Composite preservative, preparation method thereof and preservation method of aquatic products

Abstract

The invention provides a compound preservative, a preparation method thereof and a preservation method of aquatic products, and the compound preservative comprises the following components in parts by weight: 2-10 parts of soluble pearl powder, 0.5-3 parts of sipunculus nudus antibacterial peptide and 0.2-1 part of chitosan oligosaccharide. The solubility of the soluble pearl powder in water at 25 DEG C is 15wt%-25wt%. The compound preservative acts on microorganisms on the surface of the aquatic product to reduce the microorganism level on the surface of the aquatic product, so that the overall microorganism environment of the aquatic product is in a low level under the preservation condition (0-4 DEG C), the total number of bacterial colonies and increase of volatile basic nitrogen in the preservation process of the aquatic product are effectively reduced, and the purpose of prolonging the storage time is achieved. Meanwhile, the compound preservative does not contain chemical substances, is natural in raw materials, has no toxic or side effect, has good bacteriostatic and antioxidant activity, and meets the food safety standard.

Description

Technical Field

[0001] The present application relates to the technical field of aquatic product preservation, and in particular to a composite preservative and a preparation method thereof, and a method for preserving aquatic products. Background Art

[0002] Aquatic products have the characteristics of high water activity, rich nutrition, carrying a large number of microorganisms, and being extremely easy to spoil and deteriorate, so timely and effective preservation measures are needed. The preservation of aquatic products is becoming increasingly important in the entire aquatic product processing, transportation, storage and consumption.

[0003] Existing preservation technologies mostly use chemical reagents or composite methods to prepare preservatives to prevent spoilage of aquatic products. Judging from the preservation effect of oyster meat on the market, the existing preservation technologies have no obvious commercial applications, the storage time does not achieve the expected effect, and the safety indicators basically exceed the national standards, posing food safety risks. Summary of the invention

[0004] Based on this, the present application provides a composite preservative and a preparation method thereof, and a method for preserving aquatic products, so as to provide a preservation technology that can preserve freshness for a long time and is safe and effective.

[0005] The first aspect of the present application provides a composite preservative, which comprises, by weight, 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide and 0.2-1 part of chitosan oligosaccharide; the solubility of the soluble pearl powder in water at 25° C. is 15wt%-25wt%.

[0006] In some embodiments, the sandworm antimicrobial peptide comprises a first peptide segment with a molecular weight of 1211Da-2382Da and a second peptide segment with a molecular weight of 2382Da-7295Da, wherein the mass proportion of the first peptide segment in the sandworm antimicrobial peptide is 56.06wt%, and the mass proportion of the second peptide segment in the sandworm antimicrobial peptide is 37.05wt%.

[0007] In some embodiments, the soluble pearl powder has at least one of the following characteristics (1)-(4):

[0008] (1) The water content of the soluble pearl powder is 20wt%-25wt%;

[0009] (2) The ash content of the soluble pearl powder is 20wt%-30wt%;

[0010] (3) The total protein content of the soluble pearl powder is 0.75wt%-1.5wt%;

[0011] (4) The content of glycine in the soluble pearl powder is 0.13 wt %, and the content of alanine is 0.11 wt %.

[0012] The second aspect of the present application provides a method for preparing a composite preservative, comprising the following steps:

[0013] The composite preservative is prepared by mixing 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide and 0.2-1 part of chitosan oligosaccharide in parts by weight; wherein the solubility of the soluble pearl powder in water at 25° C. is 15wt%-25wt%.

[0014] In some embodiments, the method for preparing the soluble pearl powder comprises:

[0015] After mixing pearl powder raw material, water and composite acid, adding acidic protease to perform hydrolysis reaction 1 to prepare intermediate material;

[0016] After mixing the intermediate material with the composite protease, a hydrolysis reaction 2 is performed to prepare the soluble pearl powder;

[0017] Wherein, the composite acid comprises lactic acid and propionic acid, and the composite protease comprises endo-enzyme and exo-enzyme.

[0018] In some embodiments, the method for preparing the soluble pearl powder includes at least one of the following conditions (1)-(8):

[0019] (1) The mass ratio of lactic acid to propionic acid in the composite acid is 1:(1-2);

[0020] (2) The mass volume ratio of the pearl powder raw material to water is (100-200) g:100 ml, and the mass ratio of the pearl powder raw material to the composite acid is (100-200):(10-20);

[0021] (3) The mesh size of the pearl powder raw material is 20-400 mesh;

[0022] (4) The amount of the acidic protease used is 0.1wt%-0.5wt% of the amount of the pearl powder raw material used;

[0023] (5) The temperature of the hydrolysis reaction 1 is 50°C-55°C, and the time is 2h-4h;

[0024] (6) The mass ratio of endo-enzyme to exo-enzyme in the composite protease is 1:(2.5-3.5);

[0025] (7) The amount of the composite protease used is 0.1wt%-0.6wt% of the amount of the pearl powder raw material used;

[0026] (8) The temperature of the hydrolysis reaction 2 is 45°C-55°C, the time is 2h-4h, and the pH value of the mixed system is 4.5-6.5.

