Mussel oil, preparation method and application

Abstract

The invention relates to the technical field of mussel oil preparation, in particular to mussel oil, a preparation method and application. The preparation method of the mussel oil comprises the following steps: preparing mussel meat into mussel freeze-dried powder, and mixing the mussel freeze-dried powder with water to obtain a mixture; protease is added for enzymolysis, and enzymatic hydrolysate is obtained; carrying out centrifugal separation on the enzymatic hydrolysate to obtain supernate and precipitate, and respectively carrying out freeze drying to obtain freeze-dried raw materials; carrying out ultrasonic extraction on the freeze-dried raw material by using an organic solvent to obtain a crude extract; performing alkali refining and deacidification on the crude extract to obtain deacidified oil; and carrying out adsorption decoloration on the deacidified oil to obtain the mussel oil. According to the method, protease treatment and solvent extraction are combined, and ultrasonic treatment is adopted for assistance in the solvent extraction process, so that the extraction efficiency is improved. And through the refining process under specific conditions, phospholipid in the mussel oil is effectively retained, and the quality of the mussel oil is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of mussel oil preparation, and specifically relates to a mussel oil, a preparation method and an application thereof. Background Art

[0002] Mussels are delicious and nutritious, and are an important economic shellfish. The main cultured varieties include Mytilus edulis, Mytilus coruscus and Perna viridis. The contents of protein, fat and carbohydrates in the edible part of mussels account for 58.5%, 9.7% and 25.0% of their wet weight respectively. At present, mussel peptides have been industrially produced, but degreasing is required first during processing to prevent the mussel peptide products from being contaminated with grease and affecting the storage stability of the products. Although the fat content in mussels is relatively low, the content of unsaturated fatty acids is relatively high, and the content of PUFA accounts for about 32.81% - 61.14% of the total fatty acids, i.e., TFA, and the n-3 / n-6 PUFA is generally higher than 6, which makes mussel oil have excellent anti-inflammatory activity.

[0003] Currently, dried mussel powder is generally used as a raw material, and mussel oil is extracted by solvent extraction and supercritical CO2 extraction. The supercritical extraction technology has a simple process, non-toxic extractant and easy recovery, but the extraction efficiency is low and the equipment cost is high. Therefore, the solvent extraction method is still the most widely used method for mussel oil extraction. Since mussel oil contains lipids with different polarities such as phospholipids and glycerides, the choice of solvent will significantly affect the extraction efficiency. In the commonly used methods, both the Folch method and the Bligh-Dyer method use chloroform, methanol and water as extraction media. In addition, ethanol and n-hexane are also commonly used organic solvents. However, the extraction efficiency and quality of the organic solvent method are low.

[0004] Therefore, it is necessary to develop a preparation method for mussel oil. Summary of the Invention

[0005] To solve the above problems, the present invention provides a mussel oil, a preparation method and an application thereof.

[0006] The present invention is realized by the following technical solutions: A preparation method for mussel oil, comprising the following steps: Take mussel meat to make mussel freeze-dried powder with 50 - 100 meshes, mix the mussel freeze-dried powder with water to obtain a mixture; the mass-volume ratio of the mussel freeze-dried powder to water is 1 kg: 10 L - 14 L.

[0007] Adjust the pH value of the mixture to acidic and then add protease for enzymatic hydrolysis to obtain an enzymatic hydrolysate; the addition amount of the protease accounts for 0.10% - 0.20% of the mass of the mussel freeze-dried powder.

[0008] The supernatant and precipitate separated by centrifugation of the enzymatic hydrolysate are freeze-dried to obtain freeze-dried raw materials; the freeze-dried raw materials are ultrasonically extracted with an organic solvent to obtain a crude extract; the mass-volume ratio of the freeze-dried raw materials to the organic solvent is 1 kg: 10 L to 14 L.

[0009] The crude extract is heated to 30°C to 70°C in an N2 atmosphere, and a NaOH solution is added for alkali refining and deacidification for 10 min to 50 min to obtain deacidified oil; the addition amount of the NaOH solution accounts for 15% to 25% of the mass of the crude extract.

[0010] The deacidified oil is heated to 30°C to 70°C in N2, and activated carbon is added for adsorption and decolorization for 10 min to 50 min to obtain mussel oil; the addition amount of activated carbon accounts for 2% to 10% of the mass of the deacidified oil.

[0011] Preferably, the protease is any one of pepsin and acidic protease.

[0012] Preferably, the organic solvent is any one of anhydrous ethanol, a mixed solution of ethanol and n-hexane, and ethanol with a volume fraction of 95%.

[0013] In the mixed solution of ethanol and n-hexane, the volume ratio of ethanol to n-hexane is 1 to 4: 1 to 4.

[0014] Preferably, the temperature of the enzymatic hydrolysis treatment is 30°C to 37°C, and the enzymatic hydrolysis time is 4 hours to 5 hours.

[0015] Preferably, the time of ultrasonic extraction is 4 hours, and the power of ultrasonic extraction is 400 W to 800 W.

[0016] Preferably, the pH value of the mixture is adjusted to 2.0 using HCl with a molar concentration of 2 mol / L.

[0017] Preferably, the mass concentration of the NaOH solution is 5% to 25%.

[0018] The mussel oil prepared by the above method.

[0019] The application of the mussel oil in the preparation of anti-inflammatory drugs.

