Extraction process and application of atractylodes macrocephala polysaccharide

Abstract

The invention relates to an extraction process and application of atractylodes macrocephala polysaccharide, and the extraction process comprises the following steps: step 1, taking 10-15g of atractylodes macrocephala powder, feeding the atractylodes macrocephala powder into a Soxhlet extractor, and adding ethanol for reflux degreasing for 5-6 hours; 2, after degreasing is completed, the bighead atractylodes rhizome powder is exposed in air and stands for 2-3 h, then 50-65 mL of water is added, and the enzymolysis condition is set; and step 3, setting the enzymolysis pH value to be 4.5-5.0, the enzymolysis temperature to be 48-55 DEG C, the compound enzyme amount to be 0.65-0.8% and the enzymolysis time to be 45-60 minutes. According to the extraction process and the application of the atractylodes macrocephala polysaccharide, the atractylodes macrocephala polysaccharide is used as a feed additive to be applied to micropterus salmoides culture for the first time, the research on the influence of the atractylodes macrocephala polysaccharide on the growth performance and the immune function of micropterus salmoides is discussed, and the influence of the atractylodes macrocephala polysaccharide on the intestinal micro-ecological environment of micropterus salmoides is comprehensively analyzed by adopting a gene sequencing analysis technology for the first time; the change of microbial flora is analyzed, and the action mechanism of the atractylodes macrocephala polysaccharide on the growth promoting function and the immune enhancement of the micropterus salmoides is preliminarily discussed.

Description

Technical Field

[0001] The invention relates to the technical field of seabass growth research, and in particular to an extraction process and application of atractylodes macrocephala polysaccharide. Background Art

[0002] Atractylodes macrocephala polysaccharide is an extract of Atractylodes macrocephala, which is the core active ingredient of Atractylodes macrocephala. It has anti-tumor, immunomodulatory, nervous system protection, gastrointestinal mucosal protection, liver protection, antibacterial, hypoglycemic, and antioxidant pharmacological effects. It is a high-efficiency, low-toxic, and clearly defined pharmacological Chinese medicine polysaccharide. Atractylodes macrocephala polysaccharide is a feed additive and is widely used in animal production. Studies have shown that within a certain dosage range, Atractylodes macrocephala polysaccharide has significant effects on promoting growth, enhancing macrophage phagocytosis, and enhancing cellular and humoral immunity in livestock and poultry. In the aquaculture industry, recent studies have also found that Atractylodes macrocephala extract can improve the growth performance and antioxidant capacity of Japanese prawns and improve disease resistance.

[0003] California bass has the advantages of strong adaptability, fast growth, easy catching, and short breeding cycle. It is very popular among breeders. In addition, its meat is delicious, nutritious, has no intramuscular spines, and has a beautiful shape. It is suitable for consumers to eat. It has a broad market prospect and has become one of the main freshwater fish breeding varieties in my country. California bass is prone to disease under artificial high-density breeding conditions. It is necessary to strengthen disease prevention, focusing on the prevention and treatment of enteritis, gill rot, hemorrhagic disease, saprolegniasis and parasitic diseases. It is necessary to adhere to the principle of "prevention is more important than treatment" and take comprehensive prevention and control measures to kill pathogens in the breeding environment while enhancing their disease resistance, reducing disease losses, and protecting the economic interests of farmers. Therefore, it is of great significance to add appropriate intestinal probiotics and immune polysaccharides to the feed to enhance the intestinal function of California bass, improve its absorption rate of feed, and inhibit the growth of pathogenic bacteria.

[0004] In order to pursue higher production efficiency and increase production capacity of California bass, the California bass farming industry is constantly increasing its degree of intensification, and this trend of intensification is also accompanied by an increase in the incidence of animal diseases. In the past, antibiotics and other antimicrobial drugs were usually relied on to control and prevent diseases. However, as the food safety risks and environmental pollution problems caused by drug residues have gradually emerged, antibiotic resistance has become a global health problem.

