Sandwich enzyme-linked immunosorbent assay for the detection of bovine blood in animal feed

The feeding of ruminant proteins to ruminants is prohibited in most countries because the practice is thought to be responsible for the spread of bovine spongiform encephalopathy. However, currently available methods to detect ruminant blood products in rendered feedstuffs are inadequate because they lack species specificity, tissue specificity, and are not based on a thermostable analyte. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for this study that provides reliable and sensitive (0.05-0.5% v/v) detection of bovine blood materials in animal feed. The new sandwich ELISA employs two previously developed monoclonal antibodies (MAbs), Bb6G12 as the capture antibody and biotinylated MAb Bb3D6 as the detecting antibody, and is bovine-specific and blood-specific. The assay is based on the detection of a 60 kDa thermostable protein in bovine blood and provides a useful regulatory tool for monitoring fraudulent labeling or contamination of bovine blood in both heat-processed feedstuffs and unprocessed raw materials. Keywords: Bovine; blood; monoclonal antibody; sandwich ELISA.     

Production of monoclonal antibody for the detection of meat and bone meal in animal feed

For the detection of prohibited meat and bone meal (MBM) in animal feed, monoclonal antibodies (MAbs) were raised against heat-stable h-caldesmon purified from bovine intestinal smooth muscle. The obtained hybridoma cells were screened against extracts of the bovine MBM and heat-treated smooth muscle, and MAb 5E12 was identified as having the best performance. Antibody 5E12 did not react with animal feed, milk product, plant proteins, and other ingredients used for commercial animal feed except for the gelatin. This antibody diluted to 100-fold was able to detect MBM mixed in animal feed at 0.05% in an ELISA, and it showed strong affinity toward bovine smooth muscle autoclaved at 130 degrees C. Therefore, this antibody can be used in the ELISA system for field testing of the presence of MBM in animal feed.     

Detection of ruminant meat and bone meals in animal feed by real-time polymerase chain reaction: result of an interlaboratory study

The commercialization of animal feeds infected by prions proved to be the main cause of transmission of bovine spongiform encephalopathy (BSE). Therefore, feed bans were enforced, initially for ruminant feeds, and later for all feeds for farmed animals. The development and validation of analytical methods for the species-specific detection of animal proteins in animal feed has been indicated in the TSE (Transmissible Spongiform Encephalopathies) Roadmap (European Commission. The TSE (Transmissible Spongiform Encephalopathy) roadmap. URL: http://europa.eu.int/comm/food/food/biosafety/bse/roadmap_en.pdf, 2005) as the main condition for lifting the extended feed ban. Methods based on polymerase chain reaction (PCR) seem to be a promising solution for this aim. The main objective of this study was to determine the applicability of four different real-time PCR methods, developed by three National expert laboratories from the European Union (EU), for the detection and identification of cattle or ruminant species in typical compound feeds, fortified with meat and bone meals (MBM) from different animal species at different concentration levels. The MBM samples utilized in this study have been treated using the sterilization condition mandatory within the European Union (steam pressure sterilization at 133 degrees C, 3 bar, and 20 min), which is an additional challenge to the PCR methods evaluated in this study. The results indicate that the three labs applying their PCR methods were able to detect 0.1% of cattle MBM, either alone or in mixtures with different materials such as fishmeal, which demonstrates the improvement made by this technique, especially when compared with results from former interlaboratory studies.     

Determination of sulfur amino acids and tryptophan in foods and food and feed ingredients: collaborative study

Samples of 4 foods, 1 animal feed, isolated soy protein, and beta-lactoglobulin were analyzed by 9 laboratories to determine concentrations of cysteine as cysteic acid, methionine as methionine sulfone, and tryptophan. Sulfur amino acids were determined by AOAC method 43.A08-43.A13 for food and feed ingredients, in which samples are oxidized with performic acid before protein hydrolysis with 6N HCl. Tryptophan was determined after protein hydrolysis with 4.2N NaOH. In both methods, free amino acids were separated by ion-exchange or reverse-phase chromatography. Each laboratory was provided with detailed methods and with sealed vials containing solutions of standards. Samples were analyzed in duplicate, and variation between laboratories was determined. Coefficients of variation between laboratories for the 6 samples ranged from 5.50 to 11.8% for methionine as methionine sulfoxide, 8.59 to 17.3% for cysteine as cysteic acid, and 3.87 to 16.1% for tryptophan. Amino acid recoveries were determined by analysis of beta-lactoglobulin and were based on expected levels of each amino acid obtained from amino acid sequence data. The mean recovery of cysteine was 97% with a range of 88-119%. For methionine, mean recovery was 98% (range 89-115%) and for tryptophan, 85% (range 59-102%). Method 43.A08-43.A13 for food and feed ingredients has been adopted official first action for determination of cysteine and methionine in processed foods. The alkaline hydrolysis method has been adopted official first action for determination of tryptophan in foods and food and feed ingredients.     