[0020] Preferably, the anti-inflammatory drug is used for preventing or treating rheumatoid arthritis, asthma or inflammatory bowel disease.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing mussel oil, comprising the following steps: taking mussel meat to prepare mussel freeze-dried powder with a mesh size of 50 to 100, mixing the mussel freeze-dried powder with water to obtain a mixture; the mass-volume ratio of the mussel freeze-dried powder to water is 1:10 to 14; adjusting the pH value of the mixture to 2.0 and then adding protease for enzymatic hydrolysis to obtain an enzymatic hydrolysate; the addition amount of the protease accounts for 0.10% to 0.20% of the mass of the mussel freeze-dried powder; freeze-drying the supernatant and precipitate separated by centrifuging the enzymatic hydrolysate to obtain freeze-dried raw materials; subjecting the freeze-dried raw materials to ultrasonic extraction with an organic solvent to obtain a crude extract; the mass-volume ratio of the freeze-dried raw materials to the organic solvent is 1:10 to 14; heating the crude extract to 30°C to 70°C in N2, adding NaOH solution for alkali refining and deacidification for 10 min to 50 min to obtain deacidified oil; the addition amount of the NaOH solution accounts for 15% to 25% of the mass of the crude extract; heating the deacidified oil to 30°C to 70°C in N2, adding activated carbon for adsorption and decolorization for 10 min to 50 min to obtain mussel oil; the addition amount of the activated carbon accounts for 2% to 10% of the mass of the deacidified oil. In the present invention, a method combining protease treatment and solvent extraction is used. By enzymatic hydrolysis, the connection between proteins and lipids is broken, and ultrasonic treatment is used as an auxiliary during the solvent extraction process, which can improve the extraction efficiency of mussel oil. Conventional oil refining processes aim to retain triglycerides. Since most of the PUFAs in mussel oil exist in the form of phospholipids, through the refining process under specific conditions in the present invention, the phospholipids therein are effectively retained, improving the quality of mussel oil. The acid value of the detected mussel oil meets the requirements in GB10146-2015 "National Food Safety Standard Edible Animal Oils". In addition, after using the supernatant part to extract the crude extract in the present invention, its by-products can also be used to extract active peptides, greatly avoiding resource waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 It is a graph showing the influence of protease types in the present invention on the enzymatic hydrolysis effect of mussels and the oil extraction rate; Figure 1 In it, A is the graph showing the influence result on the oil extraction rate; B is the graph showing the influence result on the degree of hydrolysis; C is the graph showing the influence result on the peptide yield; D is the graph showing the influence result on the anti-superoxide anion ability of the enzymatic hydrolysate; E is the graph showing the influence result on the DPPH scavenging rate of the enzymatic hydrolysate.

[0024] Figure 2Results graph of the influence of the ratio of mussel freeze-dried powder to distilled water on the enzymatic hydrolysis effect and oil extraction rate of mussels in the present invention; Figure 2 Among them, A is the results graph of the influence on the oil extraction rate; B is the results graph of the influence on the degree of hydrolysis; C is the results graph of the influence on the peptide yield; D is the results graph of the influence on the superoxide anion scavenging ability of the enzymatic hydrolysis product; E is the results graph of the influence on the DPPH scavenging rate of the enzymatic hydrolysis product.

[0025] Figure 3 Results graph of the influence of pH value on the enzymatic hydrolysis effect and oil extraction rate of mussels in the present invention; Figure 3 Among them, A is the results graph of the influence on the oil extraction rate; B is the results graph of the influence on the degree of hydrolysis; C is the results graph of the influence on the peptide yield; D is the results graph of the influence on the superoxide anion scavenging ability of the enzymatic hydrolysis product; E is the results graph of the influence on the DPPH scavenging rate of the enzymatic hydrolysis product.

[0026] Figure 4 Results graph of the influence of enzyme addition amount on the enzymatic hydrolysis effect and oil extraction rate of mussels in the present invention; Figure 4 Among them, A is the results graph of the influence on the oil extraction rate; B is the results graph of the influence on the degree of hydrolysis; C is the results graph of the influence on the peptide yield; D is the results graph of the influence on the superoxide anion scavenging ability of the enzymatic hydrolysis product; E is the results graph of the influence on the DPPH scavenging rate of the enzymatic hydrolysis product.

[0027] Figure 5 Results graph of the influence of enzymatic hydrolysis temperature on the enzymatic hydrolysis effect and oil extraction rate of mussels in the present invention; Figure 5 Among them, A is the results graph of the influence on the oil extraction rate; B is the results graph of the influence on the degree of hydrolysis; C is the results graph of the influence on the peptide yield; D is the results graph of the influence on the superoxide anion scavenging ability; E is the results graph of the influence on the DPPH scavenging rate.

[0028] Figure 6 Results graph of the influence of enzymatic hydrolysis time on the enzymatic hydrolysis effect and oil extraction rate of mussels in the present invention; Figure 6 Among them, A is the results graph of the influence on the oil extraction rate; B is the results graph of the influence on the degree of hydrolysis; C is the results graph of the influence on the peptide yield; D is the results graph of the influence on the superoxide anion scavenging ability; E is the results graph of the influence on the DPPH scavenging rate.

[0029] Figure 7 Results graph of the influence of each single factor on the solvent extraction effect of mussel oil in the present invention; Figure 7 Among them, A is the results graph of the influence of the ratio of enzymatic hydrolysis product / absolute ethanol on the solvent extraction effect of mussel oil; B is the results graph of the influence of the ratio of ethanol / n-hexane on the solvent extraction effect of mussel oil; C is the results graph of the influence of ultrasonic time on the solvent extraction effect of mussel oil; D is the results graph of the influence of ultrasonic power on the solvent extraction effect of mussel oil.

