A squid cartilage hydrolyzate, large-scale preparation method and application thereof

Abstract

The present invention relates to a squid cartilage hydrolyzate, a large-scale preparation method and its application. Using squid cartilage discarded from aquatic product processing plants as raw material, first use acid solution to remove calcium salts, then use high-temperature acid hydrolysis reaction to quickly hydrolyze the squid cartilage into a mixture of oligosaccharides and oligopeptides, and then use Maillard reaction and ethanol to remove fishy smell to improve the flavor, and further use continuous flow centrifugation synergistic membrane separation technology to separate and purify the crude extract of squid cartilage hydrolyzate to obtain a squid cartilage hydrolyzate solution with a content of ≥95%. Compared with the enzymatic hydrolysis method, the method of the present invention has a higher yield and consumes less time. At the same time, the squid cartilage hydrolyzate produced by the present invention has better antibacterial activity than potassium sorbate and sodium benzoate; has better water absorption performance than glycerol; has better antioxidant activity than butylated hydroxyanisole (BHA) and vitamin E, and its flavor is better.

Description

Technical Field

[0001] The invention relates to a large-scale preparation method of squid cartilage hydrolyzate, belonging to the field of chemical extraction technology and food. Background Art

[0002] Squid, a common cephalopod mollusk, is a valuable raw material for aquatic product processing. my country is one of the world's leading squid catchers and processors. During squid processing, a large amount of squid cartilage is often produced as a waste byproduct. A portion of this cartilage is processed into fishmeal feed, but little of it is effectively utilized. Due to its high risk of spoilage, it is typically disposed of in landfills, which can cause significant environmental pollution. Therefore, enhancing the efficient and comprehensive utilization of this waste byproduct, squid cartilage, can not only transform waste into valuable resources and increase its technological value, but also reduce waste disposal costs, significantly impacting the development of the squid industry and environmental protection.

[0003] Related research shows that squid cartilage accounts for about 2% of its body weight, and its active ingredients are proteins (collagen, core protein, etc.) and carbohydrates (chondroitin sulfate, chitin, etc.). At present, the literature and patents related to squid cartilage all separate the active ingredients of squid cartilage into collagen, chondroitin sulfate, chitin, etc., and then use them separately for processing (for example, CN202110439789.0, CN202011521963.8, CN202110439798.X, CN201811558088.3, etc.). Although such a processing method can obtain a certain active ingredient, the price paid is that other active ingredients are treated as impurities and discarded, which will undoubtedly cause a waste of resources. Such a technical process that does not focus on the comprehensive utilization of the active ingredients in squid cartilage is also difficult to promote large-scale production.

[0004] At the same time, squid cartilage has a strong and unpleasant fishy smell that most people cannot tolerate, which is why it is used in fishmeal feed. How to effectively remove the fishy smell has become a difficult problem that must be solved in order to use squid cartilage, especially to make it acceptable for consumption as a new food ingredient. However, the literature and patents related to squid cartilage currently have almost no attention to this key issue. Summary of the Invention

[0005] In view of the two major problems of comprehensive utilization of squid cartilage and treatment of fishy odor, the present invention provides a large-scale preparation method of squid cartilage hydrolyzate with simple production process, low production cost, high product content, molecular weight distribution below 1500Da, excellent antibacterial activity and water absorption performance, good antioxidant activity and flavor, and suitable for consumption, so as to realize the high-value comprehensive utilization of marine biological resources.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A large-scale preparation method of squid cartilage hydrolysate comprises the following steps:

[0008] (1) Pretreatment

[0009] The squid cartilage is first washed with clean water to remove surface attachments and impurities such as mucus. After rinsing, it is placed in a reactor, and an acid solution 5 to 10 times the weight of the squid cartilage is added. The mixture is stirred for 15 minutes to 1 hour, and then washed with deionized water. The mass volume concentration of the acid solution is 1 to 5%.

