Background

The body of the anoectochilus roxburghii (Nemipterus virgatus) belongs to the order Perciformes, the family Goniorhidae and the genus Anoectochilus roxburghii, is oval, slightly elongated, flat on the side, less in thick meat, fine and smooth in meat quality, and widely distributed in the sea areas such as Philippines, Indonesia, Vietnam, Thailand, Australia and the like, wherein the yield of the south east China and the northern gulf sea area is the highest in China, and the method becomes one of the local and even national important marine economic fishes. The fish meat of the golden thread fish has rich protein content and is often used as a main raw material of minced fillet products. However, the essential amino acid content of the anoectochilus formosanus protein is low, and the nutritional value is low. In addition, myosin, which is a main protein in fish, is easy to generate a 'gel softening' phenomenon in thermal processing, and restricts the application of myosin in the field of deep processing in fishery. Therefore, by carrying out enzymolysis on the goldfish protein by using the exogenous protease, the peptide segment with potential biological activity in the primary structure of the protein can be released, and a feasible way can be provided for high-value utilization of low-value fish products.

Immunomodulatory peptides modulate the inflammatory process of cells, organs or tissues primarily by interacting with cell membrane immunoreceptors or participating in free radical reactions. Research has shown that local chronic inflammation is related to cancer, cardiovascular diseases, etc., and such degenerative diseases may also exacerbate in vivo inflammation, leading to a dysregulation of immune homeostasis in vivo. However, traditional anti-inflammatory drugs such as silymarin, acipimox, etc. are associated with long-term potential side effects, and excessive use of them easily causes liver function damage. The immune regulatory peptide is generally an oligopeptide consisting of 5-30 amino acids, and has the functions of inhibiting nitric oxide synthase and m-TOR receptor and clearing active oxygen in mitochondrial inner membrane; can also destroy cell membrane of pathogenic bacteria, inhibit bacteria, and improve local inflammation microenvironment. In addition, the immunoregulation peptide has the characteristics of high activity, good safety, low preparation cost and the like. Related researches prove that the bioactive peptide has the characteristic of multi-targeting, and the related immunoregulation peptide can prevent and inhibit inflammation from multiple ways such as oxidation resistance, blood pressure reduction, proinflammatory factor inhibition and the like, and has certain efficacy on inflammatory reactions induced by different ways. Therefore, compared with commercial anti-inflammatory drugs, the immunomodulatory peptides not only are more in line with consumption trends, but also have better comprehensive anti-inflammatory effects than traditional anti-inflammatory drugs.

However, the enzymatic method currently used for the preparation of biologically active peptides is inefficient, especially for animal muscle proteins, where the myosin head forms a highly alpha-helical structure by intramolecular hydrogen bonding, on the basis of which protein multimers are formed by intermolecular forces. The protease can not interact with the active site in the region, so that not only can the specific polypeptide fragment not be released, but also a great number of peptide chains with beta-turn and irregular structures at the tail part are excessively enzymolyzed. Therefore, the simple enzymolysis method is not favorable for preparing the bioactive peptide with high efficiency. At present, physical means such as ultrasonic waves, microwaves and the like are commonly adopted at home and abroad to process original protein, so that the molecular structure of the protein is expanded, the interaction between protease and enzyme cutting sites is promoted, and the yield of bioactive peptides is further improved. For example. Patent 201010188418.1 discloses an ultrasonic-assisted enzymolysis method for preparing oat antihypertensive peptide, which comprises pretreating oat protein by 250-1250W/100mL ultrasonic field, turning off ultrasonic after 5-10min and transferring into alkaline protease for enzymolysis, so as to improve the yield and activity of the antihypertensive peptide. However, for proteins with strong hydrophobicity such as myosin, hydrophobic aggregation between myosin heavy chains and oxidation of sulfydryl can be caused by traditional treatment methods such as ultrasonic wave or microwave, so that protein gel is generated, and subsequent enzymolysis is not facilitated. The high-pressure homogenization treatment can rapidly dissociate intermolecular hydrogen bonds, promote rapid unfolding of a protein structure, convert alpha-helix into beta-fold and irregular structures, and fully expose active sites of high alpha-helix heads. Thereby accelerating the subsequent proteolysis efficiency and simultaneously improving the activity of the immunoregulation peptide. And no relevant report is found about the preparation of the antioxidant peptide by using a high-pressure homogenization technology.

Ultrafiltration membrane separation is the most common technique for fractionation of bioactive peptides, and usually uses a carbon fiber membrane as a filtering medium, wherein small solute and solution can permeate through the membrane, and relatively large solute molecules can be trapped under a certain pressure. However, in the process of large-scale separation of active peptides, peptide fragments with large molecular weight are easily adsorbed on the membrane surface to block the membrane pores, which leads to reduction of membrane flux and is not favorable for recycling of ultrafiltration membranes. The polypeptide molecules are quickly polarized by an external electric field, and the molecules are improved to be fully unfolded into a rod-shaped structure, so that the polypeptides quickly pass through the ultrafiltration membrane and cannot be gathered on the surface of the membrane. In addition, the flow rate of the polypeptide can be improved by applying a pulse electric field, and the separation efficiency of the ultrafiltration membrane is improved. At present, no report is found on the literature about the application of the applied pulse electric field to assist membrane separation and bioactive peptides.