Hydrophilic chromatographic determination of carnosine, anserine, balenine, creatine, and creatinine

A new HPLC procedure based on hydrophilic interaction chromatography (HILIC) has been developed for the simultaneous determination of carnosine, anserine, balenine, creatine, and creatinine in meat. This is the first time that HILIC has been directly applied to the study of meat components, having the advantage of not requiring complex cleanup and/or sample derivatization procedures. The chromatographic separation has been developed using a silica column (4.6 x 150 mm, 3 microm), and the proposed methodology is simple, reliable, and fast (<13 min per sample). The method has been validated in terms of linearity, repeatability, reproducibility, and recovery and represents an interesting alternative to methods currently in use for determining the mentioned compounds and other polar substances. The detection limits are 5.64, 8.23, 3.66, 3.99, and 0.06 microg/mL for carnosine, anserine, balenine, creatine, and creatinine, respectively.     

Interaction of soluble peptides and proteins from skeletal muscle with volatile compounds in model systems as affected by curing agents

The effect of curing agents (salt, glucose, nitrate, nitrite, and ascorbic acid) on the binding of skeletal peptides (carnosine and anserine) and a sarcoplasmic protein (myoglobin) with key flavor compounds (hexanal, octanal, 2-pentanone, 2-methylbutanal, and 3-methylbutanal) has been studied by solid-phase microextraction (SPME). Curing agents had an effect on the interaction process between carnosine and volatile compounds, which was higher than the interactions observed with anserine and myoglobin. Sodium chloride decreased the interaction of volatiles with carnosine except for octanal, which was increased, and 2-pentanone, which was unaltered. Ascorbic acid exerted the highest effect by decreasing the interaction of carnosine with all of the volatile compounds except for octanal and 2-pentanone. The interaction with anserine was affected by sodium chloride, nitrate, and nitrite, producing a decrease in the interaction with hexanal, octanal, and methional. Finally, sodium chloride, glucose, and nitrite increased the interaction of myoglobin with hexanal, octanal, and methional. The effect of simulated stages of the curing process on the binding was also studied. A combined effect of the curing agents resulted in a change in the relative proportions of volatile compounds that can lead to different flavor perceptions of dry-cured meat products.     

Near-infrared reflectance spectroscopy enables the fast and accurate prediction of the essential amino acid contents in soy, rapeseed meal, sunflower meal, peas, fishmeal, meat meal products, and poultry meal

Near-infrared reflectance spectroscopy (NIRS) calibrations were developed to enable the accurate and fast prediction of the total contents of methionine, cystine, lysine, threonine, tryptophan, and other essential amino acids, protein, and moisture in the most important protein-rich feed ingredients. More than 1000 samples of global origin collected over four years were analyzed on amino acids following the official methods of the United States and the European Union. Detailed data and graphics are given to characterize the obtained calibration equations. NIRS was validated with independent samples for soy and meat meal products and compared to the amino acid predictions using linear crude protein regressions. With a few exceptions, validation showed that 85-98% of the amino acid variance in the samples could be explained using NIRS. NIRS predictions compared to reference results agree excellently, with relative mean deviations below 5%. Especially for meat and poultry meals, NIRS can predict amino acids much better than crude protein regressions. By enabling the amino acid analysis of many samples to be completed in a short time, NIRS can improve the accuracy of feed formulation and thus the quality and production costs of mixed feeds.     

Nonfeed application of rendered animal proteins for microbial production of eicosapentaenoic acid by the fungus Pythium irregulare

Rendered animal proteins are well suited for animal nutrition applications, but the market is maturing, and there is a need to develop new uses for these products. The objective of this study is to explore the possibility of using animal proteins as a nutrient source for microbial production of omega-3 polyunsaturated fatty acids by the microalga Schizochytrium limacinum and the fungus Pythium irregulare. To be absorbed by the microorganisms, the proteins needed to be hydrolyzed into small peptides and free amino acids. The utility of the protein hydrolysates for microorganisms depended on the hydrolysis method used and the type of microorganism. The enzymatic hydrolysates supported better cell growth performance than the alkali hydrolysates did. P. irregulare displayed better overall growth performance on the experimental hydrolysates compared to S. limacinum. When P. irregulare was grown in medium containing 10 g/L enzymatic hydrolysate derived from meat and bone meal or feather meal, the performance of cell growth, lipid synthesis, and omega-3 fatty acid production was comparable to the that of culture using commercial yeast extract. The fungal biomass derived from the animal proteins had 26-29% lipid, 32-34% protein, 34-39% carbohydrate, and <2% ash content. The results show that it is possible to develop a nonfeed application for rendered animal protein by hydrolysis of the protein and feeding to industrial microorganisms which can produce omega-3 fatty acids for making omega-3-fortified foods or feeds.     

Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.     

Composition and protein efficiency ratio of meat samples partially defatted with petroleum ether, acetone, or ethyl ether

Freeze-dried beef samples were partially defatted with either petroleum ether, acetone, or ethyl ether before determination of protein efficiency ratio (PER) to study the extraction effects on the composition and protein nutritional quality of the extracted beef. Defatting a protein source, such as meat or a meat product, may often be necessary to produce a test diet that contains 10% protein and 8% fat. Amino acid, carnosine, anserine, creatine, creatinine, inosine, and proximate compositions were determined on the extracted samples. Resulting data were compared to the composition and PER data of the beef that had no solvent treatment. Although the chemical analysis data from the study showed some variation between the proteins and other nitrogenous components of the unextracted and the extracted beef, these variations were too small to affect the protein nutritional quality of the beef as measured by PER.     

Quantitation of carnosine in humans plasma after dietary consumption of beef

Carnosine (beta-alanyl-L-histidine) is a dipeptide found in the muscle foods that has been postulated to be a bioactive food component. The objective of this research was to determine the concentration of carnosine in human plasma after ingestion of beef. Nine males and nine females were recruited for the study. Food devoid of meat products was given to the subjects so that they did not consume carnosine for 48 h prior to the test. Subjects fasted for 12 h and then had blood withdrawn prior to a meal containing 200 g of ground beef. Additional blood samples were collected over the following 24 h and carnosine concentrations were determined by HPLC. The cooked ground beef used in the study contained 52% water, 24% protein, 22% fat, and 124 mg of carnosine/100 g of beef. No plasma carnosine was detected in subjects before the consumption of the beef. Carnosine was detected in plasma 15 min after beef consumption. Plasma carnosine concentrations continued to increase with a maximum (32.7 mg of carnosine/L of plasma) being recorded 2.5 h after consumption. Carnosine concentrations then decreased until no carnosine could be detected at 5.5 h postconsumption. These results indicate that dietary carnosine is absorbed into human plasma after the consumption of beef. Since carnosine has several potential health benefits, evidence of its bioavailability suggests that it could be a bioactive food component.     

Determination of crude protein in animal feeds, using block digestion followed by steam distillation: collaborative study.

A method consisting of digesting animal feeds in a block digestor and determining ammonia by steam distillation followed by titration has been evaluated and compared with the official final action Kjeldahl method, 7.016. Fifteen laboratories analyzed 5 feed samples and lysine monohydrochloride. Statistical analysis showed that results from the 2 methods were comparable. The distillation technique has been adopted as official first action as an alternative technique for ammonia determination from the digest of the official final action block digestor method, 7.B11.     

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.     

Collaborative study of a screening method for the detection of aflatoxins in mixed feeds, other agricultural products, and foods.

A screening method for aflatoxins was collaboratively tested on 11 different agricultural and food products: white and yellow corn, peanuts, peanut butter, pistachio nuts, peanut meal, cottonseed meal, chicken, pig, and turkey starter rations, and dairy cattle feed. The method involves a rapid extraction and cleanup procedure followed by the detection of total aflatoxins (B1 + B2 + G1 + G2) as a fluorescent band on the Florisil layer of a Velasco-type minicolumn. The results of 32 collaborators from 10 different countries are presented. Samples containing 0, 5, 10, 15, 20, and 25 mug aflatoxins/kg were analyzed. Eighty-four per cent of the negative samples and 89% of the samples containing 10-25 mug total aflatoxins/kg were correctly identified. This method has been adopted as official first action for the detection of aflatoxins in corn, peanuts, peanut butter, peanut meal, cottonseed meal, mixed feeds, and pistachio nuts.     

Effects of carnosine on volatile generation from Maillard reaction of ribose and cysteine

Carnosine occurs naturally in meat and meat products in significant quantity, and it possesses strong antioxidant activity that inhibits lipid oxidation and enhances shelf life. In this study, the effects of carnosine on thermal flavor generation were investigated using the model system of cysteine and ribose, which was heated to the roasting temperature of 180 degrees C for 2 h at pH 5 and pH 8.5. The results indicated that carnosine affected volatile formation in a complex manner. Volatiles identified from the liquid phase of the reaction systems of ribose and cysteine showed that the sulfur-containing compounds such as thiophenes, thiazoles, and polysulfides were the most abundant compounds. The addition of carnosine into the reaction mixtures in general caused a reduction in contents of thiophenes and some important meaty flavor compounds such as 2-methyl-3-furanthiol, 2-furfurylthiol, and their associated dimers. On the other hand, it facilitated the generation of several important nitrogen-containing volatiles such as pyrazine, methylpyrazine, 2,6-dimethylpyrazine, and other alkyl pyrazines and thiazoles, which are known to elicit roasty and nutty flavor notes. The results suggested that carnosine acts as a nitrogenous source to facilitate the formation of nitrogen-containing compounds, possibly by degradation to form ammonia.     

