Sandwich ELISA for detection of horse meat in raw meat mixtures using antisera to muscle soluble proteins

A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of defined amounts of horse meat (1-50%) in unheated meat mixtures. The assay uses horse-specific antibodies obtained by immunoadsorption of the crude horse antisera onto immobilised sarcoplasmic extracts from chicken, beef and pig to remove cross-reacting antibodies. The purified antibodies bound to a solid support sequester horse muscle soluble proteins from meat mixtures. Further immunorecognition was made with the same antibodies conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of minced beef and pig containing variable amounts of horse meat.     

A preliminary note on the detection and partial characterization of chicken muscle soluble proteins by immunoelectrophoresis in agarose gels

Immunoelectrophoresis in agarose gels has been used to detect and partially characterize specific protein precipitin bands of chicken muscle soluble proteins (CHSP), free of crossreactions with muscle soluble proteins of cow (CSP), pig (PSP) and horse (HSP). Six precipitin bands were obtained by reacting CHSP against an anti-CHSP antiserum produced by a rabbit. These precipitin bands did not appear when the protein extracts from cow, pig and horse were analyzed against the same anti-CHSP antiserum. The six precipitin bands were specific of the chicken muscle soluble proteins. This technique may have the potential to detect the presence of chicken meat in unheated ground meat products.     

The detection and partial characterization of horse muscle soluble proteins by imnmnoelectrophoresis in agarose gels

Immunoele ctrophoresis in agarose gels has been used to detect and partially characterize specific protein precipitin hands of horse muscle soluble proteins (HSP), free of cross reactions with the muscle soluble proteins of cow (CSP) and pig (PSP). Of seven precpptin bands obtained by reacting HSP against an anti-HSP antiserum produced by a rabbit, only one was observed to appear by reacting PSP against the same anti-HSP antiserum. This band, and two more, were detected when CSP were analyzed against an anti-HSP antiserum. The other four bands were specific f or the horse muscle soluble proteins. This technique may have the potential to detect the presence of horse meat in unheated ground meat products.     

Detection of chicken meat in raw meat mixtures by a sandwich enzyme immunoassay

A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of low levels of chicken meat (1–30%) in unheated meat mixtures. The assay uses chicken-specific antibodies, obtained by immunoadsorption of the crude chicken antisera onto immobilized sarcoplasmic extracts from beef, pig and horse, to remove cross-reacting antibodies. The purified antibodies, bound to the wells of a microtitre plate, sequester chicken muscle soluble proteins from saline extracts of meat mixtures. Immuno-recognition is made with similar purified antibodies conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of substrate gives clear optical density differences, when assaying minced beef and pig containing variable amounts of chicken meat.     

Investigations of Soybean 11S Protein and Myosin Interaction by Solubility, Turbidity and Titration Studies

Solubility tests, turbidity tests, and titration experiments were employed to study the possible protein-protein interactions between purified soybean 11S protein and skeletal muscle myosin and the involvement of protein subunits in the interactions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used as an analytical tool for identification of the protein species. These tests indicate that these proteins interacted at temperatures between 85°C and 100°C. Solubility and titration experiments showed that acidic subunits of soybean US protein had little or no interaction with myosin heavy chain subunits. In contrast, soybean 11S basic subunits interacted with myosin heavy chains. The SDS-PAGE method indicated that eight commercial soy protein isolates had a similar protein species composition, but certain proteins in some isolates had lost their availability for water extraction. This may account for different functional properties exhibited by differen soy protein isolates.     

Production and partial characterization of monoclonal antibodies specific to cooked poultry meat

Monoclonal antibodies (MAbs) specific to cooked poultry muscle proteins have been developed for the detection of poultry adulterants in cooked mammalian meat. Saline (0.85% NaCl) extract of heat-treated (100 °C, 15min) chicken muscle proteins was used to immunize mice for MAb development. The specificity of MAbs was tested against chicken antigen and protein extracts from seven other meat species (pork, beef, lamb, deer, horse, turkey and duck) by indirect enzyme-linked immunosorbent immunoassay (ELISA). The immunogenic components in the poultry protein extracts were determined by SDS-PAGE followed by immunoblotting. A total of six hybridoma cell lines that secrete IgG class MAbs have been developed: MAbs 3E12 and 1A5 were able to distinguish between cooked poultry and mammalian meats, MAbs 9C6 and 6F7 reacted strongly with cooked chicken only, and MAbs, 5D2 and 6G8, reacted with both cooked turkey and chicken but not other species. All six MAbs demonstrated a proportional increased ELISA response to respective adulterated poultry samples in pork over a 0-100% range of aduleration.     

