Analysis of Pork Adulteration in Commercial Burgers Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by TaqMan Probe Real-Time Polymerase Chain Reaction

A TaqMan probe real-time polymerase chain reaction assay was developed for the determination of pork adulteration in commercial burgers. The assay combined porcine-specific primers and TaqMan probe for the selective amplification and detection of a 109-bp fragment of swine cytochrome b (cytb) gene. Specificity test with 10 ng DNA of 11 different meat-providing animal and fish species yielded a quantification cycle (Cq) of 15.5 ± 0.20 for the pork and negative results for the others in a 40-cycle reaction with a change of analysts and sources. Analysis of beef burger formulations with spiked pork showed the assay can determine 100–0.01% contaminated pork with a PCR efficiency (E) of 93.8% and a correlation coefficient (R ²) of 0.991. A plot of actual value against real-time PCR-predicted value also yielded a good linear regression, R ² 0.998, and small root mean square error of calibration, RMSEC 0.42. A strong correlation was found between the partial least square (PLS)-predicted values and real-time PCR-determined values. The accuracy of the method was ≥90% in all determinations of the standard set. Residual analysis also revealed a high precision in all determinations. Finally, a random analysis of 10 ng DNA of commercial burgers from pork, beef, chicken, mutton, and chevon yielded a Cq of 15.56 ± 0.22 to 16.24 ± 0.35 from pork burgers, and negative results from the others, showing the suitability of the assay to determine pork in commercial burgers with a high accuracy and precision.     

Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products

Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chip-based capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.     

Identification of meat species by TaqMan-based real-time PCR assay

In this study, a convenient, sensitive and specific real-time PCR assay was described for the species identification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respectively, and the performance of the method was tested. In the results, no cross-reaction was observed between the donkey and pork species specific primer-probe systems and non-target species (bovine, ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific primer-probe set and 100 ng pork DNA at the ct 33.01 level (corresponding to 0.01 ng horse DNA). The real-time quantitative assay used in this study allowed the detection of as little as 0.0001 ng template DNA from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method for the routine meat species identifications studies in raw or cooked meat products.     

Evaluation of Protease-producing Ability of Fish Gut Isolate Bacillus cereus for Aqua Feed

Extracellular protease production by Bacillus cereus isolated from the intestine of fish Mugil cephalus has been investigated in shake-flask experiment using different preparations of tuna-processing waste such as raw fish meat, defatted fish meat, alkali hydrolysate, and acid hydrolysate as nitrogen source. Among the tuna preparations tested, defatted fish meat supported the maximum protease production (134.57 ± 0.47 U ml⁻¹), and 3% concentration of the same was found to be optimum for maximizing the protease production (178.50 ± 0.28 U ml⁻¹). Effect of carbon sources on protease production in the optimized concentration of defatted tuna fish meat revealed that galactose aided the higher protease production (259.83 ± 0.04 U ml^–1) than the other tested carbon sources and a concentration of 1.5% galactose registered as optimum to enhance the protease production (289.40 ± 0.16 U ml⁻¹). The halotolerancy of B. cereus for protease production indicated that 3% of sodium chloride was optimum to yield maximum protease (301.63 ± 0.20 U ml⁻¹). Among the surfactants tested, protease production was high in Triton X 100-added medium (298.63 ± 0.12 U ml⁻¹) when compared to other surfactants, and its optimum concentration recorded was 0.8% (320.57 ± 0.17 U ml⁻¹) for more protease production. Partial characterization of crude enzyme revealed that pH 7.0 (278.90 ± 0.08 U ml⁻¹) and 60°C temperature (332.37 ± 0.18 U ml⁻¹) were optimum for better protease activity by B. cereus.     

Detection of pea in food by real-time polymerase chain reaction (PCR)

A qualitative 5′-nuclease real-time PCR-based method for the detection of pea (Pisum sativum) in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pea-specific primers and a TaqMan fluorescent probe. The primers and the probe are oriented to the chloroplast DNA intron located between trnL and trnF exons encoding for tRNA. The analytical parameters of the method were inclusivity 100%, exclusivity 100% and the detection limit of 0.11±0.07 ng of pea DNA corresponding to 12±7 diploid pea genome copies. Using a set of model meat patés with defined pea contents, a matrix-related detection limit of 0.05% was determined and a linear calibration line was constructed. The presented analytical method was useful for qualitative detection or semiquantitative determination of pea in food products. The method was relatively fast because the analysis could be performed in one working day.     