[0027] In some embodiments, the method for preparing the sandworm antimicrobial peptide comprises:

[0028] The sandworm wall is mixed with physiological saline, homogenized, and centrifuged to prepare a supernatant;

[0029] The supernatant is precipitated with ammonium sulfate to prepare a sandworm protein component;

[0030] The sandworm protein component is subjected to enzymatic hydrolysis to prepare the sandworm antimicrobial peptide;

[0031] The enzymes used in the enzymatic treatment include one or more of alkaline protease, papain and neutral protease.

[0032] In some embodiments, the method for preparing the sandworm antimicrobial peptide comprises at least one of the following conditions (1)-(4):

[0033] (1) The temperature of the enzymatic hydrolysis treatment is 45°C-55°C, the time is 4h-5h, and the enzyme addition amount is 5000u / g-8000u / g;

[0034] (2) The concentration of the physiological saline is 0.5wt%-1wt%;

[0035] (3) The mass volume ratio of the sandworm wall to the physiological saline is (1.0-1.6) g:1 mL;

[0036] (4) The homogenization temperature is 0°C-10°C.

[0037] The third aspect of the present application provides a method for preserving aquatic products, comprising the following steps:

[0038] Mixing the composite preservative with water to prepare a solution;

[0039] soaking or spraying the water product with the solution;

[0040] Wherein, in parts by weight, the composite preservative comprises 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide and 0.2-1 part of chitosan oligosaccharide; the solubility of the soluble pearl powder in water at 25° C. is 15wt%-25wt%.

[0041] In some embodiments, the soaking time is 1 min-5 min.

[0042] The composite preservative provided above is formed by compounding soluble pearl powder, sandworm antimicrobial peptide and chitosan oligosaccharide in a specific ratio. The composite preservative acts on the microorganisms on the surface of aquatic products to reduce the microbial level on the surface of aquatic products, so as to achieve a low overall microbial environment of aquatic products under preservation conditions (0-4°C), effectively reduce the increase of total colony count and volatile basic nitrogen during the storage process of aquatic products, thereby achieving the purpose of extending the storage time. At the same time, the composite preservative does not contain chemical substances, the raw materials are natural, non-toxic and side effects, has good antibacterial and antioxidant activity, and meets food safety standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0044] Figure 1 The inhibitory effects of soluble pearl powder solutions of different concentrations prepared by using the soluble pearl powder in Example 1-1 on Vibrio alginolyticus;

[0045] Figure 2 The inhibitory effects of soluble pearl powder solutions of different concentrations prepared by using the soluble pearl powder in Example 1-1 on Escherichia coli;

[0046] Figure 3 The inhibitory effects of soluble pearl powder solutions of different concentrations prepared by using the soluble pearl powder in Example 1-1 on Aeromonas hydrophila;

[0047] Figure 4 The antibacterial activity test results of sandworm antimicrobial peptides;

[0048] Figure 5 The effect of different concentrations of composite preservative solution on the volatile basic nitrogen content of oyster meat;

[0049] Figure 6 The effect of different concentrations of compound preservative solution on the total number of colonies in oyster meat;

[0050] Figure 7 This is the appearance of oyster meat after being soaked in a composite preservative solution with a concentration of 0.2g / L and the oyster meat in the control group. DETAILED DESCRIPTION

[0051] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0053] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present application, the technical solution undoubtedly includes technical solutions connected by "logical and", and also undoubtedly includes technical solutions connected by "logical or".

[0054] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0055] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0056] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and likewise any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0057] The temperature parameters in this application, unless otherwise specified, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0058] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0059] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0060] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0061] In the description of the application, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0062] If there is no special description, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution. If there is no special description, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0063] If there is no special explanation, all the steps of the present application can be performed sequentially or randomly, preferably sequentially.

[0064] The application effect of existing oyster meat preservatives is not good, and the actual preservation time is short. After market research, the actual preservation time is only 2-4 days. After reaching the maximum preservation time, although there is no obvious change in the sensory perception of oyster meat, the volatile basic nitrogen index, pH, total acid index, etc. of oyster meat have all undergone serious changes, seriously exceeding the national standard upper limit, and there are obvious safety hazards. Therefore, the preservation technology of oyster meat currently only exists in laboratory theoretical research and small-scale application under special conditions, and it is still impossible to achieve industrial application and cannot be widely used in the long-distance transportation and preservation of oyster meat.