[0010] (2) High temperature acid hydrolysis reaction

[0011] The pretreated and cleaned squid cartilage is placed in a reactor, deionized water 2 to 10 times the weight of the squid cartilage is added, an acid solution is added to adjust the pH to 1 to 3, the reaction temperature is 85 to 100° C., and the reaction is stirred for 2 to 6 hours;

[0012] (3) Maillard reaction and ethanol deodorization

[0013] After the acid reaction is completed, the reaction temperature is maintained, and an alkali solution is added to adjust the pH to 5.0-6.5 to start the Maillard reaction, and the reaction is stirred for 20 minutes to 2 hours. After the Maillard reaction is completed, an ethanol solution with a volume of 0.1% to 0.6% of the reaction solution is immediately added, and the temperature is slowly lowered to room temperature to obtain a crude extract of squid cartilage hydrolyzate, wherein the concentration of the ethanol solution is 10% to 60%.

[0014] (4) Separation and purification

[0015] Continuous flow centrifugation combined with a nanofiltration membrane with a relative molecular mass of 10kDa to 5kDa is used to remove solid residues and large molecular impurities in the crude extract. The resulting clear liquid is finally filtered through a nanofiltration membrane with a relative molecular mass of 200Da to remove small molecular impurities and inorganic salts, and is further concentrated to obtain a squid cartilage hydrolyzate solution with a content of ≥95%.

[0016] Preferably, the large-scale preparation method of the present invention further comprises:

[0017] (5) Rapid drying and sterilization packaging

[0018] The squid cartilage hydrolyzate solution is quickly dried by spray drying to obtain squid cartilage hydrolyzate powder, which is then quickly sterilized by light radiation and finally aseptically packaged.

[0019] The squid cartilage used in step (1) comes from Argentine squid, whose Latin name is Illex argentinus; the acid used in the decalcification treatment by adding an acid solution is hydrochloric acid or nitric acid.

[0020] In the step (2), the acid solution is added to carry out the high-temperature acid hydrolysis reaction, and the acid used is citric acid or sulfuric acid.

[0021] In the step (3), the alkali solution used to adjust the pH value is sodium bicarbonate, potassium bicarbonate, sodium hydroxide or potassium hydroxide; the ethanol solution can be a solution prepared with edible alcohol, or a solution containing ethanol prepared with white wine, yellow wine or cooking wine.

[0022] In the step (4), the high-speed centrifugal speed of the continuous flow centrifuge is 10,000 to 15,000 rpm; the nanofiltration membrane with a relative molecular mass of 10 kDa to 5 kDa has a flow rate of 5 to 14 cubic meters per hour, a membrane pressure of 0.552 to 0.931 MPa, and a temperature of 20 to 50°C; the nanofiltration membrane with a relative molecular mass of 200 Da has a flow rate of 5 to 14 cubic meters per hour, a membrane pressure of 0.483 to 2.758 MPa, and a temperature of 20 to 50°C; the molecular weight of the squid cartilage hydrolyzate is mostly distributed below 1,500, belonging to the oligosaccharide and oligopeptide level.

[0023] The spray drying process in step (5) has an air inlet temperature of 120-180° C. and an air outlet temperature of 70-95° C. The light radiation used is ultraviolet radiation, γ-rays or electron beam radiation, with a radiation dose of 0.01-40 kGy and a radiation time of 1-60 minutes.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The present invention uses squid cartilage discarded from aquatic product processing plants as raw material and adopts a high-temperature acid hydrolysis process to degrade carbohydrates (chondroitin sulfate, chitin, etc.) and proteins (collagen, core protein, etc.) in the squid cartilage in one step into a mixture of oligosaccharides and oligopeptides with a molecular weight distribution below 1500 Da. Compared with currently popular enzymatic hydrolysis methods (such as single enzymatic hydrolysis and combined enzymatic hydrolysis), the squid cartilage hydrolyzate produced by the process of the present invention has the highest yield and consumes the least time.

[0026] 2. Unlike other patents related to squid cartilage, which differentiate between the utilization of chondroitin sulfate, chitin / chitosan, and collagen, this present invention utilizes a unified high-temperature acid hydrolysis method to degrade the numerous active ingredients in squid cartilage, such as chondroitin sulfate, chitin, and collagen, into a squid cartilage hydrolyzate with a molecular weight distribution below 1500 Da. This squid cartilage hydrolyzate with such a molecular weight distribution is a mixture of oligosaccharides and oligopeptides, which is more conducive to human gastrointestinal absorption. Furthermore, this present invention achieves the ideal goal of maximizing the utilization of the active ingredients in squid cartilage waste.

