A method for collecting blood and preparing serum of macrobrachium rosenbergii

Abstract

A method for collecting blood and preparing serum from giant freshwater prawns includes: feeding the prawns a nutrient-rich diet under low-temperature conditions, collecting blood from their second walking legs, and obtaining purified serum through blood coagulation, serum precipitation, and further processing; the nutrient diet includes at least a protocatechuic acid-chitosan oligosaccharide derivative, which is obtained by grafting protocatechuic acid onto chitosan oligosaccharide. This invention preserves the life of individual giant freshwater prawns during blood collection and increases the amount of blood and serum collected.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for collecting blood and preparing serum from giant freshwater prawns. Background Technology

[0002] The giant freshwater prawn (Macrobrachium rosenbergii), commonly known as the Malaysian prawn or giant long-armed prawn, belongs to the phylum Arthropoda, class Crustacea, order Decapoda, family Palaemonidae, and genus Macrobrachium. Due to its delicious flavor and high nutritional value, the giant freshwater prawn has become one of the main freshwater aquaculture species in China and other Southeast Asian countries. The giant freshwater prawn aquaculture industry is experiencing explosive growth. However, with the expansion of aquaculture scale, the giant freshwater prawn faces increasingly serious disease threats, mainly from viruses, bacteria, fungi, and parasites. Therefore, research on the immune system, hematopoietic tissue, and hemolymphocyte regulation mechanisms of the giant freshwater prawn has become a hot topic.

[0003] The blood of giant freshwater prawns (Macrobrachium rosenbergii) can be used to analyze biomarkers, study disease mechanisms, and assess drug metabolism and toxicity, while serum is commonly used in cell culture, antibody detection, enzyme activity analysis, and other serological studies, helping to understand immune responses, disease states, and drug efficacy. Currently, the conventional method for collecting blood from giant freshwater prawns is by drawing blood from the heart or from the midpoint between the cephalothorax and abdomen using a syringe. This method not only requires highly skilled operators but also contradicts animal welfare principles. Therefore, there is an urgent need for a new method for collecting and preparing serum from giant freshwater prawns that is simple to operate and allows for large-volume blood collection. Summary of the Invention

[0004] The purpose of this invention is to provide a method for collecting blood and preparing serum from giant freshwater prawns, in order to solve the problem of low blood and serum yields in the prior art, thereby achieving the effect of both preserving the life of individual giant freshwater prawns and increasing the amount of blood and serum collected.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0006] A method for collecting blood and preparing serum from giant freshwater prawns includes: feeding the prawns a nutrient-rich diet under low-temperature conditions, collecting blood from their second walking legs, and obtaining purified serum through blood coagulation, serum precipitation, and further processing; the nutrient diet includes at least a protocatechuic acid-chitosan oligosaccharide derivative, which is obtained by grafting protocatechuic acid onto chitosan oligosaccharide. The protocatechuic acid-chitosan derivative contains a large number of active groups that can exchange energy within the animal's body, producing bioactive substances. These substances can not only regulate the animal's intestinal microecology, improve nutrient digestibility, enhance growth performance, and increase survival rate; but also increase the expression level of oxyhemocyanin, enhance the animal's antioxidant immunity, and effectively reduce the negative effects of animal stress.

[0007] The preparation method of this invention involves feeding the giant freshwater prawn with a nutritious diet and then collecting blood from its second walking legs. This not only yields a larger volume of blood but also effectively reduces the mortality rate of the giant freshwater prawn, thus helping to preserve the lives of individual prawns. Then, by cutting the coagulated blood gel, serum will be extracted from the cut, which helps to obtain more serum.

[0008] Preferably, the water temperature in the low-temperature environment is 10-16℃.

[0009] Preferably, in the preparation of protocatechuic acid-chitosan derivatives, chitosan and protocatechuic acid are used as substrates, and the reaction is mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to obtain protocatechuic acid-chitosan derivatives.

[0010] More preferably, the weight ratio of protocatechuic acid to chitosan is 1:2-5.

[0011] More preferably, the preparation of the protocatechuic acid-chitosan derivative specifically involves,

[0012] Protocatechuic acid was dissolved in anhydrous ethanol, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 2-morpholinoethanesulfonic acid buffer were added. The mixture was stirred continuously until completely dissolved, and chitosan was added. The mixture was stirred at room temperature for 8-24 hours, dialyzed with distilled water for 70-86 hours, centrifuged, and the supernatant was freeze-dried to obtain the protocatechuic acid-chitosan derivative.

