WO2012027926A1

WO2012027926A1 - Peptide extracted from squid viscera and preparation method, composition and use thereof

Info

Publication number: WO2012027926A1
Application number: PCT/CN2010/078397
Authority: WO
Inventors: 张偲; 罗雄明; 尹浩; 齐振雄; 李庆欣
Original assignee: 中国科学院南海海洋研究所
Priority date: 2010-09-01
Filing date: 2010-11-04
Publication date: 2012-03-08
Also published as: JP2012530788A; CN102180945B; CN102180945A; JP5410612B2

Abstract

Provided are a peptide extracted from squid viscera and the preparation method thereof, characterized in that the peptide comprises the amino acids fragment -ILGGSDPKHYTG-, the relative molecular mass of the peptide is in the range of 1400-5800, and the peptide is obtained by the following steps: enzymolyzing the viscera of Todarodes pacificus, filtrating with an ultrafiltration membrane with molecular mass cutoff of 18000 Da, isolating and purifying with gel chromatography, thus obtaining a peptide comprising the amino acids fragment -ILGGSDPKHYTG-, then adding potassium sorbate, stirring uniformly and drying. Also provided is a composition consisting of said peptide 6-8%, soybean meal powder 50-60%, fish powder 3-6%, corn meal powder 27-33% and starch for granulation 2-5%. The composition may be used as protein source of feed for marine aquaculture, especially protein source of feed for marine aquatic fish.

Description

鱿鱼内脏提取的肽及其制备方法、组合物和用途 Peptide extracted from squid and preparation method, composition and use thereof

技术领域 Technical field

本发明涉及一种新的小肽物质，具体来说涉及一种鱿鱼内脏提取的小肽及其制备方法，还涉及这种物质的组合物以及该组合物作为海洋水产养殖中的伺料蛋白源的用途。 The present invention relates to a novel small peptide substance, in particular to a small peptide extracted from squid viscera and a preparation method thereof, and to a composition of the substance and the composition as a source of a carrier protein in marine aquaculture the use of.

背景技术 Background technique

随着我国远洋渔业的迅速发展，鱿鱼的捕获量逐年增加， 2009年鱿鱼捕获量已超过 30万吨，成为我国主要的水产品加工原料。但是我国的鱿鱼加工技术含量低，在加工处理获得鱼花、鱼干、咸鱿鱼、罐头及调味品等产品后，一般有 20%〜 30%的废弃物（内脏、头、足、表皮和皮墨汁等），这些废弃物含有丰富的蛋白质、脂肪、多糖等营养物质，据珠海市出入境检验检疫局吴莉敏（《农产品加工》， 2007 (8)： 94-96 ) 研究表明，每 100 g鱿鱼内脏中含脂肪 21. 15 g，蛋白质 21. 24 _g，钙 51. 46 mg，铁 609. 07 μ g和磷 95. 88 μ g等。但这些物质极易腐烂变质并且含有对人体有害的重金属镉。通常对这些废物的处理方法是就地掩埋，这样不但对渔业资源是巨大浪费，而且还存在着环境污染的隐患。因此，如何有效地处理鱿鱼废弃物并进行高值化综合利用越来越受到国民与社会的重视。 With the rapid development of China's offshore fisheries, the catch of carp has increased year by year. In 2009, the catch of carp has exceeded 300,000 tons, making it the main raw material for aquatic products processing in China. However, China's squid processing technology is low in content. After processing and processing fish, dried fish, salted squid, canned food and condiments, there are generally 20% to 30% of waste (viscera, head, feet, skin and skin). Ink, etc.), these wastes are rich in nutrients such as protein, fat, and polysaccharides. According to Wu Limin, Zhuhai City Entry-Exit Inspection and Quarantine Bureau (Agricultural Products Processing, 2007 (8): 94-96), research shows that every 100 g of squid visceral fat containing 21. 15 g, protein 21. 24 _g, calcium 51. 46 mg, iron and phosphorus 609. 07 μ g 95. 88 μ g and the like. However, these substances are highly perishable and contain heavy metal cadmium harmful to the human body. Usually, the treatment of these wastes is buried on the spot, which is not only a huge waste of fishery resources, but also a hidden danger of environmental pollution. Therefore, how to effectively deal with squid waste and carry out high-value comprehensive utilization is increasingly valued by the people and society.

中国水产科学院黄海水产研究所的王彩理等（《水产养殖》， 2003， 24 ( 5 ) : 40-41 ) 研究鱿鱼内脏液化蛋白对南美白对虾的诱食性，发现鱿鱼内脏浆的诱食效果最佳，强于甜菜碱、牛磺酸和大蒜素。上海水产品加工技术开发中心的王建中等（《中国海洋药物》， 1999， 1 : 55-58 )在对鱿鱼内脏的综合利用研究中，利用鱿鱼内脏及其它废弃物进行自生酶水解，开发了鱿鱼油、内脏水解液，并解决了从水解液中去除镉的难题。中国海洋大学水生生物制品安全性实验室的刘春娥等（《食品工业科技》， 2004, 9 ( 25 )： 83-86 ) 在鱿鱼内脏蛋白质酶解工艺的研究中，以氨基酸得率为主要指标，采用不同蛋白酶对鱿鱼内脏蛋白质进行了水解工艺研究。研究表明，中性蛋白酶，碱性蛋白酶，胰蛋白酶和木瓜蛋白酶具有较好的分解效果，氨基酸转化率可达到 50%以上。浙江工商大学的励建荣等人的专利：一种秘鲁鱿鱼内脏综合利用技术（申请号： 200610053372. 6)，将鱿鱼内脏蒸煮，酶液化，加热，离心等步骤，得到的下层鱿鱼多肽制成用于水产动物伺料蛋白源的鱿鱼膏，对对虾的伺喂试验表明，添加鱿鱼膏能明显增加饵料的摄取量，提高幼虾的存活率和体重。福建融鑫海生物技术有限公司的苏祖凤的专利：一种用鱿鱼内脏制备水产伺料诱食剂的生产方法（申请号： 200810071073. 4)，通过将鱿鱼内脏蒸煮、静置、自然酶解发酵，油料分离后的沉淀进行 80〜90°C和 60〜65 °C两次低温真空浓縮。 Wang Caili of the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences ("Aquaculture", 2003, 24 (5): 40-41) studied the enthalpy of visceral liquefaction protein of the carp to the white prawn, and found that the entrails of the squid were the best. , stronger than betaine, taurine and allicin. Wang Jianzhong of Shanghai Aquatic Products Processing Technology Development Center ("Chinese Marine Drugs", 1999, 1 : 55-58) developed a squid for the self-generated enzymatic hydrolysis of squid viscera and other wastes in the comprehensive utilization of squid viscera. Oil, visceral hydrolysate, and solve the problem of removing cadmium from hydrolysate. Liu Chunwei, et al., Safety Laboratory of Aquatic Biological Products, Ocean University of China ("Food Industry Technology", 2004, 9 (25): 83-86) In the study of the enzymatic hydrolysis process of squid viscera, the amino acid yield is the main indicator. The hydrolysis process of carp visceral protein was studied by different proteases. Studies have shown that neutral protease, alkaline protease, trypsin and papain have good decomposition effects, and the amino acid conversion rate can reach more than 50%. Li Jianrong et al. patent of Zhejiang Gongshang University: a comprehensive utilization technology of viscera of Peruvian squid (application number: 200610053372. 6), will be squid The squid cooking, enzyme liquefaction, heating, centrifugation and other steps, the resulting lower squid polypeptide is made into a squid paste for the aquatic animal feed protein source, and the feeding test on the prawn shows that the addition of squid paste can significantly increase the intake of the bait, Improve the survival rate and body weight of juvenile shrimp. Su Zufeng's patent of Fujian Rongxinhai Biotechnology Co., Ltd.: A method for preparing aquatic food feeding attractant using squid viscera (application number: 200810071073. 4), by cooking squid viscera, standing, natural enzymatic fermentation The precipitate after separation of the oil is subjected to two low-temperature vacuum concentration at 80 to 90 ° C and 60 to 65 ° C.

