Molecular Identification of Commercial Spicy Pollack Roe Products by PCR-RFLP Analysis

ABSTRACT: Spicy pollack roe products are a popular seafood item made from fish eggs that should be made with salt-cured mature roes of walleye pollack Theragra chalcogramma . Because of high demand and poor catch of walleye pollack, however, spicy pollack roe products are often susceptible to substitution with roes of closely related codfish. In this study, a simple method identifying the ingredients of commercial spicy pollack roe products was developed to differentiate walleye pollack from codfish substitutes such as gray cod Gadus macrocephalus using PCR-RFLP (Restriction Fragment Length Polymorphism) analysis. PCR amplification of the mitochondrial cytochrome b gene yielded single fragments commonly from pollack and cod. Direct digestion of the PCR products with Mph 11031 restriction enzyme showed an unique restriction fingerprint only in pollack. This PCR-RFLP analysis enabled the reliable identification of commercial spicy pollack roe products made by only pollack roes from products padded with cod roes. It thus can be useful to expose substitution of pollack roes with lower valued codfish roes in commercial spicy pollack roe products.     

Rapid PCR-RFLP Method for the Identification of 5 Billfish Species

ABSTRACT: A rapid and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify billfish species Xiphias gladius (swordfish), Makaira nigricans (blue marlin), Makaira indica (black marlin), Istiophorus platypterus (sailfish), and Tetrapturus audax (striped marlin). After DNA extraction and amplifying, the 348-bp PCR products from gene encoding of cytochrome b were subjected to restriction enzyme analysis. No single enzyme tested was able to distinguish the 5 species at the same time, but the combination of results obtained from the digests of Bsa JI and Cac 81 could be used to differentiate the 5 billfish species. This sensitive, rapid, and valid method can be used to detect fraudulent substitutes.     

Species Identification and PCR-RFLP Analysis of Cytochrome b Gene in Cod Fish (Order Gadiformes) Products

ABSTRACT: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiles using the PCR product tRNA^Glu-cytochrome b of 9 standard cod fish, all of which are imported into Japan under the name "cod fish," were investigated as a means of rapid species identification of imported cod fish products. Endonuclease digestion using 4 restriction enzymes ( Alu I, Fok I, Mbo I, and Mse I) generated different digestion patterns for the standards, enabling species identification in imported cod fish products by comparison to the standards'RFLP profiles. Additionally, we conducted a more strict check by phylogenetic analysis on imported cod fish products with 13 standards (including 4 additional cod species from GenBank) using the mitochondrial cytochrome b gene (385 nt). Consequently, it was found that the PCR-RFLP method was sufficient for rapidly screening cod products, and in cases that require conclusive identification, phylogenetic analysis was the most suitable.     

利用线粒体DNA Cyt b基因PCR-RFLP分析方法鉴别羊肉和鸭肉

建立了一种利用线粒体DNA（mtDNA） Cyt b基因PCR-RFLP分析来鉴别羊肉和鸭肉的方法。采用一对通用引物扩增绵羊、山羊和鸭的mtDNACytb基因,并对扩增产物用DNA限制性内切酶Bsu36I和SpeI进行酶切,电泳分析酶切产物的变化。结果表明通用引物可扩增羊和鸭472bp的PCR产物,经两种内切酶酶切后,绵羊、山羊和鸭的PCR产物分别被切为大小不同的片段,其中绵羊和山羊被SpeI切为213bp和259bp,而鸭则被Bsu36I切为95bp和377bp。利用PCR-RFLP分析mtDNA Cyt b基因的方法操作简单,是一种快速鉴别羊肉和鸭肉的可靠方法。      

Genetic Variation Within and Among Rainbow Trout, Onchorhynchus mykiss, Hatchery Populations from Iran Assessed by PCR-RFLP Analysis of Mitochondrial DNA Segments

ABSTRACT Samples were collected from 3 hatchery populations of 2 trout inbreeding centers (Kelardasht and Jajrood) and mitochondrial DNA (mtDNA) of these samples was studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). ND_5/6 segment and a fragment that includes 12SrRNA, tRNA F, P, T, D-loop, and a part of cyt.b of mtDNA were amplified and investigated using different restriction enzymes. Two patterns were observed using Hae III, Ava II, Rsa I, Taq I, and Hinf enzymes on gel electrophoresis, resulting in 9 haplotypes in ND_5/6 segment, whereas only 1 pattern was observed for the fragment containing D-loop. As a measure of intrapopulation diversity and interpopulation diversity, both heterozygocity and genetic distance were determined, respectively.     

