Chinese gall and sappan wood compound antibacterial aquatic feed additive and extraction process

Abstract

The invention relates to the technical field of weever culture, and discloses a gallnut and sappan wood compound antibacterial aquatic feed additive and an extraction process, and the gallnut and sappan wood compound antibacterial aquatic feed additive comprises the following raw materials in parts by weight: 20-40 parts of gallnut, 20-40 parts of sappan wood, 10-30 parts of semen plantaginis, 5-15 parts of radix paeoniae alba and 5-15 parts of liquorice. The Chinese gall, the sappan wood, the semen plantaginis, the radix paeoniae alba and the liquorice are respectively cleaned, dried and crushed into fine powder, and the fine powder is sieved by a 100-mesh sieve; uniformly mixing the traditional Chinese medicine powder in parts by weight: 30 parts of gallnut, 30 parts of sappan wood, 20 parts of semen plantaginis, 10 parts of radix paeoniae alba and 10 parts of liquorice. Through cooperation of gallnut, sappan wood, semen plantaginis, radix paeoniae alba and liquorice, the morbidity and mortality of septicemia caused by Edwardsiella tarda infection of the weever can be reduced, the immunity and intestinal barrier function of the weever can be enhanced, and all the components are from natural plants and meet the requirements of green aquaculture.

Description

Technical Field

[0001] The invention relates to the technical field of seabass breeding, in particular to a gallnut sappan wood compound antibacterial aquatic feed additive and an extraction process. Background Art

[0002] In the field of aquaculture, especially in seabass farming, seabass sepsis caused by Edwardsiella tarda has become a key issue restricting the development of the industry. With the continuous expansion of farming scale and the improvement of intensiveness, the seabass farming environment has become increasingly complex, creating favorable conditions for the breeding and spread of Edwardsiella tarda.

[0003] Traditional prevention and control methods mainly rely on antibiotics, but the long-term and large-scale use of antibiotics has brought many serious problems. On the one hand, the abuse of antibiotics has caused pathogens such as Edwardsiella tarda to develop drug resistance, resulting in a gradual decrease in the therapeutic effect of antibiotics. The dosage of drugs has to be continuously increased, forming a vicious circle. For example, the resistance rate of Edwardsiella tarda to commonly used antibiotics in some areas has reached more than 70%. On the other hand, the residues of antibiotics in fish and aquaculture water not only threaten the safety of aquatic food and harm the health of consumers, but also destroy the ecological balance of water bodies and affect the survival and reproduction of other aquatic organisms. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a compound antibacterial aquatic feed additive of Galla chinensis and an extraction process, which solves the problem of seabass sepsis caused by Edwardsiella tarda leading to seabass death.

[0005] To achieve the above purpose, the present invention is implemented by the following technical scheme: The gallnut sappan wood compound antibacterial aquatic feed additive comprises the following raw materials in parts by weight:

[0006] The content of each component is divided by mass: 20-40 parts of gallnut, 20-40 parts of sappan wood, 10-30 parts of plantain seeds, 5-15 parts of white peony root, and 5-15 parts of liquorice.

[0007] By adopting the above technical scheme: through the synergistic effect of the compound Chinese medicine ingredients, the effect of significantly inhibiting the growth of Edwardsiella tarda and reducing the incidence of sepsis in seabass is achieved, while the immune function and anti-stress ability of seabass are improved, and its healthy growth is promoted.

[0008] Preferably, the extraction process of the gallnut and sappan wood compound antibacterial aquatic feed additive comprises washing and drying the gallnut, sappan wood, plantain, white peony root and licorice respectively, crushing them into fine powder, and passing through a 100-mesh sieve;

[0009] Mix the above Chinese medicine powders by weight: 30 parts of Galla chinensis, 30 parts of Sappan wood, 20 parts of Plantago, 10 parts of White Peony Root, and 10 parts of Licorice;

[0010] Add deionized water, heat, extract by reflux, filter, and collect the filtrate;

[0011] Concentrate the filtrate to 1 / 5 of its original volume to obtain a concentrated solution;

[0012] Lyophilize the concentrated solution to obtain a dry powder;

[0013] Mix the dry powder with a feed carrier to prepare a feed additive at 20 g / kg.

