Amount Of Free Amino Acids Research Articles

Enzymatic hydrolysis of poultry meal with endo- and exopeptidases

The objective of this research was to determine whether commercial proteases are capable of hydrolyzing denatured poultry by-product proteins that have gone through a rendering process. The material used for this research was low-ash poultry meal obtained from a local poultry processor. Samples of poultry meal were treated with the commercial proteases Alcalase, Flavourzyme, Protamex, and Liquipanol that were added individually or as a combination of 2 proteases, which were incorporated either simultaneously or sequentially. Temperature was controlled during the reaction to the optimal level for each enzyme, and pH was initially adjusted to the most favorable level for each enzyme and was maintained during the course of the reaction by addition of NaOH at established intervals. Consumption of NaOH was used to calculate the degree of hydrolysis. At the end of the hydrolysis, the molecular weight of selected hydrolysates was determined by size exclusion chromatography and by gel electrophoresis. In addition, amino acid analysis was performed on selected hydrolysates. Results show that the sequential treatment with Alcalase and Flavourzyme served best for the preparation of poultry meal hydrolysates with a maximum degree of hydrolysis of 11.1% and the highest hydrolyzable material recovered at 58%, which is attributed to the combined nature of the endo- and exocatalytic action of Alcalase and Flavourzyme, respectively. Hydrolysate with Flavourzyme or the combination of Flavourzyme and Alcalase were both good methods to produce significant amounts of free amino acids. This research shows the feasibility of hydrolyzing poultry by-products that went through a rendering process using different brands of commercial proteases. Findings from this research are important in the preparation of palatants, in which relatively inexpensive hydrolyzed poultry meal could be used to improve the flavor of companion animal food products.

Pleiotropic physiological consequences of feedback-insensitive phenylalanine biosynthesis in Arabidopsis thaliana

A large proportion of plant carbon flow passes through the shikimate pathway to phenylalanine, which serves as a precursor for numerous secondary metabolites. To identify new regulatory mechanisms affecting phenylalanine metabolism, we isolated Arabidopsis thaliana mutants that are resistant to the phytotoxic amino acid m-tyrosine, a structural analog of phenylalanine. Map-based cloning identified adt2-1D, a dominant point mutation causing a predicted serine to alanine change in the regulatory domain of ADT2 (arogenate dehydratase 2). Relaxed feedback inhibition and increased expression of the mutant enzyme caused up to 160-fold higher accumulation of free phenylalanine in rosette leaves, as well as altered accumulation of several other primary and secondary metabolites. In particular, abundance of 2-phenylethylglucosinolate, which is normally almost undetectable in leaves of the A. thaliana Columbia-0 accession, is increased more than 30-fold. Other observed phenotypes of the adt2-1D mutant include abnormal leaf development, resistance to 5-methyltryptophan, reduced growth of the generalist lepidopteran herbivore Trichoplusia ni (cabbage looper) and increased salt tolerance.

Effect of herbicides on the amount of free amino acids in the larval haemolymph of the silkworm, Bombyx mori L.

Effect of herbicides on the amount of free amino acids in the larval haemolymph of the silkworm, Bombyx mori L.

Analysis of free amino acids in Chinese teas and flower of tea plant by high performance liquid chromatography combined with solid-phase extraction

An effective liquid chromatographic method that involved precolumn derivatisation with o-phthaladehyde combined with solid-phase extraction has been developed for the determination of free amino acids in tea. Firstly, tea infusion was treated by C18 cartridge before derivatisation, resulting in great improvement of separation by Zorbax Eclipse XDB-C 18 column. Then, the contents of free amino acids in Chinese green, black, and Oolong teas and the flower of plant Camellia sinensis have been determined. The results showed that theanine was the most abundant amino acid in teas, and green tea contained much higher amounts of free amino acids than fermented ones. While the contents and composition of free amino acids in tea flower were quite different from those of teas. The tea flower contained much higher content of free amino acids. Furthermore, although theanine was the most abundant amino acid in tea flower as tea, histidine became the second one.