用通用引物扩增细胞色素b基因进行牦牛肉的鉴定

本研究的目的是建立牦牛和黄牛肉的基因水平鉴定方法。从牦牛、黄牛和四川几个地方黑山羊品种的肉中提取基因组DNA,采用一对通用引物扩增细胞色素b基因中一段长度为421bp的序列,并进行了克隆测序。结果显示,通用引物能很好地扩增不同动物细胞色素b基因,扩增片段序列在牦牛与黄牛之间存在差异较大,能用于这两种肉的鉴定;而在金堂黑山羊、白玉黑山羊和美姑黑山羊之间差异非常小。本研究建立了PCR-RFLP方法鉴定牦牛和黄牛肉,这对于动物保护以及来源不明的肉类产品的区分具有实际应用价值。     

应用PCR-RFLP和芯片生物分析系统鉴别河豚鱼品种

应用聚合酶链式反应-限制性片段长度多态分析和芯片生物分析系统建立5种河豚的分子生物学品种鉴定新方法。首先提取鱼的DNA进行细胞色素b基因的PCR扩增,然后用DdeⅠ、HaeⅢ和NlaⅢ三种限制性内切酶进行酶切,在Agilent DNA1000芯片上对酶切片段进行分离。该方法可成功区分5个河豚鱼品种,是一种快速、简便、有效的鱼类品种鉴定分析手段。     

Identification of Flatfish Species Using Polymerase Chain Reaction (PCR) Amplification and Restriction Analysis of the Cytochrome b Gene

Restriction site analysis of PCR products from a conserved region of the cytochrome b gene has been used for the specific identification of sole ( Solea solea ), European plaice ( Pleuronectes platessa ) and flounder ( Platichthys flesus). Polymerase chain reaction (PCR) amplification of the cytochrome b gene using universal primers produced a 359 bp fragment in all species analyzed. Digestion of the PCR products with Nci I, Sau 3AI and Hinf I endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of the fish species. This methodology should prove useful for enforcing labeling regulations in the authentication of flatfish species.     

Rapid PCR-RFLP Method for the Identification of Marine Fish Fillets (Seabass, Seabream, Umbrine, and Dentex)

A rapid and reliable PCR‐RFLP method was optimized to identify marine fish fillets. Seabass, seabream, umbrine, and dentex were considered in the study. After DNA extraction and PCR, the 359 bp amplification products, obtained from gene encoding the cytochrome b, were subjected to restriction enzyme analysis. All the enzymes tested were not able to distinguish all the 5 species at the same time, but the combination of the results obtained from the digestion HaeIII and NlaIII can be used to differentiate the fish fillets considered. The method described is sensitive, rapid, and reliable, and it could be used to expose fraudulent substitutions with less valuable fish.     

Molecular identification of cephalopod species by FINS and PCR-RFLP of a cytochrome b gene fragment

Identification of commercial species is a relevant issue to assure the correct labeling of seafood products. In this work two different molecular techniques, FINS (Forensically Informative Nucleotide Sequencing) and PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) were developed to identify 8 cephalopod species (families Loliginidae and Ommastrephidae) employing a fragment of the cytochrome b gene. DNA amplification for all of the species was carried out with a new set of specific primers designed in this study for cephalopods. FINS is a technique based on DNA sequencing, while PCR-RFLP allows direct species identification by comparing specific DNA restriction patterns. Both techniques are useful for cephalopod species identification. 17 food products (mainly "squid rings") were analyzed and the species employed for their manufacture identified by FINS.     

Co-amplification and sequencing of a cytochrome b fragment affecting the identification of cattle in PCR-RFLP food authentication studies

Food authentication studies based on PCR-RFLP analysis are frequently targeted to well-conserved mitochondrial sequences, such as certain regions of the cytochrome b (cytb) gene. The use of mitochondrial L14841/H15149 universal cytb-targeted primers in PCR-RFLP assays revealed the existence of a complex restriction pattern in three genetically-unrelated Iberian (Northern Spain) cows, this being due to the simultaneous co-amplification of two 359 bp cytb fragments. Microsatellite analysis of 11 bovine-specific loci confirmed no familiar linkage among the animals investigated. Both co-amplification products were successfully separated by specific cleavage with endonucleases RsaI and MvaI, which allowed the recovery of each amplification product, respectively. The new co-amplified cytb fragment described in this study was successfully sequenced, and exhibited a significantly high homology (95.1–99.3% range) with respect to mitochondrial sequences previously described for a Bos indicus specimen and for another two Asian Bos taurus, this underlining that its presence in cattle may be more extended than initially thought. In contrast, the homology with the cytb sequence widely accepted for B. taurus was only 89.6%. The results presented in this work imply that food authentication studies by PCR-RFLP analysis may be complicated in the case of cattle by the co-amplification of two different cytb fragments.     