由于现有的伺料蛋白源的制作工艺中主要采取酶解后高温蒸煮、浓縮的方式获得，这些工艺过程容易使蛋白质变性，产生胶原蛋白，不仅降低了营养成分，而且大大损害了产品对水产动物的诱食性。即使采用苏祖凤等人的方法，利用 SJN2-2000二效蒸发器对酶解液进行二次浓縮，使蒸发温度控制在 90°C以下，大大降低了浓縮温度，但是依然无法从根本上解决这一难题。 Since the existing processing of the source of the protein source is mainly obtained by enzymatic hydrolysis and high-temperature cooking and concentration, these processes are easy to denature proteins and produce collagen, which not only reduces the nutrient content, but also greatly impairs the product pair. The attraction of aquatic animals. Even with the method of Su Zufeng et al., the enzymatic hydrolysate was secondarily concentrated by SJN2-2000 two-effect evaporator, and the evaporation temperature was controlled below 90 °C, which greatly reduced the concentration temperature, but still could not be solved fundamentally. This problem.

发明内容 Summary of the invention

本发明的目的在于摒弃高温蒸煮方法，利用超滤膜从鱿鱼内脏提取出一种新的小肽，另一目的是开发出该小肽的制备方法，又一目的是开发出含有该小肽的组合物及其用途。 The object of the present invention is to abandon the high-temperature cooking method, extract a new small peptide from the squid of the squid by using an ultrafiltration membrane, and another purpose is to develop a preparation method of the small peptide, and another object is to develop a small peptide containing the small peptide. Composition and its use.

我们将日本鱿鱼 Todarodes pacificus 内脏经有机溶剂脱脂后，用爱尔卡酶和胰蛋白酶酶解，然后用超滤膜过滤脱脂后的酶解液，再用凝胶色谱分离纯化，得到含有氨基酸结构片段 -ILGGSDPKHYTG-的小肽物质，加入山梨酸钾搅匀、干燥。将本发明小肽、豆粕粉、玉米粉、淀粉、鱼粉等按配比制粒，得到的组合物可用作海洋水产养殖伺料蛋白源，对鱼虾贝类等有非常好的诱食作用，从而实现了本发明的目的。 We degrease the viscera of Japanese squid Todarodes pacificus with an organic solvent, then digest it with erka enzyme and trypsin, then filter the degreased enzymatic hydrolysate with ultrafiltration membrane, and then separate and purify it by gel chromatography to obtain a fragment containing amino acid structure. - Small peptide substance of -ILGGSDPKHYTG-, add potassium sorbate, stir well, and dry. The small peptide, the soybean meal, the corn flour, the starch, the fish meal and the like of the invention are granulated according to the ratio, and the obtained composition can be used as a source of protein for marine aquaculture, and has a very good attracting effect on fish, shellfish and the like. The object of the invention is thus achieved.

本发明的鱿鱼内脏提取的小肽，其特征是含有氨基酸结构片段 -ILGGSDPKHYTG-, 相对分子质量范围为 1400〜5800，并通过下述方法制备得到： ( 1 ) 将日本鱿鱼内脏搅碎，加水搅匀，用石油醚或正己烷或石油醚和正己烷的混合溶剂脱脂，去除有机溶剂层，水层在室温下晾 1〜3小时， 50〜60°C下烘 1〜3 小时，加入温度为 50〜60°C的水，调节 pH值至 8. 0〜8. 5，然后在 50〜60°C保温状态下，按每克原料 2000〜3000单位的比例用爱尔卡酶酶解 1〜3小时后，再按每克原料 2000〜3000单位的比例用胰蛋白酶酶解 1〜3小时，去除不溶物质，得到酶解液；（2 )将步骤（1 )得到的酶解液用截留分子质量 18000Da超滤膜过滤，再用凝胶色谱分离纯化，得到含有氨基酸结构片段 -ILGGSDPKHYTG-的小肽物质。 The small peptide extracted from the squid of the present invention is characterized in that it contains an amino acid structural fragment-ILGGSDPKHYTG-, and the relative molecular mass ranges from 1400 to 5800, and is prepared by the following method: (1) The squid of Japanese squid is crushed, and water is stirred. Homogenize, degrease with petroleum ether or n-hexane or a mixed solvent of petroleum ether and n-hexane, remove the organic solvent layer, and dry the water layer at room temperature for 1 to 3 hours, and bake at 50 to 60 ° C for 1 to 3 hours. 50~60°C water, adjust the pH to 8. 0~8. 5, then in the temperature of 50~60 °C, according to the ratio of 2000~3000 units per gram of raw material, enzymatic hydrolysis of erkaase 1~ After 3 hours, the solution is digested with trypsin for 1-3 hours per gram of raw material to remove insoluble matter to obtain an enzymatic hydrolysate; (2) the enzymatic hydrolysate obtained in step (1) is retained by a molecule. Quality 18000Da ultrafiltration membrane filtration, then use condensation Separation and purification by gel chromatography gave a small peptide substance containing the amino acid structural fragment -ILGGSDPKHYTG-.

