DNA barcoding for the species identification of commercially important fishery products in Indonesian markets

The DNA barcoding approach was used for the species identification of 44 Indonesian commercial fishery products. Additionally, the intronless nuclear rhodopsin gene fragment (RH1) was added to the analysis to enable the identification of species not yet barcoded and possible hybrids. The 655‐bp cytochrome C oxidase subunit I (COI) gene fragment marker was successfully amplified and used to identify 86% of the total fish samples at the species level using the BOLD and BLAST public databases. Moreover, the RH1 marker was used to complete COI analysis. For a number of fish species, the COI sequences (six species) and RH1 sequences (eight species) were the first entries submitted to GenBank. This study demonstrated that COI barcoding is a promising tool for Indonesian fishery products and confirmed that it could be adopted in the future for regular seafood control as part of the Indonesian integrated food traceability system.     

DNA条形码序列对药食两用品花椒的品种鉴定

目的:利用DNA条形码鉴定技术,快速、准确的鉴别中药花椒,区别中药花椒及其市售香料花椒的品种来源差异。方法:采用植物DNA试剂盒提取花椒及其混伪品基因组DNA,对全部收集样品的ITS2序列进行扩增和测序,计算物种种间种内Kimura2-parameter(K2P)遗传距离。采用Blast、最近距离法及基于ITS2序列构建Neighbor-joining(NJ)系统聚类树进行鉴别分析。结果:中药花椒及香料花椒的序列长度为224~227 bp,中药花椒种间平均K2P遗传距离明显大于香料花椒种内平均K2P遗传距离;ITS2分子系统树显示,中药花椒及香料花椒均聚为一单系分枝。结论:ITS2序列作为DNA条形码能准确鉴别中药花椒及其香料花椒,为中药花椒及其香料花椒的鉴别提供了依据。     

Examination of DNA extract from kernels and processed foods using silica-base resin

A rapid and simple DNA extraction method is needed to detect genetically modified recombinant DNA in soybean kernels and processed foods. However, since various kernels and processed foods differ greatly in form, a uniform DNA extraction method has proved elusive. The silica-base resin DNA extraction method does not use any organic solvent, and the operation is simple and the cost per extraction is low, although the frequency of its use is very low and few domestic reports exist. We therefore studied suitable conditions for a silica-base resin method. We also developed the method to get more pure DNA from soybean kernels. The silica-base resin method was found to be adequate for extracting DNA from various processed foods for PCR amplification with endogenous gene primers. In the case of DNA extraction from soybean kernels, pure DNA could be efficiently extracted after pre-heating the soybean suspension in TNE buffer. The extracted DNA showed higher ratios of absorption at 260 nm/280 nm and 260 nm/230 nm than those for samples obtained with previous methods. Moreover, our observations suggested that the extraction time could be reduced to within 30 min for processed foods such as tofu.     

Rheological properties of mustard mucilage isolated from raw and from processed mustard

Mucilage of yellow mustard (Sinapis alba L.) exhibits completely different rheological behaviour, depending on where it has been extracted from. Whereas mucilage isolated from intact mustard seeds or from separated brans acts like many hydrocolloids (e.g. increasing viscosity significantly at low concentrations) these and related properties (i. e. shear thinning behaviour) are lost, once mustard seeds are processed. Mucilage extracted from processed mustard only raises viscosity slightly. Although the supernatant of processed mustard consists of 3-5% mucilage (dry matter), it exhibits Newtonian flow. Changes of rheological properties of mustard mucilage were attributed to a change of molecular shape from an extended to a globular state due to processing. Chemical analysis has shown a significant raise of nitrogen content of mucilage after processing and during storage, indicating some chemical reaction taking place, possibly involving proteins.     

