Detection of enterotoxin by targeting entFM and hblA genes in Bacillus cereus BC1 strain inoculated into ready to eat food (RTF) and drink samples using polymerase chain reaction (PCR) was conducted. The B. cereus BC1 strain was confirmed as a Bacillus diarrhoeal enterotoxin (BDE) when tested by a commercially available Enzyme-linked immunosorbent assay-BDE immunoassay (ELISA-BDE immunoassay, TECRA). In the specificity study, both enterotoxin genes were detected on chromosomal DNA of B. cereus BC1 strain by showing a specific band of 1269 bp (entFM) and 874 bp (hblA), respectively. However, none of the target genes were detected for the other 15 genomic DNA bacteria (B. cereus (ATCC 11779), B. subtilis (ATCC 6633), Campylobacter jejuni (ATCC 29428), C. coli (Jabatan Kimia Malaysia, JKM), Clostridium perfringen (ATCC 13124), Enterobacter sakazaki (ATCC 51329), Escherichia coli (ATCC 43888), E. coli (ATCC 11735), Legionella pneumophila (ATCC 33152), Listeria monocytogenes (ATCC 35967), Salmonella typhi (IMR), S. enteritidis (ATCC 13076), S. typhimurium (ATCC 14028), Shigella flexeneri (ATCC 12022) and Vibrio cholerae bengal (Institute Medical Research (IMR), Malaysia) examined. The detection limit of both genes was 0.1 ng of genomic DNA. Thus, in the presence study it is evidence that the PCR analysis targeting enterotoxin of entFM and hblA genes are suitable and useful in detecting enterotoxic B. cereus in RTFs and drinks contaminated sample.
In Malaysia, halal certification status for some surimi-based product is still suspicious due to the incorporation of non-halal plasma protein additives as part of the food ingredient. This study was conducted to determine the presence of plasma protein additives that have been incorporated into surimi-based product using Polymerase Chain Reaction (PCR)-Southern Hybridization method which able to differentiate 7 type (beef, chicken, duck, goat, buffalo, lamb and pork) of species on a single chip. A random of 17 (n = 17*3) different brands of surimi-based product was purchased from Selangor local market in January 2013. Of 17 brands, 3 (n = 3*3) brands were positive for chicken DNA and 1 (n = 1*3) brand was positive for goat DNA, while remainder 13 brands (n = 13*3) has no DNA species detected. In presence study, it is evidence that PCR-Southern Hybridization analysis offered a reliable result due to its highly specific and sensitive properties in detecting plasma protein incorporation in surimi-based product.
Two high protein wheat flour samples of Red Horse (RH) and Bake with Yen (BY) were examined for predominant Lactobacillus spp. in fermented liquid sourdough. The identification of Lactobacillus spp. was based on biochemical tests of catalase test, gas carbon dioxide production, arginine test, the ability to grow at temperature of 15°C and 45°C and carbohydrate fermentation using API50CH kit. Those strains were identified as Lactobacillus spp. and confirmed using polymerase chain reaction (PCR) of 16S rRNA partial sequencing analysis. In the present study, we successfully isolated and identified the Lactobacillus plantarum and L. fermentum which were predominant bacteria in liquid sourdough of the sample RH and BY brand, respectively.
The aim of the study was to isolate and identify Bacillus cereus from meat curry and to subtype the isolated B. cereus using RAPD-PCR and antibiotic resistance pattern. Ready to eat (RTE) meat curry samples purchased from 12 different restaurants at Kajang, Serdang and KL Sentral regions located in Selangor and Kuala Lumpur, Malaysia. Twenty-four isolates biochemically identified as B. cereus. Antimicrobial resistance analysis demonstrated that B. cereus isolates were highly resistance to ceftriaxone (100%), vancomycin (87.5%), clindamycin (91.6%) and nalidixic acid (100%). None of the B. cereus isolates were resistance towards ciprofloxacin (100%), streptomycin (91.6%) and chloramphenicol (83.4%). The B. cereus isolates were examined for randomly amplified polymorphic DNA-polymerase chain reaction (RAPDPCR) using primer S30 (5’-GTGATCGCAG-3’) and discriminated into nine profiles. The antimicrobial analysis showed seven patterns and phenotypically less heterogeneous when compared to RAPD-PCR. A total number of nineteen types of B. cereus have produced by a combination of phenotype and genotype methods. These results demonstrated that both typing method provides evidence of the presence of similarity and diversity of the B. cereus strains from RTE meat curry.