本发明的鱿鱼内脏提取的小肽还可以加入防腐剂，例如山梨酸钾，最好在步骤（2) 得到的小肽物质每 lOOOg中加入 0. 025〜0. 050g山梨酸钾，搅匀，干燥。在中试制备中，小肽物质的纯度值为 51%〜63%时有利于成本控制。 025〜0. 050g potassium sorbate, stir well, add a small peptide to obtain the small peptide substance obtained in step (2), preferably 0. 025~0. 050g potassium sorbate, stir well, dry. In the pilot preparation, the purity of the small peptide material is 51% to 63%, which is advantageous for cost control.

本发明的鱿鱼内脏提取的小肽的制备方法，其特征是包括以下的步骤： A method for preparing a small peptide extracted from a squid of the present invention, which comprises the following steps:

(1) 将日本鱿鱼内脏搅碎，加水搅匀，用石油醚或正己烷或石油醚和正己烷的混合溶剂脱脂，去除有机溶剂层，水层在室温下晾 1〜3小时， 50〜60°C下烘 1〜 3小时，加入温度为 50〜60°C的水，调节 pH值至 8. 0〜8. 5，然后在 50°C〜60°C保温状态下，按每克原料 2000〜3000单位的比例用爱尔卡酶酶解 1〜3小时后，再按每克原料 2000〜3000单位的比例用胰蛋白酶酶解 1〜3小时，去除不溶物质，得到酶解液； (1) The squid of Japanese squid is chopped, mixed with water, degreased with petroleum ether or a mixed solvent of n-hexane or petroleum ether and n-hexane to remove the organic solvent layer. The water layer is allowed to air at room temperature for 1 to 3 hours, 50~60 After drying at ° C for 1 to 3 hours, add water at a temperature of 50 to 60 ° C, adjust the pH to 8. 0~8. 5, then in the temperature of 50 ° C ~ 60 ° C, according to 2000 grams per gram of raw materials The ratio of ~3000 units is enzymatically hydrolyzed with erka enzyme for 1 to 3 hours, and then digested with trypsin for 1 to 3 hours at a ratio of 2000 to 3000 units per gram of raw material to remove insoluble matter to obtain an enzymatic hydrolyzate;

(2) 将步骤（1 )得到的酶解液用截留分子质量 ISOOODa超滤膜过滤，再用凝胶色谱分离纯化，得到含有氨基酸结构片段 -ILGGSDPKHYTG-的小肽物质。 (2) The enzymatic hydrolysate obtained in the step (1) is filtered with a molecular weight cut-off molecular weight ISOOODa ultrafiltration membrane, and then separated and purified by gel chromatography to obtain a small peptide substance containing the amino acid structural fragment -ILGGSDPKHYTG-.

本发明的鱿鱼内脏提取的小肽的制备方法，还可以在步骤（2) 得到的小肽物质中加入防腐剂，例如山梨酸钾，最好在每 lOOOg小肽物质中加入 0. 025〜 0. 050g山梨酸钾，搅匀，干燥。 025〜 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 050g potassium sorbate, stir well, dry.

鱿鱼内脏提取的小肽的分子质量计算方法： Molecular mass calculation method for small peptide extracted from squid viscera:

logMw = -b Kav + c; 有效分配系数 Kav是表示被排阻的程度， Mw表示物质的分子质量， b， c为常数与凝胶柱本身性质相关，可通过标准蛋白质样品计算 b， c 值。 logMw = -b Kav + c; The effective partition coefficient Kav is the degree of exclusion, Mw is the molecular mass of the substance, and b, c is a constant related to the nature of the gel column. The b, c values can be calculated from standard protein samples. .

凝胶色谱分离纯化和分子质量测定的操作包括： The operations of gel chromatography separation and purification and molecular mass determination include:

( 1 ) 采用中大型凝胶色谱柱进行分离纯化。将日本鱿鱼内脏按上述本发明方法脱脂处理后进行酶解，截留分子质量 18000Da超滤膜过滤鱿鱼内脏酶解液。采用中大型凝胶色谱进行分离，凝胶色谱型号： Sephadex G-25, 柱型号：200 X 10 cm, 缓冲液为 0. 05 mol/L磷酸盐缓冲液（含 0. 16 mol/L NaCl)， pH= 7. 0，溶剂流速 10 mL/min, 柱床体积约 12000 mL, 将样品配成浓度为 50〜100 mg/mL的溶液，用 0. 45 m微孔滤膜过滤，取 500〜1000 mL进样分离纯化，采用下述步骤方法分析检测。 (1) Separation and purification using medium and large gel columns. The squid of Japanese squid was degreased according to the method of the present invention, and then subjected to enzymatic hydrolysis, and the molecular weight of 18000 Da was used to filter the squid enzymatic hydrolysate. Separation by medium and large-scale gel chromatography, gel chromatography model: Sephadex G-25, column type: 200 X 10 cm, buffer 0. 05 mol/L phosphate buffer (containing 0.16 mol/L NaCl) , pH= 7. 0, solvent flow rate 10 mL / min, volume of the bed is about 12000 mL, the sample is prepared into a solution with a concentration of 50~100 mg / mL, filtered with a 0. 45 m microporous membrane, take 500~ The 1000 mL injection was separated and purified, and the detection was carried out by the following steps.

