Nanoplate-based digital PCR for highly sensitive pork DNA detection targeting multi-copy nuclear and mitochondrial genes.

The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a 'rain effect' were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive 'rain effect' was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no 'rain effect'. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05% (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated.

Halal authentication using lateral flow devices for detection of pork adulteration in meat products: a review.

Lateral flow devices (LFDs) are straightforward scientific tools that have made substantial advances in recent years. They have been used in many fields including the meat industry to detect disease markers, determine meat freshness or meat species determination. They are, therefore, significant in the research of meat adulteration by mixed animal species, because food component authenticity is a serious concern encompassing health, economic, legal, and religious issues. Pork adulteration is one of the most crucial issues in the global meat industry. In this review, we discuss the various types of LFDs and recent research on the development of LFDs as an authenticity tool for detecting pig additives in meat-based products, and how regulatory authorities could adopt LFDs for their workflows. Despite the benefits of rapidity, simplicity, low cost, high sensitivity, and specificity, researchers face challenges when using LFD as a final confirmation test. Future directions are suggested for globalising the use of LFD as a halal authentication method.

Mining the genome of Bacillus velezensis FS26 for probiotic markers and secondary metabolites with antimicrobial properties against aquaculture pathogens.

Bacillus velezensis FS26 is a bacterium from the genus Bacillus that has been proven as a potential probiotic in aquaculture with a good antagonistic effect on Aeromonas spp. and Vibrio spp. Whole-genome sequencing (WGS) allows a comprehensive and in-depth analysis at the molecular level, and it is becoming an increasingly significant technique in aquaculture research. Although numerous probiotic genomes have been sequenced and investigated recently, there are minimal data on in silico analysis of B. velezensis as a probiotic bacterium isolated from aquaculture sources. Thus, this study aims to analyse the general genome characteristics and probiotic markers from the B. velezensis FS26 genome with secondary metabolites predicted against aquaculture pathogens. The B. velezensis FS26 genome (GenBank Accession: JAOPEO000000000) assembly proved to be of high quality, with eight contigs containing 3,926,371 bp and an average G + C content of 46.5%. According to antiSMASH analysis, five clusters of secondary metabolites from the B. velezensis FS26 genome showed 100% similarity. These clusters include Cluster 2 (bacilysin), Cluster 6 (bacillibactin), Cluster 7 (fengycin), Cluster 8 (bacillaene), and Cluster 9 (macrolactin H), which signify promising antibacterial, antifungal, and anticyanobacterial agents against pathogens in aquaculture. The probiotic markers of B. velezensis FS26 genome for adhesion capability in the hosts' intestine, as well as the acid and bile salt-tolerant genes, were also detected through the Prokaryotic Genome Annotation System (Prokka) annotation pipeline. These results are in agreement with our previous in vitro data, suggesting that the in silico investigation facilitates establishing B. velezensis FS26 as a beneficial probiotic for use in aquaculture.

Differentiation of lard from other animal fats based on n-Alkane profiles using chemometric analysis.

Adulteration of lard with other fats and oils in food production affects many areas including economics, religion, and health. Previous studies discriminated lard based on major components of fats, i.e. triglycerides and fatty acids. This study aimed to differentiate lard and other animal fats (beef, chicken and mutton fat) based on n-alkane profiles established by gas chromatography-mass spectrometry (GC-MS). Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) were able to initiate clustering of lard and other animal fats. Good result was obtained using Random Forest (RF) and Partial Least Squares-Discriminant Analysis (PLS-DA). Statistical models propose tetracosane (C24) as a potential n-alkane marker and it was found that C24 was the major alkane with composition of 15.72% (GC-MS) of total alkanes identified. Based on this finding, more interesting study may potentially be explored for the interest of various fats and oils consumers in vast applications especially using chemometrics analysis.

Next Generation Sequencing-based DNA metabarcoding for animal species profiling in fish feed.

