The effect of conditioning on the myofibrillar proteins of pork muscle

Storage of pork muscle caused changes in the troponin complex of myofibrillar proteins. The changes were temperature dependent and progressive as conditioning proceeded. An alteration in actin also occurred but this became apparent only when myofibrils had been extracted with 5 mM Tris pH 8.2. About 60% of the proteins in an extract of myofibrils in 5 mM Tris pH 8.2 were bound to the precipitate formed with added F-actin. After conditioning of the pork muscle, the amount of proteins in a Tris extract which were bound to added F-actin was considerably reduced. Some of these changes were also observed in myofibrils which had been treated with a Ca²⁺ activated proteinase.     

Isolation of Actomyosin and Myosin from Post-Rigor Turkey Breast and Thigh

Myosin/actomyosin (MAM) was extracted from post-rigor turkey breast and thigh muscles using Hasselbach-Schneider solution. Electrophoretic evaluation of the breast muscle protein extract showed it to be 86.4% myosin and 7% actin. Thigh muscle MAM contained 79.4% myosin and 4.7% actin. Slight variations in minor protein bands were observed between the breast and thigh MAM electrophoretic profiles. Gel filtration of MAM on a 2% agarose column separated actomyosin from myosin. Myosin purity was 97.9% and 99.2% for breast and thigh, respectively. Gel filtration data indicated that myosin, unassociated with actin, was the predominate protein extracted from the post-rigor tissue. Myosin was approximately 67-72% of the original MAM.     

Effect of ageing on the properties of myofibrils of rabbit muscle

The effect of ageing of rabbit muscle at 4° and 15–18° on the extractability and adenosine triphosphatase activity of the myofibrils has been examined. The amount of protein extracted by M-KCL-4 mM sodium glycerophosphate (pH 6.2) and by 0.1 M sodium tetrapyrophosphate-4 mM MgCl₂-10 mM-KH₂PO₄ (pH 7.2) increased as the muscle aged. By using the amount of Ca²⁺ adenosine triphosphatase extracted, i.e. the enzyme associated with myosin, as a measure of the amount of myosin in the actomyosin extracted, it was possible to show that the buffered potassium chloride did not extract all the actomyosin from the myofibrils of pre-rigor muscle. As the muscle aged, more actomyosin was extracted, together with some tropomyosin. Pyrophosphate, however, extracted all the myosin from the pre-rigor muscle, and the increase in the protein extracted from aged muscle was due to actin and tropomyosin in addition to myosin. It is suggested that these changes are caused by a disruption of the Z-band structure during ageing, perhaps due to the hydrolysis of tropomyosin by proteolytic enzymes. The specific Ca²⁺ adenosine triphosphatase activity of the myofibrils was unaltered by ageing but the specific Mg²⁺-activated adenosine triphosphatase, i.e. the enzyme associated with actomyosin, was reduced by about one-third. This latter result may be caused by a change in the mode of linkage of actin to myosin.     

Fermentation of concentrated skim-milk. Effects of different protein/lactose ratios obtained by ultrafiltration–diafiltration

Defatted milk was ultrafiltered–diafiltered in a 20 kDa polysulphone flat membranes UF unit to obtain concentrated milk with different protein/lactose ratios. Different samples of concentrated–diafiltrated milks with protein concentration between 4·7% and 8·9% and lactose concentration between 2·7 and 0·8% (protein/lactose ratios between 1·7 and 9·5) were fermented (Lactobacillus bulgaricus and Streptococcus thermophilus) to obtain yoghurt-like products. Firmness of the coagulum as well as pH, colony count and organoleptic test were considered in the final products. Concentrated-diafiltrated milk with lactose and protein contents lower than 2% and 6·5%, respectively, lead to a very liquid product. Enriched protein low-lactose fermented milks can be obtained by this technology. © 1998 SCI.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

A new process for preparing transparent alkalised duck egg and its quality

When fresh duck (Anas plotyrhyncus) eggs (pH 8·0–8·5) are heated, their albumen develops a turbid gel. Through appropriate alkalisation (pH 11·5–12·8), the gel's transparency can be increased. The transparency of the heated duck egg-white is affected by pH value, heating temperature, heating rate and salt concentration. This research deals with the process for preparing the transparent alkalised duck egg and the change in its quality when stored. If fresh duck eggs are pickled in a solution of 42 g NaOH+50 g NaCl litre^?1 (25·3°C) for 8 days, removed, put in a water bath and heated at 70°C for 10 min they become transparent, their hardness and penetration increasing with storage. Total bacterial count and volatile basic nitrogen also increase with storage. The total bacterial count and the volatile basic nitrogen were 4·6 × 10⁶ cfu g^?1, 0·32 mg g^?1 when stored at a temperature of under 25°C for 4 weeks, respectively.     