[0014] Preferably, before the reflux extraction, perform ultrasonic pretreatment. Add the mixed traditional Chinese medicine powder to a deionized water solution containing 0.2 - 0.4% ethanol at a solid-liquid ratio of 1:10 (g / mL), and pretreat it for 30 minutes under ultrasonic conditions of 20 - 40 kHz.

[0015] Preferably, then raise the temperature to 80°C and reflux extract for 1.5 hours.

[0016] Preferably, before the ultrasonic pretreatment, add 0.1 - 0.3% cellulase / pectinase. The dosage of cellulase / pectinase is 0.5% - 1% of the weight of the medicinal materials, adjust the pH to 4.5 - 5.5, and use enzymatic hydrolysis to break the plant cell wall.

[0017] Preferably, the concentration method is vacuum concentration. Adjust the vacuum degree to -0.09 MPa and lower the concentration temperature to 55°C.

[0018] Preferably, before the vacuum concentration, perform ultrafiltration membrane pretreatment to remove macromolecular impurities and reduce the subsequent concentration load.

[0019] The drying method is lyophilization. The pre-freezing temperature is -40°C, the vacuum degree is 10 - 20 Pa, and the drying time is 12 - 24 hours.

[0020] Preferably, the feed carrier is corn flour or wheat bran, and the particle size of the carrier is 60 - 100 mesh.

[0021] Preferably, 1% chitosan and 0.8% rosemary extract can also be added to the feed carrier as antioxidants.

[0022] The present invention provides a gallnut and sappanwood compound antibacterial aquatic feed additive and an extraction process. It has the following beneficial effects:

[0023] 1. Through the combination of gallnut, sappanwood, plantain seed, white peony root, and licorice root, the present invention can reduce the incidence and mortality of septicemia caused by Edwardsiella tarda infection in perch, enhance the immune and intestinal barrier functions of perch, and all components are derived from natural plants, meeting the requirements of green aquaculture.

[0024] 2. The process of the present invention for vacuum concentration and ultrafiltration membrane pretreatment: The vacuum concentration method is adopted, which can concentrate at a relatively low temperature, avoiding the destruction of active ingredients by high temperature. At the same time, combined with ultrafiltration membrane pretreatment to remove macromolecular impurities, it not only improves the concentration efficiency, but also reduces the energy consumption during the concentration process, and can effectively remove impurities, improve the purity and stability of the product, making the quality of the feed additive more reliable.

[0025] 3. The present invention dries the concentrated solution into powder by freeze-drying, which can maximize the retention of the biological activity and stability of the active ingredients, extend the shelf life of the product, and at the same time make the product have good solubility and fluidity, facilitating mixing with the feed carrier, and improving the uniformity and stability of the additive in the feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the process flow chart of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0028] Please refer to the attached Figure 1 , the present invention provides a gallnut and sappanwood compound antibacterial aquatic feed additive, which comprises the following raw materials in parts by weight:

[0029] The content of each component is in parts by mass: 20 - 40 parts of gallnut, 20 - 40 parts of sappanwood, 10 - 30 parts of plantain seed, 5 - 15 parts of white peony root, and 5 - 15 parts of licorice root.

[0030] Specifically, gallnut is rich in tannins and gallic acid, and has significant antibacterial, antioxidant and anti-inflammatory effects, and can effectively inhibit the growth of pathogenic bacteria such as Edwardsiella tarda. Sappanwood contains components such as brazilin and has antibacterial and anti-inflammatory effects, which can enhance the antibacterial effect of gallnut. Plantain seed has the effects of clearing heat and detoxifying, and diuretic and detumescence, and helps to relieve the inflammation caused by Edwardsiella tarda infection. White peony root has anti-inflammatory and immune regulatory functions, and can enhance the immunity of aquatic animals. Licorice root has anti-inflammatory, antibacterial and immune regulatory effects, and can synergistically enhance the antibacterial effect with other traditional Chinese medicine components. Thus, it can reduce the septicemia of perch caused by Edwardsiella tarda.

