Preparation of algal polysaccharide active coating and application of algal polysaccharide active coating as protective film formed on body surface of grouper in waterless keep-alive transportation of grouper

Abstract

The invention discloses an algal polysaccharide active coating which is prepared by taking algal polysaccharides as a matrix, compounding Vc, lysozyme and sorbitol, and a preparation method of the algal polysaccharide active coating comprises the following steps: step (1), stirring algal polysaccharides with different concentrations and 0.09% lysozyme at 40 DEG C until the algal polysaccharides and the lysozyme are completely dissolved to obtain a film forming solution; step (2), adding 30 mg / L VC into the membrane liquid formed in the step S1; (3) adding 0.3% of sorbitol into the membrane liquid formed in the step S2; and (4) carrying out ultrasonic treatment for 10 minutes by using an ultrasonic-assisted homogenizer at the power of 20kHz and 700W to obtain a uniform solution, and degassing in vacuum to form a membrane solution. The algal polysaccharide active coating prepared by the method forms an algal polysaccharide active film on the body surface of the grouper, and can be applied to a waterless keep-alive transportation method of the grouper, so that the survival time is prolonged, the transportation survival rate is improved, and the oxidative damage of the transportation tissue of the grouper is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of active coating preparation and application, in particular to the preparation of a seaweed polysaccharide active coating and its application as a protective film formed on the body surface of grouper in waterless live transportation of grouper. Background Art

[0002] Seaweed polysaccharides, extracted from marine algae, are a class of macromolecular natural compounds with excellent biocompatibility, biodegradability, diverse activities, and low toxicity. They are often associated with anti-tumor, anti-inflammatory, antioxidant, and immunomodulatory effects, and numerous studies have shown that these effects are closely related to regulating the immune system. Seaweed polysaccharides are characterized by structural stability, water retention, and film-forming properties, making them commonly used in aquatic products, meat products, and fruits. Vitamin C (Vc) is a natural antioxidant that scavenges reactive oxygen species (ROS), improves the body's nonspecific immune system, and plays a vital role in maintaining physiological homeostasis in fish. Vc participates in the synthesis of hormones and collagen and protects against stress responses. Lysozyme (muramidase, EC3.2.1.17) is a key defense molecule in the innate immune system, playing a crucial role in mediating protection against microbial invasion. It is a mucolytic enzyme derived from leukocytes. It is a food-safe antimicrobial enzyme that can also form gels and films. Many studies have shown that the application of lysozyme can greatly extend the shelf life of seafood or aquatic products, and can bring many conveniences for storage and transportation. Sorbitol molecules can improve the water retention of fish surface, and the degree of protease denaturation is low at low temperatures, which has an antifreeze effect.

[0003] Numerous studies have demonstrated the effectiveness of macromolecular polymers, particularly polysaccharides and proteins, in producing coatings with unique barrier properties. This ability reduces moisture diffusion and prevents the penetration of oxygen and microorganisms during preservation. Furthermore, bioactive compounds are incorporated into coatings to create interactive food packaging systems that provide antioxidant and antimicrobial properties. Active coating solutions are a method for extending the shelf life of fish in refrigerated or frozen storage. The addition of suitable additives, such as seaweed polysaccharides and silymarin, to active coating solutions can improve the coating's stability as well as its antimicrobial and antioxidant properties. Summary of the Invention

[0004] The present invention provides a preparation method of a seaweed polysaccharide active coating and its application as a protective film formed on the body surface of grouper in waterless alive transportation of grouper. By studying the effects of the seaweed polysaccharide active coating on the muscle physicochemical properties, blood biochemical indicators and immune metabolism of grouper in simulated waterless transportation, the stress generated by fish during waterless transportation is resolved, and the quality of grouper during waterless transportation is improved.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A seaweed polysaccharide active coating is prepared by using seaweed polysaccharide as a matrix and compounding vitamin C, lysozyme, and sorbitol. The preparation method specifically comprises the following steps:

[0007] Step (1), different concentrations of seaweed polysaccharides and 0.09% lysozyme are stirred at 40° C. until completely dissolved to obtain a film-forming solution;

[0008] Step (2), 30 mg / L VC was added to the membrane solution formed by S1;

[0009] Step (3), 0.3% sorbitol is added to the membrane solution formed by S2;

[0010] Step (4) is to use an ultrasonic assisted homogenizer to perform ultrasonic treatment at 20 kHz and 700 W power for 10 minutes to obtain a uniform solution and degas under vacuum to form a membrane solution.