[0013] More preferably, the liquid-to-solid ratio of protocatechuic acid and anhydrous ethanol is 1:2-10.

[0014] More preferably, the weight ratio of protocatechuic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.0-2.0.

[0015] More preferably, the weight ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1.5-2.5:1.

[0016] More preferably, the liquid-to-solid ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2-morpholine ethanesulfonic acid buffer is 1:10-20.

[0017] More preferably, the centrifugation speed is 8000-11000 rpm, the centrifugation temperature is 2-6℃, and the centrifugation time is 20-40 min.

[0018] Preferably, the nutritional feed comprises the following components in parts by weight: 13-25 parts fish meal, 11-20 parts wheat flour, 8-15 parts soybean protein concentrate, 5-13 parts soybean meal, 4-10 parts shrimp meal, 4-10 parts chicken meal, 2-7 parts shrimp paste, 2-7 parts squid paste, 2-5 parts soybean lecithin, 1-5 parts fish oil, 1-5 parts soybean oil, 1-3 parts protocatechuic acid-chitosan derivative, 1-3 parts schisandrin A, 0.5-2.5 parts sodium chloride, 0.2-1 part calcium carbonate, 0.2-1 part calcium dihydrogen phosphate, and 0.2-1 part choline chloride. Schisandrin A mainly participates in oxygen transport and storage, energy metabolism, and enzyme synthesis during animal growth, possessing dual nutritional functions. The schisandrin A in this nutritional feed can further enhance nutrient absorption to promote animal growth and improve survival rates. Meanwhile, schisandrin A has high bioavailability and can promote the expression of oxyhemocyanin, which helps to improve oxygen transport and utilization.

[0019] More preferably, the preparation of the nutritional feed specifically includes,

[0020] Weigh the raw materials according to the following proportions: 13-25 parts fish meal, 11-20 parts wheat flour, 8-15 parts soy protein concentrate, 5-13 parts soybean meal, 4-10 parts shrimp meal, 4-10 parts chicken meal, 2-7 parts shrimp paste, 2-7 parts squid paste, 2-5 parts soy lecithin, 1-5 parts fish oil, 1-5 parts soybean oil, 1-3 parts protocatechuic acid-chitosan derivative, 1-3 parts schisandrin A, 0.5-2.5 parts sodium chloride, 0.2-1 part calcium carbonate, 0.2-1 part calcium dihydrogen phosphate, and 0.2-1 part choline chloride. Mix the raw materials evenly, add 30-60% by volume of water and continue to mix thoroughly. Place the mixed feed into an automatic pellet mill to make feed pellets, and air dry naturally to obtain a nutritious feed.

[0021] Preferably, during blood collection, the second walking leg of the giant freshwater prawn is cut off to create an oblique cut, the prawn body is raised and the cut is placed at a low position, allowing the blood to flow out and drip into a culture dish to obtain fresh blood.

[0022] More preferably, the petri dish is placed on ice at an angle of 5°-10° to the horizontal plane.

[0023] Preferably, the ratio of the body weight of the giant freshwater prawn to the amount of blood collected is 60-100g: 3-5mL.

[0024] Preferably, during blood coagulation, the temperature conditions for blood coagulation are 10-16℃, the coagulation time is 3-10 min, and after blood coagulation, it is transferred to 2-6℃ and left to stand for 2-4 h to obtain a blood clot.

[0025] Preferably, in the serum precipitation process, the blood clot is placed at 2-6°C and at an angle of 5°-10° to the horizontal plane for 12-24 hours to obtain the precipitated serum.

[0026] Preferably, in the reprocessing of serum, the serum is centrifuged at a speed of 800-1000 rpm, a temperature of 10-16℃, and a time of 3-5 min, and 2 / 3 of the supernatant is taken as pure serum.

