The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.
The direct consumption of vegetable proteins in food products has been increasing over the years because of animal diseases, global shortage of animal protein, strong demand for wholesome and religious (halal) food, and economic reasons. The increasing importance of legume and oilseed proteins in the manufacturing of various functional food products is due to their high-protein contents. However, the greatest obstacle to utilizing these legumes and oilseeds is the presence of antinutrients; but these antinutrients can be successfully removed or inactivated by employing certain processing methods. In contrast, the potential negative impact of the antinutrients is partially balanced by the fact that they may have a health-promoting role. Legumes and oilseeds provide well-balanced amino acid profiles when consumed with cereals. Soybean proteins, wheat gluten, cottonseed proteins, and other plant proteins have been used for texturization. Texturized vegetable proteins can extend meat products while providing an economical, functional, and high-protein food ingredient or can be consumed directly as a meat analog. Meat analogs are successful because of their healthy image (cholesterol free), meat-like texture, and low cost. Mycoprotein is fungal in origin and is used as a high-protein, low-fat, health-promoting food ingredient. Mycoprotein has a good taste and texture. Texturized vegetable proteins and a number of mycoprotein products are accepted as halal foods. This article summarizes information regarding the molecular, nutritional, and functional properties of alternative protein sources to meat and presents current knowledge to encourage further research to optimize the beneficial effects of alternative protein sources.
Probiotics may be used to enhance the functionality and nutritional values of fermented sausages. This study aims to evaluate the physicochemical and sensory properties of beef sausages fermented by lactic acid bacteria of Lactobacillus plantarum IIA-2C12 and L. acidophilus IIA-2B4. These strains were isolated from beef cattle and have shown to display probiotic features. While the nutrient contents were not affected by the probiotics, the pH, texture, and color varied among the sausages. Further analysis on fatty acids showed different profiles of saturated (C14:0, C17:0, and C20:0) and unsaturated (C14:1, C18:1n9c, C18:2n6c, and C22:6n3) fatty acids in sausages with probiotics. Gas chromatography-mass spectrometry further revealed some flavor development compounds including acid, alcohols, aldehydes, aromatic, ketones, sulfur, hydrocarbons and terpenes, varied among the sausages. Hedonic test showed no difference in the preference toward aroma, texture, and color for untrained panelists.
Food falsification has direct impact on public health, religious faith, fair-trades and wildlife. For the first time, here we described a multiplex polymerase chain reaction assay for the accurate identification of five meat species forbidden in Islamic foods in a single assay platform. Five pairs of species-specific primers were designed targeting mitochondrial ND5, ATPase 6, and cytochrome b genes to amplify 172, 163, 141, 129 and 108 bp DNA fragments from cat, dog, pig, monkey and rat meats, respectively. All PCR products were identified in gel-images and electrochromatograms obtained from Experion Bioanalyzer. Species-specificity checking against 15 important meat and fish and 5 plant species detected no cross-species amplification. Screening of target species in model and commercial meatballs reflected its application to detect target species in process foods. The assay was tested to detect 0.01-0.02 ng DNA under raw states and 1% suspected meats in meatball formulation.
Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.
A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.
The effect of frozen storage on the physiochemical, chemical and microbial characteristics of two types of fish sausages was studied. Fish sausages developed (DFS) with a spice-sugar formulation and commercial fish sausages (CFS) were stored at -20 °C for 3 months. Fresh DFS contained 12.22% lipids and had a 3.53 cfu/g total bacteria count (TBC) whereas, CFS contained 5.5% lipids and had a 4.81 cfu/g TBC. During storage, TBC decreased significantly (p 0.05) in CFS. A peroxide value (PV) was not detectable until week four and eight of storage in CFS and DFS, respectively. The salt-soluble proteins (SSP) level was stable in DFS but in CFS it declined significantly (p 0.05) in both sausage types. This study showed that the effect of storage at -20 °C on fish sausages characteristics varied between formulations and depended on the ingredients of fish sausages.