[0011] As a preferred embodiment, the average body length of grouper is 27.15 cm and the average weight is 500±50 g.

[0012] As a preferred embodiment, before the experiment began, the grouper was fasted for 36 hours.

[0013] As a preferred embodiment, the density during the temporary maintenance period is 20kg / m 3 The seawater salinity is 20‰, the temperature is 23-25℃, the dissolved oxygen is 7-8 mg / L, and the pH is 7.5-8.5.

[0014] As a preferred embodiment, during the temporary culture period, the seawater is processed through a circulation filter, and 50% of the seawater in the temporary culture tank is replaced every 12 hours.

[0015] The seaweed polysaccharide active coating prepared by the method is used to form a seaweed polysaccharide active film on the body surface of grouper.

[0016] A method for transporting grouper without water and keeping it alive based on a seaweed polysaccharide active film, specifically comprising the following steps:

[0017] Step S1, the experiment was divided into four groups, namely, 20 mg / L seaweed polysaccharide (SP20), 25 mg / L seaweed polysaccharide (SP25), 30 mg / L seaweed polysaccharide (SP30) mixed with 0.09% lysozyme and 0.3% sorbitol, and a control group (CK) without seaweed polysaccharide active coating treatment;

[0018] Step S2: For each group of 20 samples, after the temporary incubation transition, the seawater was cooled from 23-25°C to 15°C at a rate of 3°C / h. Other parameters were the same as those for the temporary incubation water. Groupers with weaker touch stress responses in the temporary incubator were removed and packaged without water. The control group (CK) was not treated with protective liquid coating after cooling and dormancy.

[0019] Step S3: After being treated with the active coating of seaweed polysaccharides at different contents, the grouper fish were wrapped with a wet sponge and placed in a special oxygenated bag. Four fish were placed in a transport box for transportation. The simulated transportation process was as follows: 1 hour on a Class B highway (80 km / h) → 3 hours on a Class A highway (100 km / h) → 1 hour on a Class B highway (80 km / h), repeated three times.

[0020] Step S4: After 15 hours of transportation, three groupers were randomly selected and temporarily restored in normal temperature water for 12 hours.

[0021] As a preferred embodiment, in step S3, the grouper is wrapped with a wet sponge, and the wet sponge is placed in water at 15° C., a salinity of 20‰, and a pH of 7.0-7.5 in advance.

[0022] Beneficial effects: The present invention uses an appropriate concentration of seaweed polysaccharide active coating to wrap the grouper during waterless live transportation, which helps to prolong the survival time and improve the transportation survival rate, relieve the stress of the grouper, and effectively reduce the oxidative damage of the grouper's transportation tissue, and the decline in immunity and antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 1 is a schematic diagram of the survival rate of each experimental group in Example 1 of the present invention and the survival rate of each experimental group 12 hours after operation.

[0024] Figure 2 Schematic diagram of changes in muscle pH and water content in each experimental group in Example 2 of the present invention.

[0025] Figure 3 Schematic diagram of the changes in blood biochemistry (cortisol and glucose) in each experimental group in Example 3 of the present invention.

[0026] Figure 4 Schematic diagram of changes in liver function (albumin and total protein) in each experimental group in Example 4 of the present invention.

[0027] Figure 5 Schematic diagram of changes in renal function (creatinine and uric acid) in each experimental group in Example 5 of the present invention.

[0028] Figure 6 Schematic diagram of changes in immune indicators (lysozyme and immunoglobulin) in each experimental group of Example 6 of the present invention.

[0029] Figure 7 Schematic diagram of the changes in antioxidant (superoxide dismutase) in each experimental group of Example 7 of the present invention. DETAILED DESCRIPTION

[0030] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0031] Example 1

[0032] A method for transporting grouper without water and keeping it alive based on a seaweed polysaccharide active film, specifically comprising the following steps:

[0033] In step S1, 20 mg / L seaweed polysaccharide (SP20), 25 mg / L seaweed polysaccharide (SP25), and 30 mg / L seaweed polysaccharide (SP30) were respectively combined with 30 mg / L Vc, 0.09% lysozyme, and 0.3% sorbitol to form an active coating, and together with the control group, four experimental groups were formed, each with 20 samples.

