Tryptophan determination in proteins and feedstuffs by ion exchange chromatography

A chromatographic method was developed for the determination of tryptophan content in food and feed proteins. The method involves separation and quantitation of tryptophan (released from protein by alkaline hydrolysis with NaOH) by isocratic ion-exchange chromatography with O-phthalaldehyde derivatization followed by fluorescence detection. In this procedure, chromatographic separation of the tryptophan and α-methyl tryptophan, the internal standard, was complete in 15 min, without any interference from other compounds. The precision of the method was 1–4% relative standard deviation. Accuracy was validated by agreement with the value for chicken egg white lysozyme, a sequenced protein, and by quantitative recoveries after spiking with lysozyme. The method allows determination in a range of feed proteins, containing varied concentrations of tryptophan, and is applicable to systems used for routine amino acid analysis by ion-exchange chromatography.     

A comparison of silver ion HPLC plus GC with Fourier-transform IR spectroscopy for the determination of trans double bonds in unsaturated fatty acids

A rapid procedure has been developed for the isolation of methyl esters of trans-monoenoic fatty acids by silver ion high-performance liquid chromatography. The combined saturated plus trans-monoene fraction was collected for analysis by gas chromatography and comparison with the composition of the unfractionated sample. The trans-monoene contents of partially hydrogenated vegetable oils, determined in this way, are compared with data obtained by Fourier-transform infrared spectroscopy. Alternative methods of base-line correction are discussed. The infrared technique gave values that were a little higher than the chromatographic ones, presumably because the former also detected trans unsaturation in di- and polyunsaturated fatty acids.     

Improved separation of the major water-soluble proteins of soya meal by a single-step chromatographic procedure.

A method is described in which the major water-soluble proteins of soya meal—the storage globulins, glycinin and γ-conglycinin, and the anti-nutritional factors, Kunitz trypsin inhibitor and soya bean haemagglutinin—are resolved from one another in a single-step chromatographic procedure. The method can be used as a basis for the preparation of these components, and also as an analytical tool for examining soya protein-containing materials.     

Simultaneous isolation of proteins and fats from oil seeds. I. Principle of procedure]

The principle of the procedure for the simultaneous isolation of proteins and fats from oil seeds is illustrated by the isolation of these constituents from sunflower seeds. This procedure is based on the displacement of oil and the extraction of protein by an aqueous electrolyte solution after comminution of the pretreated seeds, removal of the insoluble cell components and separation of the extracts into a fat-containing and a protein-containing phase. The globulins are precipitated isoelectrically; the albumins, by using complexing agents or thermal coagulation. The liberation of the oil from the concentrated fat emulsion is achieved immediately in a mechanical way or after addition of destabilizers. The results are discussed with regard to the yields obtained and the composition of the final products.     

Multimethod for the determination of residues of chemotherapeutic drugs, antiparasitic agents and growth promotors in foodstuffs of animal origin. 1. General procedure and determination of sulfonamides

Applying this Multi-Method, it is possible to isolate more than 60 chemotherapeutics, antiparasitics and growth promoters in one procedure from eggs, milk and meat. Residues are detected by different chromatographic systems (HPLC with UV-Detection, Capillary-GC with an ECD). Depending on substance and foodstuff, the following detection limits can be achieved: (Table: see text). The observance of the legal tolerance level of 0.001 mg/kg Chloramphenicol (valid only for milk and eggs in the Fed. Rep. Germany) can be supervised. Residues of most Sulfonamides can be determined to adequately, check the proposed tolerance level of 0.1 mg/kg. The first part presents the procedure (applicable for all substances) and contains the summary of the chromatographic parameters for the detection of all substances and the detailed parameters for Sulfonamides. The conformation of residues of Sulfonamides is discussed carefully.     

