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。     

Integrated Process for Production of Galangal Acetate,the “Wasabi‐Like” Spicy Compound,and Analysis of Essential Oils of Rhizoma Alpinia officinarum (Hance) Farw

Rhizoma Alpinia officinarum (Hance) Farw, Zingiberaceae (AO), a ginger family herb exhibiting stimulant and a carminative bioactivity, is widely used in European and Asian countries as spicy condiment and medicinal uses. Allyl isothiocyanate (AITC) is the main pungent taste of native Wasabi (Wasabia japonica). The cytotoxicity of AITC has been implicated in thymus, adrenals, and white blood cells. Considering food safety, apparently a safer substitute for wasabi is worthy commercialized. Previously, we found AO crude paste to be rather feasible for use as a “Wasabi‐substitute” in fresh meat and cold salads. A process linking cold ethyl acetate (EtAc) extraction with silica gel adsorption and reversed phase high‐performance liquid chromatography (RP‐HPLC) (mobile phase, 75% methanol) was used to isolate galangal acetate, the Wasabi‐like taste constituent. AO contained abundant galangal acetate (3.84 ± 0.07%) compared to A. galangal (0.57 ± 0.16%), and as already confirmed by thin layer chromatography (TLC), gas chromatography (GC)/mass spectrometry (MS)/MS and Nuclear Magnetic Resonance Spectroscopy (NMR), galangal acetate was particularly thermally labile. The steam distilled essential oil (SDEO) of AO (0.14% on wet basis) contained 80 compounds (number of component, %): monoterpene hydrocarbon (21, 13.83%); oxygenated monoterpene (17, 27.08%); sesquiterpene hydrocarbon (20, 31.03%), and oxygenated sesquiterpene (20, 21.85%), respectively. However, no spicy wasabi‐like constituent remained in SDEO. Alternatively, n‐hexane, EtAc, and methanol extracts of AO all showed potent DPPH‐ and superoxide anion–scavenging activity. Conclusively, SDEO although contains 80 volatiles, galangal acetate is absent due to thermal instability. Galangal acetate exhibits pleasant “Wasabi‐like taste” for which we have successively developed an integrated process for mass production.     

Effects of fertilisation on the allyl isothiocyanate profile of above‐ground tissues of New Zealand‐grown wasabi

Wasabi (Wasabia japonica (Miq) Matsum) is a developing crop in New Zealand and is valued for its spicy taste and pungent smell. It is popular as a condiment for traditional and modern Japanese foods. However, the limited area suitable for wasabi production in Japan has resulted in inability to meet increasing market demand. Isothiocyanates (ITCs) are sulphur compounds responsible for the unique flavour of wasabi, with allyl isothiocyanate (AITC) being the main compound responsible for the pungency. In this study, AITC tissue concentration and yield were measured in three above‐ground tissues of the wasabi plant to investigate the effects of different fertiliser, manure and lime treatments. The highest tissue concentration of AITC was found in the rhizomes, ranging from 1564 to 3366 mg kg^?1 (fresh weight basis), while the petioles and leaves contained 254–373 and 453–643 mg kg^?1 respectively. Fertilisation with ammonium sulphate produced the highest‐quality rhizomes (72% increase in AITC yield) and petioles (64% increase), but the best response in the leaves (51%) resulted from the manure treatment. Nitrogen fertiliser alone reduced the AITC yield by up to 15%. These results should help in formulating guidelines for production of high‐quality wasabi tops containing high levels of AITC. © 2002 Society of Chemical Industry     

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.     

A novel real-time polymerase chain reaction (PCR) method for the detection of walnuts in food

A real-time polymerase chain reaction (PCR)-based method for the detection of the walnut (Juglans regia) component in food is described here. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with walnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the jug r2, a major allergen gene of walnut. The method was positive for 8 varieties of walnut and negative for all other tested plant materials used in food industry, including pecan nuts. The intrinsic detection limit of the method was 0.24 ng walnut DNA. Using a series of model pastry samples with defined walnut contents, a practical detection limit of 0.01% walnut content was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples (bakery and confectionery products), out of which two cakes were found to contain walnuts although they were not adequately labelled. The presented PCR method is useful for sensitive and selective detection of walnuts in food samples and can be performed in one working day.     

A novel real-time polymerase chain reaction (PCR) method for the detection of hazelnuts in food

A real-time PCR (polymerase chain reaction)-based method for the detection of hazelnuts (nuts of Corylus avellana or C. maxima) in confectionery and bakery products is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with hazelnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the hsp1 gene encoding for a low molecular weight heat-shock protein. The method was positive for five hazelnut varieties approved in Slovakia and negative for all other tested plant materials used in food industry including peanuts, walnuts, almonds, pistachio nuts, cashews and chestnuts. The intrinsic detection limit of the method was 13 pg hazelnut DNA, which corresponds to approximately 27 genome equivalents (1C). Using a series of model pastry samples with defined hazelnut contents, a practical detection limit of 0.01% (w/w) hazelnut was determined. Practical applicability of the PCR method was tested by the analysis of 20 food samples (confectionery and bakery products) along with ELISA. For all of the food samples, identical results were obtained by both methods, which conformed to the labelling. The presented PCR method is useful for sensitive and selective detection of hazelnuts in food samples and can be performed in one working day.     

