JP5410612B2 - Squid viscera extract and its preparation method, mixture and use as marine fishery feed protein source - Google Patents

Description

  The present invention relates to novel oligopeptides, and more particularly to oligopeptides extracted from squid viscera and methods for their preparation, as well as mixtures of these substances and their use as feed protein sources in marine aquaculture.

  With the rapid development of Japan's pelagic fishery, squid catches have increased year by year, and the squid catch in 2009 has exceeded 300,000 tons, making it the main fishery processing material in Japan. However, the technical level of squid processing in Japan is low, and after obtaining processed products such as pine squid, dried fish, squid salty, canned foods and seasonings, generally 20% to 30% of waste (internal organs, heads, feet, However, these wastes are rich in nutrients such as proteins, fats and polysaccharides, according to Zhuhai City Immigration and Quarantine Bureau's research (Non-patent Document 1). Each 100 g of squid viscera contains 21.15 g of fat, 21.24 g of protein, 51.46 mg of calcium, 609.07 μg of iron, 95.88 μg of phosphorus and the like. However, these substances are extremely susceptible to decay and alteration, and contain heavy metal cadmium which is harmful to the human body. Usually, these waste disposal methods are buried, but this is not only a huge waste of fishery resources, but also the problem of environmental pollution. Therefore, how to effectively treat squid waste, increase its value and use it comprehensively has become more important to the people and society.

  Wang Ayari et al. (Non-patent Document 2) of the Institute of Fisheries Science of China (Non-Patent Document 2) studied the feeding attraction of squid visceral liquefied protein to lobster shrimp. I found something stronger. In the comprehensive research on the use of squid internal organs, Wang Jianchu et al. (Non-patent Document 3) of the Kamikasui Products Processing Technology Development Center conducted hydrolysis using squid internal organs and other wastes by self-enzyme, Along with the development of a hydrolyzate, the challenge of removing cadmium from the hydrolyzate was solved. Liu Chun, et al. (Non-patent Document 4) of the Aquatic Product Safety Laboratory at China University of Marine Science and Technology (Non-Patent Document 4) used a variety of proteases to study squid visceral protein using the amino acid yield as the main indicator in the study of enzymatic degradation of squid visceral protein. As a result, the neutral protease, alkaline protease, trypsin, and papain have a good degradation effect, and the amino acid conversion rate reaches 50% or more. In Zhejiang University of Commerce and Industry's patent, Zhujiang Sakae et al., Which is a kind of integrated utilization technology of American giant squid viscera (Chinese patent application No. 200610053372.6), procedures for steaming, liquefaction, liquefaction, heating and centrifugation of squid viscera From the lower squid polypeptide obtained by the above, the squid paste to be used as a fish animal feed protein source was prepared, and the diet test on the prawns was carried out. It has been shown that survival and body weight increase. In the method of producing an aquatic feed feeding attractant using Chinese squid organs (Chinese patent application No. 20080081073.4), which is the patent of Soh Soebo of Fujian Yin Shin Biological Technology Co., Ltd. By performing natural enzymatic decomposition fermentation, low-temperature vacuum concentration is performed twice at 80 to 90 ° C. and 60 to 65 ° C. for the precipitate after separating the oil.

  Existing feed protein source production techniques mainly involve high-temperature steaming and concentration after enzymatic degradation, but in these processes, protein is easily denatured, collagen is produced, and nutritional components are reduced. In addition, the attractiveness of the product to aquatic animals is greatly impaired. Even if the method of Sozoen et al. Is used, the SJN2-2000 double-effect evaporator is used to perform secondary concentration on the enzyme decomposition solution, the evaporation temperature is controlled to 90 ° C or lower, and the concentration temperature is greatly reduced. The difficult problem cannot be solved fundamentally.