Authentication of gadoids from highly processed products susceptible to include species mixtures by means of DNA sequencing methods

Economic importance of gadoids such as fishing resource, and their conservation status, necessitates the development of techniques that allow unequivocal authentication of products made from them. Amplification of a fragment of mitochondrial cytochrome b (cyt b) marker and subsequent phylogenetic analysis were carried out to authenticate these products and assure their correct labeling. Also, SNP analysis that allows detection of species mixtures of Gadus genus was developed. For this, two fragments of the cyt b gene were amplified and sequenced, one of 464 bp and another internal fragment to this of 263 bp to allow the authentication of gadoid species in highly processed products. Obtained sequences were aligned and analyzed in order to assess the presence of informative variable positions and a maximum of 14 SNP were identified and selected. These allow detection and identification of species mixtures belonging to this genus. The developed methodologies were validated and applied to 25 commercial samples. The main novelty of this work lies in the fact that is the only work that allows the detection of species mixtures of the Gadus genus and is the only one that allows the authentication of highly processed products up to date. Furthermore, this methodology allows identifying more of 15 species of gadoids and can be applied to all kinds of seafood products. Therefore, this molecular tool can be applied in questions related to check the fulfillment of labeling regulations for seafood products, verify the correct traceability in commercial trade and for fisheries control.     

Chemical composition,antioxidant capacity and functionality of raw and processed Phaseolus lunatus

The nutrients, non‐nutritional components and bioactive compounds, as well as the antioxidant capacity of raw, cooked, tray and drum‐dried Phaseolus lunatus have been quantified. Likewise, the minerals, soluble carbohydrates, total polyphenols and tannins in the soaking and cooking waters were quantified. In addition, the functional properties such as the water and oil absorption indexes and the emulsifying and the foaming capacities were studied. The protein content of the raw beans was 24.98% and decreased, like calcium, magnesium and potassium, with the soaking and cooking processes; these losses are found in the soaking and cooking waters. Drum drying decreased anti‐nutritional factors like trypsin inhibitors (66.09%) and cyanhydric acid (50.36%). Similarly, soluble fiber, available starch, total starch, and soluble sugars diminished, while total and insoluble fiber and resistant starch increased. The content of total polyphenols, tannins and antioxidant capacity decreased with thermal processing, being drum drying the process that least diminished antioxidant capacity. Likewise, the water absorption index was increased by 85% and 161.5% with processing. It was shown that P. lunatus is an important source of nutrients and can be consumed in whole bean form or used as a functional ingredient to be added in the development of new products. Copyright © 2007 Society of Chemical Industry     

Authentication of butter from lard adulteration using high-resolution of nuclear magnetic resonance spectroscopy and high-performance liquid chromatography

Food authentication is an interesting issue for all parties in the food industry, including the fats and oils industry. Some unethical players try to blend high-quality foods, such as butter, with lower ones like lard, therefore, the analytical methods capable of analyzing the adulteration practices must be developed. This study used proton nuclear magnetic resonance spectroscopy in combination with high-performance liquid chromatography for the authentication of butter from lard adulteration. The identification of triacylglycerol composition of lard as a chemical marker for halal authentication is analyzed using high-performance liquid chromatography and high resolution nuclear magnetic resonance spectroscopy. The suitability of proton nuclear magnetic resonance provides a high-performance approach for determination butter adulterated with lard in their entirety of all proton bearing components. Peaks in the region of 2.60–2.84 ppm show special characteristics only present in lard. Only lard has its own unique characteristics which only polyunsaturated fatty acids would give signals 7 at δ 2.63, that corresponded to the chemical shift of the double-allylic methylene protons. In the same way, the intensity of signal at 2.63 ppm, due to methylenic protons in a position α to two double bonds, that is to say, due to the linoleic group. Furthermore, we also correlate some signals between ¹H and ¹³C-NMR spectra for the confirmation of signals.     

