阅读说明：本技术 一种安全高效新型蛋白抗冻剂及其制备方法和应用 (Safe and efficient novel protein antifreeze agent and preparation method and application thereof ) 是由 朱士臣 俞杰航 周绪霞 丁玉庭 刘书来 于 2020-07-06 设计创作，主要内容包括：本发明提供了一种安全高效新型蛋白抗冻剂及其制备方法和应用,所述蛋白抗冻剂的制备过程为：鱼鳞经干燥、预处理、蒸汽闪爆处理、脱钙、酶解、热水浸提及冻干后得到,所得蛋白抗冻剂可作为冷冻保护剂应用于水产品、酶及菌种的冷冻保藏；本发明原料来源广泛、成本低廉,工艺简单、高效,制备的新型抗冻剂具有安全营养和适用范围广等优点,能够有效控制冷冻食品及生物制品的品质劣变,增强其冻融稳定性。(The invention provides a safe and efficient novel protein antifreeze agent as well as a preparation method and application thereof, wherein the preparation process of the protein antifreeze agent comprises the following steps: the fish scale is obtained after drying, pretreatment, steam flash explosion treatment, decalcification, enzymolysis, hot water extraction and freeze-drying, and the obtained protein antifreeze can be used as a cryoprotectant to be applied to the cryopreservation of aquatic products, enzymes and strains; the invention has the advantages of wide raw material source, low cost, simple process and high efficiency, and the prepared novel antifreeze has the advantages of safety, nutrition, wide application range and the like, can effectively control the quality deterioration of frozen foods and biological products, and enhances the freeze-thaw stability of the frozen foods and the biological products.)

1. The protein antifreeze is characterized by being prepared by the following method:

(1) removing residual meat on the surface of a fresh fish scale, cleaning and drying to obtain a raw fish scale, immersing the raw fish scale into a sodium hydroxide aqueous solution, stirring for 2-3 h at 20-30 ℃ to remove impure proteins in the fish scale, cleaning, and drying to finish pretreatment;

(2) putting the fish scales pretreated in the step (1) into a reaction treatment cavity of steam flash explosion equipment, maintaining the pressure for 1-4 min under the steam pressure of 1-3 MPa, and after a bottom cover of the explosion cavity is automatically pulled down, quickly reducing the pressure in the cavity to the atmospheric pressure, so that water in the fish scales forms flash evaporation, and completing adiabatic work on materials from inside to outside to realize steam flash explosion treatment;

(3) adding the fish scales subjected to the steam explosion treatment in the step (2) into a decalcifying solution, stirring for 2-3 hours at 20-30 ℃, completing the decalcifying treatment, then adding an enzyme solution, carrying out enzymolysis for 2-5 hours at 20-40 ℃ and under the condition that the pH value is 2-3, inactivating the enzyme, cleaning, then leaching for 3-5 hours at the constant temperature of 50-70 ℃ by using water as a solvent, filtering to obtain a leaching solution, and carrying out vacuum freeze drying on the obtained leaching solution to obtain the protein antifreeze agent.

2. The protein antifreeze agent of claim 1, wherein in step (1), said fish scales are derived from a common freshwater species or a common marine species.

3. The protein antifreeze agent as set forth in claim 1, wherein in the step (1), the concentration of said aqueous solution of sodium hydroxide is 0.1 to 0.2M.

4. The protein antifreeze agent as set forth in claim 1, wherein in the step (1), the volume of the sodium hydroxide aqueous solution is 15-25 mL/g based on the mass of the raw fish scales.

5. The protein antifreeze agent according to claim 1, wherein in the step (3), the decalcifying solution is an aqueous solution of hydrochloric acid, acetic acid or citric acid, and the concentration is 0.3-0.5M.

6. The protein antifreeze agent as set forth in claim 1, wherein in the step (3), the volume of said decalcifying liquid is 20mL/g based on the mass of the raw fish scales.