Wu Jin, “Agricultural Products Processing” (China), 2007, (8): 94-96 Wang Sairi et al., “Aquaculture” (China), 2003, Vol. 24, No. 5, p. 40-41 Wang Jianchu et al., “Chinese Marine Drugs” (China), 1999, Volume 1, p. 55-58 Liu Chun, et al., “Food Industry Technology” (China), 2004, Vol. 9, No. 25, p. 83-86

  The object of the present invention is to abandon the high-temperature steaming method and extract a novel oligopeptide from the squid viscera using an ultrafiltration membrane (UF membrane), and another object is a method for preparing this oligopeptide And to develop a mixture containing this oligopeptide and its use.

We degreased viscera (Todarodes pacificus) viscera with an organic solvent, followed by enzymatic degradation with Alcalase (registered trademark) and trypsin, and then filtered the degreased enzymatic digestion solution using an ultrafiltration membrane, followed by gel chromatography. papermaking rectification in chromatography, after obtaining an oligopeptide comprising the amino acid structure fragments -ILGGSDPKHYTG-, this was uniformly stirred with potassium sorbate, dried. The mixture obtained by granulating the oligopeptide of the present invention, soybean meal powder, corn meal, starch, fish meal, etc. according to the blending ratio can be used as a protein source for marine aquaculture feed and is very good for fish, shrimp, shellfish, etc. There is a feeding attraction effect. This achieves the object of the present invention.

The oligopeptide extracted from the squid viscera of the present invention contains the amino acid structural fragment -ILGGSDPKHYTG-, has a relative molecular weight range of 1400 to 5800, and is characterized by being prepared by the method described below. (1) Crush the squid viscera, add water and mix uniformly, degrease with petroleum ether or n-hexane, or a mixed solvent of petroleum ether and n-hexane, remove the organic solvent layer, and keep the aqueous layer at room temperature 1 to 3 hours, heat at 50 to 60 ° C. for 1 to 3 hours, add water at a temperature of 50 to 60 ° C., adjust pH to 8.0 to 8.5, and then at a temperature of 50 to 60 ° C. In the holding state, after enzymatic degradation with Alcalase (registered trademark) for 1 to 3 hours at a rate of 2000 to 3000 units per gram of raw material, enzymatic degradation with trypsin at a rate of 2000 to 3000 units per gram of raw material for 1 to 3 hours, Insoluble substances are removed to obtain an enzyme decomposition solution. (2) Oligopeptide containing amino acid structural fragment -ILGGSDPKHYTG- by filtering the enzymatic degradation solution obtained in step (1) through an ultrafiltration membrane with a molecular weight cut off of 18000 Da, followed by separation and purification by gel chromatography Get.

  The oligopeptide extracted from the squid viscera of the present invention can be added with a preservative such as potassium sorbate, and 0.025 to 0.050 g of potassium sorbate is added per 1000 g of the oligopeptide obtained in the procedure (2). It is preferable to uniformly mix and dry. In the test preparation, it is advantageous for cost control when the purity of the oligopeptide is 51% to 63%.

The method for preparing an oligopeptide extracted from the squid viscera of the present invention is characterized by including the following procedure.
(1) Crush the internal organs of squid, add water and mix uniformly, degrease with petroleum ether or n-hexane, or a mixed solvent of petroleum ether and n-hexane, remove the organic solvent layer, and leave the aqueous layer at room temperature Let stand for 1 to 3 hours below, heat at 50 to 60 ° C. for 1 to 3 hours, add water at a temperature of 50 to 60 ° C., adjust pH to 8.0 to 8.5, then adjust to 50 to 60 ° C. In the temperature holding state, after enzymatic degradation with Alcalase (registered trademark) for 1 to 3 hours at a rate of 2000 to 3000 units per gram of raw material, enzymatic degradation with trypsin at a rate of 2000 to 3000 units per gram of raw material for 1 to 3 hours Insoluble substances are removed to obtain an enzyme decomposition solution.
(2) Oligopeptide containing amino acid structural fragment -ILGGSDPKHYTG- by filtering the enzymatic degradation solution obtained in step (1) through an ultrafiltration membrane with a molecular weight cut off of 18000 Da, followed by separation and purification by gel chromatography Get.