The expansion of worldwide aquaculture has been accompanied by extensive growth in the fish feed industry. However, improper labelling of many commercially available fish feeds has raised security and safety concerns over the species' origin of the ingredients. The inclusion of ruminants-derived ingredients in fish feed is prohibited according to EU legislation while porcine inclusion in fish feed has been a great concern among Muslim farmers. In contrast to the limited species that could be simultaneously determined using multiplex PCR, this study utilised Next Generation Sequencing-based DNA metabarcoding assay to determine the compositional profiles of animal species in fish feed samples in a more holistic manner. In relation to the religious issue associated with porcine-derived ingredients in fish feed, this study firstly aimed to determine the sensitivity of the methods in profiling fish feed adulterated with porcine blood and muscle tissues. Next, 10 commercially available fish feed samples were analysed. As a result, a detection limit of as low as 3% (w/w) porcine muscle and blood in the laboratory-prepared fish feed was obtained. The analysis of 10 commercial fish feeds shows surprising findings: 50% of the feeds contain Sus scrofa and 80% contain Bos taurus, a ruminant. Only one commercial fish feed was found to be solely composed of marine species. This study shows that commercial fish feeds sold in Malaysia contain undesirable animal species, and emphasises the need for accurate and legally enforced labelling of mammalian species in fish feed products.

Evaluation of three Bacillus spp. isolated from the gut of giant freshwater prawn as potential probiotics against pathogens causing Vibriosis and Aeromonosis.

Pathogens from the Vibrio and Aeromonas genera often cause detrimental effects to the aquaculture sector. Previously, antibiotics was used to resolve the infections, but this caused the spread of antibiotic resistant bacteria and antibiotic resistance genes into the environment. As an approach to address this issue, probiotic bacteria were introduced to improve the hosts' microbiome, disease protection, health condition, growth efficiency, feed consumption, stress response and general vigour. However, reports showed that some commercially available probiotics were restricted to a small number of microbial species and there are inconsistencies concerning its effectiveness. Hence, the aim of this study was to isolate and evaluate new Bacillus spp. from the gut of giant freshwater prawn as potential probiotics. Three Bacillus spp. isolates, Bacillus subtilis FS6 (MZ960135), Bacillus velezensis FS26 (MZ960133) and Bacillus pumilus FS97 (MZ960136) were characterised, and in vitro testing showed good probiotic properties which can help in dealing with diseases in aquaculture. Among the Bacillus spp., Bacillus velezensis FS26 showed higher antimicrobial activity towards Aeromonas hydrophila LMG 13658 and Aeromonas veronii clone DK-A. veronii-27 at 23.7 mm and 25 mm, respectively. Bacillus subtilis FS6 and Bacillus velezensis FS26 resulted in good adherence to both xylene and chloroform hydrocarbons. The Bacillus spp. isolated displayed high survivability towards 0.3% bile salt and exhibited amylase, protease, and lipase activities. Thus, the isolated Bacillus spp. are considered safe based on the sensitivity analysis towards antibiotics and γ-haemolytic activity.

Rapid porcine detection in gelatin-based highly processed products using loop mediated isothermal amplification.

Low DNA concentration recovered from highly processed products such as gelatin and gelatin-based products renders difficulty in detecting porcine contamination using conventional PCR techniques. We documented here a porcine-specific loop-mediated isothermal amplification (LAMP) to identify porcine traces in gelatin products. The porcine-specific primers were designed according to mitochondrial DNA of Cytochrome b gene sequence. Here we used two different reaction mixtures for LAMP assay (GENIE and MYRM) against the same DNA samples extracted from gelatin products and porcine-specific primers to detect the presence of porcine DNA. The porcine-specific primers were shown to be specific only to Sus scrofa against 14 DNA of other meat species. The analytical sensitivity of the LAMP assay for porcine DNA detection is 1 pg/µL using both GENIE (within 30 m) and MYRM (within 60 m) reaction mixtures. Analysis against 32 samples of gelatin products showed that five samples were found to contain porcine DNA; two samples out of six gelatin powder samples and three gelatin capsule samples out of nine. Out of these five positive samples, three were not labeled containing porcine gelatin. Overall, LAMP assay in this study showed an excellent specificity, sensitivity and rapidity in detection of porcine DNA in gelatin products. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s13197-020-04932-2).

The characteristics and roles of antimicrobial peptides as potential treatment for antibiotic-resistant pathogens: a review.