[0027] 3. Based on the composition characteristics of squid cartilage, the present invention mainly contains carbohydrates and proteins. After pretreatment and decalcification, a high-temperature acid hydrolysis process is used to degrade the chondroitin sulfate in the squid cartilage into low molecular weight chondroitin sulfate, glucuronic acid and glucose, while degrading chitin into chitosan oligosaccharides and glucosamine, and degrading protein into oligopeptides and amino acids. Among them, glucuronic acid, glucosamine and glucose are all reducing sugars. Under appropriate conditions, they can react with oligopeptides and amino acids to produce Maillard reaction (see Figure 2 ), thereby significantly improving the flavor of the squid cartilage hydrolyzate. Therefore, the Maillard reaction of the present invention differs significantly from other patents that use the addition of foreign substances to induce the Maillard reaction. The present invention utilizes the product characteristics of high-temperature acid hydrolysis of squid cartilage to achieve the goal of inducing the Maillard reaction in the reaction solution itself simply by adjusting the pH of the reaction solution, demonstrating its considerable innovation.

[0028] 4. Squid cartilage has a strong fishy odor, and its hydrolyzate will still have an unpleasant fishy smell if not treated with appropriate deodorization methods. In view of this, the present invention uses the Maillard reaction and subsequent ethanol deodorization method to significantly improve the flavor of the squid cartilage hydrolyzate in terms of both smell and taste, achieving the excellent effects of reducing fishy smell, enhancing fragrance, removing bitterness, and enhancing freshness.

[0029] 5. The large-scale production cycle of the squid cartilage hydrolyzate of the present invention can basically be completed within 12 hours, the process method is simpler and more practical; the process flow is more efficient and reliable, and the overall process can better meet the requirements of industrialization and large-scale production.

[0030] 6. Compared with potassium sorbate and sodium benzoate, common preservatives in food, the squid cartilage hydrolyzate produced by the present invention has better antibacterial activity; compared with glycerin, a common humectant in food, the squid cartilage hydrolyzate produced by the present invention has better water absorption performance; compared with butylated hydroxyanisole (BHA) and vitamin E, common antioxidants in food, the squid cartilage hydrolyzate produced by the present invention has better antioxidant activity, and its flavor is better, and it is easier for consumers to accept and eat; therefore, the squid cartilage hydrolyzate produced by the present invention can be used as a functional raw material for general food.

[0031] 7. The present invention uses high-temperature acid hydrolysis of squid cartilage to produce glucuronic acid, glucosamine and glucose. Among them, glucuronic acid, as an intermediate of human sugar metabolism, can combine with phenols, sterols and aromatic carboxylic acids and be excreted from the body in the form of glucuronic acid esters, playing a role in liver detoxification; glucosamine is an important nutrient for the formation of human chondrocytes, a basic substance for the synthesis of amino polysaccharides, and a natural tissue component of healthy articular cartilage. Glucosamine can promote the synthesis of cartilage, stimulate the growth of chondrocytes, inhibit the decomposition of articular cartilage, and also has anti-inflammatory effects; glucose is an indispensable nutrient for metabolism in organisms. The heat released by its oxidation reaction is an important source of energy required for human life activities. Therefore, the squid cartilage hydrolyzate produced by the present invention is very suitable for direct consumption as a clinical nutritional product, health food, sports food and nutritional supplement. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Table 1 shows the main components of the Argentine squid cartilage raw material of the present invention.