( 2 ) 采用小型凝胶色谱进行分子质量测定。分析条件选择：凝胶色谱型号： Sephadex G-25；柱型号： 100 X 1 cm, 缓冲液为 0. 05 mol/L磷酸盐缓冲液（含 0. 16 mol/L NaCl) ， pH= 7. 0，溶剂流速 0. 20 mL/min, 检测波长 280 歷，柱床体积约 60mL。 (2) Molecular mass determination using small gel chromatography. Analysis of condition selection: Gel chromatography No.: Sephadex G-25; column type: 100 X 1 cm, buffer is 0. 05 mol/L phosphate buffer (containing 0.16 mol/L NaCl), pH = 7. 0, solvent flow rate 0. 20 mL/min, detection wavelength 280 calendar, the bed volume is about 60mL.

( 3 )将标准蛋白质样品配成浓度为 5mg/mL的溶液，用 0. 30 m微孔滤膜过滤，取 0. 5mL进样，获得（Kav， logMw) 的对应点三个（28. 5, 4. 16)、（53. 7, 3. 13)、 (3) The standard protein sample was prepared into a solution having a concentration of 5 mg/mL, and filtered with a 0.30 m microporous membrane filter, and 0.5 mL of the injection was obtained, and three corresponding points (Kav, logMw) were obtained (28.5). , 4. 16), (53. 7, 3. 13),

(74. 9, 2. 26)，通过计算获得公式： logMw = -0. 041 Kav + 5. 33 ( R² = 0. 9998)。 (74. 9, 2. 26), obtain the formula by calculation: logMw = -0. 041 Kav + 5. 33 ( R ² = 0. 9998).

( 4)将步骤（1 )分离纯化的日本鱿鱼内脏酶解液经 0. 30 m微孔滤膜过滤后，调整浓度至 10mg/mL。采用小型凝胶色谱，取 0. 5mL进样，得出鱿鱼内脏提取的小肽在直线上的 ( Kav, logMw) 点分别为（53. 3， 3. 15)、（38. 2， 3. 76)，计算其对应分子质量为 1400和 5800。 (4) The snail entrail enzymatic hydrolysate separated and purified in step (1) was filtered through a 0.30 m microporous membrane, and the concentration was adjusted to 10 mg/mL. Using a small gel chromatography, taking 0.5 mL of the injection, it was found that the small peptides extracted from the viscera of the squid were in the line (Kav, logMw), respectively (53. 3, 3. 15), (38. 2, 3. 76), calculate the corresponding molecular mass of 1400 and 5800.

本发明含有鱿鱼内脏提取的小肽的组合物，其特征是按总质量分数 100%计，由鱿鱼内脏提取的小肽 6%〜8%，豆粕粉 50%〜60%，鱼粉 3%〜6%，玉米粉 27%〜33% 和制粒用淀粉 2%〜5%组成。所用的豆粕粉，鱼粉，玉米粉和制粒用淀粉为水产伺料用，从市场购买。 The invention comprises a composition of small peptide extracted from squid of the squid, characterized in that the small peptide extracted from the viscera of the carp is 6% to 8%, the soybean meal is 50% to 60%, and the fish meal is 3% to 6%, based on 100% of the total mass fraction. %, corn flour 27% ~ 33% and granulation starch 2% ~ 5% composition. The used soybean meal powder, fish meal, corn flour and granulated starch are used for aquatic products and are purchased from the market.

本发明的组合物的制备方法，是按上述比例将鱿鱼内脏提取的小肽、豆粕粉、鱼粉和玉米粉搅匀，以配料：水 =1 : 4 (质量比）的比例加入适量的水，搅匀，喷雾干燥，得干膏粉；按配方比例向干膏粉中加入制粒用淀粉，制粒，晾干得到。 The preparation method of the present invention is prepared by mixing small peptide, soybean meal powder, fish meal and corn flour extracted from squid viscera according to the above ratio, and adding an appropriate amount of water in a ratio of ingredients: water=1:4 (mass ratio). Stir well, spray dry, dry paste powder; add granulation starch to the dry paste powder according to the formula ratio, granulate, and dry.

本发明的组合物可用作海洋水产养殖中的伺料蛋白源，包括海洋鱼、虾、贝类等的伺料蛋白源，特别适合海洋水产鱼类的伺料蛋白源。 The composition of the present invention can be used as a source of a carrier protein in marine aquaculture, including a source of protein for marine fish, shrimp, shellfish, etc., and is particularly suitable for a source of protein for marine fish.

本发明中鱿鱼内脏提取的小肽的制作过程中温度不超过 60°C，干燥时采用喷雾干燥器进行干燥，时间短、效果好，保证了该物质不水解、不变性。本发明的组合物具有强的鱼类诱食性，其主要成分鱿鱼内脏提取的小肽具有浓郁的腥香味，是水产养殖伺料蛋白源的佳品。利用本发明提供的组合物养殖海洋鱼类，可大幅度的提高鱼类的摄食量，增加鱼类个体的单位重量。 In the process of the present invention, the small peptide extracted from the viscera of the squid is not more than 60 ° C in temperature, and is dried by a spray dryer during drying. The time is short and the effect is good, and the substance is not hydrolyzed or deformed. The composition of the present invention has strong fish attracting property, and the small peptide extracted from the viscera of the main component of the squid has a strong musk flavor and is a good source of aquaculture food source. The cultivation of marine fish by the composition provided by the present invention can greatly increase the food intake of fish and increase the unit weight of individual fish.

具体实施方式： detailed description:

以下实施例是对本发明的进一步说明，不是对本发明的限制。实施例中主要原材料、主要仪器设备、主要生物和化学试剂等如下： The following examples are intended to further illustrate the invention and are not to be construed as limiting. The main raw materials, main equipment, main biological and chemical reagents in the examples are as follows:

主要原材料： Main raw materials:

从广州市黄沙海产品市场收集日本鱿鱼（Todarodes pacificus ) 内脏，储存于 -15°C下备用，使用前 12小时在室温下解冻。 "大江牌"常规鱼用配合伺料，上海大江（集团）股份有限公司。所用豆粕粉，玉米粉购自济宁双华工贸有限公司，所用鱼粉购自荣成新希望鱼粉有限公司。 Collecting the internal organs of Japanese carp (Todarodes pacificus) from the Huangsha seafood market in Guangzhou, Store at -15 ° C for use and thaw at room temperature for 12 hours before use. "Dajiang Brand" conventional fish used in conjunction with the news, Shanghai Dajiang (Group) Co., Ltd. The soybean meal used, corn flour was purchased from Jining Shuanghua Industry and Trade Co., Ltd., and the fishmeal used was purchased from Rongcheng New Hope Fishmeal Co., Ltd.

