal meat such as beef can be adulterated with non-halal meat with low price to get an economical price. The objective of this research was to develop an analytical method for halal authentication testing of beef meatballs (BM) from dog meat (DM) using a non-targeted metabolomics approach employing liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and chemometrics. The differentiation of authentic BM from that adulterated with DM was successfully performed using partial least square-discriminant analysis (PLS-DA) with high accuracy (R2X = 0.980, and R2Y = 0.980) and good predictivity (Q2 = 0.517). In addition, partial least square (PLS) and orthogonal PLS (OPLS) were successfully used to predict the DM added (% w/w) in BM with high accuracy (R2 > 0.990). A number of metabolites, potential for biomarker candidates, were identified to differentiate BM and that adulterated with DM. It showed that the combination of a non-targeted LC-HRMS Orbitrap metabolomics and chemometrics could detect up to 0.1% w/w of DM adulteration. The developed method was successfully applied for analysis of commercial meatball samples (n = 28). Moreover, pathway analysis revealed that beta-alanine, histidine, and ether lipid metabolism were significantly affected by dog meat adulteration. In summary, this developed method has great potential to be developed and used as an alternative method for analysis of non-halal meats in halal meat products.

Characterization and functional properties of gelatin from goat bone through alcalase and neutrase enzymatic extraction.

BACKGROUND AND AIM: Gelatin is a dissolved protein that results from partial extraction of collagen, commonly from pig and bovine skin. There was no study on gelatin production from Kacang goat bones through enzymatic extraction. This study aimed to evaluate the chemical, physical, and functional properties of gelatin from bones of Kacang goat using alcalase and neutrase enzymes. MATERIALS AND METHODS: Male Kacang goat bones aged 6-12 months and two commercial enzymes (alcalase and neutrase) were used for this study. Descriptive analysis and completely randomized design (one-way analysis of variance) were used to analyze the chemical, physical, and functional properties of gelatin. Kacang goat bone was extracted with four concentrations of alcalase and neutrase enzymes, namely, 0 U/g (AG-0 and NG-0), 0.02 U/g (AG-1 and NG-1), 0.04 U/g (AG-2 and NG-2), and 0.06 U/g (AG-3 and NG-3) with five replications. RESULTS: The highest yield of gelatin extraction with alcalase obtained on AG-3 was 9.78%, and that with neutrase on NG-3 was 6.35%. The moisture content of alcalase gelatin was 9.39-9.94%, and that of neutrase gelatin was 9.15-9.24%. The ash and fat content of gelatin with alcalase was lower than that without enzyme treatment with higher protein content. The lowest fat content was noted in AG-1 (0.50%), with protein that was not different for all enzyme concentrations (69.65-70.21%). Gelatin with neutrase had lower ash content than that without neutrase (1.61-1.90%), with the highest protein content in NG-3 (70.89%). The pH of gelatin with alcalase and neutrase was 6.19-6.92 lower than that without enzymes. Melting points, gel strength, and water holding capacity (WHC) of gelatin with the highest alcalase levels on AG-1 and AG-2 ranged from 28.33 to 28.47°C, 67.41 to 68.14 g bloom, and 324.00 to 334.67%, respectively, with viscosity that did not differ, while the highest foam expansion (FE) and foam stability (FS) were noted in AG-1, which were 71.67% and 52.67%, respectively. The highest oil holding capacity (OHC) was found in AG-2 (283%). FS and OHC of gelatins with the highest neutrase levels in NG-2 were 30.00% and 265.33%, respectively, while gel strength, viscosity, FE, and WHC of gelatins with the highest neutrase levels did not differ with those without enzymes at all enzyme concentrations. B chain was degraded in all gelatins, and high-intensity a-chains in gelatin with alcalase and peptide fraction were formed in gelatin with neutrase. Extraction with enzymes showed loss of the triple helix as demonstrated by Fourier transform infrared spectroscopy. CONCLUSION: Based on the obtained results, the Kacang goat bone was the potential raw source for gelatin production. Enzymatic extraction can increase the quality of gelatin, especially the alcalase (0.02-0.04 U/g bone) method. This can be used to achieve the preferable quality of gelatin with a higher yield.

