INFLUENCE OF HARVEST SEASON ON THE PROTEOLYTIC ACTIVITY OF HEPATOPANCREAS AND MANTLE TISSUES FROM JUMBO SQUID (DOSWICUS GIGAS)

Jumbo squid (Dosidicus gigas) size and protease activities were evaluated after harvest in April (A) and November (N). A were smaller (560±120 g vs 6040±1130 g) and hepatopancreas was a larger percent of body weight (8.3±2.5 vs 4.6±2.1). Mantle from A had lower water (73.9±l.l vs 79.1± 1.2) and higher protein (29.0±1.1 vs 23.1±0.8). Lipid and protein contents of N and A hepatopancreas tissues did not differ (P < 0.05). Azocaseinolytic, trypsin‐like, chymotrypsin‐like, aminopeptidase, and carboxypeptidase activities were detected in mantle (ME) and hepatopancreas extracts (HPE). HPE and ME from N had higher activity than A for all substrates (P < 0.05). With azocasein substrate, HPE activity from A had a pH optimum of 5 ‐ 6 and a temperature optimum of 70C, whereas HPE from Nhad highest activity at pH 9 and 40–70C. ME from A had maximum proteolytic activity at pH 6 and 60C, whereas that from N had maximum activity at pH 10 and 40C. SDS‐PAGE zymograms of HPE from A and N revealed different patterns of activity and 1 and 2 major protease zones, respectively.     

CHARACTERIZATION OF PROTEINASE CLASSES IN LANGOSTILLA (PLEURONCODES PLANIPES) AND CRAYFISH (PACIFASTACUS ASTACUS) EXTRACTS

Proteinases (EC 3.4.21-24) in langostilla extract and crayfish hepatopancreas were classified using site specific effectors or chelators for either serine, cysteine, aspartic or metallo classes. Azocasein hydrolysis by langostilla and crayfish preparations was inhibited 50% and 40%, respectively, when assayed in the presence of serine proteinase inhibitor, phenylmethylsulfonyl fluoride. A similar degree of inhibition was observed with a trypsin inhibitor. However, no inhibition occurred with a chymotrypsin inhibitor. Less inhibition of azocasein hydrolysis, i.e., 20% and 14%, respectively, resulted with 1,10 phenanthroline. Inhibitors for cysteine and aspartic proteinases did not reduce the azocasein hydrolysis activities significantly. The amidase activity, with N-benzoyl-DL-Arg-p-nitroanilide as substrate was more sensitive, in both extracts, to serine proteinase inhibitors than the azocasein hydrolase activity. Results showed the presence of serine proteinases, i.e., trypsin but not chymotrypsin, as the major component and some minor activity of metallo dependent proteinases in the decapods extracts. Zymograms obtained after SDS-PAGE showed a dozen proteinases in each extract. Some of them were inhibited by a serine proteinase inhibitor and two to three were inhibited by 1,10 phenanthroline, supporting the results of the test tube proteinase activity assays. Furthermore, the reported technique for zymograms allowed direct comparison between extracts and provided information about their composition and the molecular weight of the targeted enzymes.     

PROTEOLYTIC ACTIVITY OF CRUDE ENZYME EXTRACTS OF SQUID ILLEX ARGENTINUS LIVER

The extract obtained from the acetone powder of frozen stored squid liver had optimum proteolytic activity against hemoglobin at pH 2.6 to 4.0. About 90% of the activity at pH 3.0 was inhibited by pepstatin and about 15% was inhibited by phenylmethanesulfonyl fluoride or iodoacetamide. The activity was increased by dithiothreitol and cysteine by about 55 and 40%, respectively. At pH 3 the enzyme extract had a temperature optimum of 45 to 50C; at 5C the activity was about 15% of that at 45 to 50C. At pH 7 the optimum activity was at about 45C. Heating of the extract without substrate at pH 3 and 7 caused a rapid decrease in proteolytic activity above 35 and 30C, respectively. A water extract of the liver was used to treat isolated bovine myofibrils at pH 5.5 and 0C. After 20 h a fragment of slightly lower m.w. than that of the myosin heavy chain was detected. After treatment at pH 7 and 0C, little degradation of the myofibrillar proteins was evident. At 20C the myofibrils were hydrolyzed to several products.     