主要仪器设备： Main equipment:

发酵罐： LABF0RS 实验室台式小型发酵罐，产地瑞士；匀浆机：予华 A8G型，产地：河南巩义；落地式离心机： Bechman J2-Hs高速冷冻离心机，产地美国；中大型凝胶色谱型号： S印 hadex G-25, 柱型号：200 X 10 cm, 缓冲液为 0. 05 mol/L 磷酸盐缓冲液（含 0. 16 mol/L NaCl)， pH= 7. 0，溶剂流速 50 mL/min, 柱床体积约 12000 mL; 835-50氨基酸自动分析仪（日本 Hitachi公司）；色谱仪 Waters 2690，质谱仪 Waters Platform ZMD 4000。 Fermentation tank: LABF0RS laboratory bench-top small fermenter, Switzerland; Homogenizer: Yuhua A8G type, Origin: Henan Gongyi; Floor-type centrifuge: Bechman J2-Hs high-speed refrigerated centrifuge, origin US; Medium and large gel chromatography Model: S-printed hadex G-25, column type: 200 X 10 cm, buffer is 0. 05 mol/L phosphate buffer (containing 0.16 mol/L NaCl), pH= 7. 0, solvent flow rate 50 mL/min, bed volume about 12000 mL; 835-50 amino acid automatic analyzer (Hitachi, Japan); chromatograph Waters 2690, mass spectrometer Waters Platform ZMD 4000.

主要生物和化学试剂： Major biological and chemical reagents:

胰蛋白酶： 100万活力单位 /g，大连保税区联合博泰生物技术有限公司，产品编号： RM00102。爱尔卡（Alcalase) 蛋白酶： 50万活力单位 /g，诺维信公司。有机试剂:有机试剂购自广州试剂公司（分析纯）。无机试剂:无机试剂购自广州试剂公司（分析纯）。纯水：实施例中用水均为纯水，使用 MiniQ纯水制备***制备。 Trypsin: 1 million viable units / g, Dalian Bonded Area and Botai Biotechnology Co., Ltd., product number: RM00102. Alcalase Protease: 500,000 vital units / g, Novozymes. Organic Reagent: The organic reagent was purchased from Guangzhou Reagent Co., Ltd. (analytical grade). Inorganic reagent: Inorganic reagent was purchased from Guangzhou Reagent Company (analytical grade). Pure water: In the examples, the water was pure water and was prepared using a MiniQ pure water preparation system.

实施例 1 : Example 1

收集 20 kg日本鱿鱼内脏，予华 A8G型匀浆机搅拌破碎，加入 2L水，再加入 20L 沸程为 60〜90°C的石油醚搅拌脱脂，去除石油醚层，将水层在室温下晾 3小时， 50 烘箱中烘 3小时；加入温度为 50°C的水 8L，搅匀，用饱和 NaOH溶液调节 pH值至接近 8. 5，再用 5 mol/L浓度的 NaOH溶液调节 pH值至 8. 5， 50°C保温状态下，将鱿鱼内脏放入发酵罐中，按 3000单位 /g原料加入 6 X 10⁷单位爱尔卡酶酶解 3小时后，再按 3000单位 /g原料加入 6 X 10⁷单位胰蛋白酶酶解 3小时；用 Bechman J2_Hs高速冷冻离心机以 5000转 /分钟离心分离，去除不溶物质；利用截留分子质量 18000Da 超滤膜过滤脱脂后的酶解液，获得富含小肽的成分，再用中大型 Sephadex G-25 凝胶色谱分离纯化，获得含有氨基酸结构片段 -ILGGSDPKHYTG-的小肽物质，按每 1000g小肽中加入 0. 025g山梨酸钾的配比加入 0. 0059g山梨酸钾；干燥后得鱿鱼内脏提取的小肽。 Collect 20 kg of Japanese squid viscera, stir and crush with A8G homogenizer, add 2L of water, add 20L of petroleum ether with boiling range of 60~90 °C, degrease, remove petroleum ether layer, and dry the water layer at room temperature. 3 hours, 50 ovens for 3 hours; add water at a temperature of 50 ° C 8L, stir well, adjust the pH with a saturated NaOH solution to close to 8.5, and then adjust the pH with a 5 mol / L concentration of NaOH solution to 8. 5, 50 ° C holding state, the squid visceral into the fermenter, according to 3000 units / g of raw materials added 6 × 10 ⁷ units of erka enzyme enzymatic hydrolysis for 3 hours, then add 3000 units / g of raw materials 6 X 10 ⁷ units of trypsin was digested for 3 hours; centrifuged at 5000 rpm using a Bechman J2_Hs high-speed refrigerated centrifuge to remove insoluble matter; filtered by degreased enzymatic hydrolysate using a molecular weight 18000Da ultrafiltration membrane to obtain a rich The composition of the small peptide is then separated and purified by medium-large Sephadex G-25 gel chromatography to obtain a small peptide substance containing the amino acid structural fragment-ILGGSDPKHYTG-, and the ratio of 0. 025g potassium sorbate is added to each 1000g small peptide. 0059g potassium sorbate; dried squid viscera Take small peptides.

称取鱿鱼内脏提取的小肽 80g，豆粕粉 600g，鱼粉 30g，玉米粉 270g，加入 4L水，搅拌均匀后喷雾干燥，得到干膏粉；再称取制粒用淀粉 20g，制粒，晾干，得到组合物。 Weigh 80g of small peptide extracted from squid of squid, 600g of soybean meal, 30g of fishmeal, 270g of corn flour, 4 L of water, stirred uniformly and spray-dried to obtain a dry paste powder; 20 g of starch for granulation was weighed, granulated, and air-dried to obtain a composition.