  In the preparation method of the oligopeptide extracted from the squid viscera of the present invention, a preservative such as potassium sorbate can be added to the oligopeptide obtained in the procedure (2), and 0.025 potassium sorbate per 1000 g of the oligopeptide. It is preferable to add ~ 0.050g, and to mix uniformly and to dry.

Molecular mass calculation method for oligopeptides extracted from squid viscera:
logMw = −bKav + c
Here, the effective partition coefficient Kav indicates the degree of exclusion, Mw indicates the molecular mass of the substance, b and c are constants and are related to the properties of the gel column itself, and b and c can be calculated from a standard protein sample. it can.

The operations of gel chromatography separation / purification and molecular mass measurement are as follows.
(1) Separation and purification are performed using medium and large gel chromatograph columns. After degreasing the viscera of cuttlefish in accordance with the above-mentioned method of the present invention, enzymatic decomposition is performed, and the enzymatic decomposition solution of squid viscera is filtered through an ultrafiltration membrane having a molecular weight cut off of 18000 Da. Separation using medium and large gel chromatograph. At this time, gel chromatograph type number: Sephadex G-25, column type number: 200 × 10 cm, buffer solution is 0.05 mol / L phosphate buffer solution (0.16 mol) / L NaCl included), pH = 7.0, solvent flow rate is 10 mL / min, bed volume is about 12000 mL, sample is prepared in a solution of 50-100 mg / mL concentration, and 0.45 μm microfiltration membrane is used. Filter, take 500-1000 mL, inject sample, separate and purify, and analyze and measure by the method described below.
(2) The molecular mass is measured using a small gel chromatograph. Analysis condition selection: gel chromatograph type number: Sephadex G-25, column type number: 100 × 1 cm, buffer solution is 0.05 mol / L phosphate buffer solution (containing 0.16 mol / L NaCl), pH = 7. 0, solvent flow rate 0.20 mL / min, measurement wavelength 280 nm, bed volume about 60 mL.
(3) A standard protein sample is prepared in a 5 mg / mL solution, filtered through a 0.30 μm microfiltration membrane, 0.5 mL is taken, and the sample is injected, and three corresponding points of (Kav, logMw) (28 .5, 4.16), (53.7, 3.13), (74.9, 2.26) and obtained by calculation: log Mw = −0.041 Kav + 5.33 (R ² = 0.9998) )
(4) After the enzyme hydrolyzate of squid viscera separated and purified in step (1) is filtered through a 0.30 μm microfiltration membrane, the concentration is adjusted to 10 mg / mL. Using a small gel chromatograph, 0.5 mL was sampled and injected, and the linear (Kav, log Mw) points of the oligopeptide extracted from the squid viscera were respectively (53.3, 3.15), (38.2, 3.76) was obtained and the corresponding molecular masses were calculated to be 1400 and 5800.

  The mixture containing the oligopeptide extracted from the squid viscera of the present invention is calculated with a total mass fraction of 100%. The oligopeptide extracted from the squid viscera is 6% to 8%, soybean meal powder 50% to 60%, and fish meal 3%. It consists of ~ 6%, cornmeal 27% ~ 33%, and granulating starch 2% ~ 5%. The soybean meal powder, fish meal, corn meal and starch for granulation used are for marine feed and are commercially available.

  In the preparation method of the mixture of the present invention, the oligopeptide extracted from the squid viscera, soybean meal powder, fish meal, and corn meal are uniformly mixed according to the above ratio, and the ratio of the blending material: water = 1: 4 (mass ratio). An appropriate amount of water is added, mixed uniformly, and spray-dried to obtain a dry extract powder, and granulated by adding starch for granulation to the dry extract powder according to the blending ratio, and dried to obtain a mixture.