Detection and partial characterization of soluble pig muscle proteins by immunoelectrophoresis in agarose gels

Immunoelectrophoresis in agarose gels has been used to detect and partially characterize specific protein precipitin bands of soluble proteins of pig muscle (PSP), free of cross-reactions with soluble muscle proteins of cow (CSP), horse (HSP) and chicken (CHSP). Out of six precipitin bands obtained by reacting PSPs against an anti-PSP antiserum produced by a rabbit, four bands were observed to appear by reacting HSPs against the same anti-PSP antiserum. Two more bands were detected by analysing CSPs against an anti-PSP antiserum and three bands were detected by analysing CHSPs against the same anti-PSP antiserum. Thus, only one band is specific to the soluble protein of pig muscle. This technique may have the potential to detect the presence of pork in unheated ground meat products.     

干莲子及其磨皮粉中蛋白质的组成特性比较

以凯氏定氮法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamidegelelectrophoresis,SDS-PAGE）和体积排阻高效液相色谱（size exclusion chromatography-high performance liquidchromatography,SEC-HPLC）法对比了干莲子与其磨皮粉中蛋白质含量及其亚基组成和分子质量分布。结果表明：干莲子与其磨皮粉蛋白含量分别为188.10、186.54 mg/g;二者水溶性、盐溶性蛋白质组分含量均超过总蛋白的60%,酸溶性、碱溶性蛋白质组分含量均不足总蛋白的23%。干莲子与其磨皮粉蛋白质的SDS-PAGE图谱几乎一致,含量较多的蛋白质亚基分子质量主要集中在15～20 kD和30～50 kD;蛋白质的分子质量分布也高度相似,各组分的HPLC洗脱峰基本一致,但莲子蛋白质中酸溶性蛋白的种类较多、碱溶性蛋白的含量较大。本研究表明莲子磨皮粉蛋白质的组成特性与莲子蛋白质基本一致,可以替代莲子用于生产莲子蛋白粉或其他含莲蛋白食品。     

Phase behaviour,rheology and microstructure of mixture of meat proteins and kappa and iota carrageenans

The phase behaviour of mixtures of salt soluble meat proteins, kappa (κ) and iota (ι) carrageenan in non-gelling conditions (45 °C) were determined at pH 5.6, 6.2 and 7.1. The concentration of meat proteins ranged from 0.1 to 1.0 percent and that of κ-carrageenan and ι-carrageenan from 0.02 to 0.3 percent in the mixtures. Mixtures separated under gravity to form soluble/liquid and gelled/complex phases. For meat proteins- κ-carrageenan mixtures, phase separations at all meat protein/carrageenan ratios were observed. For meat protein-ι-carrageenan mixtures, soluble complexes were formed at low meat protein to ι-carrageenan ratios and gels at higher ratios. The yield of the complex/gels increased with the increase in the concentration of the meat proteins and carrageenans and decreased with increase in the pH of the initial mix. The complex/gels formed became stronger with the increase in carrageenan in the mix and with κ-carrageenan compared to ι-carrageenan. Chemical analyses and scanning electron and phase contrast microscopy indicated that in phase separated mixtures, the bulk of the meat proteins and carrageenan were found in the gel compared to the liquid phase; and that meat protein interacted with carrageenan in the gel and formed soluble complexes with carrageenan in the liquid phase. SDS-PAGE showed that the meat proteins that interacted to form the complex/gels with carrageenan included myosin heavy chain, α-actinin, actin, myosin light chains and proteins with molecular weights around 150 and 50 kD. The outcomes of the present study could be used in the formulation of multi-component foods with a range of consistencies containing meat proteins.     

Effects of Antemortem Injected Crude Papain in Chicken Muscle

The effects of antemortem injected crude papain in chicken muscle on muscle protein hydrolysis were investigated. White Leghorn hens were injected intravenously prior to slaughter with crude papain or heat denatured crude papain. Collagen solubility (hydroxyproline) at 60°C was 210% of the heat denatured crude papain control. SDS-PAGE revealed no difference between treatments. Crude papain was unable to penetrate intact cells at physiological temperatures. A small but significant increase in muscle TCA soluble tyrosine was observed due to antemortem papain treatment. Postmortem papain treatment resulted in a 14 fold increase in tyrosine. Little if any intracellular muscle protein hydrolysis occurred as a result of antemortem papain treatment. Collagen hydrolysis may be the major contributor to antemortem papain induced meat tenderness.     