实施例 2 : Example 2:

收集 20 Kg日本鱿鱼内脏，予华 A8G型匀浆机搅拌破碎，加入 12 L水，再加入 20L石油醚和正己烷混合溶剂（1 : 1 )搅拌脱脂，去除石油醚层，将水层在室温下晾 1小时， 60°C烘箱中烘 1小时；加入温度为 60°C的水 8L，搅匀，用饱和 NaOH溶液调节 pH值至接近 8. 0，再用稀浓度的 NaOH溶液调节 pH值至 8. 0； 60 °C保温状态下，将鱿鱼内脏放入发酵罐中，加入 4 X 10⁷单位爱尔卡酶酶解 1小时后，再加入 4 X 10⁷ 单位胰蛋白酶酶解 1小时；用 Bechman J2_Hs高速冷冻离心机以 5000转 /分钟离心分离，去除不溶物质；利用截留分子质量 ISOOODa超滤膜过滤脱脂后的酶解液，获得富含小肽的成分，再用中大型 Sephadex G_25凝胶色谱分离纯化，获得含有氨基酸结构片段 -ILGGSDPKHYTG-的小肽物质，按每 1000_g小肽中加入 0. 050g山梨酸钾的配比加入 0. 0078g山梨酸钾，干燥后得鱿鱼内脏提取的小肽。 Collect 20 Kg of Japanese squid viscera, stir and crush with A8G homogenizer, add 12 L of water, add 20 L of petroleum ether and n-hexane mixed solvent (1:1), degrease, remove petroleum ether layer, and make the water layer at room temperature. Dry for 1 hour, bake in a 60 ° C oven for 1 hour; add 8 L of water at 60 ° C, stir well, adjust the pH to close to 8.0 with saturated NaOH solution, and adjust the pH with dilute NaOH solution. To 8. 0; at 60 °C, put the squid of the squid into the fermenter, add 4×10 ⁷ units of erka enzyme for 1 hour, then add 4×10 ⁷ units of trypsin to digest for 1 hour. Using a Bechman J2_Hs high-speed refrigerated centrifuge to centrifuge at 5000 rpm to remove insoluble matter; use the cut-off molecular mass ISOOODa ultrafiltration membrane to filter the degreased enzymatic hydrolysate to obtain small peptide-rich components, and then use medium and large Sephadex G_25 Separate and purify the gel, and obtain a small peptide substance containing the amino acid structural fragment-ILGGSDPKHYTG-, and add 0. 050g of potassium sorbate per 1000 _{g of} small peptide to 0. 0078g of potassium sorbate, dried to obtain squid extract Small peptide.

称取鱿鱼内脏提取的小肽 60g，豆粕粉 500g，鱼粉 60g，玉米粉 330g，加入 4L 水，搅拌均匀后喷雾干燥，得干膏粉；再称取制粒用淀粉 50g，制粒，晾干，得到组合物。实施例 3 : Weigh 60g of small peptide extracted from squid of squid, 500g of soybean meal, 60g of fishmeal, 330g of corn flour, add 4L of water, stir evenly and spray dry to obtain dry paste powder; then weigh 50g of starch for granulation, granulate, dry , to obtain a composition. Example 3:

称取实施例 1的鱿鱼内脏提取的小肽 65g，豆粕粉 535g，鱼粉 30g，玉米粉 330g，加入 4L水，搅拌均匀后喷雾干燥，得干膏粉；再称取制粒用淀粉 40g，制粒，晾干，得到组合物。实施例 4: Weighed 65 g of small peptide extracted from the viscera of the squid of Example 1, 535 g of soybean meal, 30 g of fish meal, 330 g of corn flour, and added 4 L of water, and then uniformly dried, and spray-dried to obtain a dry powder; and then weighed 40 g of starch for granulation. The granules were dried to give a composition. Example 4:

称取实施例 2的鱿鱼内脏提取的小肽 75g，豆粕粉 575g，鱼粉 50g，玉米粉 280g，加入 4L水，搅拌均匀后喷雾干燥，得干膏粉；再称取制粒用淀粉 20g，制粒，晾干，得到组合物。实施例 5: Weigh 75g of small peptide extracted from squid of Example 2, 575g of soybean meal, 50g of fish meal, 280g of corn flour, add 4L of water, stir evenly, and spray dry to obtain dry powder; then weigh 20g of starch for granulation. The granules were dried to give a composition. Example 5

氨基酸组成分析：将实施例 1和 2获得的鱿鱼内脏提取的小肽分别置于水解管中，加入 6 mol/L 的盐酸溶液，真空封口。在 11CTC下水解 24h，冷却后定容、过滤和蒸干，再加入Analysis of amino acid composition: The small peptides extracted from the squid of the squid obtained in Examples 1 and 2 were placed in a hydrolysis tube, and a 6 mol/L hydrochloric acid solution was added thereto, followed by vacuum sealing. Hydrolyze at 11CTC for 24h, cool down, dilute to volume, filter and evaporate, then add

0.02 mol/L的盐酸溶液在空气中放置 30 min, 采用 835-50氨基酸自动分析仪（日本 Hitachi公司），测除色氨酸以外的氨基酸含量，结果得到： The 0.02 mol/L hydrochloric acid solution was placed in the air for 30 min, and an amino acid content other than tryptophan was measured using an automatic analyzer of 835-50 amino acid (Japan Hitachi Co., Ltd.).

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: 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。 1.00.

采用液质联用（LC-MS)仪测分子质量： Molecular mass was measured by LC-MS:

液相色谱条件：色谱仪 Waters 2690,检测器： Waters 996，分析柱 Lichrospher C-18 (2.6 X 250mm), 流动相：甲醇-水 _0.5%乙酸梯度洗脱，检测波长 220nm，柱温 30°C，进样量， lO L,流速 0.3mL/min; 质谱条件：质谱仪 Waters Platform ZMD4000, 离子方式 ESI⁺，毛细管电压 4.80kV，锥孔电压 32 V， Gas Flow: 4.2L/h, 离子源温度 120°C，脱溶剂气温度 250°C，质量范围： 1000〜8000m/z，光电倍增器电压 670V， Analyser Vacuum2.6e〜5mBar。 Liquid chromatography conditions: Chromatograph Waters 2690, detector: Waters 996, analytical column Lichrospher C-18 (2.6 X 250 mm), mobile phase: methanol-water _0.5% acetic acid gradient elution, detection wavelength 220 nm, column temperature 30 ° C, injection volume, lO L, flow rate 0.3 mL/min; mass spectrometry conditions: mass spectrometer Waters Platform ZMD4000, ion mode ESI ⁺ , capillary voltage 4.80 kV, cone voltage 32 V, Gas Flow: 4.2 L/h, ion source Temperature 120 ° C, desolvation gas temperature 250 ° C, mass range: 1000 ~ 8000 m / z, photomultiplier voltage 670V, Analyser Vacuum 2.6e ~ 5mBar.