[0034] In step S2, for each group of 20 samples, after the temporary incubation period, the seawater temperature was lowered from 23-25°C to 15°C at a rate of 3°C / h. Other parameters were the same as for the temporary incubation water. Groupers with weak responses to touch stress were removed from the temporary incubator and packaged without water. The control group (CK) was not treated with protective liquid coating after the cooling and dormancy period.

[0035] In step S3, after being treated with the active coating containing different amounts of seaweed polysaccharides, the grouper were wrapped in a wet sponge and placed in a dedicated oxygenated bag. Four fish were placed in a transport box for transportation. The simulated transport process was as follows: 1 hour on a Class B highway (80 km / h) → 3 hours on a Class A highway (100 km / h) → 1 hour on a Class B highway (80 km / h), repeated three times.

[0036] Step S4: After 15 hours of transportation, three groupers were randomly selected and temporarily restored in normal temperature water for 12 hours.

[0037] Other parameters are as follows:

[0038] Groupers had an average body length of 27.15 cm and an average weight of 500 ± 50 g. Prior to the experiment, groupers were fasted for 36 hours. During this period, the density of the groupers was 20 kg / m³. The seawater salinity was 20‰, the temperature was 23-25°C, the dissolved oxygen level was 7-8 mg / L, and the pH was 7.5-8.5. During the period, the seawater was treated through a circulating filter, and 50% of the water in the holding tank was replaced every 12 hours.

[0039] The wet sponge is placed in water at 15°C, salinity 20‰, and pH 7.0-7.5 in advance.

[0040] Adding different concentrations of seaweed polysaccharide active coating has different effects on the survival rate of grouper during waterless transportation. Figure 1 As shown, the survival rates of grouper treated with 0 (CK), 20 (SP20), 25 (SP25), and 30 (SP30) mg / L seaweed polysaccharide active coating decreased to 40.0%, 60.0%, 70.0%, and 65.5%, respectively. The survival rate of the SP25-treated group was higher than that of the SP20 and SP30-treated groups. At 13 hours after transport, the survival rate of grouper in the SP25-treated group was the highest, reaching 90%. Twelve hours after transport resumed, all grouper in the 20 and 30 mg / L seaweed polysaccharide-addition groups survived. These results indicate that the survival rate of the CK-treated group, without seaweed polysaccharide active coating, was significantly lower than that of the three coated groups, indicating that the addition of seaweed polysaccharide active coating can effectively reduce the mortality of live fish during waterless transport. Therefore, coating grouper with appropriate concentrations of seaweed polysaccharide active coating during waterless transport can help prolong survival and improve transport survival.

[0041] Example 2

[0042] Refer to Example 1. The pH of fresh fish muscle is weakly alkaline, which will cause the pH of fish meat to decrease during storage and transportation. Figure 2 As shown in the figure, during the 8 hours of transport, the pH of the fish meat in the SP25 treatment group did not change much from that at 0 hours of transport and was relatively stable. After 12 hours of transport, the pH value of the SP30 treatment group was consistently higher than that of the other treatment groups. At 15 hours of waterless transport, the pH of all treatment groups was significantly different from that at 0 hours of transport. The lower pH level is generally attributed to H2O related to lactate production and ATP decomposition. + The reason why the pH of the SP30 treatment group was higher at 15h of transportation was that the respiratory intensity of the fish was not high and a large amount of lactic acid was not accumulated.

[0043] After 15 hours of water-free transportation, the muscle water content of each treatment group was significantly reduced (P<0.05).

[0044] Results after 15 hours of transport. Muscle moisture content in all treatments treated with the seaweed polysaccharide active coating decreased with increasing transport time, but the rate of decline was significantly slower than that in the CK-treated group. Muscle moisture content in the SP25-treated group only decreased significantly after 15 hours of transport (P < 0.05). Compared with the CK-treated group, grouper treated with the seaweed polysaccharide active coating demonstrated greater tolerance to dry transport, mitigating the effects of dry transport on muscle quality.