7. The protein antifreeze agent as set forth in claim 1, wherein in the step (3), the enzyme solution is an aqueous solution containing 1-5% by mass of protease selected from the group consisting of pepsin, neutral protease and alkaline protease.

8. The protein antifreeze agent as set forth in claim 1, wherein in the step (3), the volume of the enzyme solution is 20-30 mL/g based on the mass of the raw fish scales.

9. Use of the protein antifreeze of claim 1 as a cryoprotectant in the cryopreservation of aquatic products, enzymes and bacterial species.

Technical Field

The invention belongs to the field of food processing and storage, and particularly relates to a safe and efficient novel protein antifreeze agent, a preparation method and application thereof, which can be used as a cryoprotectant to be applied to cryopreservation of aquatic products, enzymes and strains.

Background

The low-temperature freezing storage is one of the main storage modes of fresh food and raw materials thereof, but the freezing treatment inevitably causes the formation of ice crystals, further leads to the rupture of raw material tissues, and causes the serial changes of food components such as lipid oxidation, protein oxidation and aggregation, and finally obviously reduces the processing characteristics and the nutritional quality of the food. The addition of an antifreeze is one of the common methods for enhancing the freezing storage stability of fresh foods. The traditional antifreeze agent is a compound formed by cane sugar, sorbitol and compound phosphate, and is widely applied to the freezing storage of food due to good antifreeze effect and lower cost. With the development of food science and the enhancement of the concept of safe nutrition for consumers, the potential safety risk of such conventional antifreeze agents raises concerns for consumers. For example, sucrose has higher calorie, can cause obesity and dental caries, and is not suitable for diabetics to eat, and the higher sweetness of sucrose can change the mouthfeel of the food; it has been shown that complex phosphates are at risk for an exacerbation of gastrointestinal disease. It follows that the safety and applicability of such conventional antifreeze agents are somewhat limited. As a typical representative of a novel antifreeze, antifreeze protein and antifreeze peptide show excellent antifreeze property, but the high production cost, harsh preparation conditions and potential safety hazards of the antifreeze also limit the wide application of the antifreeze protein and the antifreeze peptide, so that the development of the novel antifreeze with safe and efficient antifreeze capability becomes a key technical problem to be solved urgently in the frozen food industry.

At present, starting from the anti-freezing structure basis of the existing anti-freezing agents such as anti-freezing protein, anti-freezing peptide and polysaccharide colloid, the development of novel anti-freezing agents through the structural design from bottom to top becomes one of the main research and development ideas. Cryoprotectants having freezing resistance comparable to the cryoprotectants described above can be prepared by chemical modification or assembly modification of small molecule substances and have been used for cryoprotectants for biological tissues and special materials (Graham, B., Fayter, A.E.R. et al (2019). Synthesis of anticancer proteins Sequences: Effect of the End group photonic condensed polymerization on Ice crystallization inactivation Activity. biomacromolecules,20, 4611-4621). However, the antifreeze prepared by a chemical synthesis method still has certain safety risk and cannot be directly applied to the freezing protection of food. The fish scale glue derived from main byproducts of aquatic product processing has the structural characteristics (Gly-X-Y sequence distribution and PP II conformation) of antifreeze protein and the gel characteristic of polysaccharide colloid, and has a molecular structure basis required by an antifreeze agent, so that the fish scale glue is expected to be applied to the freezing protection of food as a novel antifreeze agent. In addition, the above-mentioned anti-freezing properties of fish-scale glue are closely related to the size of the molecular weight. Fish-scale small-molecule peptides have been shown to have antifreeze-like activities (Fu, W.Q., Wang, P.X., et al (2019). Preparation, primaryarchitecture and antibacterial activity of antibacterial peptides from microbial bacteria skin. Lwt-Food Science and Technology,101, 670-) -677); the gelling properties of the high molecular weight components of fish-scale gums are similar to the anti-freezing mechanism of polysaccharide gels (Gaukel, V., Leiter, A.et al. (2014). Synergism of differential fire Engineering of ice in skin solvents. journal of Food Engineering,141, 44-50). Therefore, the key for preparing the gelatin-based antifreeze agent is to control the reasonable composition of the molecular distribution of the ichthyol to exert the antifreeze property to the maximum extent. The main preparation method of the fish scale glue comprises a pretreatment and hot water extraction method, wherein the pretreatment method comprises acid, alkali and enzyme treatment. However, the efficiency of preparing the fish scale glue by adopting the method is greatly limited due to the state that the collagen of the fish scales is closely combined with the hydroxyapatite.