Rapid characterisation of (glyphosate tolerant) transgenic and non-transgenic soybeans using chromatographic protein profiles

The characterisation of transgenic and non-transgenic soybeans was performed in this work based on the examination of their protein profiles obtained by rapid chromatographic techniques. Two reversed-phase chromatographic methods using monolithic and perfusion stationary phases were applied to the separation of soybean proteins from different transgenic and non-transgenic soybeans. The development of the monolithic LC methodology was carried out through the study of the influence of different parameters such as gradient, ion-pairing reagent, and temperature on the separation of soybean proteins. Results from monolithic LC analysis were compared with those obtained by perfusion LC using a method previously developed by our research team. Perfusion and monolithic LC enabled the separation of soybean proteins in less than 3 and 8 min, respectively. In both cases, there were certain features in the chromatograms that seemed to be characteristic of transgenic samples. A deeper analysis of chromatographic profiles was then performed by the application of multivariate classification techniques. Results from these multivariate techniques showed that the two methods presented similar classification capabilities being both suitable for the characterisation of transgenic and non-transgenic soybeans. A more robust mathematical model was built with the data obtained by the perfusion method using 10 additional samples for training (a total of 26 samples), obtaining a 96.2% of correct classification. This model was validated by a cross-validation procedure (80.8% of correct classification) and by the correct classification of 15 out of 16 blind samples.     

Comparison of High Performance Liquid Chromatography and Enzymatic Method for the Measurement of Lactose in Milk

A high performance liquid chromatographic (HPLC) procedure was compared with an enzymatic method for the measurement of lactose in milk. A new rapid HPLC sample preparation technique was used for precipitating milk proteins with 2-propanol. Results indicated that lactose detected in milk (expressed as percentage) was consistently lower with the enzymatic method than the HPLC procedure. Mean difference between the two methods was 0.15% with a range of 0.04 to 0.51. Coefficient of variability for the HPLC and enzymatic methods were 1.02 and 2.75, respectively. Recovery of added lactose in milk by HPLC was 100.3% for whole milk, 97.1% for low fat milk, and 99.5% for skim milk.     

Theoretical triglyceride content of vegetable oils by a radiochemical technique—effect of sediment

The refining loss for a variety of olive, maize and cottonseed oils as determined in the laboratory by a chromatographic method was checked by a radiochemical procedure. By labeling oil samples with ¹⁴C-tripalmitate and applying the principle of isotope dilution analysis, the absolute content of neutral oil and the theoretical triglyceride content were determined. In order to find out the effect of the sediment in an oil on the deviation of the chromatographic refining loss from the theoretical triglyceride content, various oil samples with high amounts of sediment were compared with normal samples with a low or moderate percentage of sediment. Relations are proposed for the calculation of the theoretical triglyceride content from the one determined by the chromatographic method.     

ISOLATION AND CHARACTERIZATION OF ICE STRUCTURING PROTEINS FROM COLD-ACCLIMATED WINTER WHEAT GRASS EXTRACT FOR RECRYSTALLIZATION INHIBITION IN FROZEN FOODS

Antifreeze proteins have shown a great potential in improving the quality of frozen foods, and the isolation of proteins from natural sources that are readily available is deemed to be important. The raw leaf apoplastic extract from cold‐acclimated winter wheat grass ( Triticum aestivum ) containing recrystallization inhibition (RI) proteins was screened for proteolytic and lipolytic activity, and a heat‐stable RI protein was isolated. The enzymes detected could be easily removed by heat treatment without any loss in the RI activity. The RI protein was isolated after heat treatment of the raw extract, alcohol precipitation and separation on a size exclusion chromatographic column. Circular dichroism indicated that the RI protein was mainly β‐sheet and random coil, and these structures were preserved up to 75C. After trypsin digestion, mass fingerprinting and sequencing of the digests revealed that the protein belongs to the thaumatin‐like protein group.     