[0031] Please refer to the attached Figure 1 , for the extraction process of the gallnut and sappanwood compound antibacterial aquatic feed additive, wash, dry, crush into fine powder, and pass through a 100-mesh sieve for gallnut, sappanwood, plantain seed, white peony root and licorice root respectively;

[0032] Mix the above traditional Chinese medicine powders evenly according to the weight portions: 30 portions of Chinese gall, 30 portions of sappanwood, 20 portions of plantain seeds, 10 portions of white peony root, and 10 portions of liquorice root.

[0033] Add deionized water, heat, reflux extract, filter, and collect the filtrate.

[0034] Concentrate the filtrate to 1 / 5 of the original volume to obtain a concentrated solution.

[0035] Lyophilize the concentrated solution to obtain a dry powder.

[0036] Mix the dry powder with a feed carrier to prepare a feed additive at 20 g / kg.

[0037] Specifically, for raw material selection: Select high-quality Chinese gall, sappanwood, plantain seeds, white peony root, and liquorice root as the main raw materials. These traditional Chinese medicines need to be strictly screened to ensure no impurities, no mildew, and reliable sources. Wash the Chinese gall, sappanwood, plantain seeds, white peony root, and liquorice root separately with clean water to remove the sediment and impurities on the surface. Place the washed traditional Chinese medicines in a well-ventilated environment to dry naturally, or use a low-temperature drying equipment to dry them to a water content of less than 10%. Grind the dried traditional Chinese medicines into fine powders separately to ensure uniform particle size. Pass the ground traditional Chinese medicine powders through a 100-mesh sieve to remove the coarse particles and ensure the powders are delicate and uniform.

[0038] Formula ratio: Make the ratio according to the above weight portions: It can be 30 portions of Chinese gall, 30 portions of sappanwood, 20 portions of plantain seeds, 10 portions of white peony root, and 10 portions of liquorice root. Put the above traditional Chinese medicine powders into a stirring device and stir at a low speed for 10 - 15 minutes to ensure that each component is fully mixed evenly. Use deionized water as the extraction solvent to ensure no impurities interfere with the extraction process. After the extraction is completed, filter to remove the residue and collect the filtrate. Concentrate it to 1 / 5 of the original volume to obtain a concentrated solution. Use the lyophilization method to dry the concentrated solution into a powder. The dried powder should be light yellow or brownish-yellow, without caking, and have good fluidity.

[0039] Mix the dry powder with a feed carrier at a ratio of 1:9 to prepare a feed additive at 20 g / kg. Pack the prepared feed additive into a sealed bag and store it in a cool and dry place.

[0040] Please refer to Figure 1. Add ultrasonic pretreatment before reflux extraction. Add the mixed traditional Chinese medicine powders to a deionized water solution containing 0.2 - 0.4% ethanol according to a solid-liquid ratio of 1:10 (g / mL), and pretreat for 30 minutes under ultrasonic conditions of 20 - 40 kHz.

[0041] Specifically, the deionized aqueous solution containing 0.2 - 0.4% ethanol not only serves as a solvent but also can dissolve the fat-soluble components (such as polyphenols, flavonoids, etc.) in traditional Chinese medicine, enhancing the extraction effect. The addition of ethanol can also reduce the surface tension of the solution, further promoting the release of active ingredients, and perform pretreatment for 30 minutes at an ultrasonic frequency of 20 - 40 kHz. Ultrasonic waves generate strong mechanical vibrations and local high temperature and high pressure through the cavitation effect (i.e., the formation, growth, and rupture of bubbles in the liquid), which can effectively break the plant cell wall and make the active ingredients in traditional Chinese medicine more easily released into the solvent.

[0042] Please refer to the appendix Figure 1 , and then raise the temperature to 80°C for reflux extraction for 1.5 hours.

[0043] Specifically, heat the mixed traditional Chinese medicine powder pretreated by ultrasonic waves and the solvent solution to 80°C and carry out reflux extraction at 80°C for 1.5 hours. The solvent continuously evaporates and condenses and refluxes at high temperature, forming a circulating flow, enabling the solvent to fully contact with the traditional Chinese medicine powder and further promoting the release of active ingredients. At the same time, high temperature helps to dissolve the fat-soluble and water-soluble components in traditional Chinese medicine, improving the extraction efficiency. This step is usually carried out in a reflux extraction device with a condenser to ensure that the solvent will not be overly evaporated and lost at high temperature, while ensuring the stability and safety of the extraction process. During the reflux extraction process, the circulating flow of the solvent can continuously update the solvent components in the extract, avoiding the extraction equilibrium caused by too high a concentration of active ingredients in the extract, thereby improving the extraction efficiency.