[0027] More preferably, a method for collecting blood and preparing serum from giant freshwater prawns specifically includes:

[0028] Transfer 60-100g of giant freshwater prawns from the rearing pond to clean water and let them rest for 1-3 days. Add a small amount of ice to the water to slowly lower the water temperature to 10-16℃. Feed them nutritious feed. Place the giant freshwater prawns on a clean towel to absorb excess water from their bodies. Hold one pair of second walking legs of the giant freshwater prawn from the dorsal side of the cephalothorax with your left hand. Disinfect the first segment of one of the walking legs with 75% alcohol. Then, use sterile scissors to cut a slanted cut in the middle of the first segment. With your right hand, hold the giant freshwater prawn and raise its tail, placing the slanted cut at the lower end of the wound so that the blood drips naturally into a petri dish at a 5-10° angle to the water surface. Collect 3-5mL of fresh blood. After blood collection, cut the first segment of the walking leg at the junction of the first and second segments. Raise the leg and use absorbent cotton to stop the bleeding for about 30 seconds. Return it to clean water and let it rest for 3 days. Transfer fresh blood to 10-16℃ for 3-10 minutes until it coagulates completely. Then place it at 2-6℃ for 1-5 hours. Scratch the blood with a pipette tip and place it at 2-6℃ with a 5-10° angle for 12-24 hours. Collect the serum and centrifuge it at 800-1000 rpm for 3-5 minutes at 10-16℃. Take the upper 2 / 3 of the supernatant and filter it through a 0.22µm filter to obtain pure serum.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] This invention yields a protocatechuic acid-chitosan derivative containing a large number of active amino and hydroxyl groups, which can exchange energy within the animal body, producing bioactive substances that improve animal growth performance and regulate homeostasis. Adding this protocatechuic acid-chitosan derivative to feed not only regulates the animal's intestinal microecology, improves nutrient digestibility, enhances growth performance, and increases survival rate, but also increases the expression level of oxyhemocyanin, strengthens antioxidant immunity, and effectively reduces the negative effects of animal stress. Schisandrin A mainly participates in oxygen transport and storage, energy metabolism, and enzyme synthesis during animal growth, possessing dual nutritional functions. Adding schisandrin A to feed further assists the protocatechuic acid-chitosan derivative in improving the digestion and absorption of nutrients, enhancing growth performance, and increasing the expression level of oxyhemocyanin. Therefore, this invention provides a method for blood collection and serum preparation that can both preserve the life of individual giant freshwater prawns and increase the amount of blood and serum collected. Attached Figure Description

[0031] Figure 1 The infrared spectrum of the protocatechuic acid-chitosan derivative obtained in Example 1 is shown. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] Example 1:

[0035] S1. Preparation of protocatechuic acid-chitosan derivatives, including,

[0036] One part by mass of protocatechuic acid was dissolved in five parts of anhydrous ethanol, and 1.2 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.7 parts of N-hydroxysuccinimide, and 20 parts of 2-morpholine ethanesulfonic acid buffer were added. The mixture was stirred continuously until completely dissolved, and three parts of chitosan were added. The mixture was stirred at room temperature for 12 hours, dialyzed with distilled water for 72 hours, and centrifuged at 10,000 rpm for 30 minutes at 4°C. The supernatant was then freeze-dried to obtain the protocatechuic acid-chitosan derivative.

[0037] S2. Preparation of nutritional feed, including,

[0038] Weigh the raw materials according to the following proportions: 18 parts fish meal, 16 parts wheat flour, 12 parts soy protein concentrate, 10 parts soybean meal, 8 parts shrimp meal, 8 parts chicken meal, 5 parts shrimp paste, 5 parts squid paste, 4 parts soy lecithin, 3 parts fish oil, 3 parts soybean oil, 2 parts protocatechuic acid-chitosan derivative, 1.5 parts sodium chloride, 1 part calcium carbonate, 1 part calcium dihydrogen phosphate, and 0.5 parts choline chloride. Mix the raw materials evenly, add 40% water and continue to mix thoroughly. Place the mixed feed into an automatic pellet mill to make feed pellets, and air dry naturally to obtain a nutritious feed.

[0039] S3. Blood collection, including,

[0040] 60g of giant freshwater prawns were transferred from the rearing pond to clean water and kept in a quiescent state for 3 days, fed with nutritious feed daily. A small amount of ice was added to the water to slowly lower the temperature to 10℃. The prawns were then placed on a clean towel to absorb excess water. Using the left hand, the prawn's second walking legs were grasped from the dorsal side of the cephalothorax. The first segment of one of the walking legs was disinfected with 75% alcohol. A slanted cut was made in the middle of the first segment using sterile scissors. The prawn was held with the right hand, its tail raised, and the slanted cut placed at the lower end, allowing the blood to drip naturally into a petri dish at a 5-10° angle to the water surface. 3mL of fresh blood was collected. After blood collection, the first segment of the walking leg was cut off at the junction of the first and second segments. The leg was then raised and the bleeding was stopped with absorbent cotton for about 30 seconds. The prawn was then returned to clean water and kept in a quiescent state for 3 days.