[0065] In recent years, as consumers have higher requirements for the safety and freshness of aquatic products, biological preservatives extracted from animals, plants, and microorganisms or obtained using modern bioengineering technology have become a hot topic in the research of aquatic product preservation due to their good antioxidant and antibacterial activities, safety and non-toxicity, etc. Therefore, this application uses soluble pearl powder, sandworm antimicrobial peptides and chitosan oligosaccharides to form a composite preservative in a specific ratio, and through simple treatment methods such as spraying and soaking, the cold and fresh storage time of aquatic products is increased.

[0066] One or more embodiments of the present application provide a composite preservative, which includes, by weight, 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide, and 0.2-1 part of chitosan oligosaccharide; the solubility of the soluble pearl powder in water at 25° C. is 15wt%-25wt%.

[0067] As a non-limiting example, the weight proportion of the soluble pearl powder contained in the composite preservative can be but is not limited to 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts or a range between any two of the above weight proportions.

[0068] The weight proportion of the sandworm antibacterial peptide contained in the composite preservative can be but is not limited to 0.5 parts, 0.7 parts, 1 parts, 1.2 parts, 1.4 parts, 1.6 parts, 1.8 parts, 2 parts, 2.2 parts, 2.4 parts, 2.6 parts, 2.8 parts, 3 parts or the range between any two of the above weight proportions.

[0069] The weight proportion of chitosan oligosaccharide contained in the composite preservative can be, but is not limited to, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 or a range between any two of the above weight proportions.

[0070] The solubility of the soluble pearl powder in water at 25° C. may be, but is not limited to, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt% or a range between any two of the above solubilities, etc. When the solubility of the soluble pearl powder is within this range, it is beneficial to further improve the preservation effect.

[0071] Understandably, the composite preservative provided by the present application is formed by compounding soluble pearl powder, sandworm antimicrobial peptides and chitosan oligosaccharides in a specific ratio. The composite preservative acts on the microorganisms on the surface of aquatic products to reduce the level of microorganisms on the surface of aquatic products, so as to achieve a low overall microbial environment of aquatic products under preservation conditions (0-4°C), effectively reduce the increase in the total number of colonies and volatile basic nitrogen during the storage of aquatic products, and thus achieve the purpose of extending the storage time. At the same time, the composite preservative does not contain chemical substances, the raw materials are natural, non-toxic and side effects, has good antibacterial and antioxidant activity, and meets food safety standards.

[0072] In some embodiments, the sandworm antimicrobial peptide includes a first peptide segment with a molecular weight of 1211Da-2382Da and a second peptide segment with a molecular weight of 2382Da-7295Da, the mass proportion of the first peptide segment in the sandworm antimicrobial peptide is 56.06wt%, and the mass proportion of the second peptide segment in the sandworm antimicrobial peptide is 37.05wt%. Therefore, the proportion of small molecular weight peptide segments in the sandworm antimicrobial peptide is high, and since small molecular weight peptide segments have better biological activity in antimicrobial action, it is beneficial to improve the antimicrobial and fresh-keeping effect of the sandworm antimicrobial peptide.

[0073] It should be noted that in this context, "first peptide segment", "second peptide segment", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0074] In some embodiments, the antimicrobial peptide comprises 17 hydrolyzed amino acids.

[0075] In some optional embodiments, the moisture content of the soluble pearl powder is 20wt%-25wt%; for example, it can be but not limited to 20wt%, 20.5wt%, 21wt%, 21.5wt%, 22wt%, 22.5wt%, 23wt%, 23.5wt%, 24wt%, 24.5wt%, 25wt% or a range between any two of the above soluble pearl powders.

[0076] As a possible implementation manner, the ash content of the soluble pearl powder is 20wt%-30wt%; for example, it can be but not limited to 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt% or a range between any two of the above ash contents.

[0077] In some exemplary embodiments, the total protein content of the soluble pearl powder is 0.75wt%-1.5wt%; for example, it can be but not limited to 0.75wt%, 0.8wt%, 0.85wt%, 0.9wt%, 0.95wt%, 1wt%, 1.05wt%, 1.1wt%, 1.15wt%, 1.2wt%, 1.25wt%, 1.3wt%, 1.35wt%, 1.4wt%, 1.45wt%, 1.5wt% or a range between any two of the above protein contents, etc.

[0078] In some embodiments, the content of glycine in the soluble pearl powder is 0.13 wt %, and the content of alanine is 0.11 wt %.

[0079] In some embodiments, the soluble pearl powder comprises 13 hydrolyzed amino acids.

