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 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.
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 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.
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.
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.
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.
The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25-153.00%, PCR efficiency of 99-100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50-7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32-28.90 ± 0.42) and melting curves (74.63-78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.
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.
Adzuki bean is high in protein and fiber with a potential to be used as meat extender and fat replacer in the meat product. Replacement of both the corn flour and fat with different percentages of adzuki beans flour (ABF) has successfully produced acceptable reduced fat meatballs. Meatballs with 100% (w/w) ABF replacement exhibited highest cooking yield and higher moisture content compared to meatballs without the flour, which indicates its ability to bind water. Increasing the ABF content also increased the hardness and chewiness of the meatballs, whilst decreasing their lightness and yellowness. Replacing the corn flour and fat contents with ABF has obviously decreased the fat and calorie contents of the meatballs, yet their protein and carbohydrate contents remained the same compared to control. The sensory test revealed that meatball samples with 25% (w/w) and 50% (w/w) ABF showed no significant difference compared to control but received highest overall acceptability among the panelists. This indicates that replacement of corn flour and fat with ABF especially at 50% (w/w) in the production of reduced fat meatballs resulted with better physicochemical properties and acceptable sensory compared to original meatballs.
The influence of superheated steam cooking on fat and fatty acid composition of chicken sausage were investigated at various temperatures (150, 200, and 250°C) with different time domains (2-6 min). It has been found that the fat content of raw sample was higher than that of all cooked samples. The total fat content of cooked sample, showed a linear decreasing with time at all investigated temperatures. Superheated steam produce changes in saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) in which their values were found to decrease in cooked samples. When different cooking conditions (temperature, time) were applied, the fatty acids were decreased as the time and temperature increased. The PUFA and MUFA were less prone to decrease at 150°C, while at this temperature there was a remarkable loss in SFA content. This cooking method considerably reduced the level of fat and SFA which have a positive effect on health. In addition it could imply a great choice for consumers to choose the healthier technique for cooking food.
Gentiana lutea root is a medicinal herb that contains many active compounds which contribute to physiological effects, and it has recently attracted much attention as a natural source of antioxidants. The aim of this study was to evaluate the effects on the colour, pH, microbial activities, sensory quality and resistance to lipid oxidation (through the thiobarbituric acid method) during storage of beef patties containing different concentrations of G. lutea. Fresh beef patties were formulated with 0-5 g kg(-1) of G. lutea and 0 or 0.5 g kg(-1) of ascorbic acid and packed in two different atmospheres, Modified Atmosphere 1 (MAP1) and Modified Atmosphere 2 (MAP2), and stored at 4 ± 1 °C for 10 days. MAP1 contained 20:80 (v/v) O2:CO2 and MAP2 contained 80:20 (v/v) O2:CO2.
Four formulations were processed into frankfurters with different ratios of mechanically deboned chicken meat (MDCM) and cooked chicken skin (CCS) i.e. 80/0, 70/10, 60/20 and 50/30. The products were evaluated for proximate composition, cholesterol content, colour; 'L' value (lightness) and 'a' value (redness), percentage of cooking loss, physical measurements (shearforce-kgf and folding test), thiobarbituric acid value (TBA) and taste panel evaluation. The increment of CCS in the frankfurters increased the contents of moisture, ash, protein, fat, cholesterol, the lightness ('L' value) and redness ('a' value). After 3 months of frozen storage, the increment continued except for the moisture contents for formulations with 20 and 30% CCS. The lipid oxidation (TBA value) and cooking loss were lowered in formulations with CCS. After 3 months of frozen storage, TBA value decreased, while the cooking loss increased for all the formulations. The addition of CCS increased hardness of the frankfurters but affected folding ability, with formulation with 10% CCS scoring better grade. Sensory evaluation was carried out using 30 untrained panelists to evaluate aroma, colour, appearance, hardness, juiciness, chicken taste, oily taste, rancid taste and overall acceptance of the products. The addition of CCS in the frankfurters at 10 and 20% resulted in products with taste and texture that were acceptable after 3 months of frozen storage.
Research and development trends will continue to design innovative composite foods in which muscle proteins are combined with non-conventional animal products, non-meat proteins and functional food additives, many of which have lost their original inherent properties and characteristics. Composite food are products with meat, non-meat proteins, fats, carbohydrates and functional ingredients such as pre-emulsion, probiotics, enzymes, bioactives, peptides, hormones, emulsifiers, gelatin, animal fats/oils, alcohol and visceral tissues. Traceability of halal meat raw materials should start at the point of animal breeding, production to the stage of halal slaughter, processing operations and final point of consumption. Traceability of food additives used in the food industry remains a major hurdle for the Muslim community seeking halal food. The processes and technological advancements made in raw material processing, ingredient extractions, modifications, purification and resynthesized into many food ingredients make the question of traceability and solving of the materials and processes that are halal a monumental task. Food is only halal if the entire food chain from farm to table, is processed, handled and stored in accordance with the syariah and/or halal standards or guidelines, such as in the Jabatan Kemajuan Islam Malaysia (JAKIM): General guidelines, Malaysia Standards MS 1500:2009 and Codex Alimentarius (Food Labeling). Here lies the challenge and importance of traceability to verify the ‘wholesomeness’ of the sources of halal raw materials and final meat-based food products.
