Fluorodinitrobenzene Method Research Articles

Retention of Lysine in Foods Processed with Microwave and Conventional Heating

The effect of moist and dry heat processing on lysine content of selected foods was investigated. Two moist heat (open and closed vessel) and dry heat (microwave and hot air oven) methods were chosen for processing samples from cereal, oilseed and pulse groups with whey protein concentrate as reference sample. Unheated samples served as controls. The samples were analysed for protein content, available lysine by fluorodinitrobenzene method and for in vitro protein digestibility (IVPD). Results indicated that in moist heat-treated samples lysine retention was between 69 – 83%. Dry heat treatment resulted in a greater loss of lysine, which was proportional to the degree of heating. The retention of lysine in dry heat-treated samples ranged from 31 to 65 %. All heated samples had higher digestibility than their raw controls. In between methods of heating, the IVPD of dry heat-treated samples were slightly lower than moist heat-treated samples. It can be concluded that heat treatments increase the in vitro digestibility of proteins but lysine content was affected adversely.

Total and Reactive Lysine Contents in Selected Cereal-Based Food Products

The aim of the study was to determine and compare reactive and total lysine contents in a range of breakfast cereal products. Crude fiber, fat, ash, and crude protein contents of 20 breakfast cereal products ranged from 4 to 38, 14 to 144, 7 to 32, and 52 to 253 g/kg, respectively. The concentrations of glutamic acid (18.7-32.1 g/100 g protein) and proline (4.7-10.8 g/100 g protein) were high while those of the amino acids methionine (1.2-2.0 g/100 g protein) and histidine (1.2-3.3 g/100 g protein) were relatively low. There was a strong relationship between reactive lysine determined using the guanidination and fluorodinitrobenzene methods (R = 0.99). The total lysine content, determined after conventional acid hydrolysis, ranged from 0.8 to 3.7 g/100 g protein, while the reactive lysine content (guanidination) ranged from 0.4 to 2.8 g/100 g protein. Reactive lysine was 20-54% lower than total lysine in the cereal products. The large differences between total and reactive lysine suggest a considerable loss of lysine in the breakfast cereals tested.

Quality characteristics of broiler blood meal as affected by yeast, glucose oxidase, and antioxidant treatments

Effects of deglucosing and antioxidant treatments on quality characteristics of broiler blood meal were studied. Broiler blood was dried and ground with deglucosing and antioxidant addition. The non-deglucosed blood powder was darker, less red and yellow in color than those of the deglucosed. Yeast deglucosed blood meal had the lowest lysine availability as measured by the fluorodinitrobenzene method. Glucose oxidase desugarization improved ( p < 0.05) the lysine availability of blood powder. Treatment of broiler blood with glucose oxidase and antioxidant combination improved lysine availability by preventing an increase in TBA values.

Determination of available lysine in infant milk formulae by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method to determine available lysine is proposed. Available lysine was measured by an optimised fluorodinitrobenzene method on the basis of the reactivity of the free ε-amino group of the lysine. The classical acid hydrolysis has been improved and shortened from the usual time of 12 h to 2 h 30 min using an oil bath. Optimal resolution and quantitation of N ε-dinitrophenyllysine was obtained with a Nova-Pak C 18 column using an isocratiic elution with 35% methanol and 65% 0.01 M sodium acetate buffer (pH 4.5) and a flow-rate of 1 ml/min. Satisfactory results were obtained for the reliability of the method in terms of linearity from 0.1 to 5.0 mg/l of lysine-free base, precision (R.S.D. values between 4.3% and 7.8%), recovery (91.5%) and sensitivity (detection limit of 0.02 mg/l). The proposed method has also been checked for lack of interferences from other dinitrophenyl-amino acids.