Influence of Ethanol Treatment on the Taste Quality and Quantity of Free Amino Acids, IMP, and Inorganic Ions in a Hot-Water Extract of Yellowtail Muscle

Influence of Ethanol Treatment on the Taste Quality and Quantity of Free Amino Acids, IMP, and Inorganic Ions in a Hot-Water Extract of Yellowtail Muscle

Expression of alkaline proteinase gene in two recombinant Bacillus cereus feather-degrading strains

Two Bacillus cereus feather-degrading strains (23/1 and 6/2) were transformed using a recombinant plasmid p5.2 carrying the alkaline proteinase gene (aprE). A high level of the aprE gene expression was observed when the recombinant strains were grown on sporulation medium. The expression of the aprE gene proceeded during the early stationary phase and the p5.2 plasmid was segregationally and structurally stable in both strains. The two recombinant strains grown on a mineral medium with 1 % chicken feather as source of energy, carbon and nitrogen exhibited higher proteolytic activity ( approximately 6-fold and 2.4-fold higher for strains 23/1 (p5.2) and 6/2 (p5.2), respectively. Keratinolytic activity increased approximately 3.5-fold and 4.15-fold, respectively. The keratinolytic activity further increased when an optimized medium with yeast extract and corn oil was used. Considerable amounts of free amino acids were obtained after the biodegradation of feather which makes the new strains promising for application in feather-waste treatment to, e.g., transformation to animal feedstuff.

Exploring possibilities for quality improvement of meat raw materials from cattle ruminant animals by enzymatic treatment

The production of beef meat is a widely spread in Republic of Macedonia, and the beef is very abundant with binding tissue that influences certain hardness and tenacity both to the meat and the final meat product, thus diminishing its organoleptic quality. That demands for seeking adequate methods to improve the consistency and succulence of meat. For that purpose, were used a proteolytic enzyme preparations derived from the bacteria Streptomyces species 82 which have a high proteolytic activity. In this study was determined increased solubility of myofibril proteins at the meat (Muscles supraspinatus) and beef hearts when treated with enzyme preparations. Both Muscles supraspinatus and beef hearts showed a general tendency to increase the quantity of free amino acids when an enzyme preparation with activity of 110 PU/kg was added.

Physiological adaptations in the lichens Peltigera rufescens and Cladina arbuscula var. mitis, and the moss Racomitrium lanuginosum to copper-rich substrate

Two lichen species ( Peltigera rufescens and Cladina arbuscula subsp. mitis) and one moss species ( Racomitrium lanuginosum) growing on a copper mine heaps (probably 200–300 yr old) in the village of Špania dolina (Slovak Republic) were assessed for selected physiological parameters, including composition of assimilation pigments, chlorophyll a fluorescence, soluble proteins and free amino acid content. The lichen C. arbuscula subsp. mitis was collected also at a control locality where total copper concentration in the soil was approximately 3% that of the waste heaps. Concentrations of Al, Co, Cu, Ni, Sb and Zn were highest in thalli of Peltigera, while the moss Racomitrium contained the highest content of Fe and Pb. Thalli of Cladina contained less metals than the cyanolichen Peltigera, and except for Zn metal concentrations in Cladina from the control locality were lower than in thalli of the same species from copper mine heaps. Regardless of the species or locality, the composition of assimilation pigments and chlorophyll a fluorescence showed that the tested lichens and moss were in good physiological condition and adapted to increased copper levels in the soil. There were significantly different amounts of total free amino acids in Peltigera, Cladina and Racomitrium from the Cu-polluted field. However, differences in amount of free amino acids in control, as well as Cu-polluted thalli of Cladina were less pronounced.

Proteomics Approach to Identify Unique Xylem Sap Proteins in Pierce’s Disease-Tolerant Vitis Species

Pierce's disease (PD) is a destructive bacterial disease of grapes caused by Xylella fastidiosa which is xylem-confined. The tolerance level to this disease varies among Vitis species. Our research was aimed at identifying unique xylem sap proteins present in PD-tolerant Vitis species. The results showed wide variation in the xylem sap protein composition, where a set of polypeptides with pI between 4.5 and 4.7 and M(r) of 31 kDa were present in abundant amount in muscadine (Vitis rotundifolia, PD-tolerant), in reduced levels in Florida hybrid bunch (Vitis spp., PD-tolerant) and absent in bunch grapes (Vitis vinifera, PD-susceptible). Liquid chromatography/mass spectrometry/mass spectrometry analysis of these proteins revealed their similarity to beta-1, 3-glucanase, peroxidase, and a subunit of oxygen-evolving enhancer protein 1, which are known to play role in defense and oxygen generation. In addition, the amount of free amino acids and soluble sugars was found to be significantly lower in xylem sap of muscadine genotypes compared to V. vinifera genotypes, indicating that the higher nutritional value of bunch grape sap may be more suitable for Xylella growth. These data suggest that the presence of these unique proteins in xylem sap is vital for PD tolerance in muscadine and Florida hybrid bunch grapes.