Development of a Test to Predict Gelation Properties of Raw Turkey Muscle Proteins

A method for extraction and fractionation of muscle proteins into five fractions based on salt (NaCl) solubility was developed. The influence of protein extractability, solubility, muscle pH and total protein on gelation was investigated. Shear stress (gel strength) and shear strain (gel deformability) at failure of cooked (70°C) comminuted turkey breast gels were correlated with the 10 min protein extract and proteins soluble in 0.30–0.35M NaCl. Shear strain was correlated with muscle pH and shear stress was sensitive to total protein. Meat pH and the extraction/fraction method can be used on raw meat to indicate functional properties related to texture of cooked meat.     

Composition and protein fractions of different meat by-products used for petfood compared with mechanically separated chicken (MSC)

Pork by-products (lung lobes, kidneys), chicken viscera (head, feet and viscera) and mechanically separated chicken (MSC) were evaluated for proximate composition, protein distribution and connective tissue. Proximate composition varied among meat by-products and MSC. Pork by-products contained the most crude protein (p<0.05). Low levels of high ionic strength soluble (HIS) proteins were obtained from meat by-products. Pork lungs and chicken viscera contained the greatest amounts of insoluble (IN) proteins (p<0.05). Total collagen values were positively correlated to IN proteins, intramuscular collagen (IMC) and elastin. Types I and III collagen could not be detected by SDS-PAGE for the different meat by-products though collagen solubility appeared to be significant. These results suggest functional property differences between specific by-products are likely when used in petfood product formulations.     

Capillary electrophoresis for bovine and ostrich meat characterisation

The objective of this study was to characterise, compare and quantify the water soluble protein (WSP) and salt soluble protein (SSP) fractions from bovine and ostrich muscle by using sodium dodecyl sulphate polymer-filled capillary gel electrophoresis (CE-SDS). Samples were raw ostrich leg and eye round beef collected 24 and 48 h, respectively, after sacrifice from local slaughter houses. WSP were extracted with cold double distilled deionized water and SSP with 0.6 M NaCl/0.01 M phosphate buffer pH 6 with 0.5% polyphosphates. Separation of WSP and SSP extracts was achieved by CE-SDS. Quantitative data for individual proteins was generated by constructing a calibration curve using bovine serum albumin (BSA) as a standard. The WSP profiles showed differences for bovine and ostrich meat, both qualitatively and quantitatively and could be employed for species differentiation. Quantitative data derived for WSP and SSP from bovine and ostrich muscle showed significant differences among individual proteins. A comparison of protein profiles form ostrich and bovine meat should be useful for meat species differentiation and muscle characterisation for establishing relations to meat quality.     

11S大豆球蛋白对鸡肌球蛋白凝胶品质特性的影响

为了能够更好地揭示大豆蛋白质在肉制品加工中的作用,为肉糜类制品加工提供理论基础,本文将鸡胸脯肉和大豆蛋白质中的含量最多和最重要的肌球蛋白与11S大豆球蛋白分别进行了提取,并利用十二烷基磺酸钠凝胶电泳对蛋白质进行了鉴定;采用质构仪、低场核磁共振仪、扫描电镜等现代生物技术的方法和手段研究了11S大豆球蛋白添加浓度对肌球蛋白凝胶品质特性的影响,结果表明:所提取的两种蛋白质纯度都达到了90%以上;低浓度的11S大豆球蛋白对肌球蛋白凝胶的保水性有提高作用,较高浓度的添加显著降低凝胶保水性;11S大豆球蛋白能对肌球蛋白凝胶强度影响不大;高浓度的11S的添加能够使肌球蛋白凝胶形成空洞直径较大的不均匀的微观结构。综合考虑,4%11S的添加量能够获得理想的肌球蛋白凝胶品质。     

Detection and partial characterisation of soluble proteins of beef muscle by immunoelectrophoresis in agarose gels

Immunoelectrophoresis in agarose gels has been used to detect and partially characterise specific protein precipitin bands of beef proteins (CSP), free of cross-reactions with proteins of pig (PSP) and horse (HSP). Out of the six precipitin bands obtained by reacting CSP's against an anti-CSP antiserum produced by a rabbit, only one band was produced by interaction of the anti-CSP antiserum with PSP and HSP. The other five bands were specific beef muscle soluble proteins. This technique may be useful in detecting the presence of beef in unheated ground meat products.     