[0030] Figure 8This is the response surface and contour map of the interaction of various factors in the enzymatic hydrolysis process of the present invention affecting the oil extraction rate; Figure 8 Among them, A is the response surface and contour map of the interaction between enzyme addition amount and enzymatic hydrolysis time affecting the oil extraction rate during the enzymatic hydrolysis of mussels; B is the response surface and contour map of the interaction between enzyme addition amount and enzymatic hydrolysis temperature affecting the oil extraction rate during the enzymatic hydrolysis of mussels; C is the response surface and contour map of the interaction between enzymatic hydrolysis time and enzymatic hydrolysis temperature affecting the oil extraction rate during the enzymatic hydrolysis of mussels.

[0031] Figure 9 This is the response surface and contour map of the interaction of various factors in the enzymatic hydrolysis process of the present invention affecting the degree of hydrolysis; Figure 9 Among them, A is the response surface and contour map of the interaction between enzyme addition amount and enzymatic hydrolysis time affecting the degree of hydrolysis during the enzymatic hydrolysis of mussels; B is the response surface and contour map of the interaction between enzyme addition amount and enzymatic hydrolysis temperature affecting the degree of hydrolysis during the enzymatic hydrolysis of mussels; C is the response surface and contour map of the interaction between enzymatic hydrolysis time and enzymatic hydrolysis temperature affecting the degree of hydrolysis during the enzymatic hydrolysis of mussels.

[0032] Figure 10 This is the response surface and contour map of the interaction of various factors in the solvent extraction process of the present invention affecting the oil extraction rate; Figure 10 Among them, A is the response surface and contour map of the interaction between enzyme addition amount and enzymatic hydrolysis time affecting the oil extraction rate of mussel oil during solvent extraction; B is the response surface and contour map of the interaction between enzyme addition amount and enzymatic hydrolysis temperature affecting the oil extraction rate of mussel oil during solvent extraction, and C is the response surface and contour map of the interaction between enzymatic hydrolysis time and enzymatic hydrolysis temperature affecting the oil extraction rate of mussel oil during solvent extraction.

[0033] Figure 11 This is the influence diagram of the NaOH concentration of the present invention on the deacidification effect of mussel oil; Figure 11 Among them, A is the result diagram of the influence on acid value; B is the thin layer chromatography result diagram using Dittmer-Lester reagent; C is the thin layer chromatography result diagram developed with Dargendorf reagent.

[0034] Figure 12 This is the influence of the NaOH addition amount of the present invention on the deacidification effect of mussel oil; Figure 12 Among them, A is the result diagram of the influence on acid value; B is the thin layer chromatography result diagram using Dittmer-Lester reagent; C is the thin layer chromatography result diagram developed with Dargendorf reagent.

[0035] Figure 13 This is the influence of the deacidification time of the present invention on the deacidification effect of mussel oil; Figure 13 Among them, A is the result diagram of the influence on acid value; B is the thin layer chromatography result diagram using Dittmer-Lester reagent; C is the thin layer chromatography result diagram developed with Dargendorf reagent.

[0036] Figure 14 For the present invention, the effect of deacidification temperature on the deacidification effect of mussel oil; Figure 14 In it, A is the result graph of the effect on acid value; B is the thin-layer chromatography result graph using Dittmer-Lester reagent; C is the thin-layer chromatography result graph developed with Dargendorf reagent.

[0037] Figure 15 For the present invention, the result graph of the effect of each single factor on the adsorption and decolorization effect of mussel oil; Figure 15 In it, A is the result graph of the effect of adsorbent type on the adsorption and decolorization effect of mussel oil; B is the result graph of the effect of activated carbon addition amount on the adsorption and decolorization effect of mussel oil; C is the result graph of the effect of decolorization temperature on the adsorption and decolorization effect of mussel oil; D is the result graph of the effect of decolorization time on the adsorption and decolorization effect of mussel oil.

[0038] Figure 16 For the present invention, the effect of mussel oil on the paw thickness and arthritis score of arthritic rats, n = 10; Figure 16 In it, A is the graph of paw thickness change during feeding; B is the paw thickness graph on the 16th day; C is the paw thickness graph on the 28th day; D is the paw morphology graph on the 28th day; E is the arthritis score graph on the 28th day.

[0039] Figure 17 For the present invention, the result graph of the effect of mussel oil on the levels of related diagnostic factors in the serum of RA rats, n = 10; Figure 17 In it, A is the result graph of the effect of mussel oil on rheumatoid factor RF; B is the result graph of the effect of mussel oil on anti-cyclic citrullinated peptide antibody anti-CPP.

[0040] Figure 18 For the present invention, the result graph of the effect of mussel oil on the levels of serum inflammatory factors in RA rats, n = 10; Figure 18 In it, A is the result graph of the effect of mussel oil on TNF-α; B is the result graph of the effect of mussel oil on IL-1β; C is the result graph of the effect of mussel oil on IL-6; D is the result graph of the effect of mussel oil on IL-17.

[0041] Figure 19 For the present invention, the result graph of the effect of mussel oil on the ankle joint tissue morphology of RA rats, n = 10; Figure 19 In it, A is the hematoxylin-eosin H&E staining graph; B is the safranin-fast green staining graph. Detailed implementation manners

[0042] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0044] The index for evaluating the extraction effect in the present invention is detected as follows: 1. Oil extraction rate Taking the oil extraction rate as the main evaluation index. Take the mussel oil sample, weigh it after rotary evaporation to remove the organic solvent at 40 °C and 90 mbar, and calculate the extraction rate according to the following formula:

[0045] Extraction rate = mass of mussel oil / mass of raw mussel powder × 100.