Response of amino acid changes in Aphis gossypii (Glover) to elevated CO2 levels

AbstractEffects of elevated CO2 levels on the amino acid constituents of cotton aphid, Aphis gossypii (Glover), fed on transgenic Bacillus thuringiensis (Berliner) (Bt) cotton [Cryl A(c)], grown in ambient and double‐ambient CO2 levels in closed‐dynamics CO2 chambers, were investigated. Lower amounts of amino acids were found in cotton phloem under elevated CO2 than under ambient CO2 levels. However, higher amounts of free amino acids were found in A. gossypii fed on elevated CO2‐grown cotton than those fed ambient CO2‐grown cotton, and the contents of amino acids in honeydew were not significantly affected by elevated CO2 levels. A larger amount of honeydew was produced by cotton aphids feeding on leaves under elevated CO2 treatment than those feeding on leaves under ambient CO2 treatment, which indicates that A. gossypii ingests more cotton phloem because of the higher C:N ratio of cotton phloem under elevated CO2 levels. Moreover, the amino acid composition was similar in bodies of aphids ingesting leaves under both CO2 treatments, except for two alkaline amino acids, lysine and arginine. This suggests that the nutritional constitution of the phloem sap was important for A. gossypii. Our data suggest that more phloem sap will be ingested by A. gossypii to satisfy its nutritional requirement and balance the break‐even point of amino acid in elevated CO2. Larger amounts of honeydew produced by A. gossypii under elevated CO2 will reduce the photosynthesis and result in the occurrence of some Entomophthora spp.

Effect of corn gluten and its hydrolysate consumptions on weight reduction in rats fed a high-fat diet

This study examined the effects of corn gluten (CG) and its hydrolysate consumptions on weight reduction in rats fed a high-fat diet. Eight-month-old male Sprague-Dawley rats (n=40) were fed a high-fat diet (40% calorie as fat) for 4 weeks. They were then randomly divided into four groups and fed the isocaloric diets with different protein sources for 8 weeks. The protein sources were casein (control group), intact CG (CG group), CG hydrolysate A (CGHA group, 30% of protein as peptides and 70% as free amino acids) and CG hydrolysate P (CGHP group, 93% of protein as peptides and 7% as free amino acids). Body weight gain, adipose tissue weights, nitrogen balance, absorptions of energy, protein and fat, lipid profiles in plasma, liver and feces and hepatic activities of carnitine palmitoyl transferase (CPT), fatty acid synthase (FAS), malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PDH) were assessed. The CGHA diet had the highest amount of BCAAs, especially leucine, and most of them existed as free amino acid forms. The CGHA group showed significant weight reduction and negative nitrogen balance. Protein absorption and apparent protein digestibility in the CGHA group were significantly lower than those in other groups. Adipose tissue weights were the lowest in the CGHA group. Activity of CPT tended to be higher in the CGHA group than in other groups and those of FAS, ME and G6PDH were significantly lower in the CGHA group than in other groups. In conclusion, the CGHA diet which had relatively high amounts of free amino acids and BCAAs, especially leucine, had a weight reduction effect by lowering adipose tissue weight and the activities of FAS, ME and G6PDH in experimental animals, but it seemed to be a negative result induced by lowering protein absorption, increasing urinary nitrogen excretion and protein catabolism.

A Novel Fermented Food Product Prepared from Soybean Protein-Calcium-Gel with Monascus Fungus: Changes in Protein and Nitrogen Compounds during Fermentation

The aim of the present study was to investigate the changes in protein and nitrogen compounds in a novel fermented product generated from soybean protein-calcium-gel (SP-Cal-Gel) and Monascus fungus during fermentation. Slab SDS-polyacrylamide gel electrophoresis and measurement of nitrogen contents clearly revealed the occurrence of protein digestion in the form of degradation of α'-, α-, and β-subunits in β-conglycinin and of acidic polypeptide in glycinin; however, the band of basic polypeptide in glycinin still remained. The ratios of water-soluble nitrogen and TCA-soluble nitrogen to total nitrogen reached 52.4 or 46.6% after 60-days ripening, respectively. Moreover, involvement of a large amount of free amino acids, especially aspartic and glutamic acids, were shown in this novel fermented SP-Cal-Gel food product.