The original compact structure of the fish scales is destroyed by adopting a certain pretreatment means to realize subsequent high-efficiency enzyme action or one of effective means for preparing the fish scale glue. The steam explosion technology is an advanced biomass pretreatment technology and is generally concerned by scholars in recent years. It has the advantages of simple process, high efficiency, energy saving, no pollution, low treatment cost, etc. The steam explosion technology is that the material is placed in a pressure container, the material surface layer micropores are utilized to permeate high-pressure saturated steam into the material tissue, wherein the partially saturated steam forms high-temperature liquid water, because the material surface layer micropores are smaller, when the outside of the material is instantly reduced to atmospheric pressure, only a few parts of the high-pressure steam in the material can be released into the atmosphere through the micropores in the material surface layer, most of the saturated steam expands at a high speed, and simultaneously the high-temperature liquid water is rapidly exploded to boil to form flash evaporation, a large amount of steam completes adiabatic expansion acting in a very short time to convert heat energy into mechanical energy, and shock waves generated by the steam expansion acting generate shearing force on the material, so that the purpose of destroying the internal structure of the material tissue is realized. Zhao Li Ming discloses a clean production process of chitin, which carries out instantaneous high-pressure steam flash explosion pretreatment on raw materials by a high-density steam flash explosion device, and dissolves and extracts most of protein in the raw materials into food-grade hydrolyzed protein by enzymolysis, and the whole process has the advantages of simple process, short working procedure and convenient operation (CN 201310056441.9); houxiuli discloses a method for comprehensively utilizing cotton stalk skins by combining alkali pretreatment, low-pressure steam flash explosion and biological enzyme treatment (CN 201410298683.3). Therefore, the steam flash explosion technology can be used as a novel technology, and can destroy the original compact tissue structure of the fish scales so as to enhance the efficient and controllable preparation capacity of the fish scale glue.

Disclosure of Invention

Aiming at the defects of various antifreeze agents used for the freezing protection of fresh foods at present, the invention provides a safe and efficient novel protein antifreeze agent, and a preparation method and application thereof. The preparation process of the protein antifreeze agent comprises the following steps: the fish scale is obtained by drying, pretreating, steam flash explosion treating, decalcifying, enzymolysis, hot water leaching and freeze-drying.

The technical scheme of the invention is as follows:

a protein antifreeze agent is prepared by the following method:

(1) removing residual meat on the surface of a fresh fish scale, cleaning and drying to obtain a raw material fish scale, immersing the raw material fish scale into a sodium hydroxide aqueous solution, stirring for 2-3 h (preferably 2.5h) at 20-30 ℃ (preferably 20 ℃) to remove impure protein in the fish scale, cleaning, and drying to finish pretreatment (storage for later use);

the fish scales are derived from common freshwater fish species (such as green, grass, common carp, crucian carp, silver carp, bighead carp, bream and tilapia) or common seawater fish species (such as large yellow croaker, small yellow croaker, snakehead, cod, hairtail, grouper, sea bass and turbot);

the concentration of the sodium hydroxide aqueous solution is 0.1-0.2M, preferably 0.1M;

the volume consumption of the sodium hydroxide aqueous solution is 15-25 mL/g, preferably 20mL/g, based on the mass of the raw material fish scales;