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM -MgCl₂, 0·5 mM -EDTA and 0·1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at ?20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, K_m values were 1·7 × 10^?4, 1·5 × 10^?5 and 8·8 × 10^?7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca²⁺ or Cu²⁺, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co²⁺ > Mg²⁺ > Mn²⁺ > Fe²⁺ > Zn²⁺. Polyphosphatase was completely inhibited by 1 mM -ammonium molybdate and 50 μM -Zn²⁺ or Cu²⁺ (in the presence of Mg²⁺).     

CHARACTERIZATION OF PHYTASE ACTIVITY IN LUPIN SEED

Lupin phytase (myo-inositol hexaphosphate phosphohydrolase) and the degradation of its substrate phytic acid were studied during seed germination. Phytase and acid phosphatase activities were detected in nongerminated seeds and increased from days 1 to 8 of germination, while the concentration of phytic acid decreased during the same period. Phytase was extracted with 2% CaCl₂ solution and partially purified by ammonium sulfate fractionation and HPLC-gel permeation chromatography. Substrate specificites were determined using pnitrophenylphosphate or phytic acid. The pH and temperature optima for the fraction obtained by gel permeation were 5.0 and 50C respectively, while the Km and V _max values were 0.33 mM and 0.13 μmol·mL^?1·min^?1, respectively. It was not possible to separate phytase from phosphatase by use of gel permeation chromatography due to similarities in apparent molecular mass, however resolution of the two enzymes was achieved by DEAE-cellulose chromatography.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

Oxidation of methionine. Effects of hydrogen peroxide alone and in combination with iodide and selenite

The oxidation of methionine by hydrogen peroxide, and the influence of iodide, pH, amino acids and selenite were studied with free methionine and with casein and fish fillet protein. The concentration levels tested ranged from 0·05 mm to 3·0 mm. Hydrogen peroxide oxidation was not influenced by pH in the range 5·0 to 8·0; at pH 8·5 the rate of oxidation was increased. When iodide was added in amounts equivalent to or less than the amounts of H₂O₂, the reaction was accelerated with free but not protein-bound methionine. At higher levels iodide inhibited the oxidation. An amino acid mixture and proteins inhibited the effect of iodide; this effect seemed to be due to tryptophan. Selenite also accelerated the effect of H₂O₂, both with free and with protein-bound methionine. Cu⁺⁺ catalysed the oxidation by H₂O₂ at low reactant concentration but not at the higher levels. The reaction between methionine and H₂O₂ seemed to be of first order with respect to both reactants.     

Chemical and physico-chemical characterisation of fibres from Laminaria digitata (kombu breton): A physiological approach

The content and chemical composition of soluble and insoluble dietary fibres from the brown marine alga Laminaria digitata (kombu breton) were determined. Two enzymic-gravimetric methods were used to determine (1) the content of soluble and insoluble fibres according to a modification of the AOAC procedure, and (2) the distribution of the soluble fibres in saline buffer at 37°C and at pH 2·0 and 7·5 used to simulate the gastric and intestinal phases of digestion, respectively. The total dietary fibre contents obtained by the two methods were similar (37·3 and 40·0%) and of these 84·8–87·4% was soluble. A partitioning of soluble fibres may occur during digestion since 49·3% was recovered in saline buffer at pH 2·0 whereas 50·7% was recovered in saline buffer at pH 7·5. Solubility of dietary fibres was related to the chemistry of brown algal polysaccharides. Fucans and laminarans were essentially soluble at pH 2·0 and alginate at pH 7·5, and insoluble fibres consisted primarily of cellulose. Oil adsorption and hydration properties (uptake, retention and swelling) in water, 154 mM NaCl, and 38 mM CaCl₂ at 20 and 37°C of three particle sizes of L digitata were measured. Oil adsorption was low (0·16–0·41 g g^?1) and was related to the particle size of the fibres. Hydration properties were more important with small particles except in CaCl₂ solution and followed the order water > NaCl > CaCl₂. Water uptake and swelling were greater at 37°C than at 20°C. The overall decrease in hydration properties observed with solutions of ionic strength ～0·15 was interpreted as reflecting the decrease in the electrostatic repulsion between charged polysaccharides. The lowest water uptake, water retention and swelling were obtained with solutions of CaCl₂, and were related to the known selective afinity of alginate for calcium. Thus, L digitata is especially rich in soluble dietaryfibres, and these have physico-chemical properties characteristic of the polysaccharides present. Water absorption and uptake and swelling can be modulated according lovarious physical and chemical parameters.     