PURIFICATION AND CHARACTERIZATION OF AMINOPEPTIDASE FRACTIONS FROM SQUID (ILLEX ILLECEBROSUS) HEPATOPANCREAS

Atlantic short finned squid hepatopancreas (HP) aminopeptidases (APs) were partially separated from endoproteinases to determine their usefulness in accelerating Cheddar cheese ripening. Squid HP endoproteinases were predominantly cysteine proteases. The main peptidases identified in squid HP were APs. Several of the APs identified were metalloproteases and were activated by Ca²⁺, Mn²⁺, Zn²⁺ or Mg²⁺ salts. Squid HP homogenate was held at pH 7 at OC for 20 h, incubated with 5 mM ZnSO₄, fractionated by ammonium sulfate (20–80%) and dialyzed against 25 mM ZnSO₄. The procedure resulted in a 3–186 fold increase in the ratio of exopeptidase to endoproteinase activity with different AP substrates. Recovery of specific APs ranged between 14–47%. The partially purified squid HP peptidases had a higher ratio of exopeptidase to endoproteinase activity than two commercial products with AP activity, Flavozyme and Neutrase.     

USE OF CYSTEINE PROTEINASE INHIBITORS FROM INJURED TOMATO LEAVES IN WHITING SURIMI

Cooking surimi paste from Pacific whiting results in a gel with poor texture due mainly to myosin degradation caused by a cysteine proteinase. Cysteine and serine proteinase inhibitors were isolated from injured and methyl jasmonate treated tomato leaves. Tomato cysteine proteinase inhibitor was stable at 60C but inactivated at 90C, making it suitable for use in surimi. Tomato proteinase inhibitors (TPI), having 7.9 papain inhibitor units, inhibited autolysis about 95% in 10 g of Pacific whiting surimi. Gel strength of Pacific whiting surimi was improved by adding only 0.0 27% of TPI to the surimi formulation. Addition of TPI did not affect the color of whiting surimi gel, while egg white needed to prevent gel weakening caused the gels to have more yellow hue (P<0.05). SDS-PAGE showed that myofibrillar protein degradation was prevented during cooking when 0.027% of TPI was included in the surimi. TPI extracted from tomato plants has potential for use as food grade additive in Pacific whiting surimi.     

ANTIOXIDANT ACTIVITY OF ROSEMARY AND SAGE EXTRACTS IN RAPESEED OIL

Rosemary and sage leaves were extracted with hexane, acetone, ethyl acetate and ethanol. Yields of extracts and of carnosic acid contents were higher in solvents of medium polarity. Both carnosic acid and carnosol showed medium activity in deodorized rapeseed oil, about the same as that of butylated hydroxyanisole. Carnosic acid was moderately more active than carnosol, and the resulting oxidative stability of extracts was correlated with the content of carnosic acid, but not with the content of carnosol. The antioxidant activity was about the same in rapeseed oil as in sunflower oil, but substantially lower than that in lard. Antioxidant activities, expressed as protection factors, were higher under Schaal oven test conditions at 40 or 60C than when determined in Oxipres at 100C. The dependence of the concentration of antioxidants and the protection factor showed similar negative exponential relations in all tests.     

DEHYDROGENASE ACTIVITY FOR HIGHER ALCOHOLS IN CELL-FREE EXTRACTS OF SACCHAROMYCES CEREVISIAE

Alcohol dehydrogenase activities were determined in cell-free extracts of Saccharomyces cerevisiae with fusel oil-alcohols and other higher alcohols (including straight and branch-chain aliphatic alcohols, cinnamyl alcohol and phenethanol) as substrates. A fluorometric measurement of the formation of reduced coenzyme (NADH) was needed because of the necessity for increased sensitivity with the required use of very low concentrations of substrates. For the first time, dehydrogenase activities were measured in cell-free extracts for active-pentanol, n-hexanol and n-heptanol as substrates. Optimal molar concentration of the substrates was found to decrease with increasing carbon number. Activity measurements were made in extracts of yeasts grown under various conditions of oxygen induction and catabolite repression. Dehydrogenase activities for all substrates were higher in extracts of yeast grown aerobically as compared to those grown anaerobically. A comparison of the activities with the fusel oil-alcohols as substrates suggests a plurality of alcohol dehydrogenase activities in the last enzymic step in fusel oil formation.     