Application of magnetic polymethacrylate‐based microspheres for the isolation of DNA from raw vegetables and processed foods of plant origin

A method for the microextraction of DNA from raw vegetables and highly processed foods of plant origin suitable for PCR analysis was developed. It is based on non‐selective binding of DNA in the presence of PEG 6,000/NaCl to hydrophilic magnetic nonporous poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) ‐ P(HEMA‐co‐GMA) microspheres covered by carboxyl groups. The quantitative polymerase chain reaction (qPCR) by in‐house designed primers targeting a highly repetitive rDNA locus allowed for detection of plant DNAs in a wide range of concentrations (0.1 pg/μL to 10 ng/μL). The described procedure is fast and simple. We further demonstrate that relatively mild acidic treatment of vegetable at elevated temperatures resulted in a dramatic reduction of PCR efficiency indicating extensive degradation of DNA during pickling. The described method is suitable for the analysis of highly degraded DNA from pickled food products.

Practical applications

DNA‐based methods, such as the polymerase chain reaction (PCR), represent an important genetic approach for identification of plant species composition of foods. DNA from processed foods varies in both quality and quantity between the protocols used. Plant samples are generally characterised by a complex composition containing various inhibitors of PCR. Current methods have been tailored for specific samples while no universal protocol is available. Development of a simple universal procedure leading to PCR‐ready DNA would be beneficial. Isolation strategies based on solid phase systems have become popular for PCR‐ready DNA isolations from complex matrices. Here we applied magnetic hydrophilic P(HEMA‐co‐GMA) microspheres to extract DNA from raw vegetables and highly processed foods of plant origin. Proposed small scale PCR‐ready DNA extraction protocol was comparable with a silica‐based DNeasy Plant Mini Kit (Qiagen) and classical CTAB extraction methods in quality and amplificability of DNA while it is less costly and time consuming.     

Duplex PCR approach for the detection and quantification of donkey,horse and mule in raw and heat‐processed meat products

Donkey‐related products have been paid more attention for their high nutritional value to human beings. Due to donkey resource scarcity, coupled with gradually increasing market demand, adulterated donkey meat products with other low‐cost animal meat, especially with the similar species horse and mule, are often found in market. Therefore, detection of species fraud in donkey meat products is important for consumer protection and food industries. In this study, a simple and highly specific duplex PCR method, based on the simultaneous amplification of fragments of the mitochondrial ATP synthase subunit 8/6 and ND2, was developed and optimised for the identification of horse, donkey and mule species in raw and heat‐processed meat products. To the best of our knowledge, it was the first time for this strategy applied to these three genetically related animal meat products differentiation to date. The duplex PCR generated a 153‐bp and 83‐bp amplification products for horse and donkey, respectively. While for mule, both of the two length amplification products are appeared on the agarose gel. Target meat species could be detected at a level of 1%, and the results indicated that the duplex PCR assay could be used in the authentification of donkey‐related products with high specificity, cost‐effectiveness and simplicity.     

Microbial quality of raw aquacultured fish fillets procured from Internet and local retail markets

The microbial quality of raw fillets of aquacultured catfish, salmon, tilapia, and trout was evaluated. A total of 272 fillets from nine local and nine Internet retail markets were tested. Mean values were 5.7 log CFU/g for total aerobic mesophiles, 6.3 log CFU/g for psychrotrophs, and 1.9 log most probable number (MPN) per gram for coliforms. Differences in these microbial levels between the two kinds of markets and among the four types of fish were not significant (P > 0.05), except that Internet trout fillets had about 0.8-log higher aerobic mesophiles than did trout fillets purchased locally. Although Escherichia coli was detected in 1.4, 1.5, and 5.9% of trout, salmon, and tilapia, respectively, no sample had > or = 1.0 log MPN/g. However, E. coli was found in 13.2% of catfish, with an average of 1.7 log MPN/g. About 27% of all fillets had Listeria spp., and a positive correlation between the prevalence of Listeria spp. and Listeria monocytogenes was observed. Internet fillets had a higher prevalence of both Listeria spp. and L. monocytogenes than did those fillets purchased locally. L. monocytogenes was present in 23.5% of catfish but in only 5.7, 10.3, and 10.6% of trout, tilapia, and salmon, respectively. Salmonella and E. coli O157 were not found in any sample. A follow-up investigation using catfish operation as a model revealed that gut waste exposed during evisceration is a potential source of coliforms and Listeria spp.     