Determination of dihydroergosine in sorghum ergot using an immunoassay

Dihydroergosine (DHES) is the principal toxic alkaloid produced by sorghum ergot (Claviceps africana). It has recently been shown that DHES levels as low as 1 mg/kg in animal feed can cause significant production losses. Quantitative immunoassays for detecting the related rye ergot alkaloid, ergotamine, are described in the literature, but those assays are relatively insensitive for DHES. This paper describes competitive enzyme-linked immunosorbent assays (ELISA) for measuring the DHES concentration in grains and mixed animal feed. The assays were developed using a DHES specific mouse monoclonal antibody and rabbit polyclonal antibodies raised against DHES conjugated to bovine serum albumin. Recoveries of between 77 and 103% were obtained from spiked grain using a simple, one step extraction with 70% methanol. Both the monoclonal and the polyclonal assays are capable of detecting DHES concentrations above 0.01 mg/kg, but quantification is most reliable at concentrations of 0.1 mg/kg or higher.     

转基因产品可追溯管理及溯源技术研究进展

家畜生产链中的转基因技术作为改善农业技术,提高农业经济效益的新技术,是21世纪不可回避的并需要积极应对的技术领域。鉴于转基因食品、动物饲料及其动物产品的安全与风险并存,本文阐述了转基因生物及转基因动物的概念内涵,总结了世界各国及其消费者对转基因技术及其产品的接受态度,其中欧盟对于GM食品、饲料及GM动物的开发持谨慎的态度,在相关标识及立法上起步早,并随着技术进步不断完善立法。对于食品和饲料的GM的标识阈值欧盟为0.9%,澳大利亚和新西兰则为1%,日本与韩国则对此标识未作要求,美国、加拿大和阿根廷等则对GM产品标识采取自愿的方法,而我国目前的相关立法着重于对转基因产品安全审批,但对终端产品管理和标识尚无界定。其次,本文总结了中国在转基因动物溯源技术方面所开展的工作,一是基本构建了基于基因改良的转基因动物数据库及溯源网络平台(www.gmanimal.com.cn);二是提出了转基因动物产品溯源的全程标识技术路线;三是使用Net及网络数据库技术开发了转基因动物(猪)及其产品全程溯源模式系统,可实现从是否使用转基因饲料和(或)使用转基因动物及其产品生产的正向跟踪及方向溯源,为满足政府的监管及消费者的知情权和选择权在技术上做好了储备。     

Pea detection in food and feed samples by a real-time PCR method based on a specific legumin gene that allows diversity analysis

Real-time polymerase chain reaction is currently being used for the identification and quantification of plant and animal species as well as microorganisms in food or feed samples based on the amplification of specific sequences of low copy genes. We report here the development of a new real-time PCR method for the detection and quantification of the pea (Pisum sativum) based on the amplification of a specific region of the legS gene. The specificity was evaluated in a wide range of plant species (51 varieties of Pisum sp., and 32 other plant species and varieties taxonomically related or nonrelated). The method allows the detection and quantification of as low as 21.6 pg of DNA, which corresponds to 5 haploid genome copies. The system has been shown to be sensitive, reproducible and 100% specific for the rapid detection and quantification of pea DNA in processed food and feed samples, being therefore suitable for high-throughput analysis.     

Crop residue management and tillage methods for conserving soil and water in semi-arid regions

Soil degradation reduces soil productivity and is a serious problem on much of the land in semi-arid regions. To avert continued degradation, the soil productivity balance must be shifted from degrading processes to conservation practices. Crop residue management and conservation tillage are on the positive side of the balance. When adequate residues are available and conservation tillage is used, soil erosion is greatly reduced and water conservation is enhanced. Water conservation is important for improving crop yields in semi-arid regions, especially where irrigations is not used. A major constraint to residue management in many countries is low production and widespread use for other purposes. In such cases, clean tillage and appropriate support practices such as contouring, furrow diking, strip cropping and terracing may provide adequate soil and water conservation benefits. Where these are not adequate, alternative management practices should be implemented to ease the demand for residues, thus permitting more of them to be retained on the land for soil and water conservation purposes. Some alternative practices include limited or selective residue removal, substituting high quality forages for residues as animal feed, alley cropping, using wasteland areas more effectively, improving the balance between feed supplies and animal populations, and using alternative fuel sources.     

