α;2-Macroglobulin Inhibition of Endogenous Proteases in Fish Muscle

Kinetic parameters for α;₂-macroglobulin inhibition of selected proteases (collagenase, cathepsin D, trypsin and chymotrypsin) were determined along with the extent of inhibition of the corresponding fish muscle proteases. Protease inhibition by α;₂-macroglobulin occurred in the presence of large macromolecular substrates but not in the presence of synthetic substrates. The inhibitor remained active at refrigerated temperatures (4–7°C). The α;₂-macroglobulin did not have any inhibitory effects on proteases in intact fish muscle tissue possibly due to lack of penetration of the tissues, but showed various levels of inhibition of proteases in tissue homogenates. Inhibitor concentration of 0.1% per weight of tissue homogenate resulted in about 17% loss of proteolytic activity while 0.4% inhibitor concentration caused complete loss of protease activity.     

HYDROLYSIS OF SALMON MUSCLE PROTEINS BY AN ENZYME MIXTURE EXTRACTED FROM ATLANTIC SALMON (SALMO SALAR) PYLORIC CAECA

An extract of serine proteases from thepyloric caeca of Atlantic salmon (Salmo salar) was recovered by (NH₄)₂SO₄ and stabilized in 20% gfycerol. Proteolytic activity was determined using Azocoll and hydrofytic efficiency evaluated on salmon muscle mince. Activity assays identified chymotrypsin as the most active serine protease component, followed by trypsin and elastase. The caeca extract more efficiently hydrolyzed a salmon muscle mince substrate at 40C compared to 21.5C, and was very efficient compared to four commercial protease preparations in terms of degrees of hydrolysis (DH) achieved after a 180 min reaction. Salmon protein hydrotysates made from the enzymatic hydrolysis with the pyloric caeca enzymes had different peptide profiles at the same DH when the reaction took place at 2I.5C compared to 40C, suggesting that relative activities of these serine proteases may be different at different reaction temperatures.     

Collagenase activity and protein hydrolysis as related to spoilage of iced cod (Gadus morhua)

Collagenase activity and changes in muscular protein of iced Atlantic cod stored for 9 days were studied. The crude fish muscle extract showed maximum collagenase-like activity against bovine insoluble tendon collagen at 48 h of incubation at 37 °C. Collagenase activity against synthetic substrate increased (P<0.05), especially for fish in initial and advanced stages of decomposition. These results suggest that endogenous collagenases and other proteases may be responsible for the destruction of fine collagenous fibrils in the skeletal muscle of cod. The content of titin 1 decreased when decomposition was advanced. Moreover, a progressive degradation of sarcoplasmic proteins with a molecular weight of 100, 94, 85 and 80 kDa was observed. Results suggest that softening of cod muscle during iced storage is caused more by collagenase activity than by proteolysis of myofibrils.     

The effects of salt concentration on conformational changes in cod (Gadus morhua) proteins during brine salting

The effects of different salt concentrations (6%, 15%, 18% and 24% NaCl (w/w)) on the conformational changes of cod muscle proteins during brine salting were examined in this study. Proteins were extracted from the brine salted samples with solutions of 1 M (5.9%) and 4 M (23.4%) NaCl and the quantity of salt soluble proteins (SSP), disulfide bond (S–S), total sulfhydryl (SH) and available SH content in the soluble fraction were determined. Increased salt concentration in cod muscle promoted protein denaturation and aggregation. The SSP and total SH content decreased, whereas the S–S bond and available SH content increased with increased salt concentration in cod muscle. Disulfide bond formation correlated (r = −0.6) with a decrease in total SH groups. Higher SSP and available SH groups of the samples at lower brine concentrations was explained by smaller concentration gradients and salt diffusion rates, resulting in stronger salting-in at early stages of the brining process. There was a significant difference in conformational changes in proteins extracted with a salt solution of 1 and 4 M, mainly due to a different degree of protein aggregation.     