[0045] Example 3

[0046] Refer to Example 1. During transportation, the cortisol (COR) concentration of each group showed an upward trend. However, the growth trend of the group treated with seaweed polysaccharide active coating was significantly slowed down, indicating that the protective liquid coating treatment alleviated the stress during water-free transportation. Figure 3 As shown, compared with the SP groups, blood glucose (GLU) levels in the CK-treated fish were significantly elevated 15 hours after waterless transport (P < 0.05). The COR and GLU levels in the SP25-treated group increased more slowly (P < 0.05), reaching their lowest levels approximately 15 hours after waterless transport. GLU is an essential energy source for various life activities in fish. Energy is stored as glycogen in muscle and liver, and as glucose in blood. Blood glucose levels can influence stress levels by regulating glycogen metabolism and gluconeogenesis, leading to the release of cortisol. Hyperglycemia in the CK-treated group 15 hours after transport indicates increased metabolic rate and energy expenditure in response to stress. Therefore, it can be concluded that blood glucose may be more sensitive to waterless transport. In contrast, blood glucose levels in the SP25-treated group were lowest after 15 hours, suggesting that SP25 treatment mitigates tissue damage under stress and maintains low levels of proinflammatory cytokine expression.

[0047] Example 4

[0048] See Example 1. Protein is synthesized by the liver and acts as a carrier in the blood. Figure 4 As shown, after transport, serum total protein (TP) and albumin (ALB) concentrations in grouper treated with CK were significantly lower than those in the protective fluid-treated group. This suggests that the addition of a seaweed polysaccharide active coating can improve liver function and reduce stress responses in grouper, with the most significant effect occurring at a seaweed polysaccharide concentration of 25 mg / L. Adding different concentrations of seaweed polysaccharide active coating reduced serum protein in grouper. Eight hours before transport, ALB and TP concentrations increased due to cold stress and decreased due to prolonged dehydration and hypoxia. Grouper treated with the protective fluid coating had a delayed stress response, with a smaller magnitude of change than in the CK-treated group. TP and ALB concentrations in the SP25 and SP30-treated groups showed little change.

[0049] Example 5

[0050] Blood creatinine (Cre) and uric acid (UA) levels are important indicators reflecting renal function metabolism. Figure 5As shown, Cre levels increased with increasing transport time during waterless transport. The Cre levels in the serum of the CK-treated group were significantly higher than those in the other groups during waterless transport (P < 0.05). Cre levels in groupers treated with the protective solution remained low throughout 15 hours of live transport, indicating that the protective solution coating effectively delayed the increase in blood creatinine levels and reduced intrarenal pressure. Variations in seaweed polysaccharide concentrations had no significant effect on grouper blood creatinine levels (P < 0.05). This indicates that throughout the entire waterless transport process, UA levels in the CK-treated group were significantly higher than those in the other treatment groups (P < 0.05), indicating that protective solution coating prior to waterless transport can slow the rise in UA in grouper. After 15 hours of waterless transport, the UA content in the SP25 and SP30 treatment groups was significantly lower than that in the other groups, and the level in the SP25 treatment group was the lowest. This indicates that the treatment with the protective liquid coating can reduce the damage to the kidney tissue during waterless transport of fish. However, with the increase of seaweed polysaccharide concentration, the protective effect on the kidney tissue was not significant (P<0.05).

[0051] Example 6

[0052] As the first barrier of immunity, fish skin contains a large number of natural immune factors, such as lysozyme (LZM) in skin mucus, which has a protective effect against infection by exogenous pathogens. Figure 6 As shown in the data, during the simulated waterless transport process, the LZM activity in the serum of all groupers first increased and then decreased. The LZM activity in the CK-treated group reached a peak at 4 hours, but was significantly lower than that in the other treatment groups (P<0.05). Subsequently, LZM activity decreased, falling below the initial level at 15 hours. LZM activity in the other groups showed an upward trend, reaching a peak of 8.56 μg / mL at 8 hours of transport. LZM activity did not show a significant decrease until 12 hours later and remained above the initial level, indicating that the polysaccharide active coating can effectively inhibit the decline in the immune function of grouper during waterless transport. Therefore, adding a seaweed polysaccharide active coating before waterless transport can effectively inhibit the decline in the immune ability of grouper, reduce the sensitivity of grouper to the external environment, and reduce stress damage.

[0053] Immunoglobulin (IgM) is the most important component of the humoral immune system of bony fish, secreted by B lymphocytes. It is one of the most important anti-pathogen antibodies in fish and the main immunoglobulin mediating the humoral adaptive immunity of fish. Therefore, the concentration of IgM is usually used as an important indicator to evaluate the immune response of fish. Figure 6As shown in (B), serum IgM levels first increased significantly and then decreased with increasing transport time. After the start of waterless transport, IgM levels in the CK-treated group were significantly lower than those in the other treatment groups, while those in the SP25-treated group were significantly higher than those in the other treatment groups. As transport time increased, IgM levels in the CK-treated group remained at the lowest level. These results indicate that adding a seaweed polysaccharide protective coating before waterless transport can stimulate immune factors and increase immune protein levels. The seaweed polysaccharide concentration in the SP25-treated group was the most effective in this study and is therefore suitable for use in waterless transport of grouper.