[0046] 2. Degree of hydrolysis Referring to the ninhydrin method, using the completely hydrolyzed solution of the protein sample to be measured as the standard sample for ninhydrin colorimetry to determine the degree of hydrolysis of the protein. Calculate according to the number of peptide bonds h cleaved per gram of protein after hydrolysis and the weight W of the mussel raw material according to the following formula:

[0047] Degree of hydrolysis = h / W × 100%.

[0048] 3. Peptide yield Using the biuret method, detecting the polypeptide mass concentration C in the mussel peptide by referring to the standard curve, and calculating according to the peptide solution volume V and the weight W of the mussel raw material according to the following formula: Polypeptide yield = C × V / W × 100.

[0049] 4. Antioxidant capacity of the enzymatic hydrolysis product Using a commercial kit, detect the anti-superoxide anion capacity and the DPPH scavenging rate of the enzymatic hydrolysis product according to the method described in the instruction manual.

[0050] 5. Optimization of process parameters by response surface method On the basis of the above single-factor experiments, according to the central composite design principle of Box-Behnken, select the factors that can significantly affect the extraction effect as independent variables, take the extraction rate as the response value, design a three-level experiment, establish a model and conduct a significance test to determine the optimal extraction process conditions.

[0051] The mussel meat used in the present invention is frozen mussel meat, purchased from Guangzhou Huayi Frozen Food Co., Ltd.

[0052] The beneficial effects of the present invention will be illustrated by specific embodiments as follows.

[0053] Example 1. A preparation method of mussel oil Take mussel meat, vacuum freeze-dry it under the conditions of 10 Pa and -50 °C, and then grind it into mussel freeze-dried powder with a mesh size of 80. Using the mussel freeze-dried powder as raw material, add distilled water thereto, and adjust the mass-volume ratio of the mussel freeze-dried powder to distilled water to be 1 kg: 10 L to obtain a mixture.

[0054] Use 2 mol / L HCl to adjust the pH value of the mixture to 2.0, and then add pepsin with an enzyme activity of 250 U / mg for enzymatic hydrolysis. The addition amount of the pepsin accounts for 0.10% of the mass of the mussel freeze-dried powder, and heat and oscillate it in a shaking incubator at 30 °C for 4 h to catalyze the hydrolysis of the protease to obtain an enzymatic hydrolysate.

[0055] Centrifuge the enzymatic hydrolysate at 4000 rpm for 20 min to separate the supernatant and the precipitate, and vacuum freeze-dry the supernatant and the precipitate respectively to obtain freeze-dried raw materials; use ethanol with a volume fraction of 95% to perform ultrasonic extraction on the freeze-dried raw materials at room temperature. The mass-volume ratio of the freeze-dried raw materials to ethanol with a volume fraction of 95% is 1 kg: 10 L. The power of the ultrasonic extraction is 400 W, and the time of the ultrasonic extraction is 4 h to obtain a crude extract.

[0056] After passing N2 through the crude extract, stir it in a water bath at 50 °C, add a NaOH solution with a mass concentration of 10%, and continuously stir it in a water bath at 40 °C for 20 min for alkali refining and deacidification. Centrifuge it at 4200 r / min for 10 min, and collect the upper oil phase to obtain deacidified oil; among them, the addition amount of the NaOH solution accounts for 15% of the mass of the crude mussel oil.

[0057] After passing N2 through the deacidified oil, heat it to 50 °C, add activated carbon, and stir it for 40 min for adsorption and decolorization. Let it stand and cool, centrifuge it at 4200 rpm for 15 min, and collect the upper oil phase to obtain mussel oil; the addition amount of the activated carbon accounts for 6% of the mass of the deacidified oil.

[0058] It should be noted that the particle size of the mussel freeze-dried powder can be used within the range of 50 mesh to 100 mesh.

[0059] In Example 2, acidic protease with an enzyme activity of 50,000 U / g is used, and the remaining steps are exactly the same as those in Example 1.

[0060] In Comparative Examples 1 to 6, neutral protease with an enzyme activity of 50,000 U / g, alkaline protease with an enzyme activity of 200,000 U / g, flavor protease with an enzyme activity of 30,000 U / g, trypsin with an enzyme activity of 250 U / mg, bromelain with an enzyme activity of 600 U / mg, and papain with an enzyme activity of 80,000 U / g are used respectively, and the remaining steps are exactly the same as those in Example 1.

[0061] The extraction rate, degree of hydrolysis, antioxidant performance of the enzymatic hydrolysate, etc. in Example 1, Example 2, Comparative Example 1 to Comparative Example 6 were calculated respectively. The results are as Figure 1 shown. Under the action of pepsin, the extraction rate of mussel oil was the highest, followed by acid protease and alkaline protease.

[0062] In Example 3 to Example 4, the mass-to-volume ratio of mussel freeze-dried powder to distilled water was 1 kg: 12 L and 1 kg: 14 L respectively, and the remaining steps were exactly the same as those in the example.

[0063] In Comparative Example 7 to Comparative Example 8, the mass-to-volume ratio of mussel freeze-dried powder to distilled water was 1 kg: 6 L and 1 kg: 8 L respectively, and the remaining steps were exactly the same as those in Example 1.