Daily balance of leaf sugars and amino acids as indicators of common bean (Phaseolus vulgaris L.) metabolic response and drought intensity

Leaf soluble sugar, starch and free amino acid amounts were evaluated in two bean genotypes, Carioca and Ouro Negro, grown in 10 L pots in a greenhouse. This was realized during a single day for Carioca and during ten days of water deficit for both genotypes, at 06:00 and 18:00 h. During the day, an increase in all parameters occurred up to midday, while in the afternoon, carbohydrate amounts varied in opposition to amino acids amounts. Under water deficit, the leaf soluble sugars at 18:00 and their daily balance (amount at 18:00 minus amount at 06:00) increased on the 2(nd) day, with starch and amino acid amounts and their respective daily balances decreasing. On the 8(th) and 10(th) days, the soluble sugar daily balance was negative and the starch daily balance approached zero or was negative, while the amino acid daily balance was positive. During rehydration, soluble sugar daily balance was close to zero and starch daily balance increased, whereas amino acid daily balance was negative. The carbohydrate daily balance, strongly related to free amino acid daily balance, were good indicators for evaluating the stage of water deficit and metabolic capacity to respond to the stress in common bean.

Influence of Processing Conditions on Acrylamide Content in Black Ripe Olives

The presence of acrylamide was investigated in different presentations of commercial black ripe olives, a well-known sterilized alkali-treated product. The analysis was performed by gas chromatography-mass spectrometry (GC-MS) after bromination of acrylamide, using (13C3)acrylamide as internal standard. In-house validation data for commercial ripe olives showed good precision and accuracy of the method, with repeatability below 3% and recoveries between 94 and 105%. Acrylamide was detected in all samples, but its concentration varied significantly from 176 to 1578 microg/kg of pulp. The effects of different processing conditions (two preservation methods and three darkening methods), cultivar (Hojiblanca or Manzanilla), and presentation form (pitted or sliced olives) on acrylamide content were evaluated in experiments performed in an olive-processing plant. All canned samples were sterilized at 121 degrees C for 30 min. Statistical analysis of the data indicated that the effects of darkening method and olive cultivar were the most pronounced. Acrylamide contents did not significantly differ after 6 months of storage. The small amounts of free amino acids and reducing sugars found in olives before sterilization did not significantly correlate with the acrylamide formed.

Influence of Sulfur Fertilization on the Amounts of Free Amino Acids in Wheat. Correlation with Baking Properties as well as with 3-Aminopropionamide and Acrylamide Generation during Baking

Sulfur (S) fertilization has been long-known to influence the amounts of total free amino acids in plants. To determine the impact of S deficiency in wheat on the concentration of, in particular, free asparagine, the spring wheat cultivar 'Star' was grown in a laboratory scale (5 L pot) at five different levels of S fertilization. After maturity, the kernels were milled into white flours (1-5) and analyzed for their contents of total S and total nitrogen as well as for free amino acids and glucose, fructose, maltose, and sucrose. Extremely high concentrations of free asparagine (Asn; 3.9-5.7 g/kg) were determined in flours 1 and 2 (30 and 60 mg of S), whereas much lower amounts (0.03-0.4 g/kg) were present in flours grown at higher S levels. The amounts of the reducing carbohydrates were, however, scarcely affected by S fertilization. In agreement with the high amount of Asn in flours 1 and 2, heating of both flours led to the generation of very high amounts of acrylamide (1.7-3.1 mg/kg) as well as of 3-aminopropionamide (40-76 mg/kg). Similar concentrations were measured in crispbread prepared from both flours. Application of rheological measurements on doughs prepared from each flour and a determination of the loaf volume of bread baked therefrom clearly indicated that flours 1 and 2 would be excluded from commercial bread processing due to their poor technological properties. Two commercial flours showed relatively low concentrations of acrylamide after a thermal treatment.