Determination of feline meat in food products is an important issue for social, health, economic and religious concern. Hence this paper documented the application of species specific polymerase chain reaction-restriction fragment length polymorphism (SP-PCR-RFLP) assay targeting a short-fragments (69 bp) of mitochondrial cytochrome b (cytb) gene to screen feline meat in commercial meat products using lab-on-a-chip. The SP-PCR assay proved its specificity theoretically and experimentally while testing with different common animal, aquatic and plant species of DNA. The feline specific (69 bp, 43- and 26-bp) characteristic molecular DNA pattern was observed by SP-PCR and RFLP analysis. For assay performance, it was tested in three different types of commercial dummy meat products such as frankfurters, nuggets and meatballs and digested with AluI-restriction enzyme. The highest sensitivity of the assay using lab-on-a-chip was as low as 0.1 pg or 0.01 % (w/w) in commercial dummy meat products. We have also applied this assay to screen three important commercial meat products of six different brand from six supermarket chains located at three different states of Malaysia. Thus total 378 samples were tested to validate the specificity, sensitivity, stability of the assay and utilization of it for commercial meat product screening.
The goal of this study was to validate the commercial feasibility of a novel casing formed from chitosan containing cinnamaldehyde (2.2%, w/v), glycerol (50%, w/w) and Tween 80 (0.2% w/w) under traditional sausage manufacturing conditions. Meat batter was stuffed into both chitosan and collagen (control) casings and cooked in a water bath. Before and after cooking, both casings were compared for mechanical, barrier, and other properties. Compared to collagen, the chitosan casing was a better (P≤0.05) barrier to water, oxygen, liquid smoke, and UV light. In mechanical and other properties, the chitosan casing had higher (P≤0.05) tensile strength, lower (P≤0.05) elongation at break and tensile energy to break, and better (P≤0.05) transparency whereas a similar (P>0.05) water solubility to the collagen casing. Overall, the chitosan casing was less affected by sausage manufacturing conditions than the collagen casing, indicating that chitosan casing has potential as an alternative to the current collagen casing in the manufacture of sausages.
This study describes the development of a rapid and sensitive Loop-mediated isothermal
amplification assay for detection of swine DNA in adulterated meat and meat products. The
need to protect consumer’s right to eat foods of their choices, has made it imperative for
researchers to develop efficient means of screening and certification of food products. Six sets
of LAMP primers designed based on porcine tRNA lysine gene and ATPase subunit 8 genes
were used for the assay. Amplification was carried out under constant temperature (630C), using
a simple laboratory water bath. Average time spent in amplification and detection of results was
25 min. All results were visually detected and confirmed by electrophoresis. Detection limit of
the assay was 0.03 femtogram (fg) much high than the PCR assay, and detection probability of
the assay was 100%. Detection of 0.5% of pork spiked with 99.5% of cattle beef is indicative
of the sensitivity and robustness of the assay. This could serve as a prototype for development
of a sensitive and inexpensive Swine DNA LAMP detection kit.
The aim of this study was to examine vegetarian burger patties manufactured by two producers in Malaysia for the presence of Listeria monocytogenes. Brand A was produced by an established food manufacturer
while Brand B was produced by a small-scaled food producer. A total of 108 samples of vegetarian burger
patties produced by both manufacturers were sampled from retail market and were analyzed by combined
MPN-PCR and MPN plating method. Of all the samples tested, ten (9.3%) were found to be contaminated with L. monocytogenes. The L. monocytogenes contamination level in vegetarian burger patties manufactured by producer A (20.9% of the samples were contaminated with 3-1100 MPN/g of L. monocytogenes) was significantly higher (P
Ten selected brands of commercial chicken burgers were analysed for their proximate composition, texture profiles, colour and sensory properties. Results show commercial chicken burgers consisted of moisture, proteins, fat and ash in the range of 46.72-69.37%, 11.08-18.77%, 9.08-20.54%, and 1.50-2.96%, respectively. Meanwhile, texture profiles comprised of hardness ranging from 8003.25-19038.15g, while chewiness had the value ranging from 650.78-1275.78 g. On the other hands, cohesiveness had the value ranging from 0.223-0.371, while springiness recorded the value in the range from 0.141-0.443. Colour analysis of cooked burgers resulted in lightness (L*) ranging from 48.21-73.59, redness (a*) from 0.75-9.08, and yellowness (b*) from 21.56-31.24. In sensory evaluation, the most acceptable colour of chicken burger was the one which had the medium lightness (L*) with the value of 63.96), medium redness (a*) with the value of 7.00) and the highest yellowness (b*) intensity value at 31.24. In addition, the most acceptable texture was the one with medium hardness value of 12590 g, high chewiness value of 1195.42 g, high cohesiveness value of 0.371, and medium springiness value of 0.254. It can be concluded that the Malaysian commercial chicken burgers complied with the Food Act of Malaysia and contained different levels of chemical compositions, textural characteristics and colour properties.