DNA Barcoding for Discriminating the Economically Important Cinnamomum verum from Its Adulterants

Traded forms of spice and spice powders are often subjected to admixing with inferior substances by design or default, affecting public health and national prestige. Cinnamomum verum (true cinnamon), a high-value spice, is often adulterated with its inferior species such as C. cassia and C. malabatrum. The presence and detection of the spurious species in traded barks (whole or powder) of true cinnamon is posing problems. DNA markers are now used to detect such adulteration. Here we report the application of a DNA barcoding method to detect these adulterants in traded market samples of true cinnamon using the barcoding loci rbcL, matK and psbA-trnH. The PCR success rate, sequencing efficiency, inter and intra specific divergence, and occurrence of single nucleotide polymorphisms (SNPs) were utilized to assess the potential of each barcode loci to authenticate C. verum from its related adulterants. The amplification and sequencing success was 100% for rbcL and psbA-trnH while matK failed to amplify in the market samples. The locus of rbcL showed higher interspecific divergence while psbA-trnH exhibited lower interspecific divergence. SNPs specific to C. cassia were detected in rbcL locus in seven out of the ten market samples studied thereby confirming the presence of C. cassia adulteration in commercial samples of true cinnamon. Out of the three loci, rbcL locus proved to be efficient in tracing out adulterants in traded cinnamon. The SNP sites in this locus can be exploited in designing C. cassia specific primers, enabling kit development for easy detection of adulterants at the band level itself thereby bypassing the cost of sequencing.     

EFFECTS OF DRYING ON THE ANTIOXIDANT PROPERTIES OF HERBAL TEA FROM SELECTED VITEX SPECIES

ABSTRACT

This paper focuses on the effects of three thermal drying methods (microwave‐, oven‐ and sun‐drying) and one nonthermal drying method (freeze‐drying) on the AOP of leaves of Vitex negundo and Vitex trifolia, which are consumed traditionally as herbal tea. Microwave‐drying and freeze‐drying were found to be able to maintain the AOP of the leaves but oven‐drying and sun‐drying resulted in deterioration of AOP. Microwave‐drying has the advantage of short drying time and low water activity. AOP of Vitex herbal tea leaves that are microwave‐dried and freeze‐dried were not affected by storage up to 30 days.

PRACTICAL APPLICATIONS

Tea, one of the most widely consumed beverages apart from water, has long been known for its health‐promoting benefits in terms of its antioxidant properties due to its high phenolic content. Much of such studies, however, focus on green tea (Camellia sinensis). Although V. negundo and V. trifolia have been consumed traditionally as herbal tea, understanding of its antioxidant properties remains scarce. Drying serves as a vital part of tea processing, affecting its antioxidant content and appearance which in turn affects the commercial value of the tea. This study therefore serves as an important work in providing insights into the antioxidant properties of Vitex species and the best drying method of its leaves as herbal tea for commercial purpose. In addition, this study also provides insights into the effect of different drying methods on the storage of the leaves which is of value to the tea processing industry.     

Pyrrolizidine alkaloids (PAs) in honey and pollen‐legal regulation of PA levels in food and animal feed required

Pyrrolizidine alkaloids (PAs) are secondary plant constituents that comprise about 400 different structures and occur in two major forms, a tertiary form and the corresponding N‐oxide. PAs containing a 1,2‐double bond are pre‐toxins and metabolically activated by the action of hepatic P‐450 enzymes to toxic pyrroles. Besides the acute toxic effects, the genotoxic and tumorigenicity potential of PAs was demonstrated in some eukaryotic model systems. Recently, the potential PA contamination of food and feeding stuff attracted recurrent great deals of attention. Humans are exposed to these toxins by consumption of herbal medicine, herbal teas, dietary supplements or food containing PA plant material. In numerous studies the potential threat to human health by PAs is stated. In pharmaceuticals, the use of these plants is regulated. Considering the PA concentrations observed especially in authentic honey from PA producing plants and pollen products, the results provoke an international regulation of PAs in food.     

PCR-RFLP Analysis of the Internal Transcribed Spacer (ITS) Region for Identification of 3 Clam Species

ABSTRACT Restriction site analysis of the internal transcribed spacer (ITS) region amplified by the polymerase chain reaction (PCR) was used for the specific identification of 3 clam species: Ruditapes decussatus (grooved carpet shell), Venerupis pullastra (pullet carpet shell), and Ruditapes philippinarum (Japanese carpet shell). PCR amplification using primers based on nucleotide sequences of Mytilus ITS regions produced a fragment of 1195 bp in R. decussatus, 1074 bp in V. pullastra, and 1188 bp inR. philippinarum. Digestion of the PCR products with endonucleases HinfI and Rsa I, followed by agarose gel electrophoresis of the digested products, yielded specific restriction profiles that enabled direct visual identification of the species analyzed.     