[0005] Therefore, we urgently need to find suitable alternative antibiotic products to meet this challenge to ensure the sustainable development of aquaculture. We selected the traditional Chinese medicine Atractylodes macrocephala polysaccharide with immune-enhancing activity to explore its effects on growth performance, immune function and intestinal flora during the growth of California bass as a feed additive, and preliminarily explored its mechanism of action, providing a theoretical basis for the application of Chinese medicine alternative antibiotics in aquaculture, and providing a reference for the development and application of other immune polysaccharides. This is a difficult problem that needs to be solved in this field. Research on the application of Atractylodes macrocephala polysaccharide in animal production mainly focuses on livestock and poultry farming, and there are few studies in the field of aquaculture. The research stays at the study of its effects on growth performance and serum immune indicators, lacking in-depth analysis of its immune regulation mechanism and intestinal flora, and there is no report on the application of Atractylodes macrocephala polysaccharide in California bass farming. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides an extraction process and application of Atractylodes macrocephala polysaccharide, wherein Atractylodes macrocephala polysaccharide is added to the feed for feeding California seabass, and the body weight gain, specific growth rate, feed coefficient, survival rate and organ index of California seabass are used as growth indicators to explore the effect of Atractylodes macrocephala polysaccharide on the growth performance of California seabass, and on this basis, the appropriate dosage and method of adding Atractylodes macrocephala polysaccharide are found.

[0007] To achieve the above object, the present invention provides the following technical solution: a process for extracting Atractylodes macrocephala polysaccharide, comprising the following steps:

[0008] Step 1, take 10-15g of Atractylodes macrocephala powder, put the Atractylodes macrocephala powder into a Soxhlet extractor and add ethanol to reflux and defatted for 5-6h;

[0009] Step 2: After defatting, expose the Atractylodes macrocephala powder to the air, let it stand for 2-3 hours, then add 50-65 mL of water and set the enzymatic hydrolysis conditions;

[0010] Step 3, setting the enzymatic hydrolysis pH value to 4.5-5.0, the enzymatic hydrolysis temperature to 48-55°C, the compound enzyme amount to 0.65-0.8%, and the enzymatic hydrolysis time to 45-60min;

[0011] Step 4: Finally, quickly raise the temperature to 90-110°C to inactivate the enzyme for 5-8 minutes, add water to 180-220 mL, and extract at 90°C for 2-2.5 hours. After the water temperature drops to below 35°C, extract the Atractylodes macrocephala polysaccharide solution and filter it.

[0012] Preferably, the Atractylodes macrocephala powder is ground into a diameter of 1.5-2 mm by drying in a drying oven at 40-55° C. for 8-10 h, and stored in a desiccator for later use.

[0013] An application of Atractylodes macrocephala polysaccharide extracted by the above extraction process in California seabass farming comprises the following steps:

[0014] Step 1, 600 healthy California bass with similar weight were selected as experimental animals, and a control group was set up without adding Atractylodes macrocephala polysaccharide, and three Atractylodes macrocephala polysaccharide addition groups with concentration gradients, 0, 0.2g / Kg, 0.4g / Kg, and 0.6g / Kg as gradients, a total of 4 groups, 150 fish in each group, and 3 parallel groups were set up in each group, each parallel group had 50 fish, and the formal experiment began after the experimental California bass were acclimated for 1 week to simulate the intensive breeding environment;

[0015] Step 2: Feed the fish twice a day, morning and evening, in sufficient quantities, for 42 consecutive days;

[0016] Step 3: weigh and record the body weight of each animal before and after feeding. After feeding, randomly select 10 animals from each parallel group, remove the liver, spleen, and kidney after anesthesia, wash away the floating blood, absorb the water, and weigh and record the weight.

[0017] Step 4: Randomly select 7 fish from the control group and each experimental group for blood collection. The collected blood was centrifuged and the upper serum was collected for testing of serum biochemical indicators. The intestinal contents were collected, quickly frozen with liquid nitrogen and stored in a -80°C refrigerator for intestinal flora analysis.