Papaya (Carica papaya L. cv. Hongkong) is an economically important fruit crop grown in Malaysia. During its ripening stages, (C. papaya L.) exhibits different physicochemical properties, antioxidant capacities, and sensory quality results. The objective of this study was to elucidate in detail the antioxidant capacity of C. papaya as determined by total phenol content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power (FRAP),2,2-diphenyl-1-picrylhydrazyl (DPPH) and scavenging systemand (ABTS). The study also aimed to study physicochemical changes of papaya fruits based on measured pH, titratable acidity (TA), total soluble solids (TSS), moisture and fruit color at five different stages of ripening. The fruits were harvested at five different, stages RS1, RS2, RS3, RS4, and RS5 corresponding to 12, 14, 16, 18, and 20 weeks after anthesis, respectively. Significant differences were found at different stages of ripening. The pH of the fruit decreased significantly (P < 0.05), whereas TA, moisture, and TSS increased significantly (P < 0.05) during the ripening process. The redness (a*) and yellowness (b*) values of fruit color both increased significantly (P < 0.05), whereas
lightness (L*) varied. The total phenol content TPC, TFC, FRAP, DPPH and ABTS values increased significantly (P < 0.05) with the ripening process. Sensory evaluation based on the color, sweetness, sourness, flavor, and overall acceptance for the last three maturity stages was also performed. RS5 had a better score than RS3 or RS4. The results showed the important role of the ripening stage in increasing the antioxidant content of papaya fruits.
Ninety one leaf samples of Josapine pineapple cultivar (Kelantan, n=8; Pahang, n=20; Perak, n=11; Sabah, n=15; Johor, n=37) showing symptoms of heart rot disease were collected to determine the incidence of Erwinia chrysanthemi. Sixteen strains of E. chrysanthemi were isolated from 13 leaf samples from Pahang (n=4), Sabah (n=2) and Johor (n=7). All of the E. chrysanthemi strains displayed resistance to bacitracin with two strains showing resistance to sulfamethoxazole. None of the E. chrysanthemi strains were resistant toward ampicillin, carbenicillin, cephalothin, ceftriaxone, cefuroxime, gentamicin, kanamycin, nalidixic acid, penicillin G, streptomycin and tetracycline. All of the E. chrysanthemi strains were plasmidless. The dendrogram generated from the ERIC-PCR fingerprinting showed that the E. chrysanthemi strains formed 4 clusters and 7 single isolates at 80% similarity level. The restriction fragment length polymorphism (RFLP) analysis for 16 strains of E. chrysanthemi with HinfI and HaeIII endonuclease, 2 and 4 restriction profiles were obtained, respectively. The combinations of the four techniques were able to differentiate the 16 E. chrysanthemi strains into 14 genome types, suggesting a wide diversity of strains examined. ERICPCR fingerprinting method is found to be more discriminating and useful for the determination of the E. chrysanthemi strains relatedness.
A technique to isolate DNA from ghee was developed for the authentication of beef fat product. The method was based on pre-mixed ghee with phosphate buffer solution (PBS) prior to DNA extraction using Epicentre extraction method. The recovery of beef DNA was then analysed by polymerase chain reaction (PCR) using beef species-specific oligonucleotide primers which targeted the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene. The amplicon was 274 bp in size. The developed ghee extraction method offers a high yield of DNA providing 100 ng per μl and useful for validating beef fat product.
This study was conducted to investigate the sensitivity and detection of porcine DNA in raw materials, ingredients and finished bakery products by polymerase chain reaction (PCR) - southern hybridization on chip analysis. A total of 20 samples (n=20* 3) with three replicates for each samples were obtained from a bakery factory located in Bangi, Selangor from January to December 2012. The sensitivity level of PCR-southern hybridization on chip was 0.001 ng. The species-specific oligonucleotide primers used in PCR-southern hybridization were targeted on the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene sequence, namely cty b biotin-labeled oligonucleotide primers. The amplicon from PCR amplification was 276 bp in size. None of the raw materials, ingredients and finished bakery product samples was positive towards porcine DNA, except for the positive control. The results in the present study demonstrated that the PCR- southern-hybridization technique on the gene chip (OliproTM Porcine gene chip) is a sensitive tool for monitoring the porcine component in highly processed ingredients and finished bakery products.