[0080] One or more embodiments of the present application provide a method for preparing a composite preservative, which can be used to prepare the above-mentioned composite preservative, and the preparation method comprises the following steps: mixing 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide and 0.2-1 part of chitosan oligosaccharide by weight to prepare the composite preservative; wherein the solubility of the soluble pearl powder in water at 25°C is 15wt%-25wt%.

[0081] It can be understood that the composite preservative prepared in the present application has a good preservation effect, natural raw materials, no toxic side effects, good antibacterial and antioxidant activity, and can effectively reduce the increase in the total colony count and volatile basic nitrogen during the storage process of aquatic products, thereby extending the shelf life, providing a theoretical basis for further developing the application of pearl layer powder in the field of aquatic product preservation.

[0082] In some embodiments, the method for preparing soluble pearl powder comprises:

[0083] After pearl powder raw material, water and composite acid are mixed, acidic protease is added to perform hydrolysis reaction 1 to prepare intermediate material; after the intermediate material is mixed with composite protease, hydrolysis reaction 2 is performed to prepare soluble pearl powder; wherein the composite acid includes lactic acid and propionic acid, and the composite protease includes endo-enzyme and exo-enzyme.

[0084] In some optional embodiments, the mass ratio of lactic acid to propionic acid in the composite acid is 1:(1-2); for example, it can be, but not limited to, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2 or a range between any two of the above mass ratios. When the mass ratio of lactic acid to propionic acid is within the above range, it is beneficial to improve the reaction effect of the hydrolysis reaction.

[0085] As a non-limiting example, pearls or pearl shells are washed, dried, and crushed to prepare the pearl powder raw material.

[0086] As a possible implementation manner, the mass volume ratio of the pearl powder raw material and water is (100-200) g:100 mL; for example, it can be but not limited to 100 g:100 mL, 105 g:100 mL, 120 g:100 mL, 125 g:100 mL, 130 g:100 mL, 135 g:100 mL, 140 g:100 mL, 145 g:100 mL, 150 g:100 mL, 155 g:100 mL, 160 g:100 mL, 165 g:100 mL, 170 g:100 mL, 175 g:100 mL, 180 g:100 mL, 185 g:100 mL, 190 g:100 mL, 195 g:100 mL, 200 g:100 mL or the range between any two of the above mass volume ratios.

[0087] In some optional embodiments, the mass ratio of pearl powder raw material to composite acid is (100-200):(10-20); for example, it can be but not limited to (100-190):(10-19), (100-180):(10-18), (100-170):(10-17), (100-160):(10-16), (100-150):(10-15), (100-140):(10-14), (100-130):(10-13), (100-120):(10-12), (110-200):(11-20), (120-200):(12-20), (130-200):(13-20), (140-200):(14-20), (150-200):(15-20), (160-200):(16-20), (170-200):(17-20), (180-200):(18-20) or (190-200):(19-20), etc.

[0088] When the mass volume ratio of the pearl powder raw material to water and the mass volume ratio of the pearl powder raw material to the composite acid are within the above ranges, it is beneficial to improve the reaction effect of the hydrolysis reaction.

[0089] In some embodiments, the mesh size of the pearl powder raw material is 20-400 mesh; for example, it can be but not limited to 20 mesh, 50 mesh, 80 mesh, 100 mesh, 120 mesh, 150 mesh, 180 mesh, 200 mesh, 220 mesh, 250 mesh, 280 mesh, 300 mesh, 320 mesh, 350 mesh, 380 mesh, 400 mesh or a range between any two of the above mesh sizes.

[0090] In some optional embodiments, the amount of acidic protease is 0.1wt%-0.5wt% of the amount of pearl powder raw material; for example, it can be but not limited to 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt% or a range between any two of the above amounts.

[0091] In some exemplary embodiments, the temperature of the hydrolysis reaction 1 is 50°C-55°C; for example, it can be but not limited to 50°C, 51°C, 52°C, 53°C, 54°C, 55°C or a range between any two of the above temperatures.

[0092] As a possible implementation manner, the time of the hydrolysis reaction 1 is 2 h-4 h; for example, it may be but is not limited to 2 h, 2.5 h, 3 h, 3.5 h, 4 h or a range between any two of the above times.

[0093] It should be noted that the temperature and time of the hydrolysis reaction 1 can be combined in any appropriate manner, and both can be selected from any temperature and time of the hydrolysis reaction 1 described herein.

[0094] In some optional embodiments, the mass ratio of endonuclease to exonuclease in the composite protease is 1:(2.5-3.5); for example, it can be but not limited to 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5 or a range between any two of the above ratios, etc. When the mass ratio of endonuclease to exonuclease is within the above range, it is beneficial to improve the reaction effect of the hydrolysis reaction.