[0018] Preferably, the serum biochemical index detection uses a kit to detect blood indexes, and measures the SOD, MDA, complement C3 and complement C4 indexes of each group.

[0019] Preferably, during the intestinal flora analysis, intestinal genomic DNA is extracted and sequenced.

[0020] Preferably, during the intestinal flora analysis, genomic DNA is extracted from the sample using a DNA extraction kit, and then the DNA concentration is detected using agarose gel electrophoresis and a NanoDrop spectrophotometer. The extracted genomic DNA is used as a template to delineate the genomic sequencing region, and specific primers are added to the DNA template to allow for thorough mixing.

[0021] Preferably, after the step 4 is completed, the recorded data are sorted using Excel, and the recorded data results are expressed as Avg±SD. The normality of the data is tested by SPSSAU, and then Duncan is used to perform one-way analysis of variance on the obtained data to find significant differences between the data.

[0022] Preferably, the raw data obtained from the intestinal flora analysis is in FASTQ format, and the raw sequence is processed using cutadapt software to cut off the primer sequence, and then the raw sequence is processed using DADA2 according to the Qiime2 setting program to obtain high-quality sequences and ASV abundance tables.

[0023] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0024] 1. This invention is the first to apply Atractylodes macrocephala polysaccharide as a feed additive to California sea bass farming, and to explore its effects on the growth performance and immune function of California sea bass.

[0025] 2. For the first time, gene sequencing analysis technology was used to comprehensively analyze the effects of Atractylodes macrocephala polysaccharides on the intestinal microecological environment of California seabass, analyze the changes in its microbial flora, and preliminarily explore the mechanism of action of Atractylodes macrocephala polysaccharides on the growth-promoting function and immune enhancement of California seabass.

[0026] 3. Keeping up with the comprehensive ban on antibiotics in the breeding industry and actively exploring research on alternative antibiotics for traditional Chinese medicine will have far-reaching significance for expanding and deepening the application scope of Chinese medicine resources and achieving green development of the breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the technical roadmap of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Embodiment 1

[0030] A process for extracting Atractylodes macrocephala polysaccharide comprises the following steps:

[0031] Step 1, take 12g of Atractylodes macrocephala powder, put the Atractylodes macrocephala powder into a Soxhlet extractor and add ethanol to reflux and defatted for 5h;

[0032] Step 2: After defatting, expose the Atractylodes macrocephala powder to the air, let it stand for 2 hours, then add 50 mL of water and set the enzymatic hydrolysis conditions;

[0033] Step 3, setting the enzymatic hydrolysis pH value to 4.5, the enzymatic hydrolysis temperature to 48°C, the complex enzyme amount to 0.65%, and the enzymatic hydrolysis time to 45 min;

[0034] Step 4: Finally, quickly raise the temperature to 90°C to inactivate the enzyme for 5 minutes, add water to 180 mL, and extract at 90°C for 2 hours. After the water temperature drops to below 35°C, extract the Atractylodes macrocephala polysaccharide solution and filter it.

[0035] Embodiment 2

[0036] A process for extracting Atractylodes macrocephala polysaccharide comprises the following steps:

[0037] Step 1, take 15g of Atractylodes macrocephala powder, put the Atractylodes macrocephala powder into a Soxhlet extractor and add ethanol to reflux and defat for 6 hours;

[0038] Step 2: After defatting, the Atractylodes macrocephala powder was exposed to air and allowed to stand for 3 h, then 65 mL of water was added and the enzymatic hydrolysis conditions were set;

[0039] Step 3, setting the enzymatic hydrolysis pH value to 5.0, the enzymatic hydrolysis temperature to 55°C, the compound enzyme amount to 0.8%, and the enzymatic hydrolysis time to 60 min;

[0040] Step 4: Finally, quickly raise the temperature to 110°C to inactivate the enzyme for 8 minutes, add water to 220 mL, and extract at 90°C for 2.5 hours. After the water temperature drops below 35°C, extract the Atractylodes macrocephala polysaccharide solution and filter it.