PTR-TOF-MS Analysis for Influence of Milk Base Supplementation on Texture and Headspace Concentration of Endogenous Volatile Compounds in Yogurt

In the present study, the effects of milk fat (0.3% and 3.5% w/w), solids non-fat (8.4% and 13% w/w), and modified tapioca starch (0%, 0.5%, 1.0%, 1.5%, and 2.0% w/w) concentrations on the textural and physicochemical properties as well as the concentration of several endogenous flavor compounds in the headspace of set and stirred yogurts were investigated. The novel proton transfer reaction time-of-flight mass spectrometry technique was implemented for the non-invasive determination of the amounts of volatile organic compounds in the samples headspace. Milk fat and skim milk powder supplementation of the milk samples increased significantly the firmness and adhesiveness of yogurts (p < 0.001) and improved the stability of the formed gels by increasing their water holding capacity and reducing the amounts of expulsed whey (3.94 and 5.1 g for the milk fat and SNF-fortified samples). Acetaldehyde was significantly (p < 0.001) higher in the low fat-unfortified systems (6.15 ± 0.48 and 5.6 ± 0.60 ppmv, respectively). A similar trend was also reported in the case of 2-propanone (0.91 ± 0.11 and 1.13 ± 0.07 ppmv), diacetyl (334 ± 37 and 350 ± 34 ppbv), 2,3-pentanedione (54 ± 6 and 55 ± 6 ppbv), and 2-butanone (56 ± 7 and 68 ± 5 ppbv) for the same systems. In contrast, the concentration of flavor compounds in the headspace with hydroxyl groups (ethanol and acetoin) increased (p < 0.001) by solid non-fat fortification of milk base (350 ± 32 and 206 ± 7 ppbv, respectively, for the systems fortified with skim milk powder). Modified tapioca starch addition improved the textural properties and gel stability of yogurts whereas affected only the ethanol concentration (222 ± 16 and 322 ± 55 for the control and 2.0% w/w containing systems, respectively). Our data suggested that the reinforcement of textural and structural properties combined with the protein binding affinity of the flavor compounds seemed to be responsible for the aforementioned observations. In the case of stirred yogurts, the gel breakdown did not provoke significant changes in the headspace concentration of the most compounds, with the exception of ethanol, acetoin, and 2,3-pentanedione being significantly (p < 0.05) higher in the stirred yogurts (267 ± 29, 153 ± 11, and 38 ± 1 ppbv, respectively) than set style ones (232 ± 19, 134 ± 9, and 45 ± 3 ppbv, respectively).     

Detection and Quantification of Enterobacter sakazakii by Real-time 5′-Nuclease Polymerase Chain Reaction Targeting the palE Gene

Enterobacter sakazakii is an emerging food-borne pathogen causing invasive infection with high mortality rates in neonates and infants. The aim of this study was to develop, optimize, and evaluate real-time 5′-nuclease polymerase chain reaction (PCR) for the specific detection and quantification of E. sakazakii. Original primers and TaqMan probe targeting a sequence of E. sakazakii palE gene were designed. The developed real-time PCR system was highly specific for E. sakazakii with 100% inclusivity determined using 54 E. sakazakii strains and 100% exclusivity determined using 99 other strains. Detection limits of 4 × 10² and 4 × 10¹ CFU ml⁻¹ were determined with 100% and 90% probability, respectively. The response of the 5′-nuclease PCR system was linear (correlation coefficient ≥ 0.997) in the range of 10¹ to 10⁸ CFU ml⁻¹. Five methods of DNA sample preparation were compared. The methods of DNA preparation using the InstaGene Matrix and the simple lysis by boiling with the Triton X-100 were the most sensitive with calibration lines applicable for quantification. The developed real-time PCR targeted to the palE gene provides an alternative possibility for the detection and quantification of E. sakazakii after the suitable sample preparation.     