Characterisation of trypsin and α-chymotrypsin inhibitors in Australian wattle seed (Acacia victoriae Bentham)

Crude water extracts from Australian wattle seed (Acacia victoriae Bentham) and their salt (ammonium sulphate)-precipitated fractions were analysed for trypsin and α-chymotrypsin (chymotrypsin) inhibitor activity, using gel electrophoresis and spectrophotometric methods. Three different bands with molecular weight 30.20, 38.03 and 39.81 kDa were active, with the 50% salt-precipitated fraction exhibiting highest activity and number of active bands. The same proteins also appeared to be responsible for both trypsin and chymotrypsin inhibitor activity. To establish conditions for the inactivation of these inhibitors, whole seed and uncoated (dehulled) cotyledon were subjected to different heat treatments. Moist heat treatment at 100 °C for 30 s was sufficient to inactivate both protease inhibitors although the trypsin inhibitor was found to be more heat-resistant than was the chymotrypsin inhibitor. Soaking overnight, before thermal treatment, improved the trypsin inhibitor activity but enhanced the efficiency of thermal inactivation in both inhibitors.     

Effects of limited proteolysis on broad bean polyphenoloxidase

Partially purified broad bean polyphenoloxidase was treated with a variety of proteases. Enzyme activity increased after treatment with chymotrypsin, pepsin, subtilisin, thermolysin, trypsin, and Staphylococcus aureus V₈ protease but this activation was masked when assayed in the presence of sodium dodecylsulfate. The enzyme was resistant to proteolysis when treated with chymotrypsin, elastase, pepsin, thermolysin, and V₈ protease. Proteinase K, subtilisin and trypsin treatment resulted in partial degradation of the enzyme but only trypsin generated an active enzyme form with a slightly smaller molecular weight. Electrophoresis, followed by enzyme staining and or Western blotting, showed that trypsin treatment converted the active 45 kd enzyme into a 43 kd enzyme within 20 min. Treatment with proteinase K, subtilisin and trypsin also produced inactive higher molecular weight forms of purified polyphenoloxidase that were identified by Western blotting. The use of antibodies coupled with Western blotting also identified inactive, active, and latent polyphenoloxidase after protease treatment, and suggests that immunological methods can be used to establish the types and amounts of polyphenoloxidase present in a given tissue.     

MARINE FISH DIGESTIVE PROTEASES—RELEVANCE TO FOOD INDUSTRY AND THE SOUTH-WEST ATLANTIC REGION—A REVIEW

The biological diversity of marine species provides a wide array of enzymes with unique properties. For this reason, there is great potential for the recovery and use of digestive proteases from fish provessing wastes. Fish digestive proteolytic enzymes most commonly studied include pepsin, trypsin, chymotrypsin, gastricsin and elastase. Enzymes from cold adapted ectothermic organisms have application in the food industry because their temperature and other characteristics differ from homologous proteases from warm-blooded animals. The South-West Atlantic ocean is an enormous reservoir of different fish species. The digestive proteases from these organisms can be isolated as a by-product and used for processing aids in the food industry.     

In Vitro Digestibility of Dry Bean (Phaseolus Vulgaris L.) Proteins: The Role of Heat-Stable Protease Inhibitors

In vitro susceptibility to trypsin of whole bean extracts, and waterand salt-soluble proteins of 11 dry bean (Phaseolus vulgaris L.) varieties were investigated. Whole bean proteins were rapidly digested at a 10:l protein:trypsin ratio. Salt-soluble proteins of beans were more readily and completely hydrolyzed by trypsin than their watersoluble proteins. Of the 8 fractions obtained by DEAE-cellulose column chromatography of Great Northern bean albumins, three were identified to contain heat-stable trypsin and chymotrypsin inhibitory activity. Proteins in these fractions seemed to protect phaseolin from rapid hydrolysis by proteases by virtue of both their inhibitory activity as well as protein:protein interactions.     