Membrane separation processes for enrichment of bovine and caprine milk oligosaccharides from dairy byproducts

Breast milk is an ideal source of human milk oligosaccharides (HMOs) for isolation and purification. However, breast milk is not for sale and at most is distributed to neonatal intensive care units as donor milk. To overcome this limitation, isolating HMOs analogs including bovine milk oligosaccharides (BMOs) and caprine milk oligosaccharides (CMOs) from other sources is timely and significant. Advances in the development of equipment and analytical methods have revealed that dairy processing byproducts are good sources of BMOs and CMOs. Enrichment of these oligosaccharides from dairy byproducts, such as whey, permeate, and mother liquor, is of increasing academic and economic value. The commonly employed approach for oligosaccharides purification is chromatographic technique, but it is only used at lab scale. In the dairy industry, chromatographic methods (large-scale ion exchange, 10,000 L size) are currently routinely used for the isolation/purification of milk proteins (e.g., lactoferrin). In contrast, membrane technology has been proven to be a suitable approach for the isolation and purification of BMOs and CMOs from dairy byproducts. Therefore, this review simply introduces BMOs and CMOs in dairy processing byproducts. This review also summarizes membrane separation processes for isolating and purifying BMOs and CMOs from different dairy byproducts. Finally, the technological challenges and solutions of each processing strategy are discussed in detail.     

Effect of Lipid-Protein Interactions on Hydration Characteristics of Defatted Offal Protein Isolates

Water-protein interactions of rumen and lung protein isolates defatted by different solvents were studied by means of water sorption isotherms. Two isolation procedures were employed to obtain the isolates: (1) alkaline solubilization and isoelectric precipitation of proteins; and (2) SDS solubilization and FeCl₃ precipitation of proteins. Water monolayer values of the protein fraction showed a marked dependence on the dielectric constant of the solvent used for lipid extraction. In lung isolates they increased up to a peak when solvents of intermediate polarity were employed whereas in rumen isolates they were initially constant, decreasing with increase of polarity of solvent. Affinity of water to protein support, showed a more complex pattern and was dramatically affected by the isolation procedure used.     

Simultaneous process to isolate actomyosin and actin from post-rigor porcine skeletal muscle

A simultaneous actomyosin and actin isolation procedure from post-rigor porcine muscle was developed, based on differential solubility, gel filtration chromatography and extraction steps. The isolation process was evaluated by SDS-PAGE analysis and silver staining. Actomyosin and actin were isolated in a simultaneous process yielding 0.14 mg and 2.5 mg/g of meat, respectively, using a shorter purification process than others reported in the literature but with similar recoveries. Furthermore, actin preserves its polymerisation ability and both proteins, actomyosin and actin, could be used in further studies.     

Fatty hormones in cotton fibers 1.—Their isolation and characterisation of the constituent fatty acids

A method for the large-scale isolation of plant hormones from cotton fibers is described. Chemical euamination revealed that these are fatty substances. The constituent fatty acids in the hormones were determined by chromatographic and spectroscopic analyses which showed various fatty acids with 14–22 carbon atoms.     

Characterization of Actin and Myosin Extracts Obtained Using Two Improved Laboratory Methods

Actin and myosin are the most important contractile proteins in the muscle. Numerous authors developed different methods for isolation and purification of myofibrillar proteins. The aim of this study was to obtain suitable actin and myosin extracts from post-rigor porcine muscle to be used in further studies, such as testing the proteolytic activity of microbial cultures. Actin and myosin were quantified in the extracts using spectrophotometric methods, yielding 0.17 and 0.22 mg/mL, respectively. The isolation methods proposed in this study provided low contaminated extracts, showing purity percentages of 74.36 % in the case of actin and 65.43 % for myosin, as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Subsequently, these extracts were sterilized through a 0.22-μm polyvinylidene difluoride filter with no significant retention observed. In conclusion, the procedures described in this work for actin and myosin isolation can be recommended for microbiological studies requiring sterilized pure muscle proteins extracts.     

Observations on the detection of dimethyl- and diethyl-nitrosamines in foods with particular reference to alcoholic beverages

Simple polarographic and gas chromatographic methods commonly used for the detection of nitrosamines in model systems have been found to be inadequate when applied to a potable spirit. Although these methods indicated the possible presence of nitrosamines in the spirit, gas chromatography combined with mass spectrometry showed that compounds other than nitrosamines were responsible for the putative chromatographic peaks. After isolation by preparative gas chromatography, the compound responsible for the polarographic activity, interpreted as nitrosamine in the unconcentrated spirit, was shown to be furfural.     