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.     

Polymerase chain reaction (PCR) for the detection of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis>) in food

A method based upon polymerase chain reaction (PCR) for the detection of celery (Apium graveolens) in food was developed. The method involves DNA isolation by chaotropic or non-chaotropic solid-phase extraction and PCR with primers oriented to the sequence of the nuclear gene encoding mannitol dehydrogenase. The PCR method was shown to be specific for celery, producing a 279 bp fragment with four celery varieties and negative results with other species commonly present together with celery in food products (16 samples). The detection limit of PCR was 490–1530 pg DNA, which corresponds to 10² genome copies. When evaluated with model samples of celery in meat pâtés, a detection limit of 0.1% (w/w) was determined. When used to analyse food products from the market (dried vegetable seasonings, dehydrated bouillons), all four products declared to contain celery were correctly identified as positive and all three products in which celery was not declared were identified as negative.     

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.     

山葵的风味物质及其功能应用研究进展

山葵的风味物质是植物组织中的硫葡糖苷经内源性芥子苷酶水解而产生具有辛辣风味的异硫氰酸酯.山葵的基本概况、风味前体及风味产生机理以及异硫氰酸酯的结构及降解方面的研究进展做一综述,对山葵的功能及其在食品行业中的应用进行了报道,并对山葵的发展前景进行了展望.     

Anti‐influenza virus activity of extract of Japanese wasabi leaves discarded in summer

BACKGROUND: Japanese wasabi (Wasabia japonica) is now habitually used as a spice in some kinds of Japanese foods, and its pungent taste and flavor are preferred. Generally, rhizomes and winter leaves are used as a spice and for processed foods such as pickled wasabi. Since the leaf area of summer leaves is far greater than that of winter leaves, they are not used for food, and are discarded. Thus, we need to develop an effective use for summer leaves. We investigated anti‐influenza virus activity in these summer leaves as a new function. RESULTS: Seventy percent ethanol extracts of leaves harvested in July exhibited a high replication inhibition rate (98% or higher) in the type A strain (AH1N1, A/shimane/48/2002), its subtype (AH3N2, A/shimane/122/2002), and type B strain (B/shimane/2/2002). The extracts of summer leaves exhibited the same anti‐influenza virus activity as winter leaves, and showed a stronger activity than stems, roots, and rhizomes. CONCLUSION: A potent anti‐influenza virus activity was discovered in summer leaves of Japanese wasabi. The ethanol extracts inhibited influenza virus replication regardless of the hemagglutinin antigen type. Therefore, such extracts are expected to be a promising source of a novel anti‐influenza virus agent. Copyright © 2008 Society of Chemical Industry     

A novel real-time polymerase chain reaction method for the detection of macadamia nuts in food

A real-time PCR-based method for the detection of macadamia nuts (fruits of Macadamia integrifolia or M. tetraphylla or their hybrids) in food products is described. The method consists of DNA isolation by chaotropic solid phase extraction and subsequent PCR with macadamia-specific primers and a TaqMan fluorescent probe. The primers and the probe were targeted to the gene encoding for vicilin precursor. The method was positive for M. integrifolia and M. tetraphylla and negative for 16 other plant species used in food industry, including peanuts, walnuts, hazelnuts, almonds, pistachio nuts, cashew nuts, Brazil nuts, and chestnuts. The DNA-based detection limit of the method was 1.45 pg. Using a series of model samples with defined macadamia nut contents, a practical detection limit of 0.02% (w/w) macadamia nuts was determined. Practical applicability of the PCR method was tested by the analysis of 14 confectionery samples. For all of the samples, results conforming to the labeling were obtained. The presented PCR method is useful for relatively fast, highly selective, and moderately sensitive detection of macadamia nuts in food samples.     

Rapid method for controlling the correct labeling of products containing European squid (<Emphasis Type="Italic">Loligo vulgaris</Emphasis>) by fast real-time PCR

The squids are a group of cephalopods widely distributed and with high commercial value. In the present work, a fast real-time PCR was developed for the authentication of the European squid (Loligo vulgaris). This method is based on a specific primer/probe set that amplifies a fragment of the Internal Transcribed Spacer 1 (ITS 1) ribosomal DNA region. This technique is notable for its conceptual and practical simplicity, together with its combination of speed, sensitivity and specificity in a homogeneous assay. To all this must be added the time savings produced by the fast real-time PCR due to shorter runs. The presented methodology was validated to check how the degree of food processing affects the applicability of this technique and therefore the detection of L. vulgaris. It was demonstrated that the technique can be applied to all kinds of processed products. The commercial denomination of some cephalopods, including European squids, is an important issue due to the legal gaps, since the same species has different commercial name depending on the format in the market. The methodology herein developed was applied to 42 commercial samples to evaluate the situation regarding the labeling of products made from these species. Moreover, the method can be applied to all kinds of products regardless of the degree of processing.     