The emergence of antibiotic-resistant bacteria has become a significant and ever-increasing threat to global public health, increasing both morbidity and mortality rates, and the financial burden on health services. Infection by drug-resistant bacteria is anticipated to contribute to the demise of almost 10 million people by the year 2050 unless a competent and effective response is devised to engage with this issue. The emergence and spread of resistance are commonly caused by the excessive or inappropriate use of antibiotics and substandard pharmaceuticals. It arises when pathogens adapt to different conditions and develop self-defence mechanisms. Currently, novel antimicrobial peptides (AMPs) have been reported to be the sole cure for some clinical cases of infectious diseases such as sepsis and skin infections, although these agents may, on occasion, require administration together with an adjunctive low-dose antibiotic. Although AMPs are a promising alternative form of anti-microbial therapy and easily applied in the medical sector, they still have limitations that should not be taken lightly. Hence, this review explores the characteristics, advantages and disadvantages of AMPs for their potential in treating antibiotic-resistant pathogens.

Hydroxyproline determination for initial detection of halal-critical food ingredients (gelatin and collagen).

Gelatin and collagen are considered halal-critical ingredients as they are typically derived from either bovine or porcine animals. Current analytical methods for determining the sources of gelatin and collagen suffer from limitations in terms of robustness and false positives in peptide matching. Thus, the aim of this study was to investigate the utility of monitoring hydroxyproline, a signature amino acid for gelatin and collagen, for identifying potentially haram foodstuffs. To determine the hydroxyproline profiles among animal- and plant-based samples, one-way univariate analysis of variance followed by pair-wise comparison was used to establish statistical significance. Multivariate chemometric analysis through principal component analysis revealed a discrete distribution pattern among 59 samples due to hydroxyproline variability. Finally, inter- and intra-laboratory comparisons demonstrated the validity and robustness of hydroxyproline determination according to ISO 17025. Thus, this preliminary identification technique will aid the identification of potentially haram foodstuffs.

Alteration of the Gut Microbiome in Normal and Overweight School Children from Selangor with Lactobacillus Fermented Milk Administration.

Childhood obesity is a serious public health problem worldwide. Perturbations in the gut microbiota composition have been associated with the development of obesity in both children and adults. Probiotics, on the other hand, are proven to restore the composition of the gut microbiome which helps reduce the development of obesity. However, data on the effect of probiotics on gut microbiota and its association with childhood obesity is limited. This study aims to determine the effect of probiotics supplement intervention on gut microbiota profiles in obese and normal-weight children. A total of 37 children, 17 normal weight, and 20 overweight school children from a government school in Selangor were selected to participate in this study. Participants were further divided into intervention and control groups. The intervention groups received daily probiotic drinks while the control groups continued eating their typical diet. Fecal samples were collected from the participants for DNA extraction. The hypervariable V3 and V4 regions of 16S rRNA gene were amplified and sequenced using the Illumina MiSeq platform. No significant differences in alpha diversity were observed between normal weight and obese children in terms of the Shannon Index for evenness or species richness. However, a higher intervention effect on alpha diversity was observed among normal-weight participants compared to obese. The participants' microbiome was found to fluctuate throughout the study. Analysis of the taxa at species level showed an increase in Bacteroides ovatus among the normal weight cohort. Genus-level comparison revealed a rise in genus Lachnospira and Ruminococcus in the overweight participants after intervention, compared to the normal-weight participants. The probiotics intervention causes an alteration in gut microbiota composition in both normal and overweight children. Though the association could not be defined statistically, this study has provided an improved understanding of the intervention effect of probiotics on gut microbiome dysbiosis in an underrepresented population.

Biological control of Erwinia mallotivora, the causal agent of papaya dieback disease by indigenous seed-borne endophytic lactic acid bacteria consortium.

Dieback disease caused by Erwinia mallotivora is a major threat to papaya plantation in Malaysia. The current study was conducted to evaluate the potential of endophytic lactic acid bacteria (LAB) isolated from papaya seeds for disease suppression of papaya dieback. Two hundred and thirty isolates were screened against E. mallotivora BT-MARDI, and the inhibitory activity of the isolates against the pathogen was ranging from 11.7-23.7 mm inhibition zones. The synergistic experiments revealed that combination of W. cibaria PPKSD19 and Lactococcus lactis subsp. lactis PPSSD39 increased antibacterial activity against the pathogen. The antibacterial activity was partially due to the production of bacteriocin-like inhibitory substances (BLIS). The nursery experiment confirmed that the application of bacterial consortium W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 significantly reduced disease severity to 19% and increased biocontrol efficacy to 69% of infected papaya plants after 18 days of treatment. This study showed that W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 are potential candidate as biocontrol agents against papaya dieback disease.