[0041] Embodiment 3

[0042] A process for extracting Atractylodes macrocephala polysaccharide comprises the following steps:

[0043] Step 1, take 10 grams of Atractylodes macrocephala powder, put the Atractylodes macrocephala powder into a Soxhlet extractor and add ethanol to reflux and defatted for 5 hours;

[0044] Step 2: After defatting, expose the Atractylodes macrocephala powder to the air, let it stand for 2 hours, then add 50 mL of water and set the enzymatic hydrolysis conditions;

[0045] Step 3, setting the enzymatic hydrolysis pH value to 5.0, the enzymatic hydrolysis temperature to 50°C, the compound enzyme amount to 0.7%, and the enzymatic hydrolysis time to 50 min;

[0046] Step 4: Finally, quickly raise the temperature to 90°C to inactivate the enzyme for 5-8 minutes, add water to 200 mL, and extract at 90°C for 2 hours. After the water temperature drops to below 35°C, extract the Atractylodes macrocephala polysaccharide solution and filter it.

[0047] The experimental results showed that the optimal extraction conditions for Atractylodes macrocephala polysaccharide by the composite enzyme method were as follows: enzymatic pH of 5.0, composite enzyme amount of 0.7%, enzymatic hydrolysis time of 50 min, and enzymatic hydrolysis temperature of 50°C. The yield of Atractylodes macrocephala polysaccharide was 43.00% after alcohol precipitation of the water extract extracted under the optimized conditions, which was 13.10% higher than the yield of Atractylodes macrocephala polysaccharide extracted once with water under the same conditions. Moreover, the yield of polysaccharide extracted once by composite enzyme extraction could reach 97.95% and 95.34% of that of polysaccharide extracted twice by water and twice by composite enzyme extraction. In terms of time saving, energy saving and high efficiency, the composite enzyme method has great advantages.

[0048] refer to Figure 1 , an application of Atractylodes macrocephala polysaccharide extracted by the above-mentioned extraction process in California seabass farming.

[0049] Research content

[0050] (1) Effects of Atractylodes macrocephala polysaccharide on the growth performance of California seabass

[0051] A concentration gradient feeding experiment of Atractylodes macrocephala polysaccharide was carried out by adding it to feed. The body weight gain, specific growth rate, feed coefficient, survival rate, organ index and other growth indicators of California bass were used to explore the effect of Atractylodes macrocephala polysaccharide on the growth performance of California bass, and on this basis, the appropriate dosage and addition method of Atractylodes macrocephala polysaccharide were found.

[0052] (2) Study on the effect of Atractylodes macrocephala polysaccharide on the immune function of California sea bass Blood samples were collected and serum biochemical indicators of the control group and the experimental group were measured. SOD, MDA, complement C3, C4, etc. were used as

[0053] Evaluation indicators were used to study the effects of Atractylodes macrocephala polysaccharide on the antioxidant and nonspecific immune functions of California seabass.

[0054] (3) Effect of Atractylodes macrocephala polysaccharide on intestinal flora of California sea bass

[0055] Intestinal samples were collected, intestinal genomic DNA was extracted and sequenced, and the intestinal microbial populations of the control group and the experimental group of California seabass were analyzed to explore the effect of Atractylodes macrocephala polysaccharide on the changes in the intestinal microbial flora of California seabass.