The quality change of fish sausage (keropok lekor) coated in sago starch-gelatine coating with
papaya seed extract (PSE) during chill storage (7°C) was determined. During storage, pH,
thiobarbituric acid value (TBA), colour, moisture, and the total plate count were evaluated. pH
of samples significantly dropped (p < 0.05) during storage, and the highest decrease was in
control sample. The moisture content in control sample had an increasing trend while that of
samples with 5 and 7% PSE coatings significantly decreased, and only a slight change for
samples with 0% PSE coating. All samples had significant increase in their TBA values during
storage. The presence of the coating provided a positive effect on the colour of the fish sausages since no significant colour changes were observed during storage. TPC of control and
coated sausage in 0, 5, and 7% PSE exceeded the recommended microbial standard after 2, 6,
8, and 4 d of storage, respectively. Overall, coating with 5% of PSE was the most effective in
retarding the quality deterioration of the fish sausages.
While there is good epidemiological evidence for foods as vehicles for norovirus transmission, the precise means of spread and its control remain unknown. The feline calicivirus was used as a surrogate for noroviruses to study infectious virus transfer between hands and selected types of foods and environmental surfaces. Assessment of the potential of selected topicals in interrupting such virus transfer was also made. Ten microliters of inoculum of feline calicivirus deposited onto each fingerpad of adult subjects was allowed to air dry and the contaminated area on individual fingerpads was pressed (10 s at a pressure of 0.2 to 0.4 kg/cm2) onto 1-cm-diameter disks of ham, lettuce, or brushed stainless steel. The virus remaining on the donor and that transferred to the recipient surfaces was eluted and plaque assayed. Virus transfer to clean hands from experimentally contaminated disks of ham, lettuce, and stainless steel was also tested. Nearly 46 +/- 20.3, 18 +/- 5.7, and 13 +/- 3.6% of infectious virus was transferred from contaminated fingerpads to ham, lettuce, and metal disks, respectively. In contrast, approximately 6 +/- 1.8, 14 +/- 3.5, and 7 +/- 1.9% virus transfer occurred, respectively, from ham, lettuce, and metal disks to hands. One-way analysis of variance test showed that pretreatment (washing) of the fingerpads either with water or with both topical agent and water significantly (P < 0.05) reduced virus transfer to < or = 0.9%, as compared with < or = 2.3 and < or = 3.4% transfer following treatments with either 75% (vol/vol) ethanol or a commercial hand gel containing 62% ethanol, respectively. Despite wide variations in virus transfer among the targeted items used, intervention agents tested reduced virus transfer significantly (P < 0.05) when compared with that without such treatments (71 +/- 8.9%). These findings should help in a better assessment of the potential for cross-contamination of foods during handling and also assist in developing more effective approaches to foodborne spread of norovirus infections.
Cinnamon deodorised aqueous extract (CinDAE) was prepared and evaluated for its total phenolic (315.3 ± 35.4 mg GAE/g) and flavonoid (99.3 ± 9.6 mg RE/g) contents. Stabilizing efficiency of CinDAE, for chicken meatballs, was measured against oxidative deterioration as function of storage time under chilled conditions. For this purpose, oxidative stability [2-thiobarbituric acid reactive substances (TBARS); peroxide value (PV)], colour and sensory acceptability were measured in the control meatballs (C), and those stabilized with 200 ppm of: CinDAE (T1), ascorbic acid (T2), BHA/BHT (50/50; w/w) (T3). In comparison to "C", induction period (IP) and redness (a* value) of the stabilized samples (T1, T2 and T3) were increased, while PV and TBARS were decreased throughout storage (8 ± 1 °C) significantly (p meat products.
The antioxidant and microbial stabilities of galangal (Alpinia galanga) extract in raw minced beef were examined at 4 +/- 1 degree C. Raw minced beef containing galangal extracts (0 to 0.10%, wt/wt) were prepared. Lipid oxidation during refrigerated storage was assessed by monitoring malonaldehyde formation, using the thiobarbituric acid reactive substances method. In minced beef, added galangal extract improved oxidative stability. Galangal extract at higher concentrations of 0.05% and 0.10% (wt/wt) were also found to extend the shelf-life of minced beef. Addition of alpha-tocopherol (0.02%, wt/wt) to galangal extract (0.05%, wt/wt) were observed to increase the oxidative but not the microbial stability of minced beef during the storage of 7 days. Galangal extract may prove useful in inhibiting lipid oxidation and increasing microbial stability of minced meat.