[0044] Please refer to the appendix Figure 1 , before ultrasonic pretreatment, add 0.1 - 0.3% cellulase / pectinase, the dosage of cellulase / pectinase is 0.5% - 1% of the weight of the medicinal materials, adjust the pH to 4.5 - 5.5, and use enzymatic hydrolysis to break the plant cell wall.

[0045] Specifically, before ultrasonic pretreatment, adding 0.1 - 0.3% of cellulase and pectinase can decompose the cellulose in the plant cell wall, while pectinase can decompose pectin. The two work synergistically to effectively break the structure of the plant cell wall. Adjust the pH value of the mixed traditional Chinese medicine powder and the solvent solution to 4.5 - 5.5. This pH range is the optimal activity range of cellulase and pectinase, which can ensure the efficient catalytic action of the enzymes. Usually, citric acid or phosphate buffer solution is used to adjust the pH value to maintain a stable acidic environment. This enzymatic hydrolysis can make the active ingredients (such as polyphenols, flavonoid compounds) inside the cells more easily released into the solvent. The enzymatic hydrolysis treatment is usually carried out for 30 - 60 minutes, and the specific time is optimized according to the types of medicinal materials and the activity of the enzymes. The enzymatic hydrolysis treatment is especially suitable for the extraction of Chinese gallnut (containing tannin) and sappanwood (containing brazilin). The enzymatic hydrolysis treatment is a green and efficient extraction technology that does not require the use of a large amount of chemical reagents and meets the environmental protection requirements.

[0046] Please refer to the appendix Figure 1 , and the concentration method is vacuum concentration. The vacuum degree is adjusted to -0.09 MPa, and the concentration temperature is reduced to 55 °C.

[0047] Specifically, during the concentration process, the vacuum degree is precisely adjusted to -0.09 MPa. This vacuum degree can significantly reduce the boiling point of the solvent, enabling the solvent to evaporate at a lower temperature, thereby avoiding the destruction of active ingredients by high temperature. The concentration temperature is controlled at 55 °C. This temperature can not only ensure the rapid evaporation of the solvent but also prevent the degradation of heat-sensitive components (such as polyphenols and flavonoids). 55 °C is an optimized temperature that can maximize the retention of the activity of active ingredients while ensuring the concentration efficiency. A vacuum concentration device (such as a rotary evaporator or a vacuum concentration tank) is used. By evacuating and heating, the solvent can rapidly evaporate under a low-pressure environment. The rotating or stirring device inside the device can ensure uniform heating of the solution and improve the concentration efficiency. During the vacuum concentration process, the solvent rapidly evaporates under low pressure and at a lower temperature to form steam. The steam is cooled by a condenser and re-liquefied, and then collected in a receiving bottle. The concentrated solution is gradually concentrated to about 1 / 5 of the original volume to obtain a high-concentration extract. By reducing the vacuum degree and controlling the temperature, vacuum concentration can avoid the destruction of active ingredients by high temperature. Many traditional Chinese medicine components (such as polyphenols, flavonoids, tannins, etc.) are sensitive to high temperature and are prone to degradation or denaturation at high temperature. The vacuum concentration technology can complete the concentration process at a lower temperature, maximizing the retention of the activity of these components. Vacuum concentration significantly improves the concentration efficiency by reducing the boiling point of the solvent and accelerating the evaporation rate of the solvent.

[0048] Please refer to the appendix Figure 1 , and an ultrafiltration membrane pretreatment is added before vacuum concentration to remove macromolecular impurities and reduce the subsequent concentration load.

[0049] Specifically, an ultrafiltration membrane with a molecular weight cut-off of 5000 - 10000 Da is used. This membrane can effectively retain macromolecular impurities (such as proteins and polysaccharides), while allowing small-molecule active ingredients (such as polyphenols, flavonoids, tannins, etc.) to pass through. The filtration process of the ultrafiltration membrane is usually carried out at room temperature or slightly above room temperature to avoid damage to the membrane by high temperature or the destruction of active ingredients. By removing macromolecular impurities, the ultrafiltration pretreatment can significantly reduce the subsequent vacuum concentration load. This means that during the concentration process, the amount of solvent to be processed is reduced, and the concentration time is shortened, thereby improving production efficiency.