THE RELATIONSHIP OF DIMETHYL SULPHIDE LEVELS IN MALT,WORT AND BEER

The half-life for the conversion of malt dimethyl sulphide (DMS) precursor to free DMS has been determined at various temperatures and pH values. At pH 5·2 the half-life of the precursor in wort (S.G. 1·060) at its boiling point is 38 min, and is doubled for each 6°C fall in temperature. At pH 5·5 the half-life at the boiling point is 32·5 min. Knowing the stability of the precursor at the various temperatures in the brewing process, the extent of conversion to free DMS in wort at pitching can be predicted for malt of a given precursor content and for a given set of process conditions. The results of DMS analyses of samples taken during brewery trials are in reasonable agreement with the predicted values. This work involved infusion mashing only, but the same principles apply to decoction mashing. The fate of precursor and free DMS during fermentation and conditioning has been followed on a production scale. With some brews, where high levels of free DMS were present at pitching, much free DMS was lost during fermentation. Also, precursor DMS reappeared in the beer after a few days and there was some increase in the level of free DMS. The DMS precursor in green malt has been isolated by ion-exchange and gel-filtration chromatography. A preparation has been obtained which has 0·6 mol potential DMS per mol amino nitrogen. Thin layer chromatography showed that the preparation and its hydrolysis product had the same R_f values as S-methylmethionine and homoserine respectively.     

Preliminary studies on the mucilages extracted from Okra fruits,Taro tubers,Jew's mellow leaves and Fenugreek seeds

Okra fruits, Taro tubers, Jew's mellow leaves and Fenugreek seeds are commonly used in Egypt to prepare popular diets with desired slimy consistency.The mucilages were extracted and preliminary studies conducted to characterise them physically. The pH values of a 1% solution of the mucilages varied from 6·9-7·5 for Okra and Taro, 7·1–7·8 for Jew's mellow, and 5·9-6 for Fenugreek, depending upon extraction conditions. The highest viscosity was observed in Okra solutions, followed by Fenugreek, Jew's mellow and Taro mucilages. Okra and Jew's mellow mucilages are acidic polysaccharides which contain higher amounts of ash than the Taro and Fenugreek mucilages which are neutral polysaccharides. All mucilages are associated with protein. Gel chromatography indicated strong interaction of protein with the polysaccharide. The acid hydrolysis of the mucilages followed by paper chromatography revealed that all mucilages contain methyl pentose, glucose, galactose, and fructose, in different proportions. Taro and Fenugreek mucilages are free of rhamnose. All mucilages are devoid of arabinose and mannose except Fenugreek which contained these two sugars.     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

II. Yeast sequencing reports. The two genes encoding yeast ribosomal protein S8 reside on different chromosomes,and are closely linked to the hsp70 stress protein genes SSA3 and SSA4

A 7·4 kb segment of chromosome II was sequenced and analysed. This segment is part of the 25 kb insert of cosmid clone α1004.10 which is located on the left arm of chromosome II. Sequence analysis revealed four open reading frames (ORFs), of which two had been characterized previously (SSA3, AAR2) and one was not identified. The other ORF was precisely 600 bp long and the deduced protein sequence predicted a very basic protein (pI=11·1; molecular weight=22·5 kDa). Evidence was found that the ORF is the S40 ribosomal protein gene (RPG) S8. Consensus splice signals were found in the 5′ leader sequence and also potential RPG-specific sequences. Chromoblot analysis revealed a second copy of the S8 RPG on chromosome IV or VIII. This copy is also closely linked to an hsp70 protein gene, SSA4. The sequence has been deposited in the EMBL data library under accession number Z26879.     