Angiotensin-converting enzyme inhibitor activity of peptides derived from Kacang goat skin collagen through thermolysin hydrolysis.

BACKGROUND AND AIM: Angiotensin-converting enzyme (ACE) is one of the inhibitory enzymes isolated from animals for the treatment of hypertension. ACE inhibitor (ACE-I) peptides can be obtained by hydrolyzing proteins from various animal tissues, including muscle and connective tissues. However, the study on ACE-I activity from collagen of Kacang goat skin has not been conducted. This study explores the potency of collagen from Kacang goat skin as a source of an antihypertensive agent through ACE inhibition. Thermolysin will hydrolyze collagen and produce the peptide classified antihypertensive bioactive peptides. This study aimed to determine the potential of thermolysin to hydrolyze collagen of Kacang goat skin for ACE-I peptide production and to identify the production of ACE-I peptides. MATERIALS AND METHODS: Collagen from Kacang goat skin was hydrolyzed with thermolysin and incubated at 37°C for 1 h. Molecular weight (MW) evaluation was performed by SDS PAGE; fractionation peptides at <5 kDa, 3-5 kDa, and <3 kDa were performed by ultrafiltration and ACE-I activity determined by IC50 measurement. RESULTS: Collagen was hydrolyzed by thermolysin, resulting in protein with MW of 117.50-14.60 kDa. The protein content of fractionation at >5 kDa was 3.93±0.72 mg/mL, content of 3-5 kDa was 3.81±0.68 mg/mL, and that of <3 kDa was 2.33±0.38 mg/mL. Fractionation was performed 3 times and one of the results was selected for the ACE-I test. The selected fraction was tested by IC50 measurement with three repetitions and it showed an average enzyme activity at 0.83 mg/mL or 82.94 mg/mL. CONCLUSION: Thermolysin hydrolysis of collagen from Kacang goat skin showed the potential to produce bioactive peptides, such as ACE-I.

The employment of q-PCR using specific primer targeting on mitochondrial cytochrome-b gene for identification of wild boar meat in meatball samples.

OBJECTIVE: The objective of this study was to employ real-time or quantitative polymerase chain reaction (q-PCR) using novel species specific primer (SSP) targeting on mitochondrial cytochrome-b of wild boar species (CYTBWB2-wb) gene for the identification of non-halal meat of wild boar meat (WBM) in meatball products. MATERIALS AND METHODS: The novel SSP of CYTBWB2-wb was designed by our group using PRIMERQUEST and NCBI software. DNA was extracted using propanol-chloroform-isoamyl alcohol method. The designed SSP was further subjected for validation protocols using DNA isolated from fresh meat and from meatball, which include specificity test, determination of efficiency, limit of detection and repeatability, and application of developed method for analysis of commercially meatball samples. RESULTS: The results showed that CYTBWB2-wb was specific to wild boar species against other animal species with optimized annealing temperature of 59°C. The efficiency of q-PCR obtained was 91.9% which is acceptable according to the Codex Allimentarius Commission (2010). DNA, with as low as 5 pg/µl, could be detected using q-PCR with primer of CYTBWB2-wb. The developed method was also used for DNA analysis extracted from meatball samples commercially available. CONCLUSION: q-PCR using CYTBWB2-wb primers targeting on mitochondrial cytochrome-b gene (forward: CGG TTC CCT CTT AGG CAT TT; Reverse: GGA TGA ACA GGC AGA TGA AGA) can be fruitfully used for the analysis of WBM in commercial meatball samples.

Universal mitochondrial 16s rRNA biomarker for mini-barcode to identify fish species in Malaysian fish products.

Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96-100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers' health and economic interests.