  The mixture of the present invention can be used as a protein source for marine aquaculture (including marine fish, shrimp, shellfish, etc.) feed, and is particularly suitable as a feed protein source for marine marine fish.

  The temperature in the preparation process of the oligopeptide extracted from the squid viscera of the present invention does not exceed 60 ° C., and when dried, it is dried using a spray drying device and is effective in a short time, so that this substance does not hydrolyze or denature. Guarantee. The mixture of the present invention has a strong diet-attracting property for fish, and the oligopeptide extracted from the squid viscera, which is the main component, has a strong fishy odor and is optimal as a protein source for aquaculture feed. When a marine fish is cultivated using the mixture provided by the present invention, the amount of fish consumed can be greatly increased, and the unit weight of the individual fish can be increased.

Specific implementation method:
The following examples are further illustrations of the present invention and are not a limitation on the present invention. Main raw materials, main instrumentation, main biological and chemical reagents and the like in the examples are as follows.

Main raw materials:
The squid (Todarodes pacificus) internal organs were collected from the Guangzhou Huangsha Seafood Market, stored at -15 ° C, and thawed at room temperature 12 hours before use. “Oe 牌” regular fish feed was purchased from Shanghai Oe (Group) Co., Ltd. The used soybean meal powder and corn meal were purchased from Jining Shuanghua Industrial Trade Co., Ltd., and the used fish meal was purchased from Rongcheng New Hope Fish Powder Co., Ltd.

Main instrument equipment:
Fermenter: LABFORS laboratory tabletop small fermenter, production area; Swiss homogenous equipment: Preliminary A8G type, production area; Shenyang city stationary type centrifuge: Bechman J2-Hs high-speed freezing centrifuge, production area; US medium / large gel chromatograph model number: Sephadex G-25, column model number: 200 × 10 cm, buffer is 0.05 mol / L phosphate buffer (containing 0.16 mol / L NaCl), pH = 7.0, solvent flow rate 50mL / min, bed volume about 12000mL
835-50 amino acid automatic analyzer (Hitachi, Japan)
Chromatograph Waters 2690, Mass Spectrometer Waters Platform ZMD 4000

Main biological and chemical reagents:
Trypsin: 1 million units (enzyme activity) / g, Dalian Bonded Zone Hirogoe Biotechnology Co., Ltd., product number: RM00102
Alcalase (registered trademark) (Alcalase): 500,000 units (enzyme activity) / g, Novozymes organic reagent: Organic reagent purchased from Guangzhou Reagents Co., Ltd. (analytical reagent)
Inorganic reagents: Inorganic reagents purchased from Guangzhou Reagents (analytical reagents)
Pure water: All of the water used in the examples was pure water, and was adjusted with a MiniQ pure water preparation system.

Collect 20 kg of Japanese squid viscera, stir and crush it with a precious A8G homogenizer, add 2 L of water, add petroleum ether (20 L) with a boiling range of 60-90 ° C, stir and degrease, and remove the petroleum ether layer The aqueous layer was left at room temperature for 3 hours and heated in an oven at 50 ° C. for 3 hours. Add 8 L of water at a temperature of 50 ° C., mix evenly, adjust the pH to 8.5 with a saturated NaOH solution, adjust the pH to 8.5 with a 5 mol / L NaOH solution, and maintain the temperature at 50 ° C. In this state, the squid viscera was placed in a fermenter, 6 × 10 ⁷ units of Alcalase (registered trademark) was added at 3000 units / g of raw material, and enzymatically decomposed for 3 hours, and then 6 × 10 ⁷ at 3000 units / gK of raw material. Unit trypsin was added and enzymatically degraded for 3 hours. The insoluble material was removed by centrifugation at 5000 rpm using a Bechman J2-Hs high-speed refrigerated centrifuge. When the defatted enzyme digestion solution is filtered through an ultrafiltration membrane with a molecular weight cut off of 18000 Da, a component rich in oligopeptides is obtained, and then separated and purified by a medium / large Sephadex G-25 gel chromatograph, the amino acid structure An oligopeptide containing the fragment -ILGGSDPKHYTG- was obtained. 0.0059 g of potassium sorbate was added at a rate of 0.025 g of potassium sorbate per 1000 g of oligopeptide, and after drying, an oligopeptide extracted from the squid viscera was obtained.