Effect of Acetic Acid and Commercial Protease Pretreatment on Salting and Characteristics of Salted Duck Egg

Salting of duck egg pretreated with 5% acetic acid and different commercial proteases (flavourzyme, protamex, alcalase, and neutrase) was studied. After 2 weeks of salting, duck eggs soaked in 5% acetic acid for 30 min, followed by soaking in 5% (w/v) flavourzyme and neutrase had the highest hardening ratio (90.14 ± 2.43%, 90.25 ± 1.23%) with the coincidental increase in salt content in egg white and decrease in moisture content of yolk, compared with those from other treatments (p < 0.05). However, similar hardening ratio of the sample treated with alcalase was obtained to that of sample treated with either flavourzyme or neutrase (P > 0.05). After 1 week of salting, protamex showed the similar effect on hardening ratio to other proteases (p > 0.05). When eggs were pretreated with neutrase at different concentrations (0.25%, 0.5%, and 0.75%, w/v) for different times (30, 60, and 90 min), those pretreated with 0.25% (w/v) neutrase for 90 min had the shorter salting time, while soaking time did not have the impact on hardening ratio for egg treated with 0.5% and 0.75% neutrase (P > 0.05). The oil exudation of egg treated with 0.25% neutrase had the higher oil exudates than the control at week 2 and 3 of salting (P < 0.05). Treatment of neutrase had no impact on viscosity of egg white, regardless of salting time (P > 0.05). Microstructure study revealed that shell of salted egg pretreated with acetic acid had rough and porous surfaces when compared with control, whereas no changes in microstructure and FTIR spectra of shell membrane were found. Therefore, pretreatment of egg with 5% acetic acid for 30 min, followed by 0.25% neutrase for 90 min prior to salting could expedite the salting process and yielded egg yolk with hardening ratio and oil exudates, comparable to the traditionally salted egg.     

Real-time PCR based on single-copy housekeeping genes for quantitative detection of goat meat adulteration with pork

Adulteration of goat meat with cheaper meat such as pork has been frequently found. Conventional PCR methods to distinguish goat meat from adulteration are qualitative, which easily produce false-positive results due to contamination but not adulteration. To address this problem, real-time PCR based on single-copy housekeeping genes encoding replication protein A1 was developed for goat meat and pork. By calculating the Ct ratio of goat meat/pork, the goat meat content in a suspected adulteration could be deduced by a good linear correlation (R² = 0.9868) in a range from 5% to 80%. Analysis of the simulated samples of goat meat showed high accuracy with recoveries of 104.91% and 105.00% for the goat meat contents of 40% and 60%, respectively, and coefficient of variations were as low as 9.24% and 9.09%. Thus, the developed assay supplied a useful tool for food regulation authorities to achieve quantitative authentication of goat meat.     

Optimization of Conditions for Enzymatic Production of ACE Inhibitory Peptides from Collagen

Enzymatic condition for producing angiotensin I-converting enzyme (ACE) inhibitory peptides from collagen was optimized with the aid of response surface methodology, which also derived a statistical model for experimental validation. The results showed that the optimal condition for the hydrolysis by pepsin was at pH 2, temperature 37 °C, and in enzyme to substrate ratio (E/S) of 2 when 8.23% collagen (w/v) and 3.82 h of hydrolysis time were applied. Through the single-enzyme hydrolysis, the ACE inhibitory activity could reach an average of 78.06%. In contrast, when a combination of pepsin and trypsin was used for a multiple-proteases hydrolysis, the ACE inhibitory activity could be significantly improved to an average of 88.25%. Furthermore, the IC₅₀ (μg/mL) value of the enzyme combination by pepsin and trypsin (141.64 ± 22.11) was significantly lower than that of the combinations of pepsin and papain (438.59 ± 84.37) or pepsin and protease M (336.76 ± 87.88; p<0.05). Our results have shown that collagen can be used for enzyme-mediated production of ACE inhibitory peptides.     