[0033] Table 2 is a comparison of the extraction rates of squid cartilage hydrolyzates obtained by different hydrolysis methods. Among them, the high-temperature acid hydrolysis method of the present invention (sulfuric acid solution, pH = 1.5, 95 ° C, 3 hours); single enzymatic hydrolysis method (enzymatic hydrolysis conditions): 1. papain (material ratio 2%, pH = 6.5, 55 ° C, 24 hours); 2. bromelain (material ratio 2%, pH = 6.0, 53 ° C, 24 hours); 3. neutral protease (material ratio 2%, pH = 7.0, 50 ° C, 24 hours); 4. alkaline protease (material ratio 2%, pH = 9.0, 55 ° C, 24 hours) ); 5. Acidic protease (material ratio 2%, pH = 2.5, 50 ℃, 24 hours); 6. Pepsin (material ratio 2%, pH = 1.5, 37 ℃, 24 hours); 7. Trypsin (material ratio 2%, pH = 8.0, 37 ℃, 24 hours); 8. Flavor protease (material ratio 2%, pH = 6.5, 53 ℃, 24 hours); Composite enzymatic hydrolysis method (enzymatic hydrolysis conditions): 1. First, acidic protease (material ratio 2%, pH = 2.5, 50 ℃, 12 hours), then Papain (material ratio 2%, pH = 6.5, 55 ℃, 12 hours); 2. Acidic protease (material ratio 2%, pH = 2.5, 50 ℃, 12 hours), then bromelain (material ratio 2%, pH = 6.0, 53 ℃, 12 hours); 3. Acidic protease (material ratio 2%, pH = 2.5, 50 ℃, 12 hours), then neutral protease (material ratio 2%, pH = 7.0, 50 ℃, 12 hours); 4. Acidic protease (material ratio 2%, pH = 7.0, 50 ℃, 12 hours) H=2.5, 50℃, 12 hours), then alkaline protease (material ratio 2%, pH=9.0, 55℃, 12 hours); 5. Acidic protease (material ratio 2%, pH=2.5, 50℃, 12 hours), then flavor protease (material ratio 2%, pH=6.5, 53℃, 12 hours); 6. Pepsin (material ratio 2%, pH=1.5, 37℃, 12 hours), then trypsin (material ratio 2%, pH=8.0, 37℃, 12 hours).

[0034] Table 3 shows the flavor evaluation criteria for squid cartilage hydrolysate, squid cartilage raw material and undeodorized squid cartilage hydrolysate, and Table 4 shows the flavor evaluation results for squid cartilage hydrolysate, squid cartilage raw material and undeodorized squid cartilage hydrolysate.

[0035] Figure 1 The present invention is a process flow chart of a method for rapidly preparing squid cartilage hydrolyzate on a large scale.

[0036] Figure 2 This is a flow chart of the principle of further implementing the Maillard reaction on pre-treated decalcified squid cartilage after completing the high-temperature acid hydrolysis reaction.

[0037] Figure 3This is the chromatogram of squid cartilage hydrolysate.

[0038] Figure 4 The invention discloses a method for studying the functional activity and evaluating the flavor of squid cartilage hydrolyzate.

[0039] Figure 5 These are the antibacterial activity results of squid cartilage hydrolysate. a—Antibacterial activity of squid cartilage hydrolysate against Escherichia coli; b—Antibacterial activity of squid cartilage hydrolysate against Bacillus subtilis; c—Antibacterial activity of squid cartilage hydrolysate against Staphylococcus aureus; d—Antibacterial activity of squid cartilage hydrolysate against Pseudomonas aeruginosa; e—Antibacterial activity of squid cartilage hydrolysate against Shewanella spp. The content of squid cartilage hydrolysate, potassium sorbate, and sodium benzoate was 1 mg / mL.

[0040] Figure 6 It is the result of the water absorption performance of squid cartilage hydrolyzate (1 - squid cartilage hydrolyzate; 2 - glycerol).

[0041] Figure 7 These are the antioxidant activity results of squid cartilage hydrolysate (a - FRAP method to determine the iron ion reduction / antioxidant capacity of squid cartilage hydrolysate; b - ABTS method to determine the antioxidant capacity of squid cartilage hydrolysate; c - T-AOC method to determine the total oxidative capacity of squid cartilage hydrolysate; d - Fenton method to determine the hydroxyl free radical content of squid cartilage hydrolysate). DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0043] Example 1:

[0044] A large-scale preparation method of squid cartilage hydrolysate comprises the following steps:

[0045] (1) Pretreatment

[0046] The squid cartilage was first washed with clean water to remove surface attachments and impurities such as mucus. After rinsing, it was placed in a reactor and a hydrochloric acid solution 10 times the weight of the squid cartilage was added. The mixture was stirred for 30 minutes and then washed with deionized water. The mass volume concentration of the hydrochloric acid solution was 3%.

[0047] (2) High temperature acid hydrolysis reaction

[0048] The pretreated and cleaned squid cartilage was placed in a reactor, deionized water 5 times the weight of the squid cartilage was added, sulfuric acid solution was added to adjust the pH to 1.5, the reaction temperature was 95°C, and the reaction was stirred for 3 hours;

[0049] (3) Maillard reaction and ethanol deodorization

[0050] After the acid reaction is completed, the reaction temperature is maintained, and a sodium hydroxide solution is added to adjust the pH to 5.5 to initiate a Maillard reaction, and the reaction is stirred for 1 hour. After the Maillard reaction is completed, an ethanol solution (the concentration of the edible alcohol solution is 50%) with a volume of 0.12% of the reaction solution is immediately added, and the temperature is slowly lowered to room temperature to obtain a crude extract of squid cartilage hydrolyzate.