  Take 80 g of oligopeptide extracted from squid viscera, 600 g of soybean meal powder, 30 g of fish meal, 270 g of corn meal, add 4 L of water, mix uniformly, and then spray dry to obtain dry extract powder for granulation Weighed 20 g of starch, granulated and dried to obtain a mixture.

Collect 20 kg of cuttlefish viscera and stir and crush it with a precious A8G type homogenizer, add 12 L of water, add 20 L of petroleum ether and n-hexane mixed solvent (1: 1), stir and degrease, petroleum ether The layer was removed and the aqueous layer was left at room temperature for 1 hour and heated in an oven at 60 ° C. for 1 hour. 8 L of water at a temperature of 60 ° C. was added, mixed uniformly, the pH was adjusted to about 8.0 with a saturated NaOH solution, and the pH was further adjusted to 8.0 with a dilute NaOH solution. With the temperature maintained at 60 ° C., the squid viscera was placed in the fermenter, 4 × 10 ⁷ units of Alcalase (registered trademark) was added and enzymatically decomposed for 1 hour, and then 4 × 10 ⁷ units of trypsin was added. Enzymatic degradation was allowed for hours. The insoluble material was removed by centrifugation at 5000 rpm using a Bechman J2-Hs high-speed refrigerated centrifuge. When the defatted enzyme digestion solution is filtered through an ultrafiltration membrane with a molecular weight cut off of 18000 Da, a component rich in oligopeptides is obtained, and then separated and purified by a medium / large Sephadex G-25 gel chromatograph, the amino acid structure An oligopeptide containing the fragment -ILGGSDPKHYTG- was obtained. 0.0078 g of potassium sorbate was added at a rate of 0.050 g of potassium sorbate per 1000 g of oligopeptide, and after drying, an oligopeptide extracted from the squid viscera was obtained.

  Weigh 60 g of oligopeptide extracted from squid viscera, 500 g of soybean meal powder, 60 g of fish meal and 330 g of cornmeal, add 4 L of water, mix uniformly, and then spray dry to obtain a dry extract powder for granulation. 50 g of starch was weighed out, granulated and dried to obtain a mixture.

  65 g of the oligopeptide extracted from the squid viscera of Example 1, 535 g of soybean meal powder, 30 g of fish meal, and 330 g of cornmeal were added and mixed with 4 L of water, mixed uniformly, and then spray-dried to obtain a dry extract powder. Then, 40 g of starch for granulation was weighed, granulated and dried to obtain a mixture.

  75 g of the oligopeptide extracted from the squid viscera of Example 2, 575 g of soybean meal powder, 50 g of fish meal, and 280 g of cornmeal were added and mixed with 4 L of water, mixed uniformly, and then spray dried to obtain a dry extract powder. Then, 20 g of starch for granulation was weighed, granulated and dried to obtain a mixture.