[0064] The extraction rate of mussel oil, etc. in Example 1, Example 3 to Example 4, Comparative Example 7 to Comparative Example 8 were calculated respectively. The results are as Figure 2 shown. When the mass-to-volume ratio of mussel powder to distilled water was 1 kg: 10 L, 1 kg: 12 L and 1 kg: 14 L, the extraction rate of mussel oil, peptide yield and DPPH scavenging rate of the enzymatic hydrolysate were higher than those with the ratios of 1 kg: 6 L and 1 kg: 8 L. In Comparative Example 9 to Comparative Example 12, the pH value of the mixture was adjusted to 2.5, 3.0, 3.5, 4.0, and the remaining steps were exactly the same as those in Example 1.

[0065] The extraction rate of mussel oil, etc. in Example 1, Comparative Example 9 to Comparative Example 12 were calculated respectively. The results are as Figure 3 shown. When the pH was 2.0, the extraction rate of mussel oil, peptide yield and DPPH scavenging rate of the enzymatic hydrolysate were the largest; as the pH increased, the extraction rate of mussel oil decreased, indicating that pH = 2.0 was the optimal pH for the action conditions of pepsin.

[0066] In Example 5 to Example 6, the mass ratio of the added amount of pepsin to mussel freeze-dried powder was 0.15% and 0.20% respectively, and the remaining steps were exactly the same as those in Example 1.

[0067] In Comparative Example 13, the mass ratio of the added amount of pepsin to mussel freeze-dried powder was 0.05%, and the remaining steps were exactly the same as those in Example 1.

[0068] The extraction rate of mussel oil in Example 1, Example 5 to Example 6, Comparative Example 13 were calculated respectively. The results are as Figure 4 shown. When the mass ratio of the added amount of pepsin to mussel freeze-dried powder was 0.10%, 0.15% and 0.20%, the extraction rate of mussel oil and degree of hydrolysis were both higher than those with the mass ratio of 0.05%, and there was no significant difference among 0.10%, 0.15% and 0.20%.

[0069] In Example 7, the enzymatic hydrolysis temperature was 37 °C, and the remaining steps were exactly the same as those in Example 1.

[0070] In Comparative Examples 14 to 16, the enzymatic hydrolysis temperatures were 40 °C, 45 °C, and 50 °C respectively, and the remaining steps were exactly the same as those in Example 1.

[0071] The extraction rates of mussel oil in Example 1, Example 7, and Comparative Examples 14 to 16 were calculated respectively. The results were as Figure 5 shown. When the enzymatic hydrolysis temperatures were 30 °C and 37 °C, the extraction rates were better, and at this time, the temperature was the most suitable active temperature for the enzyme. After exceeding 37 °C, the extraction rate decreased.

[0072] In Example 8, the enzymatic hydrolysis time was 5 h, and the remaining steps were exactly the same as those in Example 1.

[0073] In Comparative Examples 17 to 19, the enzymatic hydrolysis times were 1 h, 2 h, and 3 h respectively, and the remaining steps were exactly the same as those in Example 1.

[0074] The extraction rates of mussel oil in Example 1, Example 8, and Comparative Examples 17 to 19 were calculated respectively. The results were as Figure 6 shown. When the extraction time was 4 h, the protease hydrolysis and extraction effects were better, and the effect at 5 h was the second best.

[0075] In Examples 9 to 11, the mass-to-volume ratios of the freeze-dried raw material to absolute ethanol were 1 kg:10 L, 1 kg:12 L, and 1 kg:14 L respectively, and the remaining steps were exactly the same as those in Example 1.

[0076] In Comparative Examples 20 to 21, the mass-to-volume ratios of the freeze-dried raw material to absolute ethanol were 1 kg:6 L and 1 kg:8 L respectively, and the remaining steps were exactly the same as those in Example 9.

[0077] The extraction rates of mussel oil in Example 1, Examples 9 to 11, and Comparative Examples 20 to 21 were calculated respectively. The results were as Figure 8 shown in A below. When the mass-to-volume ratios of the freeze-dried raw material to absolute ethanol were 1 kg:10 L to 1 kg:14 L, the yield of mussel oil was higher than when the mass-to-volume ratios were 1 kg:6 L and 1 kg:8 L.

[0078] In Examples 12 to 17, absolute ethanol, an ethanol-n-hexane mixed solution with a volume ratio of 4:1, an ethanol-n-hexane mixed solution with a volume ratio of 2:1, an ethanol-n-hexane mixed solution with a volume ratio of 1:1, an ethanol-n-hexane mixed solution with a volume ratio of 1:2, and an ethanol-n-hexane mixed solution with a volume ratio of 1:4 were respectively selected to ultrasonically extract the freeze-dried raw material at room temperature, and the remaining steps were exactly the same as those in Example 1.

[0079] In Comparative Example 22, n-hexane was selected for ultrasonic extraction of the freeze-dried raw material at room temperature, and the remaining steps were exactly the same as those in Example 1.

[0080] The extraction rates of mussel oil in Example 1, Examples 12 to 17, and Comparative Example 22 were calculated respectively, and the results are as Figure 7 shown in B of the figure. Ethanol with a volume fraction of 95% and absolute ethanol are the best for extracting mussel oil. The ethanol-n-hexane mixed solutions with a volume ratio of 4:1, 2:1, 1:1, 1:2, and 1:4 are the second best.

[0081] In Comparative Examples 23 to 26, the ultrasonic extraction times were 1 h, 2 h, 3 h, and 5 h respectively, and the remaining steps were exactly the same as those in Example 1.

[0082] The extraction rates of mussel oil in Example 1 and Comparative Examples 23 to 26 were calculated respectively, and the results are as Figure 8 shown in C of the figure. As the ultrasonic time prolongs, the oil extraction rate first increases and then decreases, reaching the maximum at 4 h. 4 h was selected as the extraction condition.