Effects of extrusion on the dye-binding, fluorodinitrobenzene-reactive and total lysine content of soyabean meal and peas

Commercial soyabean meal (hexane extracted and toasted) and peas were processed with a single- and twin-screw extruder, respectively, to test the effects of initial moisture level and die product temperature on the reactive lysine contents. For soyabean meal, extrusion temperatures ranged from 90 to 140°C with moisture levels of 25–40%. Extrusion of peas was carried out at temperatures ranging from 105 to 140°C at moisture levels of 15–30%. Extruded and non-extruded samples were analysed for total lysine, and for reactive lysine, determined by the fluorodinitrobenzene (FDNB) method and the dye-binding lysine (DBL) method. Extrusion of soyabean meal at moderate temperatures (140°C) and high moisture levels (27–40%) resulted in a reduction in FDNB-reactive lysine ranging from 10 to 14%. Increased moisture levels in the soyabean meal resulted in slightly lower FDNB-reactive lysine values; the major decrease was caused by the processing temperature. Extrusion of peas gave a 16% reduction in FDNB-reactive lysine only when the temperature was 140°C and the moisture level was low (15%). Although ther was a marked reduction in reactive lysine units, caused by the heat treatment, this was not detected by total lysine analysis, while DBL analysis gave a significant correlation ( r = 0.97; P < 0.001) with the FDNB-reactive lysine procedure for the extruded soyabean meal but not for the extruded peas. For unextruded samples there was close agreement between the total and FDNB-reactive lysine content. Overall, and based on the present findings, it is concluded that the FDNB method is useful for measuring the reduction in nutritional value after extrusion of soyabean meal and peas whereas the rapid DBL procedure gives inconsistent results. The extrusion of soyabean meal and peas at moderate temperatures can lead to loss of reactive lysine units.

EFFECT OF BAKING AND TOASTING ON THE PROTEIN QUALITY AND LYSINE AVAILABILITY OF BREAD

The protein efficiency ratio (PER) and net protein ratio (NPR), the protein digestibility in vivo (in rats) and in vitro (multienzyme digestion), and the lysine availability in vivo (in rats) and in vitro (fluorodinitrobenzene method) of unbaked bread mix, bread, crust, crumb, and bread toasted at two levels of darkening were determined. Compared to an adjusted PER of 0.75 for the unbaked bread ingredients, the PER of the entire loaf was 0.46, but that of the crumb was 0.91, while the crust showed a negative PER, —0.23. The corresponding NPR's were 1.51, 1.22, 1.62, and 0.69. Light toasting reduced the PER of the bread to 0.40 (NPR 1.11) and dark toasting to 0.16 (NPR 0.95). The protein digestibility decreased as the bread ingredients were exposed to greater amounts of heat (baking, light and dark toasting), but the crumb protein of untoasted bread was slightly more digestible in vivo than the protein of the raw bread ingredients. The total lysine content also decreased with increasing exposure to heat, the crumb lysine content being an exception. The available lysine content, as measured by either the biological or the chemical methods, suffered significant decrease as the heat exposure increased, and correlated very highly with PER (available lysine in vivo vs PER, r = 0.988; available lysine in vitro vs PER, r = 0.979).

Effect of heat treatment of legumes on available lysine

AbstractThe newer dye‐binding capacity difference method of measuring available lysine in legumes was shown to agree extremely well with the established but laborious fluorodinitrobenzene method. This was true both with raw legumes and with heated samples in which there had been a considerable reduction in the amount of available lysine present. Nine samples of commercially obtained dried legumes were cooked for 1, 2 and 8 h at atmospheric pressure and for 30 min under 15 lb pressure. Losses of available lysine varied with the length and severity of heat treatment and with the type of legume. The variation with the type of legume appears to be due to the amount of reducing sugars in the legumes since both reducing sugars and available lysine fell during heat treatment, and the amount of lysine lost appears to be linked with the amount of reducing sugars initially present or added as glucose.

Chemical composition and properties of soybean beta-amylase.

The molecular weight of soybean beta-amylase [EC 3.2.1.2] was determined to be 57,000 daltons by the sedimentation equilibrium method, and the enzyme was found to consist of 494 amino acid residues. No difference was found in molecular weight or composition between two components of beta-amylase separated by ion-exchange chromatography. The N-terminus of the enzyme was not detectable by the fluorodinitrobenzene method or phenylisothiocyanate method, and the C-terminus was determined to be alanine by the carboxypeptidase [EC 3.4.12.2] method. Five half-cystine residues were found in the form of cysteine; all the sulfhydryl groups could be titrated by p-chloromercuribenzoate after denaturation of the enzyme with guanidine hydrochloride, but only some in the native enzyme. The rates of mercaptide formation of these groups were dependent on pH and were different from each other, all being much lower than the rate for the free sulfhydryl group in mercaptoethanol. Differential titration experiments at different pH's and in the presence of maltose showed that mercaptide formation by only one sulfhydryl group caused loss of activity, and the reaction was accompanied by changes in the environment around aromatic side chains in the enzyme, which were detected by difference spectra and fluorescence emission spectra. These facts suggest that modification of the sulfhydryl groups causes a conformational change of the enzyme. Some preliminary crystallographic data for crystals formed at pH 4.0 were obtained, and inactivation by heavy metal salts was examined in relation to the preparation of isomorphous heavy atom derivatives.