ANTIOXIDANT ACTIVITY OF PHENOLIC EXTRACTS OF EVENING PRIMROSE (OENOTHERA BIENNIS): A PRELIMINARY STUDY

Phenolic compounds from ethanolic extracts of evening primrose ( Oenothera biennis ) were separated into ten fractions using Sephadex LH-20 column chromatography. For individual fractions, UV spectra were recorded and the content of total phenolics determined. Antioxidant activity of each isolated fraction was examined in a β-carotene-linoleate model system. Fractions were further characterized for the number of phenolic compounds, presence of proanthocyanidins and their antioxidant activity by TLC analysis. Strong antioxidant properties were noted for fractions with high content of total phenolics (fractions IV-X). Vanillin-positive compounds were observed in fractions VI-X and one compound was tentatively identified as (+)catechin/(−)epicatechin. From fraction IV, two phenolic compounds were separated by preparative TLC; compound A was most likely an isoflavone and the UV spectrum of compound B was similar to that of 2-hydroxychalcone.     

CHARACTERISTICS OF TMAO DEGRADING SYSTEMS IN ATLANTIC SHORT FINNED SQUID (ILLEX ILLECEBROSUS)

The decomposition of trimethylamine oxide (TMAO) to dimethylamine (DMA) was studied in the mantle of squid, Illex illecebrosus. The production of DMA in either whole or ground mantle stored at – 20°C was low or nonexistent. However, significant DMA was produced from TMAO during heating of squid or squid extracts. Conversion of TMAO to DMA was shown to reside in the soluble fraction of the squid mantle which catalyzed the breakdown of TMAO in an assay system consisting of FeCl₂ and ascorbate at room temperature. On the average, some 10–15% of the activity was accounted for by a high molecular weight, thermolabile substance, presumably an enzyme. The rest of the activity was in a small molecular weight, thermostable fraction. The low molecular weight fraction produced significant quantities of TMA as well as DMA, had low activity with flavin-NADH, and was not activated by methylene blue.     

ANTIMICROBIAL ACTIVITY OF PU-ERH TEA EXTRACTS IN VITRO AND ITS EFFECTS ON THE PRESERVATION OF COOLED MUTTON

The inhibitory effect of the extract of pu-erh tea (PETs) against six strains by paper-diffusion methods and the characterization of the mutton treated with different concentrations of the PETs during storage at 7 ± 1C were evaluated in this article. The results showed PETs could significantly inhibit the growth of Listeria monocytogenes, Salmonella typhimurium, Streptococcicosis faecalis, Escherichia coli and Bacillus anthraci, and their minimum inhibitory concentration were 0.07, 0.18, 0.50, 0.42 and 0.48 mg/mL, respectively. PETs showed weak inhibition for S. aureus. The cooled mutton treated with PETs resulted in suppression in the increase of total volatile base nitrogen and thiobarbituric acid reactive substance, inhibition of Enterobacteriaceae and Pseudomonas growth. The cooled mutton treated with 2% PETs showed highest color and overall acceptability. These results suggest the high potential in using PETs as a means of enhancing freshness and quality in cooled meats.

PRACTICAL APPLICATIONS

Antimicrobial activities of microbial fermented tea are much less known than its health beneficial properties. Pu-erh tea is consumed by people as a daily healthy drink all over the world, extracts of pu-erh tea may be safe to use in food systems to extend the shelf life. With the trend of increasing use of natural and biological preservatives in food products, natural antimicrobial agents from pu-erh tea may offer an innovative and interesting measure for such applications.     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM OYSTER (CRASSOTREA GIGAS)

Proteases in oyster (Crassotrea gigas) were extracted with 10 mM Tris-HCl buffer solution and purified by ammonium sulfate fractionation, Sephadex G-150 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Three fractions with caseinolytic activity, named I, II and III, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The three proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that protease I was a carboxypeptidase A-like enzyme; II and III were trypsin-like enzymes. The optimal pH of protease I for hydrolysis of hippuryl-L-phenylalanine was 9.0, II and III for hydrolysis of p-toluenesulfonyl-L-arginine methyl ester (TAME) was 8.0. The temperatures which inactivated 50% of enzymes were 78°C for protease I in 30 min; 50 and 52°C for protease II and III, respectively, in 5 min. The molecular weights of proteases I, II and III were 23,000, 34, 400 and 31, 000, respectively.     