The novel trypsin Y from Atlantic cod (Gadus morhua) – isolation,purification and characterisation

This report describes the isolation and partial characterization of the novel group III trypsin Y from the pyloric caeca of Atlantic cod. Other Atlantic cod trypsins have been used as food processing aids with good results. Trypsin Y was purified by p-aminobenzamidine affinity chromatography and characterized by SDS–PAGE and western blot analysis, as well as by activity measurements towards synthetic substrates. Identification of trypsin Y was done with polyclonal antibodies raised towards the recombinant form of the enzyme and by MALDI-TOF mass spectrometry. Trypsin Y is the only group III trypsin isolated from its native source and characterized by biochemical methods. In accordance with the r-trypsin Y, the native enzyme shows dual substrate specificity, i.e. towards trypsin and chymotrypsin specific substrates. This, along with the high cold-adapted character of trypsin Y, may be valuable for its use as a processing aid for sensitive products such as seafood.     

Partial characterisation of gelatinolytic activities in herring (Clupea harengus) and sardine (Sardina pilchardus) possibly involved in post-mortem autolysis of ventral muscle

The present work aims at identifying enzymatic activities that may contribute to the post-mortem autolysis of the ventral muscle, known as belly bursting, in herring (Clupea harengus). Gelatinolytic proteases extracted from several herring tissues and also from a sardine (Sardina pilchardus) tissue were partially characterised using gelatin zymography, inhibitor analysis, immunodetection with anti-trypsin antibody and MALDI-TOF/TOF peptide sequencing. The results indicate the presence of gelatinolytic trypsin-like serine proteases and metalloproteases in several samples including the ventral muscle of herring. MS/MS analysis gave partial sequences of peptides from some of the proteases, and these were identified as elastase, trypsin and aspartyl aminopeptidase. It is likely that belly bursting in herring is caused by leakage of enzyme-rich fluids from the intestine and/or pyloric caeca which may also contain exogenous proteases from the digestive systems of the prey.     

Proteolysis of the barley protein, hordein

Gordein isolated from barley according to Baxter's method was exposed to proteolytic enzymes at optimal medium pH. As regards the capability of gordein splitting the enzymes can be arranged in the following way: pronase greater than elastase greater than pepsin greater than papain greater than chymotrypsin greater than trypsin. A mixture of three enzymes (trypsin, chymotrypsin and elastase) taken in the amount 1/3 of the usually used concentration provoked a more powerful splitting than each of the enzymes used alone. According to the electrophoresis in polyacrylamide gel data the action of elastase led to the splitting of the high-molecular-weight fraction of gordein with accumulation of the low-molecular weight one. Preheating of gordein for 30 min at 100 degrees C increased its papain-induced splitting by 3.3 times, chymotrypsin-induced by 2.6 times, trypsin-induced by 30%, and pepsin-induced splitting by 23%. Heating did not affect the rate of gordein splitting by elastase. Pretreatment of gordein with pepsin increased over 2 times its splitting under the effect of trypsin and chymotrypsin.     

Effect of curing salt and phosphate on the activity of porcine muscle proteases

The effect of curing salt on the activity of porcine muscle proteases was evaluated, within the salt concentration range found in the manufacturing process of Spanish cured ham. Salt (NaCl) acts as a strong inhibitor of proteolytic activity; sodium nitrate and potassium nitrate do not affect cathepsin D activity; cathepsin L is inhibited at levels of the salts not found in cured ham, and Ca-dependent proteolytic activity is enhanced by nitrate concentrations below 800 ppm.

The appearance of phosphate precipitates in several areas of ham has led to the study of phosphate effect on enzymatic activity. Results show that phosphate is an inhibitor of proteolytic activity.     

Purification and characterisation of protease inhibitors from pigeon pea (cajanus cajan (l) millsp) seeds

Two protease inhibitors from Cajanus cajan seeds have been purified to homogeneity by trichloroacetic acid (TCA) solubilisation, ion-exchange and gel-filtration chromatography followed by preparative polyacrylamide gel electro-phoresis. One of the inhibitors, Cajanus trypsin-chymotrypsin inhibitor (CTCI), inhibits both bovine trypsin and chymotrypsin while the other, Cajanus trypsin inhibitor(CTI), inhibits only bovine trypsin. The two inhibitors contained no carbohydrate and had an isoelectric point of 6. CTCI and CTI had average molecular weights of 15000 and 10500, respectively. The purified inhibitors in solution were stable to heat at 80°C for 15 min and pH 7–10. In the pH range 3–5, 80% of the activity was retained. Autoclaving totally destroyed the inhibitor activity. CTCI had two sites for trypsin binding and one site for chymotrypsin binding while CTI had only one site for trypsin binding. The inhibitors were very specific towards mammalian serine proteases and did not inhibit other proteases or serine proteases of bacterial origin.     