Identification of Ostrich Meat by Restriction Fragment Length Polymorphism (RFLP) Analysis of cytochrome b Gene

ABSTRACT: To enforce labeling regulations in the authentication of ostrich meat, it might be of importance to evaluate a method to identify the ostrich meat. A restriction site of the polymerase chain reaction (PCR) product has been used for the specific identification of ostrich meat. In the present study the part of the gene encoding cytochrome b was amplified and sequenced. The digestion of the PCR products using specific estriction enzymes Hae III, Hinf I, Rsa I, and Tru 91 were used to yield specific restriction profiles that allowed a direct identification of ostrich meat in raw and heat-treated samples from meat of other food animal species.     

Genetic and chemical diversity of Eleutherococcus senticosus and molecular identification of Siberian ginseng by PCR-RFLP analysis based on chloroplast trnK intron sequence

Siberian ginseng (SG), the rhizome and root of Eleutherococcus senticosus, has been used as a tonic and anti-fatigue agent in northeastern Asia from ancient time. In recent years, SG has been becoming fairly popular as dietary supplements and health foods worldwide. In order to establish a convenient and sensitive method for authentication, chloroplast trnK intron sequences of 6 Eleutherococcus species were determined and compared. Genetic polymorphism, representing by 14 types of trnK intron sequence, in E. senticosus was observed. However, characteristic nucleotide markers stable within this species enabled clear discrimination of it from other congeners. A PCR-RFLP method was further developed, which was demonstrated to be efficient for authentication of crude drugs as well as health foods. Quantitative evaluation of three main bioactive constituents indicated chemical diversity in E. senticosus collected from northeast China and the results suggested good producing areas of SG. The chemical data clearly revealed that E. sessliflorus was unsuitable to be used as SG.     

Sarcoplasmic Reticulum Ca2+-ATPase and Cytochrome Oxidase as Indicators of Frozen Storage in Cod (Gadus morhua)

ABSTRACT: Activities of 2 membrane-bound enzymes, Ca²⁺-ATPase from the sarcoplasmic reticulum and cytochrome oxidase from the inner mitochondria membrane, were measured during frozen storage of cod. Enzyme activities were higher in cod muscle samples frozen at −30°C than at −20°C. Freezing-induced activation of both enzymes was observed and the activation was amplified by ice storage prior to freezing. Sensory evaluation conducted at 9 mo of frozen storage showed differences between the sensory properties of cod frozen immediately after catch and frozen after 3 d of storage on ice. These results indicated that the enzymes might be useful as indicators of quality changes by frozen storage.     

Survey of the authenticity of prawn and shrimp species in commercial food products by PCR-RFLP analysis of a 16S rRNA/tRNA mitochondrial region

A novel PCR-RFLP method was evaluated as a tool to assess the incidence of incorrect labelling of prawns and shrimps in commercial food products. The whole method can be performed in less than 8 h in only one day of work. PCR amplification with primers 16Scru4/16Scru3, targeted to the amplification of a ca. 530 bp region of 16S rRNA and tRNA^Val mitochondrial genes, was coupled to restriction analysis with AluI, TaqI or HinfI. Forty-one commercial food products were considered. The molecular method considered allowed the identification of up to 17 different prawn and shrimp species in all the processed products considered. Seven (28%) of the 25 food products declaring one or more species in their labels were incorrectly labelled. Authentication was successfully assessed in commercial peeled products subjected to industrial processing, in which none of the products displayed labelling at species level. Overall, incorrect labelling was detected in 10 (24.4%) of the 41 commercial products tested, while another 16 samples (39%) exhibited incomplete labelling. The molecular method evaluated in this study proved to be a rapid and easy-to-perform two-step analytical approach to achieve species identification of commercial whole specimens of frozen prawns and shrimps and in peeled processed products where such raw materials are included as added-value ingredients.     

Characterization of a Non-electrophoretic Genetic Variant of β-Casein by Peptide Mapping and Mass Spectrometric Analysis

From a total of 634 Holstein milk samples, 158 were identified as homozygous β-casein A¹ according to PAGE. All the β-casein A¹ samples were isolated in pure form by anion exchange chromatography followed by tryptic hydrolysis and peptide mapping of the hydrolysates by reversed-phase HPLC. A comparison of the chromatogram revealed that the β-casein A¹ samples could be classified into two groups based on differences in elution times of fragment 114–169 due to a change in hydrophobicity caused by amino acid substitution. Mass spectrometric analysis showed that 7 β-casein samples with more hydrophobic 114–169 fragment had a higher molecular mass (difference of 16 Da) than the remaining 151 samples with less hydrophobic fragment. Amino acid composition and sequence analysis confirmed the difference in mass of peptide 114–169 by a Pro→Leu substitution at position 137. It is proposed to identify the newly found variant as β-casein G.     