[0050] Please refer to the appendix Figure 1 , and the drying method is freeze-drying. The pre-freezing temperature is -40 °C, the vacuum degree is 10 - 20 Pa, and the drying time is 12 - 24 hours.

[0051] Specifically, place the concentrated extract or extract in the freeze-drying chamber of a freeze dryer, and set the pre-freezing temperature to -40°C. This temperature can ensure that the extract or extract is completely frozen, forming a stable solid structure, providing a good foundation for subsequent sublimation drying. During the drying process, precisely control the vacuum degree at 10 - 20 Pa. This low vacuum degree can effectively reduce the boiling point of water, enabling ice to directly sublimate into water vapor at low temperature, thus realizing the drying process. The drying time is adjusted according to the nature and thickness of the extract, usually 12 - 24 hours. Freeze-drying is carried out under low temperature and low vacuum conditions, which can effectively avoid the destruction of active ingredients (such as polyphenols, flavonoids, tannins, etc.) by high temperature. The low vacuum degree can reduce the contact with oxygen and lower the probability of oxidation reactions. At the same time, the low temperature condition can also reduce the occurrence of chemical degradation reactions, further protecting the stability of active ingredients.

[0052] Please refer to the appendix Figure 1 , the feed carrier is corn flour or wheat bran, and the particle size of the carrier is 60 - 100 mesh.

[0053] Specifically, corn flour is a commonly used feed carrier with good adsorption and stability. It can effectively adsorb the dried additive powder, ensuring the uniform distribution of the additive in the feed. Corn flour is rich in carbohydrates and can provide a certain amount of energy for aquatic animals. Wheat bran is a by-product of wheat processing, with a high fiber content and good adsorption. It can not only carry the additive but also improve the texture and palatability of the feed. The fiber component of wheat bran contributes to the digestive health of aquatic animals. A particle size of 60 - 100 mesh means that the carrier powder is fine enough to be fully mixed with the dried additive powder, while avoiding uneven distribution of the additive caused by too large particle size or dust flying and waste caused by too small particle size.

[0054] Please refer to the appendix Figure 1 , 1% chitosan (molecular weight 50,000 - 100,000 Da) and 0.8% rosemary extract can also be added to the feed carrier as antioxidants.

[0055] Specifically, chitosan is a natural polysaccharide prepared by deacetylating chitin, with a molecular weight between 50,000 and 100,000 Da. It has good biocompatibility, biodegradability, and antibacterial properties. Chitosan binds to the anionic components of the bacterial cell wall through its cationic properties, interfering with the growth and reproduction of bacteria, thereby exerting an antibacterial effect. In addition, chitosan can also adsorb moisture in the feed, keep the feed dry, and further extend the shelf life of the feed. Rosemary extract is a natural plant extract, and its main components include polyphenolic compounds such as rosmarinic acid and carnosic acid, which have good antioxidant and antibacterial properties. The polyphenolic compounds in rosemary extract can scavenge free radicals and inhibit lipid peroxidation reactions, thereby protecting the nutritional components and active ingredients in the feed from oxidative damage. When preparing the feed additive, first mix the dried additive powder evenly with corn flour or wheat bran, and then add chitosan and rosemary extract in proportion. Ensure that all components are fully mixed through a stirring or mixing device to form a uniform feed additive.

[0056] The following is an introduction in combination with specific embodiments:

[0057] Example 1: Verification of the disease resistance and growth promotion effects of the additive in bass farming

[0058] I. Experimental animals and grouping

[0059] Experimental subjects: Healthy California bass with a body weight of 50 ± 5 g were selected and purchased from a certain aquaculture farm in Guangdong. After 7 days of acclimation to the environment, the bass were grouped according to the following design. Grouping design: 500 bass were randomly divided into 2 groups - a control group (basic feed) and an experimental group (basic feed + 20 g / kg of the additive of the present invention). Each group had 5 replicates, with 50 fish in each replicate, and they were cultured in 10 recirculating aquaculture tanks with specifications of 100 cm × 60 cm × 80 cm, the water depth was 60 cm, and the stocking density was controlled at 25 fish / m 3 .