Grain Yield,Quality, and Nutrient Concentrations of Feed,Food, and Malt Barley

Barley (Hordeum vulgare L.) is a cereal grown for animal feed, human consumption, and malting. Nutrient concentrations are important as they provide information regarding the dietary values of barley consumed by animals or human beings. In addition, grain nutrient removal may be useful for refining fertilizer recommendations. A study was conducted in 2015 and 2016 investigating the cultivar effects on grain yield, quality, and grain nutrient concentrations and removal under irrigated conditions for two-row barley cultivars. Adjunct and feed cultivars produced the highest yields compared with the all-malt and food cultivars. Specific quality and nutrient values were greater than or equal to in the food cultivar compared to the malt or feed cultivars. Variations in nutrient concentrations were measured among the adjunct and all-malt cultivars, which could potentially affect the malting and brewing qualities. Grain yield, quality, nutrient concentrations and nutrient removal varied among cultivars grown under identical environmental conditions, which may influence end-use.     

Inulin determination for food labeling

Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection.     

Quantification and Characterization of Food Processing Wastes/Residues

A survey was undertaken to determine the wastes/residues generated by the food processing industry in the state of Iowa by sending a questionnaire to food processors with more than ten employees. Food processors were asked to provide a company profile, information about food waste/residue generated, and information about current disposal methods. Samples of wastes/residues were obtained from food processors willing to provide them, and proximate analysis was done. The overall response rate was 82.2 percent or 365 respondents out of 444 questioned. Companies under different category of standard industrial code (SIC) could produce similar wastes/residues that were classified in the same waste/residue streams. Within the meat and seafood category, the majority (87.7 percent) of the wastes/residues were generated by SIC codes 2077 (animal, marine fats and oils) and 2015 (poultry slaughtering, dressing, and processing). Among the waste management practices reported, excluding “other,” land application accounted for 14.0 percent of the waste stream, followed by rendering with 11.4 percent, landfill with 6.7 percent, sewage on-site with 5.0 percent and animal feed production off-site with 4.3 percent. Other waste management practices (combinations of major practices or others not clearly defined) account for 55.1 percent of the allocation of the waste stream.     

High performance liquid chromatographic determination of clocapramine in feed and plasma

A high performance liquid chromatographic method is described for the determination of clocapramine in animal feed and plasma. Samples are made alkaline and then extracted with chloroform containing opipramol as internal standard. For plasma samples, the organic phase is evaporated to dryness under a stream of nitrogen, and the residue is dissolved in dichloromethane-methanol. Extracts are chromatographed on silica gel with dichloromethane-methanol-ammonia (100 + 10 + 0.25) as eluant, and quantitated using an internal standard. Within-day precision for plasma extracts (n = 15) was 3.39, 5.7, and 4.13% at 5, 10, and 15 mg clocapramine/L plasma, respectively, and day-to-day precision was 4.6, 6.8, and 4.4% at the same levels. The detection limit was 0,5 mg/L. Recovery from feed over the concentration range 2-6 g/kg was greater than 96%.     

Supercritical fluid extraction of atrazine and other triazine herbicides from fortified and incurred eggs

Triazines are a class of important pre-emergent weed herbicides. Some members of this class of herbicides exhibit carcinogenic and immunotoxicity properties, which make their use controversial in areas where animal feed crops are grown. It is therefore important to determine if triazine residues are transported to animal food products in order to ascertain the extent of human exposure. Most of the current herbicide residue extraction methods are time-consuming and solvent intensive. Supercritical fluid extraction (SFE) using CO(2) has been used as a alternative for other residue extraction methods as a replacement for hazardous organic solvents. In this study, 10 triazines were extracted from eggs fortified at 100 ppb using unmodified supercritical CO(2) at a pressure of 10000 psi and a temperature of 50 degrees C with off-line collection on a solid phase extraction cartridge containing Florisil. Atrazine recovery averaged 90.4% with an RSD of 3.3%. The other triazines were recovered at mean levels >73%. In a separate feeding study, atrazine and two of its dealkyl metabolites were detected in the egg. The results indicate that SFE is a viable technique for isolating triazine residues from eggs, requiring only 8 mL of solvent for each analysis.     

Methodology for Creating an Information Tool for Animal Food Facilities

The U.S. Food Safety Modernization Act, administered by the Food and Drug Administration (FDA), requires animal feed and pet food facilities to conduct a hazard analysis by identifying and evaluating potential hazards in animal food. This paper describes the creation of an information tool designed to help animal food facilities comply with this requirement. A thorough search of the scientific literature and FDA recall data identified the occurrence of hazards in animal food, assessed the severity of the hazards, and provided a scoring system useful for the identification and prioritization of specific animal food-hazard-species combinations.     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.     