Amino acid composition analysis:
The oligopeptides extracted from the squid viscera obtained in Examples 1 and 2 were each put into a hydrolysis tube, a 6 mol / L hydrochloric acid solution was added, and the mixture was vacuum-sealed. Hydrolyzed at 110 ° C. for 24 hours, cooled, then fixed volume, filtered, steam dried, added 0.02 mol / L hydrochloric acid solution and placed in air for 30 min, 835-50 amino acid automatic analyzer (Nippon Hitachi, Ltd.) ) Was used to measure the content of amino acids other than tryptophan and the following results were obtained.
I: L: G: S: D: P: K: H: Y: T = 1.05: 1.02: 3.01: 0.96: 0.98: 1.00: 0.97: 0. 98: 0.95: 1.00

Measurement of molecular mass using liquid chromatography / mass spectrometer (LC-MS):
Liquid chromatographic conditions: chromatograph Waters 2690, detector: Waters 996, analytical column Lichlorosphere C-18 (2.6 × 250 mm), mobile phase: methanol-water-0.5% acetic acid gradient elution, measurement wavelength 220 nm, Column temperature 30 ° C., sample injection volume 10 μL, flow rate 0.3 mL / min. Mass spectrometry conditions: Mass spectrometer Waters Platform ZMD 4000, ionization method ESI ⁺ , capillary voltage 4.80 kV, cone voltage 32 V, Gas Flow: 4.2 L / h, ion source temperature 120 ° C., solvent degassing temperature 250 ° C. Mass range: 1000-8000 m / z, photomultiplier tube (PMT) voltage 670V, Analyzer Vacuum 2.6e-5mBar.

  When the molecular mass of the oligopeptide was measured using a liquid chromatography / mass spectrometer (LC-MS), no molecular ion peak appeared, and only a fragment peak after removing some groups was obtained.

  Amino acid sequence measurement: The amino acid sequence of the squid oligopeptide was measured by the Edman degradation method. Sequence analysis showed that the oligopeptide had a linking structure in the order of -ILGGSDPKHYTG-.

  By performing amino acid composition analysis, gel chromatography analysis, oligopeptide molecular mass measurement and amino acid sequence measurement for amino acids of oligopeptide extracted from squid viscera, it contains -ILGGSDDPKHYTG-oligopeptide structural fragment, The result was that the molecular mass was between 1400 and 5800.

Bora Feeding Experiment April 2009, 50 groups of 1 year old mullet, 10 control groups to which the feed protein source of the present invention was not added, 10% of the mixture of Examples 1 to 3 (each 10 groups) Tail) and an experimental group (10 fish) to which 10% of fresh squid visceral fluid was added, and the same general feed (“Oe Kashiwa” normal fish formula feed) was fed. Put fish in 5 ponds of about 1.5 cubic meters, change the water by 1/5 every day for the first month, and change the water by 1/3 every day for the next half month, after 7, 15, 30, and 45 days The total body weight and the total consumption of feed during the corresponding time interval were recorded, and the change in weight and the consumption of feed were analyzed. Of these, the control group was killed on the 17th day, so the data is the data after conversion to 10 fish. The results are shown in Table 1. Fresh squid visceral fluid is a raw material obtained by degrading squid viscera with autoenzymes for 3 hours. As can be clearly seen from Table 1, compared to the control group to which the feed protein source of the present invention was not added and the experimental group to which a general fresh squid visceral enzyme digestion solution was added, the oligopeptide extracted from the squid viscera was included. The mixture was able to greatly increase the food intake of mullet as a feed protein source and increase the unit weight of mullet individuals.

Feeding experiment of bass In August 2009, 50 largemouth basses on the 45th day of birth were added to the control group (10 fishes) to which the feed protein source of the present invention was not added, and to the experimental group (each of which was 10% of the mixture of Examples 1 to 3). 10 fish) and an experimental group (10 fish) to which 10% of fresh squid visceral fluid was added, and the same general feed ("Oe tsumugi" normal fish mixed feed) was fed. Put fish in 5 ponds of about 1.5 cubic meters, change 15% of water every day for the first month, change 20% of water every day for the following month, 7 days, 15 days, 30 days and 60 days later The total body weight and the total consumption of the feed within the corresponding time interval were recorded, and the change of the weight and the consumption of the feed were analyzed. Of these, one bus in the control group died on the 5th day, so the data is data after conversion to 10 fish. The results are shown in Table 2. Fresh squid visceral fluid is a raw material obtained by degrading squid viscera with autoenzymes for 3 hours. As can be clearly seen from Table 2, compared to the control group to which the feed protein source of the present invention was not added and the experimental group to which a general fresh squid visceral enzyme digestion solution was added, the oligopeptide extracted from the squid viscera was used. The mixture significantly increased the amount of food consumed by the bath as a feed protein source and increased the unit weight of the individual bath.