CHANGES IN MYOFIBRILLAR PROTEINS AND LIPIDS OF SQUID (ILLEX ARGENTINUS) DURING FROZEN STORAGE

The biochemical properties of actomyosin (AM) and lipid composition of the mantle from frozen‐stored whole squid were investigated. Irrespective of the sex of specimens, during the first months of storage, there was a trend of decreased protein solubility, reduced viscosity and enzymatic activities of AM. A significant decrease (P < 0.05) in the relative percentage of the myosin heavy chain and a significant increase (P < 0.05) in those of paramyosin and the 155‐kDa component were also observed. After freezing, phospholipids (PL), sterols (ST) and triacylglycerols (TAG) represented 38.6, 29.1 and 21.2%, respectively, of total lipids (TL) from the mantle of male squid. Free fatty acids (FFA) plus diacylglycerols constituted only 11% of TL. TL extract from the mantle of female squid had a higher percentage of PL and had lower ST. In the frozen‐stored male and female squid, TAG were hydrolyzed earlier than PL. At zero time of storage, the relative percentages of saturated and unsaturated fatty acid in TL extracts from the mantle of male and female squid were 39.1, 59.2 and 38.7, 61.1, respectively. Irrespective of sex, the saturated FA fraction significantly (P < 0.05) decreased and the unsaturated one significantly (P < 0.05) increased after 8 months of frozen storage.     

CHARACTERIZATION OF PROTEOLYTIC ACTIVITY IN OCTOPUS (Octopus vulgaris) ARM MUSCLE

A new proteolytic activity assay was devised to avoid the interference of paramyosin which causes gelling during the enzymatic assay. Extremely high autolytic activity was observed in octopus arm muscle, which was 40–500 fold higher than those of various other fish species. The proteinase was inhibited strongly by leupeptin and iodoacetic acid and, to a lesser degree, by transepoxysuccinyl-L-leucylamino (4-guanidono)butane (E-64), indicating the class as a thioi proteinase. The proteinase exhibited optimum activity at pH 2.5 and 40C, although it contained a sulfhydryl group in the active site. Myosin heavy chain was the primary myofibrillar protein which was hydrolyzed during the autolysis of octopus arm followed by paramyosin. Actin showed no signs of hydrolysis during the incubation of up to 8 h. Due to its high affinity for myosin, the enzyme activity should be controlled during processing octopus to ensure the functionality of myosin.     

菜籽多糖对四氧嘧啶致糖尿病小鼠降血糖作用的研究

研究菜籽多糖（RSPS）在四氧嘧啶致糖尿病小鼠体内的降血糖作用。腹膜注射200rag／ks四氧嘧啶造小鼠糖尿病模型，腹膜注射给药12d。测定给药前后小鼠血糖值、小鼠体重与免疫器官重量、血清抗活性氧单位与肝匀浆MDA含量。75mg／kg·d与400mg／kg·d RSPS可使糖尿病小鼠血糖分别下降18．38％与24．62％，能显著增加糖尿病小鼠胸腺与脾脏指数，提高血清抗活性氧单位，降低肝匀浆MDA含量。400mg／kg·d RSPS对正常小鼠血糖无显著影响。研究结果表明RSPS在糖尿病小鼠体内具有降血糖、增进免疫与抗氧化作用。     

桑叶多酚氧化酶活性的研究

采用分光光度法和扫描电子显微镜法对桑叶多酚氧化酶(PPO)的结构、活性以及不同抑制剂的抑制效果进行研究.结果表明:桑叶多酚氧化酶为纤维状蛋白;它的最适pH值为7.0;最适温度为40℃;在相同抑制剂浓度下,总体抑制效果为:抗坏血酸>亚硫酸氢钠>柠檬酸>氯化镁>氯化钠.