What Is Being Learned About Starch Properties from Multiple‐Level Characterization

A survey is given of methods to characterize the lowest three levels of starch structural features: individual chains, branched molecules, and the arrangement of branched molecules in a sample (e.g., crystalline and amorphous lamellae of starch granules in grain). The survey also covers ways of treating the results so as to understand starch structure–property correlations: for example, the structural characteristics that control the rate of digestion of a starch‐containing food. A number of studies are then examined not only to show how these techniques have been used to discover correlations between these structural characteristics and properties of importance but also to deduce reasonable causal explanations for the correlations. An overview of problems that have not yet been solved in each of these starch structural levels is also given. The applications of these characterization methods have considerable potential as tools to choose and process native starches with improved functional properties for human food, animal feed, and industrial uses, including biomaterials.     

Potential applications of water hyacinth for water,air recycling in closed systems

Russian space scientists have done much work on closed ecological life support systems (CELSS) using higher plants and algae both for recycling water and air and regeneration of food. Research in this country has been directed mostly towards physiochemical methods for recycling water and air. Development of CELSS using biological components such as aquatic vascular plants (water hyacinth) has recently been recommended. Use of water hyacinth in open systems for municipal wastewater treatment has been studied extensively along with its many other terrestrial uses (animal feed, source of fresh water through evapotranspiration, methane source, fertilizer and compost). After reviewing the research work in CELSS, controlled environment agriculture, and water hyacinth and its uses, potential applications of water hyacinth in closed ecological systems for water, air recycling are outlined.     

HPLC analysis of rosmarinic acid in feed enriched with aerial parts of Prunella vulgaris and its metabolites in pig plasma using dual-channel coulometric detection

This paper describes a sensitive isocratic HPLC/ECD method developed for the determination of rosmarinic acid (RA) in plant material, animal feed, and pig plasma. The plasma sample preparation only includes protein precipitation and adjustment of the pH. The applicability of the method was tested on plasma samples of pigs that were exposed to a 91-day oral intake of RA via feed enriched by aerial parts of Prunella vulgaris. The plasma was directly analyzed using the method described as well as after enzymatic hydrolysis. When no hydrolysis step was included, RA and caffeic acid (CA) were quantified in the plasma. In hydrolyzed plasma samples, several other metabolites were determined, including dihydrocaffeic, ferulic, and dihydroferulic acid. The dual-channel coulometric detection employed, as an alternative to mass spectrometry, offers good selectivity and sensitivity owing to the electrochemical properties of the phenolic constituents.     

Determination of spinosad and its metabolites in food and environmental matrices. 3. Immunoassay methods

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidopteran larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing a magnetic particle-based immunoassay (IA) test kit have been developed and validated for determining spinosad in environmental and food matrices. These methods involve an extraction of the residues from the matrices with appropriate solvents. For some matrices, the sample extracts can be diluted and measured directly by IA without any cleanup. For other matrices, sample extracts are purified using liquid-liquid partitioning and/or solid phase extraction prior to measurement by IA. The methods determine the total residue of spinosad, which includes the active ingredients (spinosyns A and D) and several minor metabolites, including spinosyn B, spinosyn K, and N-demethylspinosyn D. The methods have validated limits of quantitation of 0.0001 microgram/mL in water, 0.05 microgram/g in sediment, and 0.010 microgram/g in crops, crop processed commodities, and animal tissues. This paper briefly summarizes the residue methodology and method validation data for spinosad in 34 food, feed, and environmental matrices.     

Immunoassays of soy proteins

Proteins of soybeans (Glycine max) are widely used in animal and human nutrition. In addition to the bulk of the seed storage proteins, which are classified as albumins and globulins, approximately 6% of soybean proteins are classified as inhibitors of trypsin and chymotrypsin and approximately 0.5% are sugar-binding lectins. The two major classes of inhibitors are the Kunitz trypsin inhibitor, which inhibits trypsin, and the Bowman-Birk inhibitor (BBI), which inhibits both trypsin and chymotrypsin. Unless removed or inactivated, these inhibitors and lectins can impair the nutritional quality and safety of soy-based diets. On the other hand, several studies suggest that BBI can also function as an anticarcinogen, possibly through interaction with a cellular serine protease. Good-quality soybean proteins contribute to the nutritional value of many specialty foods including infant soy formulas and milk replacers for calves, and provide texture to many processed foods. However, they may also induce occasional allergic responses in humans. This paper outlines immunoassays developed to analyze for soy proteins in different soybean lines, in processed foods, and in nonsoy foods fortified with soy proteins. An assessment of the current status of immunoassays, especially of enzyme-linked immunosorbent assays for soybean inhibitors of digestive enzymes, soy globulins, and soy lectins, demonstrates the usefulness of these methods in plant and food sciences and in medicine.