[0051] (4) Separation and purification

[0052] First, solid residues in the crude extract were removed by high-speed centrifugation at 14,000 rpm using a continuous flow centrifuge. Macromolecular impurities in the crude extract were then removed using a nanofiltration membrane with a relative molecular mass of 5 kDa at a flow rate of 6 cubic meters per hour, a membrane pressure of 0.61 MPa, and a temperature of 35°C. Finally, small molecular impurities and inorganic salts were removed from the resulting clear solution using a nanofiltration membrane with a relative molecular mass of 200 Da at a flow rate of 10 cubic meters per hour, a membrane pressure of 1.85 MPa, and a temperature of 35°C. The solution was concentrated to obtain a squid cartilage hydrolyzate solution with a content of 97.85%.

[0053] (5) Rapid drying and sterilization packaging

[0054] The squid cartilage hydrolyzate solution was quickly dried by spray drying, with the inlet air temperature set at 170°C and the outlet air temperature set at 95°C to obtain a squid cartilage hydrolyzate powder; the powder was then quickly sterilized by γ-ray radiation with a radiation dose of 10 kGy and a radiation time of 15 minutes, and finally sterile packaging was performed.

[0055] Example 2:

[0056] A large-scale preparation method of squid cartilage hydrolysate comprises the following steps:

[0057] (1) Pretreatment

[0058] The squid cartilage was first washed with clean water to remove surface attachments and impurities such as mucus. After rinsing, it was placed in a reactor and a nitric acid solution 5 times the weight of the squid cartilage was added. The mixture was stirred for 1 hour and then washed with deionized water. The mass volume concentration of the nitric acid solution was 2%.

[0059] (2) High temperature acid hydrolysis reaction

[0060] The pretreated and cleaned squid cartilage was placed in a reactor, deionized water 10 times the weight of the squid cartilage was added, citric acid solution was added to adjust the pH to 2.0, the reaction temperature was 85°C, and the reaction was stirred for 6 hours;

[0061] (3) Maillard reaction and ethanol deodorization

[0062] After the acid reaction is completed, the reaction temperature is maintained, potassium hydroxide solution is added to adjust the pH to 6.0 to initiate the Maillard reaction, and the reaction is stirred for 2 hours. After the Maillard reaction is completed, a 0.1% by volume white wine solution (the concentration of ethanol in the white wine is 53%) is immediately added, and the temperature is slowly lowered to room temperature to obtain a crude extract of squid cartilage hydrolyzate.

[0063] (4) Separation and purification

[0064] First, solid residues in the crude extract were removed by high-speed centrifugation at 12,500 rpm using a continuous flow centrifuge. Macromolecular impurities in the crude extract were then removed using a nanofiltration membrane with a relative molecular mass of 5 kDa at a flow rate of 8 cubic meters per hour, a membrane pressure of 0.72 MPa, and a temperature of 35°C. Finally, small molecular impurities and inorganic salts were removed from the resulting clear solution using a nanofiltration membrane with a relative molecular mass of 200 Da at a flow rate of 5 cubic meters per hour, a membrane pressure of 0.65 MPa, and a temperature of 35°C. The solution was concentrated to obtain a squid cartilage hydrolyzate solution with a content of 97.5%.

[0065] (5) Rapid drying and sterilization packaging

[0066] The squid cartilage hydrolyzate solution was quickly dried by spray drying, with the inlet air temperature set at 160°C and the outlet air temperature set at 85°C to obtain a squid cartilage hydrolyzate powder; the powder was then quickly sterilized by ultraviolet radiation with a radiation dose of 5 kGy and a radiation time of 60 minutes, and finally sterile packaged.

[0067] Example 3:

[0068] A large-scale preparation method of squid cartilage hydrolysate comprises the following steps:

[0069] (1) Pretreatment

[0070] The squid cartilage was first washed with clean water to remove surface attachments and impurities such as mucus. After rinsing, it was placed in a reactor and a hydrochloric acid solution 8 times the weight of the squid cartilage was added. The mixture was stirred for 45 minutes and then washed with deionized water. The mass volume concentration of the hydrochloric acid solution was 4%.