A Study on the Effect of Sucrose Upon the Solubility of the Teeth

The purpose of this study was to examine the effect of sucrose upon the solubility of the dental hard tissues.Enamel and dentine powder prepared from freshly extracted bovine teeth was suspended in the following 3 solutions, namely pH 7.5 tris-buffer solution (B), mol sucrose in solution B (S) and solution S autoclaved at 125°C for 1hr (CS). After 16 days incubation at 37°C, the amounts of Ca and P dissolved in each solution were determined. Solution S increased the solubility of Ca and P in dentine, but decreased it in enamel in comparison with solution B. Solution CS significantly increased the solubility of Ca and P in both enamel and dentine.The free N-terminal groups in enamel and dentine incubated in solution B and solution CS for 16 days were determined by the use of Sanger's fluorodinitrobenzene method. No difference was found between the two solutions.In tooth powder deprived of its organic components by extraction with ethylendiamine, in natural apatite powder and in lipid-extracted teeth powder, the solubility tests were carried out under similar experimental conditions.The results indicated that the amounts of dissolved Ca and P were strongly influenced by the extraction of the lipid component.In the caramelized sucrose solution, lactate, formate, acetate and pyruvate were found. Any one of these acids was able to decrease calcium ion concentration in slightly alkalized CaCl2 solution.

Studies on the Protein Portion of Thyroglobulin: AMINO ACID COMPOSITIONS AND TERMINAL AMINO ACIDS OF SEVERAL THYROGLOBULINS

Amino acid analyses have been performed on the thyroglobulins from calf, sheep, pig, and man. These proteins contain approximately one-fifth of their amino acid residues in the form of dicarboxylic amino acids, almost one-half of these being in the amide form. The basic amino acids account for about 10% of the total amino acids. Other prominent amino acids in the thyroglobulins are serine, leucine, alanine, and glycine, and there is a substantial amount of half-cystine present, representing approximately 250 moles per mole of protein. The amino acids, which number close to 5500 per molecule, account for 87 to 91% of the weight of the proteins, the remainder being made up of 8 to 10% of carbohydrate (approximately 300 monosac-charide residues per molecule) and iodine. The terminal amino acids of the four thyroglobulins have been investigated. Amino-terminal amino acid analysis by the fluorodinitrobenzene method has indicated the presence of 2 residues of dinitrophenyl-aspartic acid per molecule. A third terminal position appears to be occupied by leucine in the sheep and calf proteins, by glycine in the pig, and by serine in the human. Each protein, in addition, has partial residues of several dinitrophenyl-amino acids which account for another mole of terminal residues per mole of protein. These findings would be consistent with the presence of four peptide chains in the thyroglobulin molecule. Carboxyl-terminal amino acid analyses, performed by hydrazinolysis, resulted in the release of 2 to 3 moles of amino acids per mole of protein after 8 hours of treatment. These amino acids included serine, glutamic acid, glycine, alanine, and leucine. Longer exposure to hydrazine was not possible because of the considerable hydrolytic cleavage of amino acid hydrazides or of peptide bonds which occurred, as evaluated by hydrazine treatment of the methylated protein. Carboxypeptidase digestion led to the release of a limited number of amino acids, most prominent among them being leucine and serine.