DNA条形码技术在淀粉掺假鉴别中的应用

基于植物DNA条形码技术建立淀粉及其加工制品成分鉴别检测方法。通过提取淀粉5大物种苜蓿、马铃薯、木薯、番薯和玉米的基因组DNA为模板,分别以ITS2、matK、rbcL和trnH-psbA基因通用引物进行聚合酶链式反应扩增,并将测序结果提交GenBank数据库BLAST比对,评价不同植物DNA条形码序列对其鉴别能力。筛选出ITS2＋trnH-psbA为较适组合序列,并对自行采购的5 个淀粉样品和4 个粉条样品进行检测。     

Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products

The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg^?1 for sesame and 1.4 mg kg^?1 for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.     

Botanical origin authentication of dietary supplements by DNA‐based approaches

Herbal products, such as dietary supplements, have become a subject of increasing global importance for their health benefits and economic considerations. However, they have also been targets of adulteration practices, being the accurate identification of botanicals in herbal products of utmost importance to protect the health and expectations of consumers. Particularly, in the case of dietary supplements, which can have different types of formulations, the identification of plant material used in their production is often a research challenge. DNA‐based techniques have played a crucial role on the development of a wide range of tools for the authentication of herbal products. Therefore, this review intends to describe their main progresses, critically discussing their advantages and drawbacks when applied to authenticate herbal products, focusing on dietary supplements. DNA barcoding is particularly emphasized because it has provided the highest number of applications, followed by the advances on high‐resolution melting analysis combined with DNA barcodes. A special emphasis is also given to the promising approaches relying on DNA metabarcoding and isothermal amplification.     

基于ITS2序列及二级结构对紫苏与其变种、紫苏子与其混伪品的鉴别

采用ITS2 一级序列联合其二级结构对紫苏与其变种、紫苏子与其混伪品进行鉴定,为紫苏药材的使用提供更科学的分类方法.收集紫苏与其近缘种、紫苏子混伪品基源植物样本,提取DNA进行PCR扩增;并结合GenBank中的序列获得所有物种的ITS2序列.应用MEGA7.0软件进行序列分析、计算种内种间遗传距离、构建NJ树,利用I...     

Authentication of raw and processed tuna from Indonesian markets using DNA barcoding,nuclear gene and character-based approach

Establishing seafood authentication methods is an important task for fisheries research laboratories and food control authorities. Nowadays, the extent of fish species substitution is suspected being greater than ever before in commercial markets. In order to provide reliable polymerase chain reaction (PCR)-based authentication systems for tunas, we collected and analyzed authentic tuna reference samples and tuna-food products from Indonesian markets. Our analytical methods mainly relied on identification using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as a genetic marker for “DNA barcoding,” as well as the rhodopsin (RH1) gene as a nuclear marker. Additionally, we identified species-specific nucleotide diagnostic positions (characters) to complete the results obtained basic local alignment search (BLAST) and phylogenetic analysis. Authentication results of tuna-food products showed relatively successful amplification for the COI gene; RH1 acted as an alternative solution for some of the samples, which had failed to react in COI-PCR. Species of the genus Thunnus could not be unambiguously differentiated by BLAST and phylogenetic analysis (neighbor-joining tree) in all cases due to the high similarity of the COI sequences. However, the character-based identification method was found to be helpful for species assignment in case of tuna-food products. Therefore, our findings demonstrated that the COI gene could be more reliable used as a tool for Indonesian commercial tuna products authentication, if the sequencing results were combined with the character-based identification using differences at certain nucleotide positions.     

DNA Barcoding to Detect Chilli Adulteration in Traded Black Pepper Powder

Value-added forms of black pepper (Piper nigrum L.) are an important item of trade globally. Adulteration by default or design of the commodity not only leads to economic loss and public health issues but also to self-respect of a nation. DNA barcoding is assuming significance as a quality assurance technique in many agri-food commodities. Three barcoding loci viz., psbA-trnH, rbcL, rpoC1 were used in the study to detect bio adulteration of traded black pepper powder. PCR amplification of P. nigrum and traded black pepper powder was performed for all the three loci. Sequence analysis and BLAST results revealed chilli adulteration in two out of nine market samples, originating probably from exhausted black pepper powder fortified with chilli. Of the three loci, psbA-trnH proved to be the best and ideal for detection of chilli adulteration in black pepper yielding amplicons of size 600 bp and 350 bp, respectively. Cloning and sequencing of the adulterant specific band of both market samples were done to confirm the results. It was further validated using simulated samples of chilli and black pepper powders in various proportions. The method proved efficient to detect adulteration even at very low levels (0.5% adulteration). HPLC analysis also supported the chilli adulteration of black pepper powder. The method is easy, reliable and efficient, and can be used by the regulatory agencies for quality assurance of black pepper powder.     