Antioxidant peptides with Angiotensin converting enzyme inhibitory activities and applications for Angiotensin converting enzyme purification

Five commercial peptides, namely, reduced glutathione (GSH), oxidized glutathione (GSSG), carnosine, homocarnosine, and anserine, were used to test angiotensin converting enzyme inhibitory (ACEI) activities using N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly (FAPGG) as a substrate. All of these peptides showed dose-dependent ACEI activities. Using 50% inhibition (IC(50)) of captopril as 0.00781 microM for the reference, the IC(50) values of GSH, carnosine, homocarnosine, and anserine were determined to be 32.4 microM, 5.216 mM, 6.147 mM, and 6.967 mM, respectively. GSH or carnosine showed mixed noncompetitive inhibition against ACE. When 0.0164 mM GSH or 0.4098 mM carnosine was added, the apparent inhibition constant (K(i)) was 49.7 microM or 3.899 mM, respectively. Commercial glutathione-Sepharose 4 fast flow, GSH-coupled CNBr-activated and GSH-coupled EAH-activated Sepharose gels were used for ACE purification. Commercial ACE could be adsorbed only by EAH-coupled GSH gels and eluted off the gels by increasing salt concentrations. These EAH-coupled GSH gels might be developed as affinity aids for ACE purification.     

Collaborative study of an automated method for the determination of crude protein in animal feeds.

An automated macro Kjeldahl instrument determines per cent protein at the rate of 20 samples/hr. The methodology involved is similar to the present official final action Kjeldahl method, sec. 7.016. The 2 methods were compared in a collaborative study. Sixteen animal feeds, 4 meats, tryptophan, ammonium dihydrogen phosphate NBS standard, and ammonium sulfate primary standard were analyzed by the participating laboratories. The data were agreement between the 2 methods. The automated method has been adopted as official first action for the determination of crude protein in feeds, plants, and cereal foods.     

Comparison of HgO and CuSO4 as digestion catalysts in manual Kjeldahl determination of crude protein in animal feeds: collaborative study

The official AOAC manual Kjeldahl method for determining crude protein in animal feeds, 7.015, uses HgO as a catalyst in the digestion step. Because of environmental considerations, there is considerable interest in alternative catalysts. A collaborative study compares the official HgO-catalyzed method and an alternative using CuSO4. Fifty-four samples consisting of blind duplicates of closely matched pairs, representing a range of animal feed materials and 2 standard materials, were analyzed once by each method. Results were returned by 22 laboratories. Means and standard deviations between methods were comparable. The CuSO4-catalyzed method has been adopted official first action.     

Determination of ergosterol in cereals, mixed feed components, and mixed feeds by liquid chromatography

A sensitive, rapid, reproducible, and reliable liquid chromatographic (LC) method is described for determination of ergosterol in feedstuffs. The sample is saponified directly and the saponified mixture is extracted with n-hexane. Ergosterol is determined without further purification or cleanup steps by using a liquid chromatograph with a 250 X 4.6 mm column packed with LiChrosorb Si 60, 5 microns, and a high pressure column prefilter. The ultraviolet detector is set at 282 nm. The limit of detection was 0.1 ppm; recovery ranged between 96.7 and 102.2%. Diode array technology is used for identification and peak purity control. Under strong UV irradiation (254 nm) and oxygen or nitrogen atmosphere ergosterol was converted almost quantitatively to ergocalciferol. Under the described conditions of the method, ergosterol proved to be stable. Ergosterol was determined in cereals, mixed feeds (e.g., for swine and poultry), and their components of plant and animal origin. It was not found in carcass meal, meat-and-bone meal, citrus pulps, or molasses; only traces were detected in fish meal.     

Collaborative study of a semiautomated method for the determination of crude protein in animal feeds.

A semiautomated method consisting of digestion of animal feeds in a block digestor and determination of ammonia by ammonia-salicylate reaction has been studied collaboratively, along with the official final action Kjeldahl method, sec. 7.016. Each collaborator analyzed 16 feed samples, tryptophan, ammonium dihydrogen phosphate NBS standard, and ammonium sulfate primary standard. Statistical analysis showed that the 2 methods agreed. The semiautomated method has been adopted as official first action.     