Halal is a term that describes substances that are deemed ‘pure and clean’ which Muslims are
allowed to consume according to Islamic law. The industrialization of food processing in the
20th and 21st centuries has exposed Muslims community to various ingredients such as blood
plasma, transglutaminase and gelatin introduced in meatballs and surimi product. Muslims
are facing difficulties to ascertain which products are permitted or not under the Islamic law.
Thus, this paper is to give knowledge of non-halal ingredients being introduced in meatballs
and surimi products for consumers, researchers and policy makers. Local halal logo issued by
Department of Islamic Development Malaysia (JAKIM) is needed to imply that all ingredients
used in the food production and processing are Syariah compliance. The scientific evidence
to substantiate any claim on Halal issue was developed based on several methods including
PCR-based methods with different mitochondria and chromosomal DNA (MtDNA and cDNA)
primers, real-time PCR with different probes and DNA binding agent, loop-mediated isothermal
amplification (LAMP) with different primers developed, PCR- RFLP, ELISA and etc.
Megabiodiversity of Malaysian’s flora and fauna which include microorganism could be conserved and served as alternative source indigenous yeast, the leavening agent of commercial bread making. This study was conducted in attempt to exploit the potential of Saccharomyces cerevisiae strains isolated from 30 different local fruits and plant parts as a leavening agent in bread making. The enrichment was carried out by fermenting the plant samples in medium containing Grape Must at 25°C for 10 days following by isolation of tentative yeasts at 30°C for 3 to 5 days. 20 out of 30 samples tested showed the presence of yeasts was then selected for identification of S. cerevisiae strains through biochemical and physiological tests. Of the 20 yeast strains examined, 13 strains were identified as S. cerevisiae and potentially used as leavening agent in bread making where 5 strains namely SN3, SMK9, SDB10, SRB11 and SS12 showed better fermentative performance compared to commercial strains. Thus, indicated that the local fruits and plant parts could be the potential source of indigenous S. cerevisiae strains for leavening agent in bread making.
Forty three (n=43) genomic DNA of Escherichia coli (11 isolates from eggs and 32 isolates from imported beef meats) were characterized by shiga toxin 1 (stx1), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplified polymorphic DNA-PCR (RAPD-PCR) analyses. In the shiga toxin 1 (stx1) gene detection with primer stx 1F (5’-TTCTTCGGTATCCTATTCCC-3’) and stx 1R (5’- CTGTCACAGTAACAACCGT-3’), 9 E. coli of beef meats isolates were positive toward sxt1 gene. The results of the ERIC-PCR and RAPD-PCR were analyzed using GelCompar II software. ERIC-PCR with primer ERIC1 (5’-CACTTAGGGGTCCTCGAATGTA -3’) and ERIC2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’) discriminated the E. coli into 6 clusters and 10 single isolates at 80% similarity. RAPD-PCR with primer Gen8 and Gen9, produced 10 clusters and 15 single isolates and 12 clusters and 14 single isolates of 80%, respectively. These results demonstrated that both ERIC-PCR and RAPD-PCR are useful and suitable tools for molecular typing of those isolates examined.
The study was conducted to detect the porcine DNA in pharmaceutical products in local market using polymerase chain reaction (PCR) and southern-hybridization on the biochip. A total of 113 (n=113) of hard (82 samples) and soft gel (31 samples) capsules from pharmaceutical products were purchased and tested for the presence of porcine DNA for Halal authentication. All capsules were gelatin-based purchased from local over the counter (OTC) markets. Of all samples tested, 37.2% (42/113) contained porcine DNA. While, none porcine DNA band was detected for 62.8% (71/113) of capsules tested. All samples which were positive toward porcine DNA were imported pharmaceutical products with none Halal logo. Results in the presence study demonstrated that the PCR techniques and southern-hybridization on the biochip is suitable tool for monitoring the Haram component in highly processed product of soft and hard capsule.