[0083] In Examples 18 to 21, the ultrasonic extraction powers were 500 W, 600 W, 700 W, and 800 W respectively, and the remaining steps were exactly the same as those in Example 1.

[0084] In Comparative Example 27, the ultrasonic extraction power was 0 W, and the remaining steps were exactly the same as those in Example 1.

[0085] The extraction rates of mussel oil in Example 1, Examples 18 to 21, and Comparative Example 27 were calculated respectively, and the results are as Figure 8 shown in D of the figure. Ultrasonic extraction significantly increased the extraction rate of mussel oil, and 400 W was selected as the extraction condition.

[0086] 1.3 Optimization of enzymatic hydrolysis and solvent extraction processes by response surface methodology 1.3.1 Optimization of enzymatic hydrolysis process by response surface methodology Table 1 Design and results of response surface optimization test for enzymatic hydrolysis process Table 1 shows the design and results of the response surface experiment for the enzymatic hydrolysis process. As can be seen from Table 2 and Figure 8 it is known that the model has a good fitting degree with the experimental results, and the experimental results can be analyzed and predicted. The order of the magnitudes of the three factors affecting the oil extraction rate is: enzymatic hydrolysis time > enzyme addition amount > enzymatic hydrolysis temperature. According to the experimental results, the quadratic terms of the enzymatic hydrolysis time and enzymatic hydrolysis temperature have a very significant effect on the oil extraction rate, p < 0.01, and the enzyme addition amount, enzymatic hydrolysis time, and their respective quadratic terms have a significant effect on the oil extraction rate,p < 0.05. The response value of the degree of hydrolysis is shown in Table 3 and Figure 9 as follows. The results show that the model has a good fitting degree with the experimental results, and can analyze and predict the experimental results. The order of the three factors affecting the degree of hydrolysis is also: enzymolysis time > enzyme dosage > enzymolysis temperature. After optimization by the response surface experiment, the optimal process parameters are enzyme dosage 0.11%, enzymolysis time 4.2 h, and enzymolysis temperature 35 °C.

[0087] Table 2 ANOVA of the response surface regression model for the enzymolysis process, oil extraction rate Note: * Intuitively shows the significance of the P value, * represents p < 0.05, ** represents p < 0.01; Note: " / " indicates that there is no such item.

[0088] Table 3 ANOVA of the response surface regression model for the enzymolysis process, degree of hydrolysis Note: * Intuitively shows the significance of the P value, * represents p < 0.05, ** represents p < 0.01; Note: " / " indicates that there is no such item.

[0089] 1.3.2 Optimization of the solvent extraction process by the response surface method Table 4 Experimental design and results of the response surface optimization of the solvent extraction process As shown in Table 4 are the experimental design and results of the response surface of the solvent extraction. The results of the response surface of the mussel oil extraction process show that the model has a good fitting degree with the experimental results and can analyze and predict the experimental results. The results are shown in Table 5 and Figure 10 as follows. It can be seen from Table 5 that the order of the three factors affecting the oil extraction rate is: ultrasonic time > solid-liquid ratio > ultrasonic temperature. The optimal process parameters are liquid-solid ratio 10.4, ultrasonic time 4.4 h, and ultrasonic power 510 W.

[0090] Table 5 ANOVA of the response surface model for the solvent extraction process Note: * Intuitively shows the significance of the P value, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001; Note: " / " indicates that there is no such item.

[0091] 1.3.3 Determination and verification of the optimal process Table 6 Verification of the optimal extraction process of mussel oil In Examples 22 to 25 of caustic refining and deacidification, the mass concentrations of the NaOH solution were 5%, 15%, 20%, and 25% respectively, and the remaining steps were exactly the same as those in Example 1.

[0092] In Examples 26 to 29 of caustic refining and deacidification, the addition amounts of the NaOH solution accounted for 17.5%, 20%, 22.5%, and 25% of the mass of the crude mussel oil respectively, and the remaining steps were exactly the same as those in Example 1.

[0093] In Examples 30 to 33, the temperatures of caustic refining and deacidification were 30°C, 50°C, 60°C, and 70°C respectively, and the remaining steps were exactly the same as those in Example 1.

[0094] In Examples 34 to 37, the times of caustic refining and deacidification were 10 min, 30 min, 40 min, and 50 min respectively, and the remaining steps were exactly the same as those in Example 1.

[0095] The deacidification conditions, namely the NaOH concentration, the NaOH addition amount, the deacidification temperature, and the deacidification time, were investigated for their effects on the deacidification effect and the phospholipid content in mussel oil. The results are as Figures 11 - 14 shown. Taking the maximum retention of phospholipids while reducing the acid value as the goal, combining the results of both, a 10% alkali concentration, a 15% alkali addition amount, and deacidification at 40°C for 20 min were selected as the optimal deacidification conditions. Through an orthogonal experiment, the optimal NaOH addition amount was determined to be 20.0%, the deacidification temperature was 50°C, and the deacidification time was 30 min. At this time, the acid value of the mussel oil was 1.33 ± 0.19 mg KOH / g.

[0096] In Comparative Examples 28 to 29, diatomaceous earth, activated clay, and macroporous resin were respectively selected for adsorption decolorization, and the remaining steps were exactly the same as those in Example 1.

[0097] Decolorization effects of activated carbon, activated clay, macroporous resin, and diatomaceous earth on deacidified mussel oil Figure 15 A. The activated carbon had the highest decolorization rate. The activated carbon had a large specific surface area, a high decolorization coefficient, and characteristics such as hydrophobicity, and had a strong adsorption effect on high-molecular substances and non-polar pigments. Therefore, the activated carbon had the best decolorization effect.

