Preventive Effect of Sugars on Denaturation of Fish Protein during Frozen Storage

The cryoprotective effect of glucose was increased in proportion to the concentration of glucose. Maltotriose was most effective on ATPase activity, protection of SH-group and solubility of actomyosin during frozen strage. It was found that hydroxyl group of sugars was an important factor for preventive effect of cryoprotectant, and that the structure of sugars was also an important factor. The preventive effects of sorbitol and glucose on the denaturation of carp muscle actomyosin during frozen storage were reported by Takashi et alp and the effects of some amino acids, peptides, acetyl amino acids, carboxylic acids and carbohydrates were investigated and discussed availably by Noguchi.2) He summerized the property of cryoprotectants from view point of molecular structure. This report describes about the preventive effects of derivatives of glucose and about the different effects due to the difference in molecular weight, number of polar group and structure of molecules of sugar. Experimental Procedure Preparation of actomyosin from carp muscle. An alive breeding carp, Cyprinus carpis, about 750 g of body weight and about 25 cm of body length, was killed instantly by electric shock in a refrigerator at about 2°C. Immediately the muscle was cut off and minced with a meat grinder. Then the actomyosin was prepared according to procedure of Table 1. All operations were performed at about 4°C. Procedure of frozen storage of carp muscle protein. The protein solution which was prepared according to the Table 1 was diluted to the concentration of 3-5 mg protein/m/. with 0.6 M KC1, and this solution was the sample for freezing storage experiment. The definite volume of additives was added to the sample, the solution was carefully stirred to avoid bubbling at about 2°C for 16 hr. At the same time, additive free sample as a control was also carried out according to the same procedure. The aliquot of 15 m/ of these samples was placed respectively into test tubes, stored in a deep freezer at —20°C for various periods, namely 2, 4 and Laboratory of Food Chemistry, Faculty of Fisheries, University of Kagoshima. 92 Mem. Fac. Fish. , Kagoshima Univ. Vol. 25, No. 1 (1976) . Table 1. Procedure of the preparation of carp actomyosin. Coarsely minced muscle of carp (50 g) washed 2 times with phosphate buffer (pH 7.5, 1=0.05) —centrifuged at 5000 G for 20 min. Residue —— extracted with 5 volumes of 0.45 M KCl-phosphate buffer (pH 7! 5) centrifuged at 5000 G for 20 min. Supernatant diluted slowly twice with 10 volumes of cold water centrifuged at 5000 G for 20 min. Precipitate dissolved in 1.2 M KC1 with equal volume of the precipitate stirred for 3 hr with magnetic stirrer centrifuged at 15000 G for 30 min. Supernatant (crude actomyosin) 8 weeks. After the storage for appropriate periods, these samples were thawed at room temperature, and the solubility, ATPase activity, the number of thiol group and the viscosity of the protein were measured. They were given as indicator of protein denaturation. Determination of ATPase activity. Assay mixture contained 2 m/ of 62.5 mM Tris-maleate buffer (pH 6.8), 2 ml of 12.5 mM CaCl2 0.5 ml of 10 mM ATP and 0.5 ml of actomyosin suspension. The enzyme reaction was initiated by the ad dition of ATP at 25°C, stopped by the addition of 2 ml of 15% perchloric acid and filtered with filter paper. The inorganic phosphate in the filtrate was determined by the method of Rockstein et a/. 3) One enzyme unit was defined as the amount of enzyme that catalized the release of one //mole of inorganic phosphate under the assay conditions per minute. Determination of residual thiol group. Thiol group of actomyosin was determined by the method described by Matsumoto.4) Determination of viscosity. Viscosity of actomyosin was measured by the use of viscometer, Ostwald's viscometer. Determination of N-acetyl-D-glucosamine. N-acetyl-D-glucosamine was de termined by the method described by Fukui.5) Determination of hydrophobic area of protein. Hydrophobic area of protein was determined by using the fluorescent pigment, l-anilinonaphthaleno-8-sulfate (ANS) at the concentration of 25 fig per ml of water. The intensity of fluorescent of standard solution (9 m/ of n-propanol+1 ml of fluorescent pigment solution) was adjusted to 100%. Then the intensity of fluorescence of the sample (1 ml of protein solution+8m/ of 0.6 M KC1, pH6.8+lm/ of fluorescent pigment solution) was read. Ooshiro •Hironaka •Hayashi : Preventive Effect of Sugars on Fish Protein Denaturation 93 Paper chromatography. Components of polymers of glucose were analyzed and identified by paper chromatography using the solvent system; pyridine: n-butanol: water=4: 6:3. After development, papers were sprayed to detect components of polymers with the solvent; 1.7 g phthalate and 0.93 g aniline in 100 ml of n-butanol saturated with water. Determination ofglucose. Glucose wasdetermined by usinganthrone reagent.6) Determination of protein. Protein was determined from the absorbance at 280 m//. Preparation of polymers of glucose. As polymers of glucose obtained as commercial reagents were dimer and trimer, polymers composed of more than four molecules of glucose was prepared from the partial decomposition of starch by aamylase and ^-amylase, which was a kind gift from Nippon Starch Co., Ltd. The separation of polymers of glucose was carried out by adsorption chromatography with activated charcoal. Preparation of 1, 2, 6-acetyl-D-glucosamine. The mixture of 50 g of acetic anhydride and 50 g of pyridine anhydride was cooled at 0°C and 10 g of N-acetyl-Dglucosamine was added to the mixture. The mixture was allowed to stand at room temperature for 2 or 3 days. The reactant was added to ice-water (200 ml) with stirring contineously and extracted 3 times with 150 ml of chloroform. The extractant was washed successively 3 times with 150 ml of 1 N HC1, and 2 times with 150 m/ of water, and dried over anhydrous sodium sulfate. The solution was con centrated under reduced pressure with rotary evaporator. The concentrated was dissolved in a few volume of hot alcohol. Ether was added to the solution and the crystal was deposited slowly. The crystal was recrystallized from same solvent. The amount of recovery was 61%. The melting point of the crystal was from 138 to 139°C and [a]1,,6'was +90°. Preparation of a-pentaacetyl-D-glucose. The mixture of 50 g of acetic anhydride and 65 g of pyridine anhydride was cooled at 0°C. Glucose (10 g) was added to the mixture and the mixture was stirred until glucose was dissolved com pletely. Then it was allowed to stand at room temperature for 18 hr. The deposited crystal was recrystallized from 95% ethyl alcohol. The yield of product was 19 g. The melting point of the crystal was from 112 to 113°C and [a]£6 was +102°. Results Effect of the concentration of glucose. In order to clarify the preventive effect -of theglucose -added to the actomyosin solution at various concentration, glucose concentration was varied as follows; 0.2,2.0,20.0 and 200.0 mM. These solutions^of protein containing glucose were frozen at —20°C and stored for 2, 4 and 8 weeks. After thawing at room temperature, solubility, ATPase activity, numbers of thiol group and reduced viscosity of protein were determined. Results were shown in Table 2. 94 Mem. Fac. Fish. , Kagoshima Univ. Vol. 25, No. 1 (1976) Table 2. Effect of the concentration of glucose on the prevention of denaturation of actomyosin. Protein Solubility