[0041] S4. Serum precipitation and reprocessing, including,

[0042] Fresh blood was transferred to 10°C for 10 minutes. After the blood had completely coagulated, it was placed at 4°C for 2 hours. The blood was then cut with a pipette tip and placed at 4°C for 12 hours. The serum was collected and centrifuged at 1000 rpm for 5 minutes at 10°C. The upper 2 / 3 of the supernatant was filtered through a 0.22 μm filter to obtain pure serum.

[0043] Example 2:

[0044] Except for changing the mass fraction of protocatechuic acid-chitosan derivative to 1 part in the formulation of the nutritional feed, the other conditions are the same as in Example 1.

[0045] Example 3:

[0046] S1. Preparation of protocatechuic acid-chitosan derivatives is the same as in Example 1.

[0047] S2. Preparation of nutritional feed, including,

[0048] Weigh the raw materials according to the following proportions: 18 parts fish meal, 16 parts wheat flour, 12 parts soy protein concentrate, 10 parts soybean meal, 8 parts shrimp meal, 8 parts chicken meal, 5 parts shrimp paste, 5 parts squid paste, 4 parts soy lecithin, 3 parts fish oil, 3 parts soybean oil, 2 parts protocatechuic acid-chitosan derivative, 2 parts schisandrin A, 1.5 parts sodium chloride, 1 part calcium carbonate, 1 part calcium dihydrogen phosphate, and 0.5 parts choline chloride. Mix the raw materials evenly, add 40% water and continue to mix thoroughly. Put the mixed feed into an automatic pellet mill to make feed pellets, and air dry naturally to obtain nutritious feed.

[0049] S3. Blood collection, same as in Example 1.

[0050] S4. The precipitation and reprocessing of serum are the same as in Example 1.

[0051] Example 4:

[0052] Except for changing the mass fraction of protocatechuic acid-chitosan derivative to 1 part in the formulation of the nutritional feed, the other conditions are the same as in Example 3.

[0053] Example 5:

[0054] Except for changing the mass fraction of schisandrin A to 1 part in the formulation of the nutritional feed, the other conditions are the same as in Example 3.

[0055] Comparative Example 1:

[0056] S1. Preparation of nutritional feed, including,

[0057] Weigh the raw materials according to the following proportions: 18 parts fish meal, 16 parts wheat flour, 12 parts soy protein concentrate, 10 parts soybean meal, 8 parts shrimp meal, 8 parts chicken meal, 5 parts shrimp paste, 5 parts squid paste, 4 parts soy lecithin, 3 parts fish oil, 3 parts soybean oil, 2 parts chitosan, 1.5 parts sodium chloride, 1 part calcium carbonate, 1 part calcium dihydrogen phosphate, and 0.5 parts choline chloride. Mix the raw materials evenly, add 40% water and continue to mix thoroughly. Put the mixed feed into an automatic pellet mill to make feed pellets, and air dry naturally to obtain nutritious feed.

[0058] S2. Blood collection, same as in Example 1.

[0059] S3. The precipitation and reprocessing of serum are the same as in Example 1.

[0060] Comparative Example 2:

[0061] S1. Preparation of nutritional feed, including,

[0062] Weigh the raw materials according to the following proportions: 18 parts fish meal, 16 parts wheat flour, 12 parts soy protein concentrate, 10 parts soybean meal, 8 parts shrimp meal, 8 parts chicken meal, 5 parts shrimp paste, 5 parts squid paste, 4 parts soy lecithin, 3 parts fish oil, 3 parts soybean oil, 2 parts chitosan, 2 parts schisandrin A, 1.5 parts sodium chloride, 1 part calcium carbonate, 1 part calcium dihydrogen phosphate, and 0.5 parts choline chloride. Mix the raw materials evenly, add 40% water and continue to mix thoroughly. Put the mixed feed into an automatic pellet mill to make feed pellets, and air dry naturally to obtain nutritious feed.

[0063] S2. Blood collection, same as in Example 1.

[0064] S3. The precipitation and reprocessing of serum are the same as in Example 1.

[0065] Experimental example:

[0066] 1. Material Characterization

[0067] The infrared spectra of the samples were determined using a Fourier transform infrared spectrometer, employing the KBr pellet method, within a wavenumber range of 400-4000 cm⁻¹. -1 Measurements were taken within the specified range, with a resolution of 0.06 cm. -1 , scanned 32 times.