[0098] In Examples 38 to 41, activated carbon was used as the decolorizing agent, and the addition amounts of the activated carbon accounted for 2%, 4%, 8%, and 10% of the mass of the deacidified oil respectively.

[0099] In Examples 42 to 45, the temperatures of adsorption decolorization were 30°C, 40°C, 60°C, and 70°C respectively, and the remaining steps were exactly the same as those in Example 1.

[0100] The adsorption decolorization times in Examples 46 to 49 were 10 min, 20 min, 30 min, and 50 min respectively, and the remaining steps were exactly the same as those in Example 1.

[0101] The effects of the amount of activated carbon added, the adsorption decolorization temperature, and the adsorption decolorization time on the decolorization effect were investigated, and the results are shown as B, C, and D in Figure 15 . The optimal adsorption decolorization conditions were as follows: the amount of activated carbon added was 6%, the adsorption decolorization temperature was 50 °C, and the adsorption decolorization time was 40 min. Through orthogonal experiments, the optimal amount of activated carbon added was determined to be 8%, the decolorization temperature was 50 °C, and the decolorization time was 50 min. At this time, the decolorization rate of mussel oil was 73.64 ± 0.48%.

[0102] Experimental Example 1, Quality Analysis of Mussel Oil The acid value, peroxide value, lipid composition, and fatty acid composition of the mussel oil prepared in Examples 1 to 49 were determined.

[0103] The acid value and peroxide value were detected according to the national standards "GB5009.229-2016 Determination of Acid Value in Foods" and "GB5009.227-2016 Determination of Peroxide Value in Foods" respectively.

[0104] The lipid composition of the mussel oil sample was analyzed by liquid chromatography-mass spectrometry, LC-MS.

[0105] The fatty acid composition of the mussel oil sample was analyzed by gas chromatography-mass spectrometry, GC-MS.

[0106] The average values of the acid value and peroxide value of the mussel oil prepared using the optimal process screened in Examples 1 to 49 are shown in Table 7. The acid value and peroxide value of the crude extract were higher than those of the finally prepared mussel oil; the acid value of the mussel oil met the requirements in GB10146-2015 "National Food Safety Standard Edible Animal Fats".

[0107] Table 7 Acid Value and Peroxide Value of Mussel Oil The lipid compositions of the crude extract and the finally prepared mussel oil in Examples 1 to 49 are shown in Table 8. The lipids in the mussel oil were mainly triglycerides and phospholipids, and the phospholipid content could reach more than 60%. In addition, the lipid compositions of the crude extract and the finally prepared mussel oil changed little, indicating that refining did not affect the quality of the mussel oil.

[0108] Table 8 Lipid Compositions of Mussel Oil before and after Refining In Examples 1 to 49, the fatty acid compositions of the crude extract and the finally prepared mussel oil are shown in Table 9. The polyunsaturated fatty acids (PUFAs) in the finally prepared mussel oil, that is, the content of PUFAs is higher than that of the crude extract, mainly manifested in the increased contents of eicosapentaenoic acid (C20:5n-3, EPA) and docosahexaenoic acid (C22:6n-3, DHA); while the content of monounsaturated fatty acids (MUFAs) in the mussel oil is not much different from that of the crude extract.

[0109] Table 9 Fatty Acid Compositions of Mussel Oil before and after Refining Experimental Example 2 Exploration of the Anti-Rheumatoid Arthritis Activity of Mussel Oil 1. Animal Grouping and Administration Method After 50 SD rats were adaptively fed for one week, they were randomly divided into five groups: a normal group, labeled as N, n = 10; a model group, labeled as M, n = 10; and a mussel oil group, labeled as MO, n = 10. During the feeding process, the rats ate the feed and drank the water freely. The rats in the N group and the M group ingested the NIH-31M standard feed throughout the process, and the rats in the MO group ingested the NIH-31M feed added with 0.5% mussel oil. The mussel oil was the mussel oil prepared in Example 1, and the addition amount of the mussel oil accounted for 0.5% of the mass of the NIH-31M feed. The addition method was to equally replace the soybean oil in the original feed.

[0110] On the 15th day of feeding, the rats in each group were subjected to modeling: in the model group and the mussel oil group, 0.1 mL of complete Freund's adjuvant (CFA) was subcutaneously injected into the right hind paw of the rats to induce rheumatoid arthritis; 0.1 mL of normal saline with the same volume as the inducer in the model group was subcutaneously injected into the right hind paw of the rats in the normal group to explore the anti-rheumatoid arthritis activity of the mussel oil.

[0111] 2. Sacrifice and Sampling of Experimental Rats The rats were anesthetized by intraperitoneal injection of 20% urethane with a dosage of 0.5 ml / 100 g. Blood was taken from the abdominal aorta into EDTA anticoagulant tubes and ordinary blood collection tubes, and centrifuged at 4°C and 3000 rpm for 10 min. The supernatant was taken and stored in a refrigerator at -80°C. The muscle tissue and the complete ankle joint of the right hind paw of the rats were taken and immediately fixed in a 4% paraformaldehyde fixative.