采用液质联用仪测定小肽分子质量时没有发现其分子离子峰，只得到其脱去部分基团后的碎片峰。 When the molecular weight of the small peptide was determined by a liquid chromatography-mass spectrometer, the molecular ion peak was not found, and only the fragment peak after the partial group was removed was obtained.

氨基酸序列测定：采用 Edman降解法测鱿鱼小肽的氨基酸序列。序列分析表明小肽具有 -ILGGSDPKHYTG-顺序结构链接。 Amino acid sequence determination: The amino acid sequence of the small peptide of the carp was measured by Edman degradation method. Sequence analysis indicated that the small peptide has a -ILGGSDPKHYTG-sequence structure linkage.

通过对鱿鱼内脏提取的小肽的氨基酸进行氨基酸组成分析、凝胶色谱分析、小肽分子质量测定和氨基酸序列测定，结果得到其中含有 -ILGGSDPKHYTG-小肽结构片段，相对分子质量在 1400至 5800之间。实施例 6: The amino acid composition of the small peptide extracted from the squid of the squid was analyzed by amino acid composition, gel chromatography, small peptide molecular mass determination and amino acid sequence determination. The result was a fragment containing -ILGGSDPKHYTG-small peptide with a relative molecular mass of 1400 to 5800. between. Example 6:

鲻鱼的伺喂实验， 2009年 4月，取 1龄鲻鱼 50尾，分成不添加本发明伺料蛋白源的对照组（10尾）、按 10%添加实施例 1〜3的组合物的实验组（各 10尾）和按 10% 添加鱿鱼内脏鲜液的实验组（10尾），而且都喂养相同的普通伺料（"大江牌"常规鱼用配合伺料）。把鱼放入 5个 1.5立方米左右的池中，前一个月每天换水 1/5，后半个月每天换水 1/3，记录其 7天、 15天、 30天和 45天后的总体重和相应时间间隔内伺料消耗总量，分析他们的重量变化情况和伺料消耗情况。其中第 17天对照组鲻鱼死亡 1尾，数据是按 10尾换算后的数据。结果见表 1，鱿鱼内脏鲜液为鱿鱼内脏经过自生酶解 3小时后获得的末经加工的物质。从表 1可以清楚看到：与不添加本发明伺料蛋白源的对照组和添加一般鱿鱼内脏酶解鲜液的实验组相比，含鱿鱼内脏提取的小肽的本发明组合物作为伺料蛋白源可大幅度的提高鲻鱼的摄食量，增加鲻鱼个体的单位重量。实施例 1〜3的组合物，鱿鱼内脏鲜液和对照组对鲻鱼生长的影响 The feeding experiment of squid, in April 2009, 50 squid of 1st instar squid, divided into the control group (10 tails) which did not add the source of the servo protein of the present invention, and the composition of Examples 1-3 was added by 10%. The experimental group (10 tails each) and the experimental group (10 tails) which added 10% of the squid visceral fresh liquid, and both were fed the same common feeder ("Dajiang brand" conventional fish used to support the feed). Put the fish into 5 pools of 1.5 cubic meters, change the water by 1/5 every day in the previous month, and change the water by 1/3 every day for the second half of the month. Record the total of 7 days, 15 days, 30 days and 45 days. The total amount of material consumed during the weight and corresponding time interval, and their weight changes and servo consumption were analyzed. On the 17th day, the control group squid died 1 time, the data is the data after 10 conversions. The results are shown in Table 1. The squid visceral fresh liquid is squid. The processed material obtained after the viscera was autolyzed for 3 hours. As is clear from Table 1, the composition of the present invention containing the small peptide extracted from the viscera of the carp was used as a feeder compared with the control group to which the source of the servo protein of the present invention was not added and the experimental group to which the general squid visceral enzymatic solution was added. The protein source can greatly increase the food intake of the squid and increase the unit weight of the carp. Effects of the compositions of Examples 1 to 3, squid fresh liquid and control group on the growth of carp

实施例 7: Example 7

鲈鱼的伺喂实验， 2009年 8月，取出生 45天的大口黑鲈鱼 50尾，分成不添加本发明伺料蛋白源的对照组（10尾）、按 10%添加实施例 1-3的组合物的实验组（各 10尾）和按 10%添加鱿鱼内脏鲜液的实验组（10尾），而且都喂养相同的普通伺料 ("大江牌"常规鱼用配合伺料）。把各组鱼放入 5个 1. 5立方米左右的池中，前一个月每天换水 15%，后一个月每天换水 20%，记录其 7天、 15天、 30天和 60天后的总体重和相应时间间隔内伺料消耗总量，分析他们的重量变化情况和伺料消耗情况。其中第 5天对照组鲈鱼死亡 1尾，数据是按 10尾换算后的数据。结果见表 2。鱿鱼内脏鲜液为鱿鱼内脏经过自生酶解 3小时后获得的末经加工的物质。从表 2 可以清楚看到：与不添加本发明伺料蛋白源的对照组和添加一般鱿鱼内脏酶解鲜液的实验组相比，含鱿鱼内脏提取的小肽的本发明组合物作为伺料蛋白源可大幅度的提高鲈鱼的摄食量，增加鲈鱼个体的单位重量。实施例 1-3的组合物，鱿鱼内脏鲜液和对照组对鲈鱼生长的影响 The feeding experiment of squid, in August 2009, 50 squid of black mullet, which was taken for 45 days, was divided into a control group (10 tails) without adding the source of the servo protein of the present invention, and a combination of Examples 1-3 was added at 10%. The experimental group (10 tails each) and the experimental group (10 tails) with 10% squid visceral fresh liquid were added, and both were fed the same common feeder ("Dajiang Brand" conventional fish was used for feeding). Put each group of fish into 5 pools of 1.5 cubic meters, change water 15% every day for the previous month, and change water 20% every day for the next month, record 7 days, 15 days, 30 days and 60 days later. The total weight and the total amount of servo consumption in the corresponding time interval, analyze their weight changes and the consumption of the feed. On the 5th day, the control group died of squid, and the data was converted to 10 times. The results are shown in Table 2. The squid visceral fresh liquid is the last processed material obtained after the squid viscera is autolyzed for 3 hours. As is clear from Table 2, the composition of the present invention containing the small peptide extracted from the viscera of the carp was used as a feeder, compared with the control group to which the source of the servo protein of the present invention was not added and the experimental group to which the general squid visceral enzymatic solution was added. The protein source can greatly increase the food intake of the squid and increase the unit weight of the carp. Effects of the compositions of Examples 1-3, squid fresh liquid and control group on the growth of carp