[0056] The specific research plan is as follows:

[0057] (1) Sample preparation

[0058] 600 California bass with similar weight and health were selected as experimental animals. A control group (without Atractylodes macrocephala polysaccharide) and three Atractylodes macrocephala polysaccharide addition groups with concentration gradients were set up, with 0, 0.2, 0.4, and 0.6 g / Kg as the gradient, a total of 4 groups, 150 fish in each group, and 3 parallel groups in each group, with 50 fish in each parallel group. The formal experiment began after the California bass was acclimated for 1 week to simulate the intensive breeding environment. They were fed in sufficient amounts twice a day in the morning and evening for 42 consecutive days. Their weights were weighed one by one before and after feeding and recorded. After the feeding, 10 fish were randomly selected from each parallel group, and the liver, spleen, and kidney were taken after anesthesia. After washing the floating blood and absorbing the water, they were weighed and recorded. At the same time, 7 fish were randomly selected from the control group and each experimental group for blood collection. After the centrifugation operation, the upper serum was collected for the detection of serum biochemical indicators. The intestinal contents were collected, frozen in liquid nitrogen, and stored in a -80℃ refrigerator for intestinal flora analysis.

[0059] (2) Sample analysis

[0060] The blood indexes were tested using a kit, and the SOD, MDA, complement C3, C4 and other indicators of each group were measured. The intestinal genomic DNA was extracted and sequenced. The genomic DNA was extracted from the sample using a DNA extraction kit, and then the DNA concentration was tested using agarose gel electrophoresis and NanoDrop spectrophotometer. The extracted genomic DNA was used as a template to delineate the genomic sequencing area, and specific primers were added to the DNA template to mix it thoroughly. It was placed in a Takara PCR instrument for testing. The relevant data of the intestinal microbial flora analysis were obtained.

[0061] (3) Data analysis

[0062] The recorded data were sorted using Excel, and the recorded data results were expressed as Avg±SD. SPSSAU was used to test the normality of the data, and then Duncan was used to perform one-way analysis of variance on the obtained data to find significant differences between the data (r<0.05). The original data obtained from the intestinal flora analysis were in FASTQ format, and the original sequence was processed using cutadapt software to cut off the primer sequence. Then, DADA2 was used to process the original sequence according to the Qiime2 setting program to obtain high-quality sequences and ASV abundance tables. The obtained data were subjected to diversity analysis, and the intestinal flora information of California bass was obtained through various algorithms.

[0063] Feasibility Analysis

[0064] (1) As an efficient, low-toxic, and non-residue immune enhancer, immune polysaccharides have a rich research basis in the application research of aquaculture. The application of Atractylodes macrocephala polysaccharide as a feed additive in livestock and poultry farming has shown that it has many effects such as promoting growth and improving immunity. Its anti-disease effect has also been confirmed in experiments on other aquatic animals. Therefore, the application of Atractylodes macrocephala polysaccharide in California bass farming has a rich research reference.

[0065] (2) Atractylodes macrocephala is one of the Chinese medicinal materials with the largest annual sales volume in my country, with an annual total domestic output of more than 10,000 tons. Therefore, the sources of Atractylodes macrocephala polysaccharides are very extensive.

[0066] (3) California bass is a high-quality meat source that is popular among the public. It is widely farmed in the country and has a stable source of fry.

[0067] In summary, the present invention selects PAMK, a traditional Chinese medicine polysaccharide with immune-enhancing activity, to explore its effects as a feed additive on the growth performance, immune function and intestinal flora of California bass during its growth, and preliminarily explores its mechanism of action, to provide a theoretical basis for the application of Chinese medicine antibiotics in aquaculture, and to provide a reference for the development and application of other immune polysaccharides. In the current environment where the country has completely banned antibiotics in the aquaculture industry, developing Atractylodes macrocephala and its extracts into mature and effective aquatic feed additive products has far-reaching significance for expanding and deepening the application scope of Chinese medicine resources and realizing the green development of the aquaculture industry. The application of immune polysaccharides in aquaculture can effectively improve the growth performance and immune function of aquatic animals, and is an important means to promote antibiotic-free aquaculture.