[0060] II. Feed and culture conditions

[0061] Basic feed: Using corn flour or wheat bran as the feed carrier, the nutritional components meet the basic growth requirements of bass. Experimental feed: The dried powder prepared by the method of this scheme was evenly mixed in the basic feed at a ratio of 20 g / kg, and it was prepared and used immediately to ensure the activity of the additive. Culture environment: The water temperature was controlled at 25 ± 2 °C, the dissolved oxygen was ≥ 6 mg / L, the pH was maintained at 7.5 ± 0.5, the ammonia nitrogen was ≤ 0.2 mg / L, the nitrite was ≤ 0.1 mg / L, and the light cycle was set to 12 h light / 12 h dark. Feed was given 3 times a day at 08:00, 12:00, and 16:00, and the feeding amount was 3% - 5% of the fish body weight, which was dynamically adjusted according to the feeding situation.

[0062] III. Artificial infection method

[0063] Bacterial suspension preparation: Inoculate Edwardsiella tarda (ATCC15947) into LB liquid medium, shake and culture at 37 °C for 24 h, then centrifuge to collect the bacteria, wash twice with sterile saline, and finally adjust the concentration of the bacterial suspension to 1×10 6 CFU / mL (verified by plate counting method). Infection method: On the 45th day of breeding, use intraperitoneal injection to infect, inject 0.1 mL of bacterial suspension into each fish (infection dose 1×10 5 CFU / fish), and the control group is injected with an equal amount of sterile saline to ensure aseptic operation during the process.

[0064] IV. Observation indicators

[0065] Record of morbidity and death: Continuously observe for 15 days after infection, record the morbidity symptoms (black body color, abnormal swimming, swollen anus, bleeding gill filaments) and dead individuals every day, and calculate the morbidity rate (number of diseased fish / total number of fish in each group × 100%) and mortality rate (number of dead fish / total number of fish in each group × 100%).

[0066] Growth performance: Weigh at the beginning and end of breeding respectively, calculate the relative growth rate (RGR, %) = [(final average weight - initial average weight) / initial average weight] × 100%, and the feed conversion rate (FCR) = total feed intake / (final total weight - initial total weight).

[0067] Immune indicators: On the 7th day after infection, randomly select 10 fish from each group, collect blood from the caudal vein and separate the serum, and use a kit to measure the activities of lysozyme (LZM) and superoxide dismutase (SOD), and operate strictly according to the kit instructions.

[0068] V. Data statistics

[0069] Independent sample t-tests were performed using SPSS 26.0. P < 0.05 indicates a significant difference between groups. The results are as follows:

[0070]

[0071] Result analysis:

[0072] The morbidity rate and mortality rate of the experimental group were reduced by 40.3% and 48.6% respectively compared with the control group (P < 0.01), indicating that the additive of the present invention can effectively inhibit the vibriosis of perch caused by Edwardsiella tarda infection, which is consistent with the in vitro antibacterial test results. This effect may be related to the additive's inhibition of pathogen adhesion, regulation of intestinal microecology, or enhancement of the surface mucosal barrier function.

[0073] Growth performance improvement: The relative weight gain rate of the experimental group was 15.6% higher than that of the control group (P<0.05), and the feed conversion rate decreased by 12.4% (P<0.05), indicating that the additive can promote the absorption of nutrients, improve the feed utilization efficiency, and is beneficial to the rapid growth of bass.

[0074] Immune index regulation: The activities of serum LZM and SOD in the experimental group were 24.1% and 17.6% lower than those of the control group respectively (P<0.01). This result suggests that the additive of the present invention does not play a role by enhancing the body's innate immune response (such as bactericidal action of lysozyme and scavenging of free radicals by antioxidant enzymes), but may maintain the body's immune homeostasis by inhibiting the excessive immune response, reducing the release of inflammatory factors, and avoiding immune damage. The synergistic effect of this regulation mode and the improvement of disease resistance reflects the comprehensive improvement effect of the additive on the health of bass.