用ITS2鉴定芥辣类调味品中山葵、辣根和芥末组分

日式饮食中的芥辣类调味品主要以同属十字花科的山葵(Eutrema wasabi)、辣根(Armoracia rusticana)、芥末(mustard)做原料加工而成。利用核糖体DNA内部转录间隔区Ⅱ(Internal transcribed spacer 2,ITS2)技术快速、准确地鉴定出山葵、芥末和辣根,为该技术应用于芥辣类产品质量检测提供参考。以3种植物材料(山葵、辣根、白芥末)为实验材料,提取基因组DNA,通过引物ITS2_S2进行PCR扩增得到ITS2片段,测序结果通过生物信息学分析进行物种鉴定。MEGA7.0(Molecular evolutionary genetics analysis)分析ITS2序列结果表明,山葵、白芥末和辣根K2P(Kimura 2-parameter)遗传距离在0.088～0.171,均大于0.01,种间变异位点有36个,初步推断利用ITS2序列能将山葵、白芥末和辣根3物种区分开来。另外,GenBank中获得山葵以及近亲西北山嵛菜、辣根、芥末的ITS2序列,MEGA7.0进行种间序列分析,计算K2P,并用邻接法(Neighbor-joining,NJ)构建进化树,山葵与近亲西北山嵛菜聚为一支,棕芥末与白芥末、黑芥末聚为一大支,而辣根单独为一支。通过分析ITS2序列,发现山葵与近亲西北山嵛菜、辣根、芥末的种间K2P遗传距离在0.030～0.105,均大于0.01。研究表明,利用ITS2技术可以鉴别山葵、辣根和芥末等近缘物种,此技术可以用于芥辣类调味品特定原料成分鉴定及定量,为食品质量控制和食品安全提供科学依据。     

Barcode High Resolution Melting (Bar-HRM) analysis for detection and quantification of PDO “Fava Santorinis” (Lathyrus clymenum) adulterants

Legumes considered as one of the most important crops worldwide. Due to high price as a PDO product, commercial products of “Fava Santorinis” are often subjected to adulterations from other legume products coming from other Lathyrus or Vicia and Pisum species. Using plant DNA barcoding regions (trnL and rpoC) coupled with High Resolution Melting (Bar-HRM) we have developed a method allowing us to detect and authenticate PDO “Fava Santorinis”. Bar-HRM proved to be a very sensitive tool able to genotype Lathyrus and its closed relative species and to detect admixtures, being sensitive enough to as low as 1:100 of non-“Fava Santorinis” in “Fava Santorinis” commercial products. In conclusion, Bar-HRM analysis can be a faster, with higher resolution and cost effectiveness alternative method to authenticate PDO “Fava Santorinis” and to quantitatively detect adulterations in “Fava Santorinis” with other relative commercial “Fava” food products.     

Molecular identification of dried squid products sold in China using DNA barcoding and SYBR green real time PCR

ABSTRACT

Dried squid products are popular in China as a snack food, side dishes, or refreshments, and the market appeal can be reflected by the high price that occasionally reaches 497 RMB per kg. However, the absence of harmonisation around the definition of squid, as well as the problems with visual inspection for processed seafood products, make alternative species substitution for dried squid products a frequent occurrence. The aim of the present study was to apply a DNA barcoding approach for species identification of 48 dried squid products collected from the largest online shopping platform in China. Moreover, we also developed a novel SYBR green real-time PCR assay (simplex and duplex followed by a melting curve analysis) specific for Illex argentinus and Todarodes pacificus based on cytochrome C oxidase subunit I (COI) gene.

Results highlighted the successful DNA extraction and PCR amplification of a 655 bp COI gene fragment from all products. A maximum similarity value in the range of 98-100% was obtained for all readable sequences using the BOLD and BLAST public databases and four species (Dosidicus gigas, Uroteuthis edulis, I. argentinus, and T. pacificus) were identified. The specificity of the designed primer sets was confirmed against 23 non-target species, and the newly developed methods were successfully applied to screen I. argentinus and T. pacificus in dried squid products.