[0071] (2) High temperature acid hydrolysis reaction

[0072] The pretreated and cleaned squid cartilage was placed in a reactor, deionized water 10 times the weight of the squid cartilage was added, sulfuric acid solution was added to adjust the pH to 1.0, the reaction temperature was 90°C, and the reaction was stirred for 2 hours;

[0073] (3) Maillard reaction and ethanol deodorization

[0074] After the acid reaction is completed, the reaction temperature is maintained, sodium bicarbonate solution is added to adjust the pH to 5.0 to start the Maillard reaction, and the reaction is stirred for 45 minutes; after the Maillard reaction is completed, a cooking wine solution (the concentration of ethanol in the cooking wine is 15%) with a volume of 0.4% of the reaction solution is immediately added, and the temperature is slowly lowered to room temperature to obtain a crude extract of squid cartilage hydrolyzate;

[0075] (4) Separation and purification

[0076] First, solid residues in the crude extract were removed by high-speed centrifugation at 15,000 rpm using a continuous flow centrifuge. Macromolecular impurities in the crude extract were then removed using a nanofiltration membrane with a relative molecular mass of 10 kDa at a flow rate of 8 cubic meters per hour, a membrane pressure of 0.64 MPa, and a temperature of 35°C. Finally, small molecular impurities and inorganic salts were removed from the resulting clear solution using a nanofiltration membrane with a relative molecular mass of 200 Da at a flow rate of 8 cubic meters per hour, a membrane pressure of 1.35 MPa, and a temperature of 35°C. The solution was concentrated to obtain a squid cartilage hydrolyzate solution with a content of 96.95%.

[0077] (5) Rapid drying and sterilization packaging

[0078] The squid cartilage hydrolyzate solution was quickly dried by spray drying, with the inlet air temperature set at 165°C and the outlet air temperature set at 90°C to obtain a squid cartilage hydrolyzate powder; the powder was then quickly sterilized by electron beam irradiation with a radiation dose of 10 kGy and an irradiation time of 30 minutes, and finally aseptically packaged.

[0079] As shown in Table 1, more than three-quarters of the components of Argentine squid cartilage are carbohydrates including chondroitin sulfate and chitin, and crude proteins including collagen and core protein. Water and ash account for approximately 15% and 7%, respectively, while the fat content is very low. Therefore, the present invention designs a process flow for the large-scale preparation of squid cartilage hydrolysate.

[0080] As shown in Table 2, compared with the single enzymatic hydrolysis method (8 types, 24 hours) and the combined enzymatic hydrolysis method (6 types, 24 hours), the squid cartilage hydrolyzate prepared by the high-temperature acid hydrolysis method of the present invention has the highest yield and the lowest time cost (3 hours).

[0081] Based on the flavor evaluation criteria in Table 3 and as shown in Table 4, in terms of smell, the squid cartilage raw material has a very strong and unpleasant fishy smell, which is unacceptable to most people. The subjects were unable to smell the squid aroma at all. Moreover, due to the offensive fishy smell of the squid cartilage raw material, the subjects in this experiment were unwilling to further taste and judge its umami and bitterness. Therefore, no taste data was obtained for the squid cartilage raw material.

[0082] Based on the flavor evaluation criteria in Table 3 and as shown in Table 4, the squid cartilage hydrolyzate produced by the present invention has significantly improved and enhanced olfactory and taste compared to the squid cartilage raw material and the squid cartilage hydrolyzate that has not been deodorized. On the one hand, such results indicate that the squid cartilage hydrolyzate produced by the present invention is more attractive to consumers and is expected to be used in the food field as a nutritional additive with good flavor. On the other hand, it also indicates that the Maillard reaction and ethanol deodorization method adopted in the process of the present invention can effectively improve the product flavor of the squid cartilage hydrolyzate, achieving the good effects of reducing fishy smell, increasing fragrance, removing bitterness and enhancing freshness.