[0095] As a possible implementation manner, the amount of the composite protease is 0.1wt%-0.6wt% of the amount of the pearl powder raw material; for example, it can be but not limited to 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, 0.55wt%, 0.6wt% or a range between any two of the above amounts.

[0096] In some optional embodiments, the temperature of the hydrolysis reaction 2 is 45°C-55°C; for example, it can be but not limited to 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C or a range between any two of the above temperatures, etc.

[0097] In some embodiments, the time of the hydrolysis reaction 2 is 2 h-4 h; for example, it may be but is not limited to 2 h, 2.5 h, 3 h, 3.5 h, 4 h or a range between any two of the above times.

[0098] In some exemplary embodiments, when the hydrolysis reaction 2 is performed, the pH value of the mixed system is 4.5-6.5; for example, it can be but is not limited to 4.5, 4.7, 5, 5.2, 5.5, 5.8, 6.3, 6.5 or a range between any two of the above pH values.

[0099] It should be noted that the temperature, time, and pH value of the mixed system of the hydrolysis reaction 2 can be combined in any appropriate manner, and the three can be selected from any temperature, time, and pH value of the mixed system of the hydrolysis reaction 2 described in this article.

[0100] In some optional embodiments, before the hydrolysis reaction 2 is performed, pearl shell powder can be used to adjust the pH value of the intermediate material to 4.5-6.5.

[0101] The properties of the soluble pearl powder prepared by the above method are the same as those of the soluble pearl powder defined in the product (composite preservative), and will not be described in detail here.

[0102] In some embodiments, the method for preparing sandworm antimicrobial peptides includes: mixing sandworm walls with physiological saline, homogenizing, and centrifuging to prepare a supernatant; precipitating the supernatant with ammonium sulfate to prepare a sandworm protein component; and enzymatically hydrolyzing the sandworm protein component to prepare a sandworm antimicrobial peptide; wherein the enzyme used in the enzymatic hydrolysis includes one or more of alkaline protease, papain, and neutral protease.

[0103] In some optional embodiments, the temperature of the enzymatic hydrolysis treatment is 45°C-55°C; for example, it can be but not limited to 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C or a range between any two of the above temperatures, etc.

[0104] As a possible implementation manner, the time of enzymatic treatment is 4h-5h; for example, it can be but not limited to 4h, 4.2h, 4.4h, 4.6h, 4.8h, 5h or a range between any two of the above times.

[0105] In some exemplary embodiments, the amount of enzyme added for enzymatic hydrolysis treatment is 5000u / g-8000u / g; for example, it can be but not limited to 5000u / g, 5500u / g, 6000u / g, 6500u / g, 7000u / g, 7500u / g, 8000u / g or a range between any two of the above amounts, etc.

[0106] It should be noted that the temperature, time and amount of enzyme added for the enzymatic hydrolysis treatment can be combined in any appropriate manner, and the three can be selected from any temperature, time and amount of enzyme added for the enzymatic hydrolysis treatment described herein.

[0107] In some embodiments, the concentration of physiological saline is 0.5wt%-1wt%; for example, it can be but not limited to 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt% or a range between any two of the above values.

[0108] As a possible implementation manner, the mass volume ratio of sandworm wall to physiological saline is (1.0-1.6) g:1mL; for example, it can be but not limited to 1.0g:1mL, 1.1g:1mL, 1.2g:1mL, 1.3g:1mL, 1.4g:1mL, 1.5g:1mL, 1.6g:1mL or a range between any two of the above mass volume ratios.

[0109] In some optional embodiments, the homogenization temperature is 0°C-10°C; for example, it can be but not limited to 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C or a range between any two of the above temperatures.

[0110] In some embodiments, the peptide content in the sandworm protein component is 80wt%-90wt%; for example, it can be but not limited to 80wt%, 81wt%, 82wt%, 83wt%, 84wt%, 85wt%, 86wt%, 87wt%, 88wt%, 89wt%, 90wt% or a range between any two of the above values.

[0111] The properties of the sandworm antimicrobial peptide prepared by the above method are the same as those of the sandworm antimicrobial peptide specified in the product (composite preservative), and will not be repeated here.

[0112] One or more embodiments of the present application provide a method for preserving aquatic products, comprising the following steps:

[0113] The composite preservative is mixed with water to prepare a solution; the solution is used to soak or spray the water product; wherein, in parts by weight, the composite preservative comprises 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide and 0.2-1 part of chitosan oligosaccharide; and the solubility of the soluble pearl powder in water at 25° C. is 15wt%-25wt%.

[0114] It should be noted that the properties of the soluble pearl powder used in the preservation method are the same as those of the soluble pearl powder specified in the product (composite preservative), and will not be described in detail here.