Identification of hare meat by a species-specific marker of mitochondrial origin

Meat species identification in food has gained increasing interest in recent years due to public health, economic and legal concerns. Following the consumer trend towards high quality products, game meat has earned much attention. The aim of the present work was to develop a DNA-based technique able to identify hare meat. Mitochondrial cytochrome b gene was used to design species-specific primers for hare detection. The new primers proved to be highly specific to Lepus species, allowing the detection of 0.01% of hare meat in pork meat by polymerase chain reaction (PCR). A real-time PCR assay with the new intercalating EvaGreen dye was further proposed as a specific and fast tool for hare identification with increased sensitivity (1 pg) compared to end-point PCR (10 pg). It can be concluded that the proposed new primers can be used by both species-specific end-point PCR or real-time PCR to accurately authenticate hare meat.     

Improved Sample Preparation for Real-Time PCR Detection of Listeria monocytogenes in Hot-Smoked Salmon using Filtering and Immunomagnetic Separation Techniques

This work evaluated the application of filtration and immunomagnetic separation (IMS) as sample pretreatments for use in combination with real-time polymerase chain reaction (PCR) to detect and quantify Listeria monocytogenes in hot-smoked salmon. Salmon was artificially inoculated with L. monocytogenes at levels ranging from 8 × 10⁰ to 8 × 10⁵ cfu/g of sample, and homogenates obtained from these samples were filtered to recover bacterial cells without a pre-enrichment step. High recovery of bacterial cells was achieved using standard coffee filters. IMS significantly reduced the co-extraction of PCR inhibitors present in the samples to increase the assay sensitivity with regression line parameters applicable for quantification. The limit of detection and quantification were equal to 2 × 10¹–4 × 10¹ and 2 × 10² cfu/g of sample, respectively. The entire detection procedure could be completed within 3.5 h. This study demonstrated that coupling filtration and IMS with real-time PCR has contributed to improve the sensitivity of L. monocytogenes detection from hot-smoked salmon.     

A novel real-time polymerase chain reaction method for the qualitative detection of pistachio in food

In order to provide an appropriate method for the detection of pistachio (Pistacia vera) in food products, a novel real-time PCR was developed. The pistachio-specific primers and the TaqMan fluorogenic probe were designed to target the internal transcribed spacer between 18S ribosomal RNA and 5.8S ribosomal RNA genes. Using dilutions of the pistachio DNA, the intrinsic detection limit of the method was determined to be 0.012 pg. At specificity testing, the method was positive for 11 pistachio varieties and negative for 26 plant and animal species used in food industry. A detection limit of 0.0004% (w/w) was determined for pistachio nuts in model pastry. Practical applicability of the elaborated method was tested by the analysis of 44 food samples, out of which 7 food products were identified as containing undeclared pistachio. The developed real-time PCR may be utilized for sensitive and selective detection of pistachio in food products.     

Effects of Soy and Corn Flour Addition on Batter Rheology and Quality of Deep Fat-Fried Shrimp Nuggets

In this paper, the effects of soy and corn flour (5 and 10% w/w) addition to the batter formulation on the quality of deep fat-fried shrimp nuggets were evaluated. Rheological properties of batters, coating pick-up, moisture content, and oil content of the samples were determined. Shrimp nuggets were fried at 150 °C, 170 °C, 190 °C, for 1, 2, 3, 4, and 5 min. The coating pick-up, oil and moisture content were found to be directly proportional to batter viscosity. All batters were found to show shear thinning behavior by exhibiting flow behavior index ≤ 1. The batters were modeled as power law fluids. Batter formulation, frying time, and temperature significantly (p < 0.01) affected moisture and oil content of shrimp nuggets. Soy flour added batters provided the highest consistency index, 7.595 and 10.635 Pa.sⁿ for 5% and 10% soy flour added, respectively. Soy flour was found to be an effective ingredient in decreasing oil content of fried nuggets. Batters containing 5% corn flour showed the lowest moisture content and the highest oil content among all the formulations. The mean moisture and fat content of shrimp nuggets coated with batter contained 10% soy and 5% corn flour, fried at 190 °C, for 5 min were 0.59 ± 0.022, 0.480 ± 0.029 and 0.149 ± 0.035, 0.346 ± 0.024 (g/g db), respectively.     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.     