Occurrence,heat and antibiotic resistance profile of Bacillus cereus isolated from raw cow and processed milk in Mezam Division,Cameroon

Bacillus cereus is the aetiologic agent of two distinct forms of food poisoning: the diarrhoeal and emetic syndromes. Little data exist on the prevalence of B. cereus in raw milk and milk products sold in Cameroonian towns. This study was aimed at investigating the occurrence, heat and antibiotic resistance of B. cereus isolated from raw milk and selected milk products in Mezam division, Cameroon. Bacillus cereus was isolated by inoculating samples onto mannitol‐egg yolk‐polymyxin B agar. Isolates were characterised morphologically and biochemically. The occurrence of B. cereus in raw milk (8.22%) was less than that in milk powder (13.33%). Bacillus cereus was not isolated from fermented milk. There was no significant difference (P < 0.05) between the B. cereus load in raw milk (2.6 × 10³ cfu/mL) and milk powder (3.0 × 10² cfu/mL). All the isolates showed haemolysin activity and were sensitive to tetracycline, gentamicin, chloramphenicol and nalidixic acid, but resistant to penicillin, ampicillin and trimethoprim/sulphamethoxazole. The detection of drug‐resistant, haemolysin‐positive isolates should serve as a warning for an impending health hazard following consumption of untreated milk. Heat resistance of isolates was assessed by determining the decimal reduction time; D‐value (time to inactivate 90% of the B. cereus spores); and the heat sensitivity, z (temperature increase leads to a tenfold reduction in the D‐value). The values for D₁₀₀ ranged from 0.5 to 3.5 min, and z‐values ranged from 10.0 to 32.6 °C. These results could be used in the dairy industry to evaluate the importance of heat treatment on B. cereus inactivation and calculation of process efficiency.     

Detection and identification of marine fish mislabeling in Guangzhou's supermarkets and sushi restaurants using DNA barcoding

In this study, DNA barcoding was applied to identify the distinct species of fish products in Guangzhou supermarkets and sushi restaurants in order to confirm whether products were correctly labeled. Samples were analyzed using mitochondrial cytochrome C oxidase subunit I (CO I) gene as the target. Our results showed that the CO I gene of all 139 samples examined was successfully amplified by PCR. When sequenced, 30 samples (21.58%) were mislabeled as the wrong species, 11 samples had insufficient information provided on the label to determine if the labeling was correct (7.91%), and four samples failed sequencing (2.88%). We also found that the use of proper labels for fish products in sushi restaurants was higher than that in supermarkets. As a simple, rapid, and efficient technology, DNA barcoding can be widely used for species identification of fish products. Our work shows that regulation of the labeling of fish products, as we evaluated in Guangzhou and other markets in China, is needed on a global scale.     

Aerobic bacterial, coliform, Escherichia coli and Staphylococcus aureus counts of raw and processed milk from selected smallholder dairy farms of Zimbabwe

A cross sectional study was conducted to enumerate total viable bacteria (TBC), coliforms, Escherichia coli and Staphylococcus aureus in raw (n = 120) and processed (n = 20) milk from individual farms from three smallholder dairy schemes of Zimbabwe between October, 2009 and February, 2010. Data on management factors were collected using a structured questionnaire. A standard pour plate technique was used to enumerate total viable bacteria, while for coliforms, E. coli and S. aureus, counts were assessed by the spread plate technique. The association of total viable bacterial counts and management factors was assessed using univariable and a linear regression model. The log₁₀ TBC for raw milk differed significantly (P < 0.05) amongst the schemes with the lowest (5.6 ± 4.7 log₁₀ cfu/ml) and highest (6.7 ± 5.8 log₁₀ cfu/ml) recorded from Marirangwe and Nharira respectively. The mean log₁₀ of TBC of processed milk (6.6 ± 6.0 log₁₀ cfu/ml) were marginally higher than those of raw milk (6.4 ± 5.6 log₁₀ cfu/ml) but not significant (P > 0.05). The coliform, E. coli and S. aureus counts for raw milk significantly differed (P < 0.05) amongst the study areas. The variation in TBC, coliforms, E. coli and S. aureus counts amongst the schemes could be attributed to differences in milking hygiene where farms with more access to training and monitoring of microbiological quality of milk had lower counts. Linear regression analysis revealed dairy scheme, delivery time and season of milking as independently associated with increased TBC of raw milk. The high TBC of raw and processed milk generally indicated low levels of milking hygienic practices, and high level of post-processing contamination, respectively. The high TBC, coliform, E. coli and S. aureus counts of both raw and processed milk may present a public health hazard. Thus, educating the farmers on general hygienic practices, quickening the delivery of milk to collection centres, or availing cooling facilities on-farm will improve the microbiological quality and safety of milk.     