[0054] Example 7

[0055] During the entire waterless and live transport process, the activity levels of superoxide dismutase (SOD) in grouper in each treatment group showed a trend of first increasing and then decreasing. SOD activity can indirectly reflect the body's ability to scavenge oxygen free radicals. Figure 7 As shown in the results, after 4 and 8 hours of simulated waterless transport, the SOD activity in the CK treatment group showed a significant upward trend and was significantly higher than that in the other treatment groups. This phenomenon may be related to changes in matrix oxygen free radicals, or it may be that the grouper did not receive the seaweed polysaccharide protective solution coating before waterless transport, resulting in strong oxidative stress in the grouper. After 12 hours of waterless transport, the SOD activity in grouper showed a downward trend in all treatment groups, among which the grouper treated with the polysaccharide protective solution showed the most gradual change trend. There was no significant difference in SOD activity in grouper treated with different seaweed polysaccharide concentrations.

[0056] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A seaweed polysaccharide active coating, characterized in that: The preparation method is prepared by using seaweed polysaccharide as a matrix, compounding vitamin C, lysozyme and sorbitol, and the preparation method specifically comprises the following steps: Step (1), different concentrations of seaweed polysaccharides and 0.09% lysozyme are stirred at 40° C. until completely dissolved to obtain a film-forming solution; Step (2), 30 mg / L VC was added to the membrane solution formed by S1; Step (3), 0.3% sorbitol is added to the membrane solution formed by S2; Step (4) is to use an ultrasonic assisted homogenizer to perform ultrasonic treatment at 20 kHz and 700 W power for 10 minutes to obtain a uniform solution and degas under vacuum to form a membrane solution.

2. The seaweed polysaccharide active coating according to claim 1, characterized in that: The average body length of grouper is 27.15cm and the average weight is 500±50g.

3. The seaweed polysaccharide active coating according to claim 1, characterized in that: Before the experiment, the grouper was fasted for 36 h.

4. The seaweed polysaccharide active coating according to claim 1, characterized in that: The density during the temporary period is 20kg / m 3 The seawater salinity is 20‰, the temperature is 23-25℃, the dissolved oxygen is 7-8 mg / L, and the pH is 7.5-8.

5.

5. The seaweed polysaccharide active coating according to claim 1, characterized in that: During the temporary holding period, the seawater is processed through a circulating filter and 50% of the seawater in the temporary holding tank is replaced every 12 hours.

6. Use of the seaweed polysaccharide active coating as claimed in claim 1 to form a seaweed polysaccharide active film on the body surface of grouper.

7. A method for transporting grouper without water and keeping it alive based on seaweed polysaccharide active film, characterized in that: The specific steps include: Step S1, the experiment was divided into four groups, namely, 20 mg / L seaweed polysaccharide, 25 mg / L seaweed polysaccharide, 30 mg / L seaweed polysaccharide mixed with 0.09% lysozyme and 0.3% sorbitol, and a control group without seaweed polysaccharide active coating treatment; Step S2: For each group of 20 samples, after the temporary incubation transition, the seawater was cooled from 23-25°C to 15°C at a rate of 3°C / h. Other parameters were the same as those for the temporary incubation water. Groupers with weaker touch stress responses in the temporary incubator were removed and packaged without water. A control group was placed in a dormant state without the protective liquid coating. Step S3: After being treated with the active coating of seaweed polysaccharides at different contents, the grouper fish were wrapped with a wet sponge and placed in a special oxygenated bag. Four fish were placed in a transport box for transportation. The simulated transportation process was as follows: 1 hour on a B-class road → 3 hours on an A-class road → 1 hour on a flat road on a B-class road, repeated 3 times. Step S4: After 15 hours of transportation, three groupers were randomly selected and temporarily restored in normal temperature water for 12 hours.

8. The method for transporting grouper without water and keeping it alive based on seaweed polysaccharide active film according to claim 7, characterized in that: In step S3, the grouper is wrapped with a wet sponge, and the wet sponge is placed in water at 15° C., a salinity of 20‰, and a pH of 7.0-7.5 in advance.

Images