(2) putting the fish scales pretreated in the step (1) into a reaction treatment cavity of steam flash explosion equipment, maintaining the pressure for 1-4 min (preferably 1min) under the steam pressure of 1-3 MPa (preferably 1.5MPa), and after a bottom cover of the explosion cavity is automatically pulled down, quickly reducing the pressure in the cavity to the atmospheric pressure, so that the water in the fish scales forms flash evaporation, and finishing adiabatic work on the material from inside to outside to realize steam flash explosion treatment;

(3) adding the fish scales subjected to the steam flash explosion treatment in the step (2) into a decalcifying solution, stirring for 2-3 h (preferably 2h) at 20-30 ℃ (preferably 20 ℃), completing the decalcifying treatment, then adding an enzyme solution, carrying out enzymolysis for 2-5 h (preferably 3h) at 20-40 ℃ and pH 2-3, inactivating the enzyme, cleaning, then leaching for 3-5 h (preferably 3h) at 50-70 ℃ (preferably 50 ℃) at constant temperature by using water as a solvent, filtering to obtain a leaching solution, and carrying out vacuum freeze drying on the obtained leaching solution to obtain the protein antifreeze agent (stored in a dark dry place);

the decalcifying liquid is an aqueous solution of hydrochloric acid, acetic acid or citric acid, and the concentration of the aqueous solution is 0.3-0.5M, preferably 0.3M hydrochloric acid;

the volume consumption of the decalcification solution is 20mL/g based on the mass of the raw material fish scales;

the enzyme solution is an aqueous solution of protease with the mass fraction of 1% -5%, the protease is selected from pepsin, neutral protease or alkaline protease, preferably pepsin, and the enzyme activity is 1: 3000A;

the volume dosage of the enzyme solution is 20-30 mL/g based on the mass of the raw material fish scales;

the preferred enzymatic conditions are: the temperature is 35 ℃, the pH value is 2.5, and the enzymolysis time is 4 h;

the enzyme deactivation method comprises the following steps: inactivating the enzyme at a constant temperature of 90 ℃ for 3-5 min.

The protein antifreeze agent prepared by the invention can be used as a freezing protective agent to be applied to freezing preservation of aquatic products, enzymes and strains.

Compared with the prior art, the invention has the following advantages:

1. the novel antifreeze agent prepared by the invention is derived from byproducts generated in aquatic product processing, and has the advantages of wide raw material source and low cost compared with the higher production cost of antifreeze protein and antifreeze peptide. In addition, the antifreeze agent is prepared by taking the aquatic product processing by-product as a raw material, and a new way is provided for high-value utilization of the aquatic product processing by-product and reduction of the environmental protection cost of the aquatic product processing industry.

2. The method combines the steam flash explosion to pretreat the fish scales, has the advantages of simple process, high efficiency, no pollution, low treatment cost and the like compared with the traditional treatment method, can greatly shorten the preparation period of the fish scale glue, and is beneficial to the high-efficiency controllable preparation of the fish scale glue with high freezing resistance.

3. The novel antifreeze prepared by the invention mainly comprises protein, has high heat and incompatibility of diabetics compared with the traditional antifreeze (sucrose, sorbitol and compound phosphate compound), and has the advantages of safety, nutrition, wide application range and the like. The product of the invention can effectively control the quality deterioration of frozen foods and biological products, enhance the freeze-thaw stability of the frozen foods and biological products, and can be widely applied to the fields of preservation of aquatic products, enzymes, strains and the like as a cryoprotectant.

Drawings

FIG. 1 shows a fish scale collagen antifreeze agent prepared by the present invention.

Figure 2 shows the freeze protection effect of fish scale glue on surimi.

FIG. 3 cryoprotective effect of fish-scale glue on catalase.

Figure 4 the cryoprotective effect of fish-scale gelatin on lactobacillus bulgaricus.

FIG. 5 cryoprotective activity of gelatin antifreeze polypeptides in the comparative examples.

FIG. 6 shows a comparison of the yields of gelatin prepared by different methods.

Detailed Description

The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the non-essential modifications and adjustments made by those skilled in the art according to the above disclosure still fall within the scope of the present invention.