[0083] like Figure 1 As shown, the present invention uses squid cartilage discarded from aquatic product processing plants as raw material, first uses acid to remove calcium salts, then uses high-temperature acid hydrolysis reaction to quickly hydrolyze the squid cartilage into a mixture of oligosaccharides and oligopeptides, then uses Maillard reaction and ethanol to remove fishy smell to improve the flavor of the squid cartilage hydrolyzate, and further uses continuous flow centrifugation and membrane separation technology to separate, purify and desalinate the crude extract of the squid cartilage hydrolyzate at room temperature to obtain a squid cartilage hydrolyzate solution with a content of ≥95% and a molecular weight of less than 1500Da, finally uses spray drying technology to quickly dry the squid cartilage hydrolyzate to form a powder, and uses light radiation technology to quickly sterilize, seal and preserve.

[0084] like Figure 2 As shown, the present invention uses a high-temperature acid hydrolysis process to degrade chondroitin sulfate in decalcified squid cartilage into low-molecular-weight chondroitin sulfate, glucuronic acid, and glucose. It also degrades chitin into chitooligosaccharides and glucosamine, and degrades protein into oligopeptides and amino acids. Glucuronic acid, glucosamine, and glucose are all reducing sugars that, under appropriate conditions, can undergo a Maillard reaction with oligopeptides and amino acids, significantly improving the flavor of the squid cartilage hydrolyzate.

[0085] like Figure 3 As shown, the molecular weight distribution of the squid cartilage hydrolyzate produced by the present invention is between 1500Da and 75Da, and it is a mixture of oligopeptides and oligosaccharides that is easily absorbed by the human gastrointestinal tract.

[0086] like Figure 4As shown, the functional activity of the squid cartilage hydrolyzate produced by the present invention was studied primarily for its antibacterial activity, water absorption capacity, and antioxidant activity. Specifically, the antibacterial activity of the squid cartilage hydrolyzate against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Shewanella was studied. The antioxidant activity of the squid cartilage hydrolyzate was studied primarily for its iron ion reduction / antioxidant capacity, ABTS antioxidant capacity, total oxidant capacity, and hydroxyl radical content. Furthermore, the flavor of the squid cartilage hydrolyzate produced by the present invention was evaluated for its squid aroma and fishy odor in terms of olfaction, and its umami and bitterness in terms of taste.

[0087] like Figure 5 As shown in the results, the antibacterial activity of squid cartilage hydrolyzate against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas and Shewanella is better than that of potassium sorbate and sodium benzoate, common preservatives in food (the larger the diameter of the inhibition zone, the better the antibacterial activity). Such results indicate that the squid cartilage hydrolyzate produced by the present invention has good broad-spectrum antibacterial activity.

[0088] like Figure 6 As shown, compared with glycerol having good water absorption performance, the squid cartilage hydrolyzate produced by the present invention has better water absorption performance.

[0089] like Figure 7 As shown, the antioxidant activity of squid cartilage hydrolysate and common food antioxidants—BHA and vitamin E—were determined using four different antioxidant activity assays (FRAP, ABTS, T-AOC, and Fenton). The results were essentially consistent; the order of antioxidant activity from strongest to weakest was: squid cartilage hydrolysate (prepared by the present method) > BHA > vitamin E. For the FRAP and ABTS methods, higher iron ion reduction / antioxidant capacity and ABTS antioxidant capacity correlated with stronger antioxidant activity. For the T-AOC and Fenton methods, lower total oxidative capacity and hydroxyl radical content correlated with stronger antioxidant activity.

[0090] Table 1: Main components of the Argentine squid cartilage raw material of the present invention

[0091]

[0092] Table 2: Comparison of extraction rates of squid cartilage hydrolysates using different hydrolysis methods

[0093]

[0094] Table 3: Flavor evaluation criteria for squid cartilage hydrolyzate, squid cartilage raw material, and undeodorized squid cartilage hydrolyzate

[0095]

[0096] Table 4: Flavor evaluation results of squid cartilage hydrolyzate, squid cartilage raw material and squid cartilage hydrolyzate without deodorization

[0097]