[0068] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0069] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for extracting Atractylodes macrocephala polysaccharide, characterized in that: The following steps are involved: Step 1, take 10-15g of Atractylodes macrocephala powder, put the Atractylodes macrocephala powder into a Soxhlet extractor and add ethanol to reflux and defatted for 5-6h; Step 2: After defatting, expose the Atractylodes macrocephala powder to the air, let it stand for 2-3 hours, then add 50-65 mL of water and set the enzymatic hydrolysis conditions; Step 3, setting the enzymatic hydrolysis pH value to 4.5-5.0, the enzymatic hydrolysis temperature to 48-55°C, the compound enzyme amount to 0.65-0.8%, and the enzymatic hydrolysis time to 45-60min; Step 4: Finally, quickly raise the temperature to 90-110°C to inactivate the enzyme for 5-8 minutes, add water to 180-220 mL, and extract at 90°C for 2-2.5 hours. After the water temperature drops to below 35°C, extract the Atractylodes macrocephala polysaccharide solution and filter it.

2. The extraction process of Atractylodes macrocephala polysaccharide according to claim 1, characterized in that: The Atractylodes macrocephala powder is obtained by grinding dried Atractylodes macrocephala into a diameter of 1.5-2 mm, drying it in a drying oven at 40-55° C. for 8-10 hours, and storing it in a desiccator for later use.

3. Application of Atractylodes macrocephala polysaccharide extracted by the extraction process according to any one of claims 1 to 2 in California seabass breeding, characterized in that: The steps include: Step 1, 600 healthy California bass with similar weight were selected as experimental animals, and a control group was set up without adding Atractylodes macrocephala polysaccharide, and three Atractylodes macrocephala polysaccharide addition groups with concentration gradients, 0, 0.2g / Kg, 0.4g / Kg, and 0.6g / Kg as gradients, a total of 4 groups, 150 fish in each group, and 3 parallel groups were set up in each group, each parallel group had 50 fish, and the formal experiment began after the experimental California bass were acclimated for 1 week to simulate the intensive breeding environment; Step 2: Feed the fish twice a day, morning and evening, in sufficient quantities, for 42 consecutive days; Step 3: weigh and record the body weight of each animal before and after feeding. After feeding, randomly select 10 animals from each parallel group, remove the liver, spleen, and kidney after anesthesia, wash away the floating blood, absorb the water, and weigh and record the weight. Step 4: Randomly select 7 fish from the control group and each experimental group for blood collection. The collected blood was centrifuged and the upper serum was collected for testing of serum biochemical indicators. The intestinal contents were collected, quickly frozen with liquid nitrogen and stored in a -80°C refrigerator for intestinal flora analysis.

4. The use of Atractylodes macrocephala polysaccharide in California seabass farming according to claim 3, characterized in that: The serum biochemical index detection adopts a kit to detect blood indexes, and measures the SOD, MDA, complement C3 and complement C4 indexes of each group.

5. The use of Atractylodes macrocephala polysaccharide in California seabass farming according to claim 3, characterized in that: During the intestinal flora analysis, intestinal genomic DNA is extracted and sequenced.

6. The use of Atractylodes macrocephala polysaccharide in California seabass farming according to claim 5, characterized in that: During the intestinal flora analysis, genomic DNA is extracted from the sample using a DNA extraction kit, and then the DNA concentration is detected using agarose gel electrophoresis and a NanoDrop spectrophotometer. The extracted genomic DNA is used as a template to delineate the genomic sequencing region, and specific primers are added to the DNA template to allow for thorough mixing.

7. The use of Atractylodes macrocephala polysaccharide in California seabass farming according to claim 6, characterized in that: After the step 4 is completed, the recorded data are sorted using Excel, and the recorded data results are expressed as Avg±SD. The normality of the data is tested by SPSSAU, and then Duncan is used to perform one-way analysis of variance on the obtained data to find significant differences between the data.

8. The use of Atractylodes macrocephala polysaccharide in California seabass farming according to claim 6, characterized in that: The raw data obtained from the intestinal flora analysis was in FASTQ format. The raw sequence was processed using cutadapt software to cut off the primer sequence, and then the raw sequence was processed using DADA2 according to the Qiime2 setting program to obtain high-quality sequences and ASV abundance tables.

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