IDENTIFICATION OF TWO MATRIX METALLOPROTEINASES IN THE SKELETAL MUSCLE OF PACIFIC ROCKFISH (SEBASTES SP.)

Two heat-stable metalloproteinases with collagenase activity have been identified in the skeletal muscle of Pacific rockfish (Sebastes sp.) and partially characterized. The 47 kDa and 95 kDa enzymes appear to be similar to mammalian matrix metalloproteinases (MMPs), with respect to molecular weight, pH-activity profile, substrate specificity, dependence on calcium for activity, and inhibition-activation profiles. The fish metalloproteinases readily hydrolyzed collagen and gelatin, but not β- or K-casein, in SDS-PAGE substrate zymograms. They also hydrolyzed collagen and gelatin in solution, but showed very low activity in solubilizing native fibrillar collagen. They did not hydrolyze solutions of β- or K-casein, azocasein, hide powder azure or synthetic substrates for trypsin, chymotrypsin or collagenase. To our knowledge, this is the first report where fish muscle proteinases have been identified as possible members of the family of MMPs. The rockfish muscle MMPs appear to be different from other currently identified fish muscle proteinases in regard to molecular weight, substrate specificity and activation-inhibition profiles. These two enzymes could be partly responsible for the degradation of collagen and other extracellular matrix proteins in fish muscle and for the texture softening of seafood products.     

Effect of Phytate on Solubility, Activity and Conformation of Trypsin and Chymotrypsin

Phytate:proteinase interactions and their effects on the solubility, activity, and conformation of trypsin and chymotrypsin were investigated in model systems. At pH 3.0, phytate at 0.167-0.250 (w/w) ratios formed insoluble complexes with both the enzymes, while at pH 7.8, Ca⁺⁺ was required for the complex formation. At pH 7.8, phytate increased trypsin and chymotrypsin activity by 5-7%. The mean chymotrypsin activity was increased by about 18% when interactions were carried out at pH 3.0, whereas under similar conditions, trypsin activity was strongly inhibited. Circular dichroism spectroscopic studies revealed conformational changes in the enzyme secondary structure that seemed to have influenced their activity. The role of phytate in protein metabolism and the possible ramifications of present findings in human nutrition were addressed.     

Protein inhibitors of hydrolases in plant foodstuffs

Abstract

In this review protein inhibitors of hydrolases occurring in plant tissues used as foods are treated, that is, proteins inhibiting digestive enzymes such as proteases (trypsin and chymotrypsin, which are discussed in more detail, elastase, pepsin, carboxypeptidases), amylases, lipases, and some other hydrolases. Topics covered are the chemistry of these inhibitors including their isolation, structure, and properties; their physiological function; their occurrence in plants and foodstuffs, with special emphasis on their inactivation during processing; their toxicology; and the uptake by human beings. In addition, analytical aspects are discussed in greater detail, and procedures for inhibitor activity determination are included.     

凡纳滨对虾内源蛋白酶对肌原纤维蛋白的降解作用

为确定引起凡纳滨对虾自溶的主要酶类及分布部位,本文测定了凡纳滨对虾各内源蛋白酶的活性,分析主要内源蛋白酶贮藏期间的变化,并比较主要内源蛋白酶对虾肌原纤维蛋白的降解作用。结果显示,虾头蛋白酶活达90.17U/mg,是虾肉的300倍。虾头中的胰蛋白酶活性最高,达13.59U/mg。全虾贮藏4d后虾肉总酶活和胰蛋白酶活增加,于第8d虾肉第一腹节总酶活增至1.66U/mg,胰蛋白酰胺酶和酯酶活分别达到0.97U/mg和1.84U/mg。SDSPAGE结果显示,随贮藏温度和时间的增大,内源酶对肌原纤维蛋白的肌球蛋白重链(MHC)和肌动蛋白(Actin)降解程度越大,其中虾头中胰蛋白酶对肌原纤维蛋白的降解作用最强。因此凡纳滨对虾胰蛋白酶很可能是肌肉软化的主要作用酶。      