Detection of celery ( Apium graveolens ), mustard ( Sinapis alba , Brassica juncea , Brassica nigra ) and sesame ( Sesamum indicum ) in food by real-time PCR

Legislation requires labelling of foods containing allergic ingredients, amongst them celery, mustard and sesame. Here we present robust quantitative and sensitive methods for real-time PCR detection of celery, mustard (Sinapis alba and Brassica sp.) and sesame in food. The development of the DNA-based assays was part of an effort to generate alternative detection methods for allergens for which effective protein-based assays are lacking. The celery and sesame methods were specific for the celery mannitol dehydrogenase gene and the sesame allergen encoding 2S albumin gene, respectively, when tested against a range of plant materials. The mustard method was specific for the allergen encoding sinA gene and its homologues present in different Brassica sp. All primer probe pairs gave high amplification efficiency and sensitivities below approximately ten molecules of purified template DNA. These DNA-based detection methods will constitute supplementary and complementary methods to the traditional protein-based methods. Laboratories may choose different analysis formats depending on the food matrix, the availability of specific tests and the performance characteristics of the tests.     

Development and validation of a novel real-time PCR method for the detection of celery (Apium graveolens) in food

The paper presents a novel real-time PCR method allowing the detection of traces of celery (Apium graveolens) in complex food matrices. The method is based on the amplification of a sequence of the gene coding for the Apium graveolens NADPH-dependent mannose-6-phosphate reductase. It allows the detection of three commonly used celery varieties, celery roots (Apium graveolens var. rapaceum), celery stalks (Apium graveolens var. dulce) and leaf celery (Apium graveolens var. secalinum) and does not show any cross-reactivity with 64 biological species, including ten members of the Apiaceae family. The limit of detection, determined by analysing serially diluted celery extracts, is 10 pg celery DNA for all three celery varieties. In spiked model sausages, the LOD is 0.005% celery. The real-time PCR method was applied to 26 commercial food products. Celery DNA was found in one out of ten samples without any information about the presence of celery.     

Detection of pig derivatives in food products for halal authentication by polymerase chain reaction –restriction fragment length polymorphism

A method for detection of the presence of pig derivatives in three types of food products—sausages and casings, bread and biscuits—using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene was developed. Genomic DNA of sausages and casings, bread and biscuits were extracted. The genomic DNA from the food products were found to be of good quality for the sausages and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs (bp). However, no genomic DNA was detected from the casing samples and poor quality of genomic DNA was extracted from bread and biscuits. No amplification of mt cyt b gene was produced from bread and biscuit samples. To differentiate between samples, the amplified PCR products were digested with restriction enzyme (RE) BsaJI, resulting in species‐specific RFLP. The cyt b PCR‐RFLP species identification assay gave excellent results for detection of pork adulteration in food products and is a potentially reliable technique to avoid species adulteration or fraudulent species substitution for halal authentication. Copyright © 2006 Society of Chemical Industry     

In-house validation of a real-time PCR method for rapid detection of<Emphasis Type="Italic"> Salmonella</Emphasis> ssp. in food products

A real-time polymerase chain reaction (PCR) method for Salmonella ssp. detection in food samples has been developed and validated in-house. The specificity of the assay was confirmed by tests with 295 different Salmonella strains, including four strains of Salmonella bongori. When tested with extracted Salmonella DNA the lowest detected amount was found to be 5 fg, which is equivalent to approximately one genome copy. The detection limit was further determined by artificial contamination of minced meat with S. Typhimurium cells and of pastry with S. enteritidis using the most probable number approach for cell dose dilutions. It was calculated that even one colony forming unit of Salmonella was still detectable in 25 g food after enrichment culture for 18 h. An additional PCR system for internal positive control, which was included in each reaction and detected in parallel via another reporter fluorescence dye, has no negative impact on the sensitivity of the assay. The method was evaluated with 1,293 naturally contaminated food samples and compared to the conventional cultural method. Of 55 positive PCR samples, 45 were confirmed by the cultural method. The statistical comparison revealed a correlation of 99.2% for specificity, of 100% for sensitivity and of 99.2% for trueness. The results of the comparative analysis and the advantages of the real-time PCR method for detection of Salmonella ssp. under routine laboratory conditions are discussed.     

A novel real-time polymerase chain reaction method for the detection of pecan nuts in food

A real-time PCR-based method for the detection of the pecan (Carya illinoiensis) component in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pecan-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the putative gene for allergenic vicilin-like seed storage protein of pecan. The method was positive for 10 pecan varieties and negative for all other tested plant materials used in food industry, including walnut. The intrinsic detection limit of the method was 1 pg pecan DNA which corresponds to 1.2 haploid genome copies. Using a series of model pastry samples with defined pecan contents, a practical detection limit of 0.01% (w/w) pecan was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples; no discrepancies between the declared and detected pecan contents were found. The presented PCR method is useful for sensitive and selective detection of pecans in food samples and can be performed in one working day.