Kinetics Modeling of Mass Transfer Using Peleg’s Equation During Osmotic Dehydration of Seedless Guava (<Emphasis Type="Italic">Psidium guajava</Emphasis> L.): Effect of Process Parameters

Peleg’s equation was used to study the effect of process parameters on kinetics of mass transfer in terms of solids gain and water loss during osmotic dehydration using 30–50% (w/w) sucrose solution at 30, 40 and 50 °C. The experimental data were successfully fitted employing Peleg’s equation with the coefficient of determination (R ²) higher than 0.88, the root mean square error, and the mean relative percentage deviation modulus (E) of less than 0.003% and 6.40% for all treatments, respectively. In all cases, initial mass transfer rate parameter (K ₁) decreased significantly (p < 0.05) as the solution concentration and solution temperature increased suggesting a corresponding increase in the initial mass transfer rate. Initial mass transfer rate followed an Arrhenius relationship which showed that solids gain had the highest temperature sensitivity (E _a = 21.93–33.84 kJ mol⁻¹) during osmotic dehydration. Equilibrium mass transfer parameter (K ₂) decreased significantly (p < 0.05) as solution concentration increased demonstrating that the equilibrium solid and water contents increased with increase in solution concentration. The equilibrium solid and water contents were also estimated adequately using Peleg’s equation (R ² > 0.78). The results of this work allow estimating the kinetics of mass transfer during osmotic dehydration in order to obtain products with determined solid and water contents.     

Structural Relaxation of Salmon Gelatin Films in the Glassy State

Mechanical relaxation of glassy carbohydrates has been reported extensively in the literature; however, little work is available on protein-based systems. This study deals with the structural relaxation of salmon (Salmo salar) gelatin in the glassy state. Skin gelatin was obtained by an acid–alkaline extraction method. Molecular weight (M _w) was determined by capillary viscometry. Films prepared by casting (7% w/v) were equilibrated to a moisture content of ~18.4% (db). The glass transition temperature (T _g) and enthalpic relaxation were determined by differential scanning calorimetry (DSC). Mechanical properties were assessed using a texture analyzer at constant temperature and moisture content. DSC showed a T _g ~34°C, and the selected storage temperature (T _a ) was 29°C (T _g − T _a = 5°C). The films were aged for 0, 4, 8, 16, and 40 h. Viscometry produced values of M _w ~90.2 kDa. The stress relaxation was modeled by the Kohlrausch–Wlliams–Watts (KWW) equation, reporting an increase in relaxation time (τ ₀) as the ageing time increased (τ ₀ ~6.41E + 03 s for 0 h; τ ₀ ~9.01E + 05 s for 40 h). β parameter was smaller for the aged films, indicating a spread of relaxation times. The derivative of KWW equation (dφ/dt) indicated a more rapid relaxation in a fresh sample compared with aged films. DSC showed an excess in enthalpy (ΔH) on the aged samples due to the non-equilibrium state of the matrix. ΔH increased with ageing time with values of ΔH ~2.42 J/g for the films aged for 40 h. This work demonstrated molecular relaxation process of gelatin in the glassy state, which must be taken into account if this material is used as a structure forming matrix.     

Kinetics of Crude Peroxidase Inactivation and Color Changes of Thermally Treated Seedless Guava (Psidium guajava L.)

Thermal treatment of seedless guava (Psidium guajava L.) cubes was carried out in the temperature range of 80–95 °C. The kinetics of peroxidase inactivation and color changes due to thermal treatments were determined. Peroxidase inactivation followed a first-order kinetic model, where the activation energy was 96.39 ± 4 kJ mol⁻¹. Color was quantified in terms of L, a, and b values in the Hunter system. The color changes during processing were described by a first-order kinetic model, except total color difference which followed a zero-order kinetic model. The temperature dependence of the degradation followed the Arrhenius relation. The activation energies (E _a) for L, a, b, and total color difference (ΔE) were 122.68 ± 3, 88.47 ± 5, 104.86 ± 5, and 112.65 ± 5 kJ mol⁻¹, respectively. The results of this work are a good tool to further optimize seedless guava thermal treatment conditions.