PURIFICATION OF REACTIVE SULFHYDRYL ENZYMES BY BIOSELECTIVE ADSORPTION ON MONOMERIC AVIDIN: PURIFICATION OF SULFHYDRYL OXIDASE1

ABSTRACT A biotinylation-bioselective adsorption procedure was developed for single-step purification of bovine sulfhydryl oxidase from solubilized skim milk membrane vesicles. Sulfhydryl oxidase was specifically biotinylated by reaction of its chemically reactive sulfhydryl group with a disulfide-containing biotinylation reagent giving a mixed disulfide between the enzyme and the biotinyl moiety. The biotinylated enzyme was then selectively adsorbed on a monomeric avidin matrix. After washing all nonspecifically bound proteins from the matrix, the enzyme was released by reduction of the disulfide with dithiothreitol. Both the biotinylation and the isolation are performed at pH 7.0 under extremely mild conditions. The resulting enzyme preparation appeared to be homogeneous by gel electrophoresis and the specific activity increased over 3000 fold in comparison to whey. The monomeric avidin matrix can be regenerated and used indefinitely. This purification procedure should be generally applicable to proteins with a chemically reactive sulfhydryl group.     

Determination of methionine by gas–liquid chromatography: Methods for the elimination of an interfering component present in some fish meals

Intact methionine residues in food proteins, including a limited number offish meals, have been determined using gas chromatography to measure methyl thiocyanate released after CNBr reaction. However, methionine values for certain fish products determined by that method have been found to be low and variable compared with those determined by ion-exchange chromatography after performic acid oxidation. This was traced to the presence of a component of many fish meals that, when reacted with CNBr, gave a product which co-eluted with the internal standard, ethyl thiocyanate. A method was developed to eliminate this interference and allow methionine determinations to be carried out using the same gas chromatographic procedure.     

Behavior of fat constitutents during refining. 8. Determination of polymeric fatty acids in rape seed oil and their behavior during refining]

1. Studies on rape-seed oil have shown that the determination of oxypolymers by means of the method according to Rost (determination of fatty acids insoluble in petroleum ether) yields unsatisfactory results. By way of contrast, the gravimetric determination (after isolation of the polymers via the non-urea adduct fatty acid methyl esters and thin-layer chromatographic separation), as proposed by the authors, permits to obtain results of good reproducibility and to determine thermopolymers and oxypolymers separately. 2. During the refining of rape-seed oil, it was observed that its polymer content decreased considerably on bleaching, and increased on deodorizing.     

Direct Gas Chromatographic Analysis of Fusel Alcohols, Fatty Acids and Esters of Distilled Alcoholic Beverages

A gas chromatographic method is proposed for the quantitative determination of the main volatile components of distilled alcoholic beverages such as whisky, brandy and rum. This gas chromatographic method enables direct analysis of 23 volatile components, 5 alcohols, 4 acids, 11 esters and 3 aldehydes by simply mixing a beverage sample with internal standards. A fused silica crosslinked 5% phenymethyl silicone capillary column (50 m × 0.31 mm i.d.) is used. The accuracy of the analysis is satisfactory, with the within-day and day-to-day coefficients of variation for most compounds being less than 10%, and the entire procedure can be completed in 40 min.     

半制备型高效液相色谱法分离刺葡萄花色苷单体

经大孔吸附树脂HP-20纯化刺葡萄花色苷的粗提物后,以半制备型高效液相色谱法分离得到高纯度的刺葡萄花色苷单体。以XCharge C18柱（20 mm×250 mm,10 μm）制备柱,考察梯度洗脱条件、流动相流速、进样量对刺葡萄花色苷分离的影响,确定了最佳制备条件为甲醇-3%甲酸溶液流动相梯度洗脱、流速15 mL/min、进样量1.2 mL,实现了2 种主要花色苷单体的分离及制备。经超高效液相色谱-四极杆飞行时间质谱鉴定,2 种花色苷单体分别是锦葵素-3,5-O-双葡萄糖苷和锦葵素-3,5-O-双葡萄糖苷-香豆酰,产品纯度分别达到了99.54%和98.28%。方法具有简单易行、经济快速、易于放大等特点,适用于刺葡萄花色苷标准品的大规模制备。