Effects of extrusion and conventional processing methods on protein and antinutritional factor contents in pea seeds

The effects of high temperature short time (HTST) treatment compared with other conventional processes on protein, phytic acid, condensed tannins, polyphenols, trypsin, chymotrypsin and α-amylase inhibitor activities and haemagglutinating activities in Renata, Solara and Ballet pea seeds were investigated. Ballet cultivar showed highest protein, phytic acid, tannin, polyphenol contents and trypsin and chymotrypsin inhibitory activities. All pea cultivars contained trypsin- and chymotrypsin-inhibiting activity and lectins but only Solara had α-amylase inhibitory activity. Under extrusion conditions (148°C, 25% moisture and 100 rpm) this thermal processing method was the most effective in condensed tannin, trypsin, chymotrypsin, α-amylase inhibitors and haemagglutinating activity reduction, without modifying protein content as occurs by dehulling, soaking and germination treatments. Trypsin and chymotrypsin inhibitors and haemagglutinating activities in peas were more readily abolished by extrusion treatment than was chymotrypsin inhibitory activity.     

Detection and partial characterisation of subtilisin inhibitors in legume seeds by isoelectric focusing

Seed extracts of ten legume species were subjected to isoelectric focusing in acrylamide gels and inhibitors of subtilisin, three other bacterial proteases, trypsin and chymotrypsin were detected by a negative-staining technique based on the chromogenic substrate, acetyl-D, L-phenylalanine-2-naphthylester. In addition to complex patterns of trypsin and chymotrypsin inhibitor zones, most legume seeds examined exhibited one major and one minor inhibitor of subtilisin, and the other bacterial proteases. However, in cowpea and black gram only the major, and in lentil and soya bean only two minor subtilisin inhibitor zones were detected. Isoelectric points of the subtilisin inhibitors range from 4.4 to 5.9. The inhibitor patterns obtained by isoelectric focusing of extracts mixed with bacterial protease confirmed that inhibitors of the bacterial proteases represent a new type of inhibitor different from the well characterised trypsin and chymotrypsin inhibitors.     

特定致腐菌对冷却猪肉的致腐作用机制

为研究特定致腐菌在冷却猪肉中的致腐作用机制,将韩国假单胞菌PS1（Pseudomonas koreensis PS1）和梭状芽孢杆菌J4（Bacillus fusiformis J4）分别接种到新鲜冷却猪肉中进行致腐实验,以不接种致腐菌的猪肉为对照,于4℃条件下贮藏,每隔1 d测定各组肉样的细菌总数、总挥发性盐基氮（total volatile basic nitrogen,TVB-N）含量、蛋白质含量以及脂肪酶、弹性蛋白酶、胰蛋白酶和胶原蛋白酶活力。结果表明：各组肉样细菌总数均呈现先上升后稳定的趋势,贮藏第5天,接种P. koreensis PS1、B. fusiformis J4的肉样细菌总数均超过8.0（lg（CFU/g））,明显大于对照组;接种特定致腐菌的2组肉样脂肪酶、弹性蛋白酶、胰蛋白酶和胶原蛋白酶活力均高于对照组,且呈现先上升后降低趋势,至贮藏第7天,弹性蛋白酶、胰蛋白酶和胶原蛋白酶活力均达到峰值,而脂肪酶活力在贮藏第5天达到峰值;同时,各组肉样蛋白质含量不断降低,产物TVB-N含量明显增加,贮藏第5天,接种特定致腐菌的2组肉样TVB-N含量均超出15mg/100g,明显高于对照组。