The Maturational Disassembly and Differential Proteolysis of Paralogous Vitellogenins in a Marine Pelagophil Teleost: A Conserved Mechanism of Oocyte Hydration1

A structural analysis of the differential proteolysis of vitellogenin (Vtg)-derived yolk proteins in the maturing oocytes of a marine teleost that spawns very large pelagic eggs is presented. Two full-length hepatic cDNAs (hhvtgAa and hhvtgAb) encoding paralogous vitellogenins (HhvtgAa and HhvtgAb) were cloned from nonestrogenized Atlantic halibut, and the N-termini of their subdomain structures were mapped to the oocyte and egg yolk proteins (Yps). The maturational oocyte Yp degradation products were further mapped to the free amino acid (FAA) pool in the ovulated egg. The deduced amino acid sequences conformed to the linear NH(2)-(LvH-Pv-LvL-beta'-CT)-COO(-) structure of complete teleost Vtgs. However, the Yps did not match the expected cleavage products of complete Vtgs. Specifically, the phosvitin subdomain of the HhvtgAa paralogue remains covalently attached to the lipovitellin light chain, while the phosvitin subdomain of the HhvtgAb paralogue remains covalently attached to a C-terminal fragment of the lipovitellin heavy chain (LvH). During oocyte hydration, the LvH of the HhvtgAa paralogue is disassembled and extensively degraded to FAA. In the HhvtgAb paralogue, the LvH is nicked in the C-sheet in a manner similar to that seen in lamprey and other teleosts. A small part of the C-terminal end of the LvH-Ab undergoes proteolysis to FAA, together with the phosvitin, beta' component, and much ( approximately 65%) of the lipovitellin light chain (LvL-Ab). The independently measured FAA pool in the ovulated egg corroborates that calculated from differential proteolysis of the Yps. Based on the 3:1 (HhvtgAb:HhvtgAa) Yp expression ratio, each paralogue contributes approximately equal amounts of FAA to the organic osmolyte pool of the hydrating oocyte during maturation.

야생 돌복숭아 씨와 과육의 아미노산 및 지방산 조성에 관한 연구

우리나라 전역에서 자생하는 야생 돌복숭아(Prunus persica Batsch var, davidiana Max.)의 씨와 과육 중의 각종 생리활성 물질을 규명하여 바이오헬스 기능성 소재 등의 자원으로서 이용 가능성을 검토하기 위한 자료로, 돌복숭아 씨와 과육 중의 일반성분, 구성 총 아미노산, 유리 아미노산 및 지질 구성 지방산 조성을 분석한 결과는 다음과 같다. 돌복숭아 씨 및 과육 중의 일반성분 함량은 탄수화물이 63.92% 와 75.11%로 대부분을 차지하였으며, 조단백질 27.85%, 12.77%, 수분 3.61%, 4.69%, 조지 방이 1.21%, 4.80% 그리고 조회분이 3.41%와 2.63% 함유되어 있는 것으로 나타났다. 돌복숭아 씨 중의 구성 총 아미노산의 함량은 3,444.35 mg%로 이 중 aspartic acid가 681.10 mg%, glutamic acid 495.48 mg%, alanine 283.66 mg%, serine 251.36 mg%, proline 229.80 mg%, lysine 192.31 mg% 및 leucine이 191.34 mg%의 순으로 조성되어 있었으며, 특히 aspartic acid 및 glutamic acid가 대부분을 차지하였다. 돌복숭아 과육의 단백질 구성 아미노산 조성의 총 함량은 1,064.02 mg%로, aspartic acid 250.15 mg%, glutamic acid는 129.63 mg%로 과육의 주요 총 아미노산이었고 lysine, proline, leucine, alanine, serine 등의 순으로 함유되어 있었다. 돌복숭아 씨 중 유리 아미노산 조성의 총 함량은 6,215.34 mg%로, 이 중 glutamic acid가 827.25 mg%로 가장 많았으며, threonine, valine, <TEX>$\beta-aminobutyric$</TEX> acid 등의 순으로 함유되어 있었다. 과육 중의 유리 아미노산 조성의 총 함량은 683.82 mg%로, glutamic acid(339.49 mg%)가 대부분 이었으며, serine, proline, alanine, <TEX>$\gamma-amino-n-butyric$</TEX> acid의 순으로 함유되어 있었다. 지질 구성 지방산 조성은 포화지방산이 12.49%, 단불포화지방산 13.11% 및 다불포화지방산이 74.36%의 함유비율을 보였으며, 필수지방산인 n-6계 linoleic acid<TEX>$(C_{18:2})$</TEX>와 n-3계 linolenic acid<TEX>$(C_{18:3})$</TEX>가 대부분을 차지하였다. 야생 돌복숭아 과육 중의 지방산 조성은 포화지방산이 16.74%, 단불포화지방산 17.51% 및 다불포화지방산이 65.73%의 함유 비율을 보였는데, 이 중 다불포화지방산인 n-6계 linoleic acid<TEX>$(C_{18:2})$</TEX>와 n-3계 linolenic acid<TEX>$(C_{18:3})$</TEX>가 지질 구성 총 지방산의 대부분을 차지하는 함유 비율을 나타내었다. Amino acid and fatty acid compositions of the physiological activity substance in the seed and pulpy substance of feral peach (Prunus persica Batsch var. davidiana Max.) were analyzed for the use as an biohealth functional processed products. The proximate compositions in the vacuum freeze dried seed and pulpy substance of feral peach were carbohydrate 63.92% and 75.11%, crude protein 27.85% and 12.77%, moisture 3.61% and 4.69%, crude fat 1.21% and 4.80%, crude ash 3.41% and 2.63%, respectively. Total amino acid contents in the protein of feral peach seed were 3,444.35 mg%, and the major amino acids were aspartic acid(681.10 mg%), glutamic acid(495.48 mg%), alanine(283.66 mg%), serine(251.36 mg%), proline(229.80 mg%), lysine(192.31 mg%) and leucine(191.34 mg%), respectively. Total amino acid contents in the protein of feral peach pulpy substance were 1,064.02 mg%, and the major amino acids followed aspartic acid(250.15 mg%), glutamic acid(129. 63 mg%), lysine, proline, leucine, alanine and serine, in a decreasing order. The richest total amino acid content contained in feral peach seed and pulpy substance was aspartic acid, followed by glutamic acid. The amount of free amino acids of feral peach seed were 6,215.34 ms%, and the major free amino acids were glutamic acid(827.25 mg%), threonine, valine and <TEX>$\beta-aminobutyric$</TEX> acid, respectively. Free amino acid contents of pulpy substance were 683.82 mg%, and the major free amino acids were glutamic acid(339.49 mg%), serine proline, alanine and <TEX>$\gamma-amino-n-butyric$</TEX> acid. Especially, in the case of glutamic acid, it was highest. The compositions of major total fatty acid in the lipid feral peach (Prunus persica Batsch var. davidiana Max.) seed and pulpy sabstance were linoleic acid(<TEX>$C_{18:2}$</TEX>, n-6) and linolenic acid(<TEX>$C_{18:3}$</TEX>, n-3), particularly.