[0068] Figure 1 The infrared spectrum of the protocatechuic acid-chitosan derivative obtained in Example 1 is shown. (3441 cm⁻¹) -1 Absorption peaks for OH and NH appear nearby, at 2923 cm⁻¹. -1 and 1428cm -1 Absorption peaks of CH appeared nearby, at 1654, 1550, and 1324 cm⁻¹. -1 Nearby, absorption peaks corresponding to C=O, NH, and CN in N-acetylglucosamine residues appeared, at 1672 cm⁻¹. -1 and 1531cm -1 Nearby, absorption peaks appeared at 1716 cm⁻¹ for the carbonyl group (C=O) and the aromatic ring (C=C) of the protocatechuic acid molecule. -1 The presence of an absorption peak at C=O nearby indicates that an ester bond has been formed between the hydroxyl groups of chitosan and the carboxyl groups of protocatechuic acid, demonstrating that protocatechuic acid has been successfully grafted onto chitosan.

[0069] 2. Survival rate

[0070] Blood was collected from giant freshwater prawns according to the methods in Examples 1-5 and Comparative Examples 1-2. After blood collection, the first segment of the walking leg was cut off at the junction of the first and second segments, the leg was raised, and hemostasis was achieved with absorbent cotton for about 30 seconds. The prawns were then placed back into clean water and kept incubated for 3 days. The survival rate of giant freshwater prawns after blood collection in Examples 1-5 and Comparative Examples 1-2 was calculated, with 10 biological replicates for each treatment group.

[0071] Table 1. Survival rate of giant freshwater prawns after blood collection

[0072]

[0073] As shown in Table 1, the survival rate of Examples 1-2 was higher than that of Comparative Example 1. The difference lay in the composition of the feed for the giant freshwater prawns. The feed for Example 1 used protocatechuic acid-chitosan derivatives, while Comparative Example 1 used chitosan. The survival rate of Example 1 was higher than that of Example 2. The difference lay in the amount of protocatechuic acid-chitosan derivatives used in the feed. This indicates that adding an appropriate amount of protocatechuic acid-chitosan derivatives to the feed to replace chitosan can effectively improve the survival rate of giant freshwater prawns after blood collection.

[0074] The survival rates of Examples 3-5 were higher than those of Examples 1-2 and Comparative Example 2. The difference was that the nutritional diets of Examples 3-5 used protocatechuic acid-chitosan derivatives and schisandrin A in combination, while the nutritional diets of Examples 1-2 only added protocatechuic acid-chitosan derivatives, and the nutritional diets of Comparative Example 2 only added schisandrin A without using protocatechuic acid-chitosan derivatives. This indicates that compared to the use of protocatechuic acid-chitosan derivatives and schisandrin A alone, the addition of schisandrin A can help protocatechuic acid-chitosan derivatives further improve the survival rate of giant freshwater prawns after blood collection.

[0075] The survival rate of Example 3 was higher than that of Examples 4-5. The difference was that the ratio of protocatechuic acid-chitosan derivative and schisandrin A in the nutrient feed was different. This shows that adding an appropriate proportion of protocatechuic acid-chitosan derivative and schisandrin A to the feed can effectively improve the survival rate of giant freshwater prawns after blood collection.

[0076] 3. Determination of oxyhemocyanin content

[0077] Blood was collected from giant freshwater prawns according to the methods in Examples 1-5 and Comparative Examples 1-2. 10 μL of blood was taken and diluted with 990 μL of deionized water, and the absorbance value (A335) at 335 nm was read. Hemocyanin content (mmol / L) = (A335 × R) / (ε × S × P). Where R is the total volume of the reaction solution (μL), and ε is the molar extinction coefficient 17.26 mmol / L. -1 ·cm -1 S is the volume of the sample to be tested (μL), and P is the light transmission length (cm).

[0078] Table 2. Oxyhemocyanin content

[0079]

[0080] As shown in Table 2, the oxyhemocyanin content of Examples 1-2 is higher than that of Comparative Example 1. This is because in the nutrient feed, Example 1 uses protocatechuic acid-chitosan derivative, while Comparative Example 1 uses chitosan. The oxyhemocyanin content of Example 1 is higher than that of Example 2 because the amount of protocatechuic acid-chitosan derivative used in the nutrient feed is different. This indicates that adding an appropriate amount of protocatechuic acid-chitosan derivative to the feed to replace chitosan can effectively improve the expression of oxyhemocyanin in Macrobrachium rosenbergii.