Influence of cell integrity on textural properties of raw, high pressure, and thermally processed onions

The integrity of onion cells and its impact on tissue texture after high pressure and thermal processing was studied. The contribution of cell membranes and the pectic component of cell walls on the texture properties of onion tissue were analyzed. Neutral red (NR) staining of onion parenchyma cell vacuoles was used for the evaluation of cell membrane integrity and microscopic image analysis was used for its quantification. The content of methanol in tissue as a result of pectin methylesterase activity was used to evaluate the pectin component of the middle lamella and cell walls and the hardening effect on the tissue after processing. High pressure treatments consisted of 5-min holding times at 50, 100, 200, 300, or 600 MPa. Thermal treatments consisted of 30-min water bath exposure to 40, 50, 60, 70, or 90 °C. In the high pressure treatments, loss of membrane integrity commenced at 200 MPa and total loss of membrane integrity occurred at 300 MPa and above. In the thermal treatments, membrane integrity was lost between 50 and 60 °C. The texture of onions was influenced by the state of the membranes and texture profiles were abruptly modified once membrane integrity was lost. Hardening of the tissue corresponded with pressure and temperature PME activation and occurred after membrane integrity loss. PRACTICAL APPLICATION: The texture of vegetables is an important quality attribute that affects consumer preference. Loss of textural integrity also indicates that other biochemical reactions that affect color, flavor, and nutrient content may occur more rapidly. In this study, we analyzed changes in the texture of onions after preservation with heat and high pressure.     

Determination of γ-glutamyl-valyl-glycine in raw scallop and processed scallop products using high pressure liquid chromatography–tandem mass spectrometry

The determination of the kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) in raw scallop and processed scallop products was carried out using high pressure liquid chromatography–tandem mass spectrometry (LC/MS/MS). The detection of γ-Glu-Val-Gly was achieved using a multiple reaction monitoring (MRM) method. The optimised condition enabled the precise determination of γ-Glu-Val-Gly. Raw scallop contained 0.08 μg/g γ-Glu-Val-Gly, and the γ-Glu-Val-Gly levels in processed scallop products, such as dried-scallop and scallop extract, were measured to be 0.64 and 0.77 μg/g, respectively. This is the first report to confirm the existence of γ-Glu-Val-Gly in foodstuff.     

Detection of roe deer,red deer,and hare meat in raw materials and processed products available in Poland

To allow detection of meat from the most popular game species in Poland, we developed a PCR-based method for identification of roe deer (Capreolus capreolus), red deer (Cervus elaphus), and hare (Lepus europaeus). The designed primers were based on the noncoding, mitochondrial D-loop region. Amplicon sizes ranged from 116 to 255 bp. The primers exhibited no cross-reactivity with the DNA from common slaughter and other game species. The detection limit of the assay was established to be below 0.001 % in raw red deer (C. elaphus) and hare (L. europaeus) meat, and below 0.01 % in raw roe deer (C. capreolus) meat, whereas <0.5 % of hare and red deer meat in processed samples could be detected. The PCR-based assay was used for authentication of 17 samples of raw game meat and 32 samples of game meat-containing products available in Polish markets. Analysis of all tested raw meat and processed products revealed the presence of DNA of investigated species in concordance with producers’ declarations.