40. Chiral effects of R/S-1-α-a-methylbenzyl-3-p-tolylurea on changes in the amounts of free amino acids in the root tips of rice and wheat

40. Chiral effects of R/S-1-α-a-methylbenzyl-3-p-tolylurea on changes in the amounts of free amino acids in the root tips of rice and wheat

マウスにおける多穀麹の蛋白質分解促進効果

We studied the effect of proteolysis promotion of grain koji in mice. Grain koji is an enzyme-rich food which is produced by solid-state fermentation with Aspergillus oryzae in a mixture of six kinds of grains (foxtail millet, Japanese barnyard millet, millet, sorghum, barley, purple-black rice). Grain koji contains higher acid-protease and neutral-protease activities than unfermented grain materials. Therefore, grain koji could digest casein in the simulated gastric juice, giving rise to higher amounts of free amino acids. When male ICR mice were fed with CE-2 diets containing 10% grain koji for 28 days, a significantly (p<0.05) higher absorption ratio was observed in comparison with that in the control group of mice reared on CE-2 diets supplemented with 10% grain materials. The serum lipid concentrations were almost the same among the various mice groups. The present study has thus clarified the proteolysis promotion of grain koji.

A Judgment on Postmortem Aging inLongissimus DorsiBased on a Peptide Substrate Library

We attempted to develop a method to determine easily and effectively the degree of postmortem aging of pork longissimus dorsi (LD) by measuring the activity of proteases in the LD using fluorogenic peptide substrates. LD was used to measure the change with time in the protease activity detected with these substrates. Determining the variations within the LD muscles, strong positive correlations were found between changes in hardness and fluorescence intensities against Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P<0.005), and strong negative correlations were found between changes in total amounts of free amino acids and Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P<0.001). Negative correlations were also observed between changes in the amounts of free Ala, Arg, Lys, Leu, Met, Phe, and Tyr and the fluorescence intensities against Ala, Arg, Lys, Leu, Met, Phe, and Tyr-MCA respectively (P<0.001).