[0075] Example 2:

[0076] Verification of the effects of vacuum concentration and ultrafiltration membrane pretreatment

[0077] I. Experimental purpose:

[0078] Verify the effects of vacuum concentration combined with ultrafiltration membrane pretreatment on the retention rate of active ingredients, impurity removal rate, and concentration efficiency.

[0079] II. Experimental design

[0080]

[0081] III. Experimental methods

[0082] Detection of active ingredients:

[0083] The contents of gallotannin (chromatographic column: C18, mobile phase: methanol - 0.1% phosphoric acid solution = 45:55, detection wavelength 254nm) and hematoxylin (mobile phase: acetonitrile - 0.1% formic acid solution = 30:70, detection wavelength 225nm) in the concentrated solution were determined by HPLC method.

[0084] Determination of impurity removal rate:

[0085] The polysaccharide content was determined by anthrone - sulfuric acid method, and the protein content was determined by Lowry method. The impurity removal rate was calculated as (impurity content before pretreatment - impurity content after pretreatment) / impurity content before pretreatment × 100%.

[0086] Evaluation of concentration efficiency:

[0087] Record the time required to concentrate 1000 mL of filtrate to 200 mL, and calculate the energy consumption per unit volume of concentration (kWh / L).

[0088] IV. Experimental results

[0089] Detection index Experimental group Control group 1 Control group 2 Concentration time (min) 65±2 120±3 85±2 Energy consumption (kWh / L) 0.32±0.02 0.85±0.04 0.45±0.03 Tannin retention rate (%) 88.5±1.2 72.3±1.5 82.1±1.0 Hematoxylin retention rate (%) 81.2±1.0 65.4±1.3 75.3±1.1 Polysaccharide removal rate (%) 68.3±2.0 22.5±1.8 35.2±1.5 Protein removal rate (%) 72.1±1.5 18.7±1.2 40.3±1.3

[0090] Result analysis:

[0091] Thermal-sensitive component protection effect: In the experimental group, the retention rates of tannin and hematoxylin were increased by 22.4% and 24.1% respectively compared with the control group 1 (normal pressure high-temperature concentration), which confirmed that the low-temperature vacuum environment at 55°C significantly reduced the thermal degradation of polyphenolic components (the degradation rate decreased from 27.7% to 11.5%), which was completely in line with Claim 6, "Avoiding the destruction of active ingredients by high temperature".

[0092] Impurity interception synergistic effect: The removal rates of polysaccharides and proteins by the ultrafiltration membrane (8000Da) were increased by 33.1% and 31.8% respectively compared with the group without pretreatment (control group 2), effectively reducing the macromolecular impurities in the concentrated solution (the total solid content decreased by 25%) and reducing the risk of wall sticking in the subsequent drying process, verifying the technical effect of Claim 7, "Improving the product purity".

[0093] Example 3

[0094] Verification of the advantages of the freeze-drying process

[0095] I. Experimental purpose

[0096] By comparing with spray drying, verify the improvement effect of freeze drying on the stability of active ingredients, the physical properties of products and the shelf life.

[0097] II. Experimental design

[0098]

[0099] III. Detection indicators

[0100] Component stability test:

[0101] Accelerated aging experiment: Store at 40°C and RH75% for 3 months, and determine the contents of tannin and hematoxylin by HPLC every month, and calculate the retention rate (the initial content is standardized to 100%).

[0102] Physical property detection:

[0103] Moisture content: Direct drying method (105°C, 4h) in GB5009.3-2016;

[0104] Solubility: Dissolution time of 1g powder in 10mL deionized water (stopwatch timing until completely dispersed) and solution turbidity (NTU, measured by turbidimeter);

[0105] Flowability: Angle of repose measurement (natural packing angle of powder, the smaller the angle, the better the flowability).

[0106] Microbial indicators: The total number of colonies (GB4789.2-2016) is detected monthly during storage, and the requirement is ≤ 1000 CFU / g.

[0107] IV. Experimental results

[0108]

[0109] Active ingredient protection mechanism: Freeze-drying forms ice crystal structures through pre-freezing at -40°C and directly sublimates and dehydrates under a vacuum of 10-20 Pa, avoiding the hydrolysis of tannins (the hydrolysis rate drops from 21.1% to 5.7%) and the oxidation of hematoxylin (the oxidation rate drops from 31.7% to 10.3%) caused by the high temperature (130°C) in spray drying, which is completely consistent with "retaining biological activity" in claim 8.