[0115] The properties of the sandworm antimicrobial peptide used in the preservation method are the same as those of the sandworm antimicrobial peptide defined in the product (composite preservative), and will not be described in detail here.

[0116] In some embodiments, the soaking time is 1 min-5 min; for example, it can be but not limited to 1 min, 2 min, 3 min, 4 min, 5 min or a range between any two of the above times.

[0117] The technical scheme of the present application is described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following embodiments are preferably referred to the guidance given in the present application, and can also be carried out according to the experimental manual or conventional conditions in the field, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0118] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0119] 1. Preparation of soluble pearl powder

[0120] Example 1-1

[0121] The pearl shells were cleaned, dried, and crushed into 400 mesh powder; 200g of pearl powder was added to 100ml of distilled water, 20g of lactic acid and propionic acid (w / w=1:2), mixed and reacted, cooled to 55°C, and then acidic protease was added for hydrolysis for 2 hours. A small amount of pearl shell powder was used to adjust the pH of the mixed system to 6.5, and composite protease (endonuclease and exonuclease with a mass ratio of 1:3) was added for hydrolysis at 55°C for 2 hours, and then the enzyme was inactivated at 100°C for 10 minutes, and the filtrate was filtered and freeze-dried to obtain soluble pearl powder. The soluble pearl powder was a white powder with good water solubility. Its solubility in water was measured to be 19wt% at 25°C; the total protein content of the soluble pearl powder was 1.42g / 100g, the ash content was 21wt%, the moisture content was 22wt%, the glycine content was 0.13wt%, and the alanine content was 0.11wt%.

[0122] Example 1-2

[0123] The pearl shells were cleaned, dried, and crushed into 400 mesh powders; 100g of pearl powder was taken, 100ml of distilled water, 10g of lactic acid and propionic acid (w / w=1:1) were added, the mixture was cooled to 50°C after reaction, acidic protease was added for hydrolysis for 4 hours, a small amount of pearl shell powder was used to adjust the pH of the mixed system to 4.5, composite protease (endonuclease and exonuclease with a mass ratio of 1:3) was added for hydrolysis at 45°C for 4 hours, enzyme was inactivated at 100°C for 10 minutes, the filtrate was filtered, and soluble pearl powder was obtained by freeze drying. The soluble pearl powder was a white powder with good water solubility. Its solubility in water was measured to be 22wt% at 25°C; the total protein content of the soluble pearl powder was 0.96g / 100g, the ash content was 20wt%, the moisture content was 21wt%, the glycine content was 0.13wt%, and the alanine content was 0.11wt%.

[0124] Examples 1-3

[0125] The pearl shells were cleaned, dried, and crushed into 400 mesh powder; 150g of pearl powder was taken, 100ml of distilled water, 15g of lactic acid and propionic acid (w / w=1:1.5) were added, the mixture was cooled to 53°C after reaction, acidic protease was added for hydrolysis for 3 hours, a small amount of pearl shell powder was used to adjust the pH of the mixed system to 5.5, composite protease (endonuclease and exonuclease with a mass ratio of 1:3) was added for hydrolysis at 50°C for 3 hours, enzyme was inactivated at 100°C for 10 minutes, the filtrate was filtered, and soluble pearl powder was obtained by freeze drying. The soluble pearl powder was a white powder with good water solubility. Its solubility in water was measured at 25°C to be 19wt%; the total protein content of the soluble pearl powder was 1.04g / 100g, the ash content was 21wt%, the moisture content was 20wt%, the glycine content was 0.13wt%, and the alanine content was 0.11wt%.

[0126] The soluble pearl powder prepared in Example 1-1 was used to prepare soluble pearl powder solutions with mass percentage concentrations of 0.5wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6.7wt% and 10wt%, respectively, and water was used as a blank control; the inhibitory effects of the soluble pearl powder solutions of different concentrations and the blank control on Vibrio alginolyticus, Escherichia coli and Aeromonas hydrophila were respectively measured; the test method was: the OD value of the sample to be tested was measured at an absorbance of 550nm, and the OD value was correlated with the logarithm of the total number of colonies to indicate the content of microorganisms in the sample. The results are shown in FIG. Figure 1-3 Among them, the inhibitory effect on Vibrio alginolyticus is shown in Figure 1 As shown in Figure 2, the inhibitory effect on Escherichia coli is as follows Figure 2 As shown in Figure 2, the inhibitory effect on Aeromonas hydrophila is as follows Figure 3 shown.