[0081] The hemocyanin content in Examples 3-5 was higher than that in Examples 1-2 and Comparative Example 2. This is because in the nutritional feed, Examples 3-5 used protocatechuic acid-chitosan derivative and schisandrin in synergy, while Examples 1-2 used only protocatechuic acid-chitosan derivative, and Comparative Example 2 used only schisandrin without protocatechuic acid-chitosan derivative. This indicates that compared to the use of protocatechuic acid-chitosan derivative and schisandrin alone, the addition of schisandrin can assist protocatechuic acid-chitosan derivative in further improving the expression of oxyhemocyanin in Macrobrachium rosenbergii.

[0082] The hemocyanin content in Example 3 was higher than that in Examples 4-5 because the ratio of protocatechuic acid-chitosan derivative and schisandrin A in the nutrient feed was different. This indicates that adding appropriate amounts of protocatechuic acid-chitosan derivative and schisandrin A to the feed can effectively improve the expression of oxyhemocyanin in giant freshwater prawns.

[0083] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0084] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for collecting blood and preparing serum from giant freshwater prawns, characterized in that: After feeding the giant freshwater prawns with a nutrient-rich diet in a low-temperature environment, blood was collected from their second walking legs. The blood was then coagulated, the serum was extracted and further processed to obtain pure serum. The nutritional feed comprises the following components in parts by weight: 13-25 parts fish meal, 11-20 parts wheat flour, 8-15 parts soybean protein concentrate, 5-13 parts soybean meal, 4-10 parts shrimp meal, 4-10 parts chicken meal, 2-7 parts shrimp paste, 2-7 parts squid paste, 2-5 parts soybean lecithin, 1-5 parts fish oil, 1-5 parts soybean oil, 1-3 parts protocatechuic acid-chitosan derivative, 1-3 parts schisandrin A, 0.5-2.5 parts sodium chloride, 0.2-1 part calcium carbonate, 0.2-1 part calcium dihydrogen phosphate, and 0.2-1 part choline chloride, wherein the protocatechuic acid-chitosan derivative is obtained by grafting protocatechuic acid onto chitosan.

2. The method for collecting blood and preparing serum from giant freshwater prawns according to claim 1, characterized in that, The water temperature in the low-temperature environment is 10-16℃.

3. The method for collecting blood and preparing serum from giant freshwater prawns according to claim 1, characterized in that, In the preparation of the protocatechuic acid-chitosan derivative, chitosan and protocatechuic acid are used as substrates, and the reaction is mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to obtain the protocatechuic acid-chitosan derivative.

4. The method for collecting blood and preparing serum from giant freshwater prawns according to claim 3, characterized in that, The weight ratio of protocatechuic acid to chitosan is 1:2-5.

5. The method for collecting blood and preparing serum from giant freshwater prawns according to claim 1, characterized in that, In the blood collection process, the second walking leg of the giant freshwater prawn is cut off to create an oblique cut. The prawn is then raised and the cut is placed at a low position, allowing the blood to flow out and drip into a culture dish, thus obtaining fresh blood.

6. The method for collecting blood and preparing serum from giant freshwater prawns according to claim 5, characterized in that, The culture dish was placed on ice at an angle of 5°-10° to the horizontal plane.

7. The method for collecting blood and preparing serum from giant freshwater prawns according to claim 1, characterized in that, The ratio of the weight of the giant freshwater prawn to the amount of blood collected is 60-100g: 3-5mL.

8. The method for collecting blood and preparing serum from giant freshwater prawns according to claim 1, characterized in that, In the blood coagulation process, the temperature conditions for blood coagulation are 10-16℃, the coagulation time is 3-10 min, and after blood coagulation, it is transferred to 2-6℃ and left to stand for 2-4 h to obtain a blood clot.

9. A method for collecting blood and preparing serum from giant freshwater prawns according to claim 1, characterized in that, In the process of serum precipitation, the blood clot is placed at 2-6°C and at an angle of 5°-10° to the horizontal plane for 12-24 hours to obtain the precipitated serum.

10. A method for collecting blood and preparing serum from giant freshwater prawns according to claim 1, characterized in that, In the reprocessing of the serum, the serum is centrifuged at a speed of 800-1000 rpm, a temperature of 10-16℃, and a time of 3-5 min. Two-thirds of the supernatant is taken as pure serum.

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