MYOSIN STABILITY IN INTACT CHICKEN MUSCLE AND A PROTEIN COMPONENT RELEASED AFTER AGING

SUMMARY– My osin from both red (myosin-R) and white (myosin-W) chicken muscle was extracted with Hasselbach-Schneider solution after the muscle had aged for 30 min and 24 hr post-mortem. Myosin-W from aged muscle contained a fast moving boundary in sedimentation velocity studies. When this fraction (component T) was separated on a DEAE-Sephadex column, the ATPase activity and sedimentation pattern of the resulting myosin were similar to those of chromatographed myosin from unaged muscle. The numbers of sulfhydryl groups in both myosin-R and myosin-W were not affected by aging. The extinction coefficient and absorption spectrum of component T were different from those of myosin, and component T had neither ATPase nor 5'-adenylic acid deaminase activity. Lowering the pH of myosin extracted from unaged muscle to the pH values found in muscle aged for 24 hr caused aggregation and loss of ATPase activity. These aggregates of myosin differed from component T in both sedimentation velocity and chromatographic pattern.     

Application of Carbohydrases in Extracting Protein from Rice Bran

Enzymatic pretreatments of rice bran, using commercial carbohydrases to improve nitrogen extractability, were investigated. The pretreatment used a response surface method which included the variations in concentration of Viscozyme L (0–120 FBG unit) and Celluclast 1.5L (0–360 NC unit), time of incubation (1–5 h) and pH (3·8–5·4). The experiments were conducted at two different temperatures, 40 and 50°C. At 40°C, the maximum nitrogen extraction predicted by the model was obtained when the Viscozyme–Celluclast mixture at pH 3·8 with incubation time of 5 h was applied, giving an extracted N of 51%. At the process temperature of 50°C, the model predicted that the use of Viscozyme alone, under similar conditions, would have a most significant effect in increasing the efficacy of the nitrogen extraction, producing a 57% extracted N. This enzymatic pretreatment method improved the nitrogen extractability of rice bran at the neutral pH. © 1997 SCI.     

Polyphenoloxidases from Williams Pear (Pyrus communis L,cv Williams): Activation,Purification and Some Properties

Activation of a crude polyphenoloxidase (PPO) preparation extracted from Williams pears was investigated. Comparison between several activation agents led to the hypothesis of a limited conformational change involved in the activation process. Using ammonium sulphate precipitation followed by hydrophobic and ion exchange chromatography, two PPO fractions, F1 and F2, were 124- and 36-fold purified. F1 contained two forms characterised by isoelectric point equal to 4·2 and 4·5 while F2 contained two other forms associated with isoelectric point of 3·8 and 4·0. The molecular mass determined by gel filtration and confirmed after SDS-page was unique (c 43 kDa). F1 and F2 showed similar apparent Km values, in the 5–11 mM range for the three main phenolic substrates in pear cortex. Chlorogenic acid appeared to be a better substrate than catechins for pear PPO. © 1997 SCI.     

Proteolytic degradation of myofibrillar components by carp cathepsin L

Carp cathepsin L, which is the best candidate to produce textural change in the arai-treated carp fillet, exhibited maximum hydrolytic activity for Z-Phe-Arg-MCA and soluble casein at pH 5·0–5·5. The proteolytic action of the enzyme was evaluated by complete degradation of various carp myofibrils at pH 5·0 over 30 min and by potent degradation of the same proteins at pH 5·5–6·0 over 20 h. All myofibrillar components were partially degraded by the enzyme at pH 6·5–7·0, but varying amounts of them remained undegraded after 20 h. These findings indicate that carp cathepsin L degrades not only carp myofibrillar components but also their resultant products between pH 5·0 and 7·0 and that it markedly acts on myosin heavy chain, α-actinin and troponin-T and -I. Carp cathepsin L likely contributes to postmortem muscle tenderisation of carp fillet over an extensive pH range during storage. © 1998 SCI.     

Biochemical changes in fermented rice-shrimp (Macrobrachium idella) mixture: Changes in protein fractions

Rice-shrimp mixture was allowed to under go natural fermentation for 10 days at 31°C. Daily biochemical tests were carried out in fermented rice-shrimp mixture for 10 days.The protein fractions, particularly soluble-nitrogen, increased from the beginning until the end of the fermentation period. The same pattern also occurred with amino-nitrogen formation concomitant with the reduction in molecular weight of shrimp protein. Ammonia-nitrogen was produced in small amounts and reached a maximum on the seventh day. Cathepsin D activity in shrimp had an optimum pH of 3·5 and an optimum temperature of 40°C. The activity of this enzyme decreased with fermentation time.