[0127] Depend on Figure 1-Figure 3 It can be seen that different concentrations of soluble pearl powder solutions have strong inhibitory activity against Escherichia coli and Aeromonas hydrophila, and the inhibitory effect on Vibrio alginolyticus is the most significant, and the mass percentage concentration is 2% to show activity. There is a linear relationship between the antibacterial effect of soluble pearl powder within a certain concentration range. The sample solution with a mass percentage concentration of 4% has a more obvious antibacterial activity within 8 hours, and the antibacterial effect begins to weaken after 8 hours.

[0128] Although the article does not provide the antibacterial effects of the soluble pearl powder prepared in Examples 1-2 and 1-3, it is not difficult to understand that the inhibitory effects of the soluble pearl powder prepared in Examples 1-2 and 1-3 on Vibrio alginolyticus, Escherichia coli and Aeromonas hydrophila are similar to the inhibitory effects of the soluble pearl powder prepared in Example 1-1 on Vibrio alginolyticus, Escherichia coli and Aeromonas hydrophila.

[0129] 2. Preparation of Sandworm Antimicrobial Peptides

[0130] Example 2-1

[0131] The fresh sandworm wall was washed and chopped, and physiological saline with a mass concentration of 0.85wt% (the mass volume ratio of sandworm wall and physiological saline was 1.3g:1mL) was added, homogenized at 4°C, centrifuged, and the supernatants (water-soluble proteins) obtained twice were combined after secondary extraction, and the sandworm protein component was obtained by ammonium sulfate precipitation; the sandworm protein component was enzymatically hydrolyzed with alkaline protease, wherein the enzymatic hydrolysis parameters included: the amount of alkaline protease added was 7000u / g, the time was 4.30 hours and the temperature was 55°C; after the enzymatic hydrolysis was completed, the enzymatic hydrolysis product was inactivated, centrifuged, concentrated and freeze-dried to obtain the sandworm antibacterial peptide powder.

[0132] Example 2-2

[0133] The difference between Example 2-2 and Example 2-1 is that papain is used to replace alkaline protease in Example 2-2, and the other aspects are the same.

[0134] Example 2-3

[0135] The difference between Example 2-3 and Example 2-1 is that neutral protease is used instead of alkaline protease in Example 2-3, and the other aspects are the same.

[0136] The antibacterial activity of the sandworm antimicrobial peptides prepared in Examples 2-1 to 2-3 against Vibrio alginolyticus, Shewanella algae, Escherichia coli and Pseudomonas aeruginosa was tested, and water was used as a blank control; the test method was: taking the sample to be tested and detecting its OD value at an absorbance of 550nm, using the property that the size of its OD value is correlated with the logarithm of the total number of colonies, the content of microorganisms in the sample was briefly indicated; the test results are as follows Figure 4 As shown. Figure 4 It can be seen that the sandworm antimicrobial peptide has excellent antibacterial effect on Vibrio alginolyticus, Shewanella algae, Escherichia coli and Pseudomonas aeruginosa.

[0137] 3. Test on the preservation effect of oyster meat

[0138] The soluble pearl powder prepared in Example 1-1, the sandworm antimicrobial peptide prepared in Example 2-1, chitosan oligosaccharide and distilled water were mixed to prepare composite preservative solutions of different concentrations (0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L), wherein the content of soluble pearl powder in each composite preservative solution was 8 wt %, the content of sandworm antimicrobial peptide was 1.9 wt %, and the content of chitosan oligosaccharide was 0.7 wt %.

[0139] The oyster meat was soaked in the above-mentioned composite preservative solutions of various concentrations (as the treatment group) for 5 minutes, picked up and drained, placed in a sterile container, sealed and packaged, and refrigerated and stored at 4°C; the oyster meat from the same batch that had not been treated in any way was used as the control group and was treated in the same way as the treatment group. The changes in the total colony count and volatile basic nitrogen content of the oyster meat in the treatment and control groups during storage were tested. The effects of different concentrations of composite preservative solutions on the volatile basic nitrogen content of oyster meat are shown in Figure 2. Figure 5 The effect of different concentrations of composite preservative solution on the total number of oyster meat colonies is shown in Figure 6 shown.

[0140] Depend on Figure 5-6 It can be seen that the volatile basic nitrogen content and total colony count of oyster meat treated with different concentrations of composite preservatives were reduced to varying degrees, among which the concentration of the composite preservative at 0.2 g / L can effectively inhibit the spoilage rate of oyster meat.

[0141] The sensory evaluation of oyster meat after being soaked in a 0.2 g / L composite preservative solution was conducted. Figure 7 As shown. Figure 7 It can be seen that the state of the oyster meat in the treatment group after 9 days of refrigerated storage (0-4℃) was basically the same as the initial state, while the control group showed surface autolysis and more surface mucus secretion after 5 days of storage. In terms of smell, the treatment group had a fresh and sweet aroma of oysters, while the control group had a smelly and corrupt smell.