[0110] Product quality optimization: The low moisture content (≤ 3%) and high porosity structure shorten the dissolution time of the freeze-dried powder to within 15 s and reduce the turbidity to below 20 NTU, meeting the rapid dissolution requirements of aquatic feeds; the angle of repose ≤ 35°, ensuring that the coefficient of variation of the mixing uniformity with 80-100 mesh carriers (corn flour / bran) ≤ 7% (in line with GB / T36866-2018).

[0111] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The Chinese gallnut and sappanwood compound antibacterial aquatic feed additive is characterized in that It comprises raw materials in the following parts by weight: The content of each component is by mass fraction: 20 - 40 parts of Chinese gall, 20 - 40 parts of sappanwood, 10 - 30 parts of plantain seed, 5 - 15 parts of white peony root, 5 - 15 parts of liquorice root.

2. The extraction process of the galla chinensis and sappanwood compound antibacterial aquatic feed additive according to claim 1, characterized in that, Wash, dry, pulverize Chinese gall, sappanwood, plantain seed, white peony root and liquorice root into fine powder respectively, and sieve through a 100 - mesh sieve; Mix the above traditional Chinese medicine powders evenly according to the weight parts: 30 parts of Chinese gall, 30 parts of sappanwood, 20 parts of plantain seed, 10 parts of white peony root, 10 parts of liquorice root; Add deionized water, heat, reflux extract, filter, and collect the filtrate; Concentrate the filtrate to 1 / 5 of the original volume to obtain a concentrated solution; Freeze - dry the concentrated solution to obtain a dry powder; Mix the dry powder with a feed carrier to prepare a feed additive of 20 g / kg.

3. The extraction process of the galla chinensis and sappanwood compound antibacterial aquatic feed additive according to claim 2, characterized in that, Before reflux extraction, add ultrasonic pretreatment. Add the mixed traditional Chinese medicine powder into a deionized water solution containing 0.2 - 0.4% ethanol according to a solid - liquid ratio of 1:10 (g / mL), and pretreat it under ultrasonic conditions of 20 - 40 kHz for 30 minutes.

4. The extraction process of the galla chinensis and sappanwood compound antibacterial aquatic feed additive according to claim 3, characterized in that, Then raise the temperature to 80 °C and reflux extract for 1.5 hours.

5. The extraction process of the Chinese gallnut and sappanwood compound antibacterial aquatic feed additive according to claim 3, characterized in that, Before ultrasonic pretreatment, first add 0.1 - 0.3% cellulase / pectinase. The dosage of cellulase / pectinase is 0.5% - 1% of the weight of the medicinal materials, adjust the pH to 4.5 - 5.5, and use enzymatic hydrolysis to break the plant cell wall.

6. The extraction process of the Chinese gallnut and sappanwood compound antibacterial aquatic feed additive according to claim 2, characterized in that, The concentration method is vacuum concentration. Adjust the vacuum degree to - 0.09 MPa and the concentration temperature drops to 55 °C.

7. The extraction process of the Chinese gallnut and sappanwood compound antibacterial aquatic feed additive according to claim 6, characterized in that, Before vacuum concentration, add ultrafiltration membrane pretreatment to remove macromolecular impurities and reduce the subsequent concentration load.

8. The extraction process of the galla chinensis and sappanwood compound antibacterial aquatic feed additive according to claim 2, characterized in that, The drying method is freeze - drying, the pre - freezing temperature is - 40 °C, the vacuum degree is 10 - 20 Pa, and the drying time is 12 - 24 hours.

9. The extraction process of the Chinese gallnut and sappanwood compound antibacterial aquatic feed additive according to claim 2, characterized in that, The feed carrier is corn flour or wheat bran, and the particle size of the carrier is 60 - 100 mesh.

10. The extraction process of the Chinese gallnut and sappanwood compound antibacterial aquatic feed additive according to claim 9, characterized in that, 1% of chitosan and 0.8% of rosemary extract can also be added to the feed carrier as antioxidants.

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