[0112] 3. Measurement of Rat Body Weight, Food Intake, Paw Thickness and Arthritis Score From the administration of the intervention to the end of the experiment, the body weight and food intake of the rats were measured every two days to observe the changes in the body weight and food intake of the rats, and the measurement time was controlled within the same time range. Starting from the day when the rats were modeled, the thickness of the right hind paw of each rat was measured every other day. The right hind paw of each rat was repeatedly detected 3 times with a vernier caliper, and the average value was taken. At the end of the experiment, the rats were scored for arthritis. The scoring criteria were as follows: no redness or swelling of the joints and toes was scored 0 points, mild but definite joint swelling was scored 0.5 points, or severe swelling was scored 1 point.

[0113] 4. Determination of Rat Serum Indexes According to the instructions of the ELISA kit, the contents of rheumatoid factor RF, anti-cyclic citrullinated peptide antibody anti-CCP, TNF-α, IL-1β, IL-6 and IL-17 in the serum of rats were detected.

[0114] 5. Observation of Histopathological Sections of Ankle Joints The ankle joint specimens of the rats were immersed in a decalcifying solution for decalcification, and then routinely dehydrated with gradient ethanol with volume fractions of 75%, 85%, 90%, and 95% ethanol. After being cleared with xylene, they were infiltrated with paraffin and embedded. The wax blocks were sectioned with a thickness of 4 μm. After the sections were baked at 60 °C, they were stored at room temperature for later use. The paraffin sections were successively placed in xylene, absolute ethanol, and 75% alcohol by volume fraction for gradient dewaxing. After the sections were stained with hematoxylin, the excess stain was washed off with tap water. After differentiation with hydrochloric acid aqueous solution, they were blued with ammonia aqueous solution; then the sections were dehydrated with gradient alcohol, namely 85% alcohol by volume fraction and 95% alcohol by volume fraction, and stained with eosin stain. After staining, the sections were successively placed in absolute ethanol, n-butanol, and xylene for treatment until they were clear, and then sealed with neutral gum after air drying.

[0115] 6. Safranin Fast Green Staining The sections were immersed in fast green stain and then washed with water to remove the excess stain, and differentiated with hydrochloric acid alcohol; then stained with safranin stain and quickly dehydrated with absolute ethanol. The sections were cleared with n-butanol and xylene, and sealed after air drying.

[0116] The experimental results are as follows: The results are as Figure 16 shown. Mussel oil can effectively improve the swelling degree of the hind paws of rats and reduce the arthritis score. In addition, mussel oil significantly reduced the levels of rheumatoid arthritis diagnostic factors and pro-inflammatory factors in the serum, as Figure 17 and Figure 18 shown, indicating that mussel oil effectively prevented the development of rheumatoid arthritis in rats and inhibited the inflammatory response in rats. Through H&E staining and safranin fast green staining, it was found that mussel oil could reduce the pathological changes of joints, cartilage and synovial tissues, and had a protective effect on the damage of joints and synovium, as Figure 19 shown.

[0117] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0118] The above-described embodiments only express several implementation manners of the present invention, and the description thereof is relatively specific and detailed. However, it should not be construed as a limitation on the scope of the invention patent. For those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A method for preparing mussel oil, characterized in that, It includes the following steps: Take mussel meat to make mussel freeze-dried powder with a mesh size of 50 to 100 meshes, and mix the mussel freeze-dried powder with water to obtain a mixture; the mass-volume ratio of the mussel freeze-dried powder to water is 1 kg: 10 L to 14 L; Adjust the pH value of the mixture to acidic and then add protease for enzymatic hydrolysis to obtain an enzymatic hydrolysate; the addition amount of the protease accounts for 0.10% to 0.20% of the mass of the mussel freeze-dried powder; Freeze-dry the supernatant and precipitate separated by centrifugation of the enzymatic hydrolysate to obtain a freeze-dried raw material; ultrasonically extract the freeze-dried raw material with an organic solvent to obtain a crude extract; the mass-volume ratio of the freeze-dried raw material to the organic solvent is 1 kg: 10 L to 14 L; Heat the crude extract to 30°C to 70°C in an N2 atmosphere, add NaOH solution for alkali refining and deacidification for 10 min to 50 min to obtain deacidified oil; the addition amount of the NaOH solution accounts for 15% to 25% of the mass of the crude extract; Heat the deacidified oil to 30°C to 70°C in N2, add activated carbon for adsorption and decolorization for 10 min to 50 min to obtain mussel oil; the addition amount of activated carbon accounts for 2% to 10% of the mass of the deacidified oil.

2. The method according to claim 1, wherein The protease is any one of pepsin and acidic protease.

3. The method according to claim 1, characterized in that, The organic solvent is any one of anhydrous ethanol, a mixed solution of ethanol and n-hexane, and ethanol with a volume fraction of 95%; In the mixed solution of ethanol and n-hexane, the volume ratio of ethanol to n-hexane is 1 to 4: 1 to 4.

4. The method according to claim 1, characterized in that, The temperature of the enzymatic hydrolysis treatment is 30°C to 37°C, and the enzymatic hydrolysis time is 4 hours to 5 hours.

5. The method according to claim 1, characterized in that The time of the ultrasonic extraction is 4 hours, and the power of the ultrasonic extraction is 400 W to 800 W.

6. The method according to claim 1, characterized in that, Use HCl with a molar concentration of 2 mol / L to adjust the pH value of the mixture to 2.

0.

7. The method according to claim 1, characterized in that, The mass concentration of the NaOH solution is 5% to 25%.

8. Mussel oil prepared by the method according to any one of claims 1 to 7.

9. Application of the mussel oil according to claim 8 in the preparation of anti-inflammatory drugs.

10. The application according to claim 9, wherein The anti-inflammatory drug is used for preventing or treating rheumatoid arthritis, asthma or inflammatory bowel disease.

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