[0142] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, 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, they should be considered to be within the scope of this specification.

[0143] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A composite preservative, characterized in that: In parts by weight, the composite preservative comprises 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide and 0.2-1 part of chitosan oligosaccharide; the solubility of the soluble pearl powder in water at 25° C. is 15wt%-25wt%.

2. The composite preservative according to claim 1, characterized in that: The sandworm antimicrobial peptide comprises a first peptide segment with a molecular weight of 1211Da-2382Da and a second peptide segment with a molecular weight of 2382Da-7295Da, wherein the mass proportion of the first peptide segment in the sandworm antimicrobial peptide is 56.06wt%, and the mass proportion of the second peptide segment in the sandworm antimicrobial peptide is 37.05wt%.

3. The composite preservative according to any one of claims 1 to 2, characterized in that: The soluble pearl powder has at least one of the following characteristics (1) to (4): (1) The water content of the soluble pearl powder is 20wt%-25wt%; (2) The ash content of the soluble pearl powder is 20wt%-30wt%; (3) The total protein content of the soluble pearl powder is 0.75wt%-1.5wt%; (4) The content of glycine in the soluble pearl powder is 0.13 wt %, and the content of alanine is 0.11 wt %.

4. A method for preparing a composite preservative, characterized in that: The steps include: The composite preservative is prepared by mixing 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide and 0.2-1 part of chitosan oligosaccharide in parts by weight; wherein the solubility of the soluble pearl powder in water at 25° C. is 15wt%-25wt%.

5. The preparation method according to claim 4, characterized in that: The preparation method of the soluble pearl powder comprises: After mixing pearl powder raw material, water and composite acid, adding acidic protease to perform hydrolysis reaction 1 to prepare intermediate material; After mixing the intermediate material with the composite protease, a hydrolysis reaction 2 is performed to prepare the soluble pearl powder; Wherein, the composite acid comprises lactic acid and propionic acid, and the composite protease comprises endo-enzyme and exo-enzyme.

6. The preparation method according to claim 5, characterized in that: The method for preparing the soluble pearl powder comprises at least one of the following conditions (1) to (8): (1) The mass ratio of lactic acid to propionic acid in the composite acid is 1:(1-2); (2) The mass volume ratio of the pearl powder raw material to water is (100-200) g:100 mL, and the mass ratio of the pearl powder raw material to the composite acid is (100-200):(10-20); (3) The mesh size of the pearl powder raw material is 20-400 mesh; (4) The amount of the acidic protease used is 0.1wt%-0.5wt% of the amount of the pearl powder raw material used; (5) The temperature of the hydrolysis reaction 1 is 50°C-55°C, and the time is 2h-4h; (6) The mass ratio of endo-enzyme to exo-enzyme in the composite protease is 1:(2.5-3.5); (7) The amount of the composite protease used is 0.1wt%-0.6wt% of the amount of the pearl powder raw material used; (8) The temperature of the hydrolysis reaction 2 is 45°C-55°C, the time is 2h-4h, and the pH value of the mixed system is 4.5-6.

5.

7. The preparation method according to any one of claims 5 to 6, characterized in that: The preparation method of the sandworm antimicrobial peptide comprises: The sandworm wall is mixed with physiological saline, homogenized, and centrifuged to prepare a supernatant; The supernatant is precipitated with ammonium sulfate to prepare a sandworm protein component; The sandworm protein component is subjected to enzymatic hydrolysis to prepare the sandworm antimicrobial peptide; The enzymes used in the enzymatic treatment include one or more of alkaline protease, papain and neutral protease.

8. The preparation method according to claim 7, characterized in that: The method for preparing the sandworm antimicrobial peptide comprises at least one of the following conditions (1)-(4): (1) The temperature of the enzymatic hydrolysis treatment is 45°C-55°C, the time is 4h-5h, and the enzyme addition amount is 5000u / g-8000u / g; (2) The concentration of the physiological saline is 0.5wt%-1wt%; (3) The mass volume ratio of the sandworm wall to the physiological saline is (1.0-1.6) g:1 mL; (4) The homogenization temperature is 0°C-10°C.

9. A method for preserving aquatic products, characterized in that: The steps include: Mixing the composite preservative with water to prepare a solution; soaking or spraying water products with the solution; Wherein, in parts by weight, the composite preservative comprises 2-10 parts of soluble pearl powder, 0.5-3 parts of sandworm antibacterial peptide and 0.2-1 part of chitosan oligosaccharide; the solubility of the soluble pearl powder in water at 25° C. is 15wt%-25wt%.

10. The preservation method according to claim 9, characterized in that: The soaking time is 1 min to 5 min.

Images