Overall, DNA barcoding is a robust tool for seafood species identification and the novel method is effective in screening I. argentinus and T. pacificus in food products.     

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.     

Authentication of Small Berry Fruit in Fruit Products by DNA Barcoding Method

Small berry fruit products are gaining an expanded market due to their high nutrition value. However, the authenticity of products is challenged by adulteration and mislabeling. To establish an accurate and robust method for identifying both known and unknown fruit species in small berry fruit products, DNA barcoding technology based on Sanger sequencing was adopted. To overcome the influence of processing conditions on DNA recovery, mini‐barcodes of rbcL and ITS and a medium‐barcode of psbA‐trnH were applied. To identify ingredients in products containing mixed species, plasmid cloning was applied to separate mixed barcodes. The method established in this paper could detect 1% to 10% target species in mixed fruit juice.     

DNA barcoding reveals high substitution rate and mislabeling in croaker fillets (Sciaenidae) marketed in Brazil: The case of “pescada branca” (Cynoscion leiarchus and Plagioscion squamosissimus)

The removal of morphological features during fish processing hinders identification to the species level, increasing the chances of species substitution and the mislabeling of marketed products. We used DNA barcoding to assess whether species substitutions occur in croaker (Sciaenidae) fillets labeled as “pescada branca” sold in the Brazilian Amazon, where two species are known under this vernacular name (Cynoscion leiarchus and Plagioscion squamosissimus). A 577-bp cytochrome C oxidase subunit I (COI) sequence was obtained from 137 fillets and compared with the sequences of whole Sciaenidae fish that were identified based on their morphology and the reference sequences of the BOLD and GenBank public databases. DNA barcoding was able to identify 90% of the samples analyzed to the species level, and the results showed a high rate of species substitution in the fillets labeled as “pescada branca”. The substitution rate was 100% if using the criterion that the fillets should be C. leiarchus and 76.6% if using the criterion that they should be P. squamosissimus. Additionally, the results show that “pescada branca” was replaced in most cases by species of lower commercial value, which clearly demonstrates economic fraud aimed at increased profits. Our data confirm that DNA barcoding is a sensitive and reliable tool that can be applied to authenticate processed fish.     

Differentiation of Cordyceps Sinensis by a PCR-Single-Stranded Conformation Polymorphism-Based Method and Characterization of the Fermented Products in Taiwan

Cordyceps sinensis (Berk.) Sacc., which is a part of traditional Chinese medicine, has been used as a tonic food and herbal medicine in Asia for centuries. C. sinensis is sold as powders, and the quality is often questionable. To confirm the quality of C. sinensis, products were purchased from different supermarkets in Taiwan. Product samples were analyzed using PCR-single-stranded conformation polymorphism (PCR-SSCP) assay based on internal transcribed spacer 2 (ITS2). PCR-SSCP patterns distinguished ten samples into six groups, but no sample was grouped with representative C. sinensis (rCS). Only two of the ten C. sinensis functional ingredient samples (CSFFMS) tested CB and ST, were similar to the reference product of C. sinensis. This in consistent result could be caused by poor fermentation control process, contamination of original marketing samples or adultration. Therefore, the use of PCR-SSCP could be used for species differentiation or the detection of mutations based on ITS2 region in C. sinensis. The method represents a potential novel means for the differentiation of Cordyceps herbal medicines/functional ingredients and could be used as a quality control in the Cordyceps fermentation industry.     

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.     

基于植物DNA条形码技术对杏仁露中花生源性成分的鉴别研究

杏仁露含有丰富的植物蛋白,深受广大消费者的喜爱,拥有广阔的消费市场,同时其成分与配料表是否相符也备受消费者关心。本研究基于植物DNA条形码技术与PCR技术,设计、筛选同时能对杏仁、花生、核桃、大豆、芝麻和榛子六个物种进行扩增的通用引物,并将其应用于杏仁露中花生源性成分的检测。试验表明引物ITS2-2和trn H-psb A-1分别对六个物种的扩增成功率和测序成功率均较高;通过计算杏仁、花生的基因组DNA提取率,设计掺假模型在提取的杏仁基因组DNA中掺入花生基因组DNA,引物ITS2-2对于掺入85.80%的花生检测结果为杏仁,trn H-psb A-1对于掺入6.94%的花生检测结果为花生。引物ITS2-2和trn H-psb A-1可作为鉴别杏仁露中花生源性成分的植物DNA条形码组合。本研究为该类食品检测提供了新的思路,可作为相关研究的参考。