Physiology and Microbiological Quality of Fresh-cut Mango Cubes as Affected by High-O2 Controlled Atmospheres

Preliminary study showed that among 40‐ to 100‐kPa O₂ atmospheres, 60‐kPa O₂ reduced the respiration of fresh‐cut ‘Carabao’ mango cubes the most when held at 5 °C or 13 °C for 42 h. Therefore, the effects of 60‐kPa O₂ on the physiology and microbial quality of fresh‐cut ‘Carabao’ and ‘Nam Dokmai’ mango cubes were determined and compared with those held in air. The high‐O₂ atmosphere reduced the respiration rate of ‘Carabao’ mango cubes stored at 5 °C but stimulated the rate after 2 d of storage at 13 °C. Browning of ‘Carabao’ cubes was accelerated by 60‐kPa O₂ at 13 °C. With ‘Nam Dokmai’ cubes, the high O₂ had no effect on respiration rate, browning, and incidence of water‐soaked appearance at 5 °C and 13 °C. The high O₂ did not affect texture or ascorbic acid content of ‘Carabao’ and ‘Nam Dokmai’ mango cubes at either temperature. Counts of lactic acid bacteria and molds were below the detection level (2.4 log colony‐forming units [CFU]/g) during storage at both temperatures. However, 60‐kPa O₂ stimulated the growth of mesophilic aerobic bacteria on ‘Carabao’ cubes and yeasts of ‘Nam Dokmai’ cubes at 13 °C. The increased microbial count may have been due to the higher pH of cubes stored in 60‐kPa O₂ at 13 °C than at 5 °C or in air. Within ‘Nam Dokmai’ mango cubes, the predominant genera in mesophilic aerobic bacteria were Enterobacter, Klebsiella, and Pantoea and in the yeasts were Candida, Cryptococcus, and Rhodotorula. These results indicate that 60‐kPa O₂ is not desirable for mango cubes when held at 13 °C.     

Synthesis of monohydroxylated inositolphosphorylceramide (IPC-C) in Saccharomyces cerevisiae requires Scs7p,a protein with both a cytochrome b5-like domain and a hydroxylase/desaturase domain

Saccharomyces cerevisiae mutants lacking Scs7p fail to accumulate the inositolphosphorylceramide (IPC) species, IPC-C, which is the predominant form found in wild-type cells. Instead scs7 mutants accumulate an IPC-B species believed to be unhydroxylated on the amide-linked C₂₆-fatty acid. Elimination of the SCS7 gene suppresses the Ca²⁺-sensitive phenotype of csg1 and csg2 mutants. The CSG1 and CSG2 genes are required for mannosylation of IPC-C and accumulation of IPC-C by the csg mutants renders them Ca²⁺-sensitive. The SCS7 gene encodes a protein that contains both a cytochrome b₅-like domain and a domain that resembles the family of cytochrome b₅-dependent enzymes that use iron and oxygen to catalyse desaturation or hydroxylation of fatty acids and sterols. Scs7p is therefore likely to be the enzyme that hydroxylates the C₂₆-fatty acid of IPC-C. © 1998 John Wiley & Sons, Ltd.     

一株牛蒡根际纤维素降解芽孢菌的分离鉴定和发酵分析

从牛蒡根际分离到一株纤维素降解菌株NP10。该菌是芽孢杆菌,其16S rDNA 的Genbank 序列号是FJ908093。分批发酵法对NP10 进行的分析显示,该菌是一株碱性纤维素降解菌。其滤纸酶(FPA)酶活力在32℃、pH8 发酵48h 达到最大为18.2U/mL,其羧甲基纤维素酶(CMCase)活力在32℃、pH7 发酵36h 达到最大,为8.1U/mL。     

Ca2+ Affects Physicochemical and Conformational Changes of Threadfin Bream Myosin and Actin in a Setting Model

The effect of Ca²⁺ on physicochemical and conformational changes of threadfin bream (TB) myosin and actin during setting at 25 and 40°C was investigated. Ca²⁺ ion at 10 to 100 mM induced the unfolding of myosin and actin as evident by an increase of surface hydrophobicity (S_o ANS) at 40 °C. Total SH groups also decreased with an increased Ca²⁺ concentration, suggesting that Ca²⁺ promoted the formation of disulfide bonds during setting at 40 °C. Both hydrophobic interactions and disulfide linkages were involved in formation of myosin aggregates at 40 °C and were enhanced by addition of 10 to 100 mM Ca²⁺. Myosin Ca‐ATPase activity decreased when Ca²⁺ was greater than 50 mM, indicating conformational changes of myosin head. Circular dichroism spectra demonstrated that Ca²⁺ reduced the α‐helical content of myosin and actin incubated at either 25 or 40 °C. Ca²⁺ induced conformational changes of TB myosin and actin incubated at 40 °C to a greater extent than at 25 °C.