Claims

权利要求 Rights request

1 . 一种鱿鱼内脏提取的小肽，其特征是含有氨基酸结构片段 -ILGGSDPKHYTG-, 相对分子质量范围为 1400〜5800，并通过下述方法制备得到： A small peptide extracted from squid of a squid, characterized by comprising an amino acid structural fragment -ILGGSDPKHYTG-, having a relative molecular mass ranging from 1400 to 5800, and prepared by the following method:

( 1 )将日本鱿鱼 Tbi/aroifespac /yc^内脏搅碎，加水搅匀，用石油醚或正己烷或石油醚和正己烷的混合溶剂脱脂，去除有机溶剂层，水层在室温下晾 1〜3小时， 50〜60°C下烘 1〜3小时，加入温度为 50〜60°C的水，调节 pH值至 8. 0〜8. 5，然后在 50〜60°C保温状态下，按每克原料 2000〜3000单位的比例用爱尔卡酶酶解 1〜3 小时后，再按每克原料 2000〜3000单位的比例用胰蛋白酶酶解 1〜3小时，去除不溶物质，得到酶解液；（2 ) 将步骤（1 ) 得到的酶解液用截留分子质量 18000Da 超滤膜过滤，再用凝胶色谱分离纯化，得到含有氨基酸结构片段 -ILGGSDPKHYTG- 的小肽物质。 (1) Crush the Japanese squid Tbi/aroifespac /yc^ viscera, add water and mix well, degrease with petroleum ether or n-hexane or a mixture of petroleum ether and n-hexane to remove the organic solvent layer, and the water layer is dried at room temperature. 5小时后后下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下下The ratio of 2000 to 3000 units per gram of raw material is digested with erka enzyme for 1 to 3 hours, and then digested with trypsin for 1 to 3 hours at a ratio of 2000 to 3000 units per gram of raw material to remove insoluble matter and obtain enzymatic hydrolysis. (2) The enzymatic hydrolysate obtained in the step (1) was filtered through a 18000 Da ultrafiltration membrane with a molecular weight cut off, and then separated and purified by gel chromatography to obtain a small peptide substance containing the amino acid structural fragment -ILGGSDPKHYTG-.

2.根据权利要求 1所述的一种鱿鱼内脏提取的小肽，其特征是在步骤（2)得到的小肽物质每 1000g中加入 0. 025〜0. 050g山梨酸钾，搅匀，干燥。 050〜0. 050g potassium sorbate, stir well, dry, a small peptide substance obtained in step (2) is added 0. 025~0. 050g potassium sorbate, stir well, dry .

3. 一种鱿鱼内脏提取的小肽的制备方法，其特征是包括以下的步骤： (1) 将日本鱿鱼 7bo¾roo½? pac / c^内脏搅碎，加水搅匀，用石油醚或正己烷或石油醚和正己烷的混合溶剂脱脂，去除有机溶剂层，水层在室温下晾 1〜3 小时， 50〜60°C下烘 1〜3小时，加入温度为 50〜60°C的水，调节 pH值至 8. 0〜8. 5，然后在 50〜60°C保温状态下，按每克原料 2000〜3000单位的比例用爱尔卡酶酶解 1〜3小时后，再按每克原料 2000〜3000单位的比例用胰蛋白酶酶解 1〜3小时，去除不溶物质，得到酶解液； 3. A method for preparing a small peptide extracted from squid of a squid, which comprises the following steps: (1) pulverizing the squid of Japanese squid 7bo3⁄4roo1⁄2? pac / c^, adding water, mixing with petroleum ether or n-hexane or petroleum The solvent mixture of ether and n-hexane is degreased, the organic solvent layer is removed, the water layer is air-dried at room temperature for 1 to 3 hours, baked at 50 to 60 ° C for 1 to 3 hours, and water at a temperature of 50 to 60 ° C is added to adjust the pH. 5 to 8. 5, and then in the temperature of 50 ~ 60 ° C, according to the ratio of 2000 ~ 3000 units per gram of raw materials with erka enzymatic hydrolysis for 1 to 3 hours, then according to 2000 per gram of raw materials The ratio of ~3000 units is digested with trypsin for 1 to 3 hours to remove insoluble matter to obtain an enzymatic hydrolysate;

4.根据权利要求 3所述的一种鱿鱼内脏提取的小肽的制备方法，其特征是在步骤（2)得到的小肽物质每 lOOOg中加入 0. 025〜0. 050g山梨酸钾，搅匀，干燥。 The smear of potassium sorbate, stirred in the amount of 0. 025~0. 050g potassium sorbate, stirred in the small peptide material obtained in step (2). Evenly, dry.

5. 一种权利要求 1所述的鱿鱼内脏提取的小肽的组合物，其特征是按总质量分数 100%计，由权利要求 2所述的鱿鱼内脏提取的小肽 6%〜8%，豆粕粉 50%〜60%，鱼粉 3%〜6%，玉米粉 27%〜33%和制粒用淀粉 2%〜5%组成。 A composition of small peptide extracted from squid viscera according to claim 1, characterized in that the small peptide extracted from the squid of claim 2 is 6% to 8% based on 100% of the total mass fraction. Soybean meal 50%~60%, fishmeal 3%~6%, corn flour 27%~33% and granulation starch 2%~5%.

6. 权利要求 5所述的组合物作海洋水产伺料蛋白源的应用。 6. Use of the composition of claim 5 as a source of marine aquatic product.