Aflatoxins in domestic and imported foods and feeds

Aflatoxins, metabolic products of the molds Aspergillus flavus and A. parasiticus, may occur in foods and feeds. These toxins cannot be entirely avoided or eliminated from foods or feeds by current agronomic and manufacturing processes and are considered unavoidable contaminants. To limit aflatoxin exposure, the U.S. Food and Drug Administration (FDA) has set action levels for these toxins in foods and feeds involved in interstate commerce. FDA continually monitors food and feed industries through compliance programs. This report summarizes data generated from compliance programs on aflatoxins for the fiscal year 1986. Commodities sampled included peanuts and peanut products, corn and corn products, tree nuts, cottonseed, milk, spices, manufactured products, and miscellaneous foods and feeds. Correlations were highest between aflatoxin contamination and geographical areas for corn/corn products and cottonseed/cottonseed meal. Higher incidences of aflatoxin contamination in corn and corn products designated for human consumption were observed in samples collected in the southeastern states (32 and 28%, respectively). A higher incidence of contamination was observed in corn designated for animal feed from Arkansas-Texas (74%) than from the southeastern states (47%). Only 3% of feed corn from corn belt states contained detectable aflatoxins. All aflatoxin-contaminated cottonseed was collected in the Arizona-California area; 80% of cottonseed meal analyzed from this area also contained detectable levels of aflatoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

纳米级兔骨粉球磨法制备工艺优化

为了探索兔骨精深加工的新方法,提高兔骨的利用率和附加值,该文以70日龄伊拉兔的脊骨、肋骨和腿骨为试验原料(其中脊骨和肋骨的总质量与腿骨的质量比为4∶1),经高压蒸煮、蛋白酶酶解、胶体磨研磨、真空冷冻干燥、标准筛过筛、球磨机球磨等处理,采用动态光散射的方法对兔骨粉的平均粒径和分布系数(PDI,particle dispersion index)进行测量,在单因素试验的基础上,用响应面法优化球磨法制备纳米级兔骨粉的工艺参数,并建立二次回归方程。最终得到球磨法的最佳制备工艺为:球磨转速558 r/min,球磨时间4.7 h,球料比为3.66∶1,在此条件下进行3次验证试验,得出兔骨粉的平均粒径为(502.5±11.7)nm,PDI为0.497±0.021,与预测值的相对误差均低于5%,表明回归模型有效。研究结果对兔骨和其他畜禽骨的精深加工具有一定的参考价值。     

胡萝卜复合火腿和香肠的质构特性及显微结构(简报)

为探索蔬菜复合肉制品品质特性的评价方法,分别用物性分析仪和扫描电镜研究了胡萝卜复合火腿和香肠的质构特性和显微结构。质构测定结果表明:与对照组相比,HLB-1复合火腿(鲜胡萝卜25%)和HLB-2复合火腿(胡萝卜粉2.5%)的硬度分别降低了70.94%和57.23%,咀嚼度分别降低了64.81%和34.86%;与对照组比较,胡萝卜复合香肠(鲜胡萝卜25%)的硬度和咀嚼度分别降低了30.84%和51.30%。感官评定结果表明:与对照组比较,胡萝卜复合火腿和香肠的口感更柔和细腻、嫩度更好。通过扫描电镜对产品结构的观察表明:在添加了鲜胡萝卜的火腿和香肠中均发现了圆形光滑的小颗粒状物质,复合肉制品内部的细微结构均匀、结构比较膨松,这在一定程度上解释了胡萝卜复合火腿和香肠的感官和质构特征。     

Determination of phylloquinone and menaquinones in animal products with fluorescence detection after postcolumn reduction with metallic zinc

A high-performance liquid chromatographic (HPLC) method for the determination of phylloquinone and menaquinones in foods of animal origin is described. The K vitamers were quantified with a fluorescence detector after postcolumn reduction with metallic zinc using K1(25) as an internal standard. Extraction was done either with 2-propanol-hexane (meat and fish products) or with acid hydrolysis method (dairy products). Prior to quantification, sample extracts were purified by semipreparative HPLC; in addition, the fats of cheese and rainbow trout samples were removed with lipase hydrolysis. By this method the phylloquinone and menaquinones (MK-4 to MK-10) present in a few representative samples of different animal food groups were determined. HPLC-MS was used to confirm the identification of K vitamers. Long-chain menaquinones were found from bovine and pig livers as well as from various cheeses. The total vitamin K contents calculated as the sum of quantified K vitamers were in general low (mean content 10-100 ng/g); the highest amount was analyzed in chicken meat (600 ng/g).