Claims (6)

Include oligopeptides comprising an amino acid structure fragments -ILGGSDPKHYTG-, relative molecular weight range Ru der 1400-5800, a squid viscera extract,
(1) The viscera of squid (Todarodes pacificus) is stirred , crushed, added with water, stirred uniformly, degreased with petroleum ether or n-hexane, or a mixed solvent of petroleum ether and n-hexane, and an organic solvent layer The aqueous layer was allowed to stand at room temperature for 1 to 3 hours, heated at 50 to 60 ° C. for 1 to 3 hours, added with water at a temperature of 50 to 60 ° C., adjusted to pH 8.0 to 8.5, Thereafter, in a state where the temperature is kept at 50 to 60 ° C., enzymatic degradation is performed with Alcalase (registered trademark) for 1 to 3 hours at a rate of 2000 to 3000 units per 1 g of the raw material, and then 1 trypsin at a rate of 2000 to 3000 units per 1 g of the raw material. A step of enzymatic decomposition for ˜3 hours to remove insoluble substances to obtain an enzymatic decomposition solution, and (2) the enzymatic decomposition solution obtained in step (1) is subjected to a molecular weight cutoff of 180 Filtered through a 00Da ultrafiltration membrane, then Sephadex G-25 was manufactured by Seiko gel chromatography, squid in 臓抽 out thereof, characterized in that it is obtained by prepared by obtaining the o Rigopepuchi de. Step (2) the oligopeptide de potassium sorbate 0.025~0.050g per 1 000 g obtained in addition, squid in 臓抽 out of claim 1, wherein the drying and uniformly stirred . Include oligopeptides comprising an amino acid structure fragments -ILGGSDPKHYTG-, relative molecular weight range is 1400 to 5800, a process for preparing a squid in 臓抽 out thereof,
(1) The viscera of squid (Todarodes pacificus) is stirred , crushed, added with water, stirred uniformly, degreased with petroleum ether or n-hexane, or a mixed solvent of petroleum ether and n-hexane, and an organic solvent layer After leaving the aqueous layer at room temperature for 1 to 3 hours, heating at 50 to 60 ° C. for 1 to 3 hours, adding water at a temperature of 50 to 60 ° C., and adjusting the pH to 8.0 to 8.5. In the state kept at 50 to 60 ° C., after enzymatic degradation with Alcalase (registered trademark) for 1 to 3 hours at a rate of 2000 to 3000 units per gram of raw material, trypsin at a rate of 2000 to 3000 units per gram of raw material. 1 to 3 o'clock enzymatic decomposition to remove insoluble substances to obtain an enzymatic degradation solution, and (2) the molecular weight of the enzyme degradation solution obtained in step (1) is 18000 Da filtered through a ultrafiltration membrane, and then refining with Sephadex G-25 gel chromatography, process for the preparation of squid viscera extract characterized in that it comprises a step of obtaining O Rigopepuchi de. Step (2) the oligopeptide de potassium sorbate 0.025~0.050g per 1 000 g obtained in addition, squid in 臓抽 out of claim 3, wherein the drying and uniformly stirred Preparation method. When calculated as the total mass fraction of 100%, squid in 臓抽 out was 6% to 8% of claim 2, soybean meal powder 50% to 60%, fish meal 3% to 6%, corn meal 27% to 33 % and a mixture containing squid in 臓抽 out of claim 1, characterized in that it comprises 2% to 5% granulation starch.   Application of the mixture according to claim 5 as marine fishery feed.