四种非肌肉蛋白对冷冻竹荚鱼鱼糜凝胶能力的影响研究

探讨了四种常用的非肌肉蛋白对竹荚鱼鱼糜凝胶特性的影响,对不同鱼糜加热过程中凝胶强度、持水力、白度、SDS-PAGE电泳图谱以及微观结构的变化进行比较。实验结果表明,血浆蛋白对冷冻竹荚鱼的蛋白酶有很强的抑制效果,最佳添加量为1%。卵清蛋白及乳清蛋白在5%高浓度时其凝胶增强效果与1%的血浆蛋白添加效果近似,大豆蛋白的效果较差,四种蛋白对鱼糜白度、持水力影响不大。非肌肉蛋白应用于竹荚鱼鱼糜的品质改善中,对凝胶劣化有一定的抑制,这对竹荚鱼鱼糜的品质控制具有一定意义。     

发酵剂及猕猴桃蛋白酶促进马肉发酵香肠蛋白质的降解

为了提高马肉发酵香肠的品质,添加发酵剂和猕猴桃蛋白酶,促进其发酵成熟和肌肉蛋白质的降解。以新鲜马肉和马脂肪为原料,分别设计三个试验组,一组为对照组,另两组为处理组;对照组不采取处理,而两个处理组添加2%的发酵剂和0.05%的猕猴桃蛋白酶,并在相应条件下进行发酵,检测其总氮、非蛋白氮等指标,并通过SDS-PAGE电泳分析其肌肉蛋白质的降解情况。结果表明,灌肠后(0 d)各组总氮含量为3.09 g/100 g左右,而加工结束后(28 d),对照组(CK组)、发酵组(A组)、猕猴桃蛋白酶组(B组)总氮含量分别是3.41 g/100 g、3.85 g/100 g及4.15 g/100 g,说明总氮含量明显上升,并且发酵剂和猕猴桃蛋白酶的影响显著;28 d CK组、A组、B组非蛋白氮含量分别为0.42 g/100 g、0.52 g/100 g、0.65 g/100 g,与0 d非蛋白氮含量0.22 g/100 g相比,CK组上升1.9倍,A组上升2.4倍,B组上升2.9倍;SDS-PAGE电泳结果表明,在整个加工过程中A组与B组肌浆蛋白逐渐发生降解,大分子条带发生了明显的变化,发酵剂对马肉发酵香肠肌浆蛋白和肌原纤维蛋白的降解有显著的促进作用,猕猴桃蛋白酶对肌浆蛋白的影响显著,并比发酵剂大,但对肌原纤维蛋白的作用不大。     

Monoclonal antibody sandwich ELISA for the potential detection of chicken meat in mixtures of raw beef and pork

A sandwich ELISA (enzyme-linked immunosorbent assay) has been developed successfully for the detection of defined amounts of chicken meat (1-100%) in beef and pork meat mixtures. The assay uses a monoclonal antibody (BC9) specific to a chicken muscle soluble protein to capture this protein from complex meat mixtures. Further immunorecognition of the captured protein was attained with rabbit polyclonal antibodies against chicken muscle proteins (anti-CHSP). A commercial goat anti-rabbit immunoglobulin conjugated to peroxidase was used to detect the anti-CHSP antibodies bound to the chicken protein. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of beef and pork meats containing variable amounts of chicken meat.     

ON THE NATURE OF COOKED MEAT HEMOPROTEIN

SUMMARY— Reflectance spectra of cooked meat and the precipitates obtained on heating aqueous muscle extracts, mixtures of bovine serum albumin (BSAl and horse heart metmyoglobin IMbl at 60°C. and Mb alone at > 80°C were all similar. BSA-Mb mixtures in 5.5 M urea atpH 6.0 yielded soluble complexes that were spectrally the same as those present in cooked meat. Sephadex chromatography showed these complexes to be denatured hemoproteins where the protein could be denatured BSA or dimers or higher aggregates of denatured apomyoglobin (ApoMb). The affinity of the hematin for denatured BSA was much greater than for the ApoMb aggregates. No evidence was obtained for complexes of a type where hematin was coupled with both denatured ApoMb and denatured BSA. It is suggested that the complexes present in cooked meat are also denatured hemoproteins, where the protein may be any of several of the denatured proteins present in cooked meat. An electron-spin resonance (ESR) spectra of cooked meat indicated that at - 196°C the ferric ion in the hematin was essentially low-spin. The mechanism of formation and the nature of the possible bonding between hematin and denatured protein are discussed.     

Identification of novel peptides for horse meat speciation in highly processed foodstuffs

There is a need for robust analytical methods to support enforcement of food labelling legislation. Proteomics is emerging as a complementary methodology to existing tools such as DNA and antibody-based techniques. Here we describe the development of a proteomics strategy for the determination of meat species in highly processed foods. A database of specific peptides for nine relevant animal species was used to enable semi-targeted species determination. This principle was tested for horse meat speciation, and a range of horse-specific peptides were identified as heat stable marker peptides for the detection of low levels of horse meat in mixtures with other species.