Measurement of Available Lysine in Heated and Unheated Foodstuffs by Chemical and Biological Methods

The availability of lysine in meat, and in egg albumin before and after heat treatment in the presence of glucose, was determined by rat growth assay and by the fluorodinitrobenzene (FDNB) method. A 25% gluten diet was used as a basal diet to which graded levels of lysine or the protein under test were added. There was a high correlation between lysine consumed and weight gain of rats fed the standard diets for 2 weeks. Autoclaving of egg albumin to which 1 to 2% glucose had been added decreased the availability of lysine as measured by the FDNB method. However, the rat-growth assay gave a far lower value. This lowering was found not to be due to the formation of toxic compounds nor to loss of methionine. Similar results were obtained using autoclaved beef. Digestibility measurements of autoclaved egg albumin gave values similar to the value obtained by rat-growth assay. Fecal analysis showed that not only was lysine excretion increased but also excretion of other amino acids as well. The FDNB method does not appear suitable for the estimation of availability of lysine in heat-treated foods. It appears from fecal excretion studies that some lysine in heat-treated proteins measured as available by the FDNB method may be excreted as part of the undigestible residue in the feces.

The differential determination of lysine in heated milk. I. In vitro methods.

AbstractLysine content and availability were determined in a set of milk samples which had sustained increasing heat treatments. Lysine content was measured after acid hydrolysis of the sample (TLV or x‐value), lysine availability by an enzymic digestion procedure (ALV‐e or y‐value) and by two modifications of the fluorodinitrobenzene method: the direct method (ALV‐f I or z‐value) and Carpenter's corrected straight acid procedure (ALV‐f II or v‐value). The four procedures gave strongly correlated, but numerically quite different results. The enzymic procedure and Carpenter's chemical method produced very similar results for lysine availability. In all heated samples, lysine content was much higher than lysine availability.The direct fluorodinitrobenzene method yielded values intermediate between those for lysine content and ‘true’ lysine availability. A nomograph is presented which accounts for the different conditions of lysine in heated milk and allows the interconversion of the x, y, z and v‐values.

Some chemical studies on a strain of white clover mosaic virus

The WDC-17 strain of white clover mosaic virus was found to contain 9.4% RNA and the protein subunit to consist of about 133 amino acid residues, each subunit having a molecular weight of approximately 14.3 × 10 3. The C-terminal amino acid residue appears to be arginine and the next, lysine. No N-terminal amino acid could be detected by the fluorodinitrobenzene method. White clover mosaic virus RNA was found to have a high adenylic acid content and low amount of guanylic acid. The ratios for Ap, Gp, Up, and Cp were approximately 33:16:22:28, respectively.

Studies on the protein of rabbit reticulocyte ribosomes

The total protein of ribosomes from rabbit reticulocytes was isolated by the method of Curry & Hersh (1962) , using lithium chloride. Amino acid analysis showed that it contains approximately equal amounts of basic and acidic amino acids; however, it is basic because of the high proportion of acidic residues that are amidated. Analysis for N-terminal amino acids by the fluorodinitrobenzene method of Sanger (1945) showed that the N-terminal groups are methionine (56%), valine (15%), alanine (13%) and glycine (9%). The molecular weight of the protein estimated from the yield of N-terminal amino acids is 12,700 ± 1000.

The preparation, purification, and amino acid sequence of a polypeptide renin substrate.

A purified preparation of a polypeptide renin substrate prepared by tryptic degradation of the protein renin substrate has been analyzed by the fluorodinitrobenzene method and after degradation with renin, carboxypeptidase, and phenylisothiocyanate, has been found to possess the amino acid sequence; asp-arg-val-tyr-ileu-his-pro-phe-his-leu-leu-val-tyr-ser. The first 10 of these amino acids constitutes hypertensin I which is released by cleavage of the leucyl-leucine bond by renin. The remaining 4 amino acids, leu, val, tyr, ser, apparently link hypertensin I to the protein renin substrate.

The amino acid sequence of hypertensin. II.

The amino acid sequence of horse hypertensin II has been determined by the use of chymotrypsin, the fluorodinitrobenzene method, and stepwise phenylisothiocyanate degradation. The results indicate that the amino acids of hypertensin II are arranged in the following order: asp-arg-val-tyr-iso-hist-pro-phe.

Reactions of glucose with lysine

1. 1. The fluorodinitrobenzene method, in conjunction with paper chromatography and ionophoresis, was used to identify the sites of reaction of glucose with lysine and to measure the rates of reaction. 2. 2. During the storage of lysine with glucose at pH 8.5, 37 °C. and 70% relative humidity, the α- and ϵ-amino groups of lysine reacted with glucose at equal rates. 3. 3. A large excess of glucose (8 moles/amino group) caused no change in reaction rates of the amino groups of lysine.