Claims

1. A large-scale preparation method of squid cartilage hydrolysate, characterized in that: The following steps are involved: (1) Preprocessing The squid cartilage is first washed with clean water to remove surface attachments and mucus impurities. After rinsing, it is placed in a reactor and an acid solution 5 to 10 times the weight of the squid cartilage is added. The mixture is stirred for 15 minutes to 1 hour and then washed with deionized water. The mass volume concentration of the acid solution is 1 to 5%. (2) High temperature acid hydrolysis reaction The pretreated and cleaned squid cartilage is placed in a reactor, deionized water 2 to 10 times the weight of the squid cartilage is added, an acid solution is added to adjust the pH to 1 to 3, the reaction temperature is 85 to 100° C., and the reaction is stirred for 2 to 6 hours; (3) Maillard reaction and ethanol deodorization After the acid reaction is completed, the reaction temperature is maintained, and an alkali solution is added to adjust the pH to 5.0-6.5 to start the Maillard reaction, and the reaction is stirred for 20 minutes to 2 hours. After the Maillard reaction is completed, an ethanol solution with a volume of 0.1% to 0.6% of the reaction solution is immediately added, and the temperature is slowly lowered to room temperature to obtain a crude extract of squid cartilage hydrolyzate, and the concentration of the ethanol solution is 10% to 60%. (4) Separation and purification Continuous flow centrifugation combined with a nanofiltration membrane with a relative molecular mass of 10kDa to 5kDa was used to remove solid residues and large molecular impurities in the crude extract. The resulting clear liquid was finally filtered through a nanofiltration membrane with a relative molecular mass of 200Da to remove small molecular impurities and inorganic salts, and further concentrated to obtain a squid cartilage hydrolyzate solution with a content of ≥95%.

2. The method for large-scale preparation of squid cartilage hydrolysate according to claim 1, characterized in that: In step (1), the squid cartilage used is from Argentinian squid, the scientific name of which is Illex argentinus ; In step (1), the acid solution is added for decalcification treatment, and the acid used is hydrochloric acid or nitric acid.

3. The method for large-scale preparation of squid cartilage hydrolysate according to claim 1, characterized in that: In step (2), the acid solution is added to carry out high-temperature acid hydrolysis reaction, and the acid used is citric acid or sulfuric acid.

4. The method for large-scale preparation of squid cartilage hydrolysate according to claim 1, characterized in that: In step (3), the alkali solution added to adjust the pH value is sodium bicarbonate, potassium bicarbonate, sodium hydroxide or potassium hydroxide; the ethanol solution is a solution prepared with edible alcohol.

5. The method for large-scale preparation of squid cartilage hydrolysate according to claim 1, characterized in that: In step (3), the alkali solution added to adjust the pH value is sodium bicarbonate, potassium bicarbonate, sodium hydroxide or potassium hydroxide; the ethanol solution is a solution containing ethanol prepared from white wine, yellow wine or cooking wine.

6. The method for large-scale preparation of squid cartilage hydrolysate according to claim 1, characterized in that: In step (4), the high-speed centrifugal speed of the continuous flow centrifuge is 10,000 to 15,000 rpm; the flow rate of the nanofiltration membrane with a relative molecular mass of 10 kDa to 5 kDa is 5 to 14 cubic meters per hour, the membrane pressure is 0.552 to 0.931 MPa, and the temperature is 20 to 50°C; the flow rate of the nanofiltration membrane with a relative molecular mass of 200 Da is 5 to 14 cubic meters per hour, the membrane pressure is 0.483 to 2.758 MPa, and the temperature is 20 to 50°C.

7. The method for large-scale preparation of squid cartilage hydrolysate according to claim 1, characterized in that: The method further includes step (5) rapid drying and sterilization packaging: using spray drying to rapidly dry the squid cartilage hydrolyzate solution to obtain a squid cartilage hydrolyzate powder, then rapidly sterilizing it by light radiation, and finally performing aseptic subpackaging.

8. The method for large-scale preparation of squid cartilage hydrolysate according to claim 7, characterized in that: The inlet air temperature of the spray drying used in step (5) is 120-180°C, and the outlet air temperature is 70-95°C; the light radiation used is ultraviolet radiation, γ-ray or electron beam radiation, the radiation dose is 0.01-10 kGy, and the radiation time is 1-60 minutes.

9. The squid cartilage hydrolyzate prepared according to the large-scale preparation method according to any one of claims 1 to 8.

10. Use of the squid cartilage hydrolyzate according to claim 9 in the preparation of an antibacterial preparation.

11. Use of the squid cartilage hydrolyzate according to claim 9 in preparing a moisturizing ingredient or an antioxidant preparation.

Images