CN113046275B - Bacillus used for prawn feed and fermented Chinese herbal medicine preparation used for prawn feed - Google Patents

Bacillus used for prawn feed and fermented Chinese herbal medicine preparation used for prawn feed Download PDF

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CN113046275B
CN113046275B CN202110457762.4A CN202110457762A CN113046275B CN 113046275 B CN113046275 B CN 113046275B CN 202110457762 A CN202110457762 A CN 202110457762A CN 113046275 B CN113046275 B CN 113046275B
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潘鲁青
王宇轩
苏晨
丁彦军
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Ocean University of China
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Abstract

The fermented Chinese herbal medicine preparation is prepared by crushing rehmannia glutinosa, angelica sinensis, radix paeoniae alba and ligusticum wallichii, mixing the crushed materials in proportion, inoculating 3-11% of bacillus safensis and bacillus stratosphericus mixed (1: 1-1: 4) bacterial solution, and fermenting under proper conditions. The traditional Chinese medicine comprises the following components in parts by weight: 12-20 parts of prepared rhizome of rehmannia, 9-25 parts of angelica, 9-12 parts of white peony root and 6-10 parts of ligusticum wallichii. The preparation method is simple and convenient, can increase the effective content of the Chinese herbal medicine and improve the utilization efficiency of the Chinese herbal medicine. The compound feed can be applied to prawn feeds, can enhance the immunity of prawn organisms, enhance the capability of prawn to resist pathogenic bacteria, reduce the application of antibiotics and chemical drugs, increase the breeding income on the premise of 'green, high efficiency and health', and has good application prospect.

Description

Bacillus used for prawn feed and fermented Chinese herbal medicine preparation used for prawn feed
Technical Field
The invention belongs to the technical field of feed additives, and particularly relates to bacillus for prawn feed and a fermentation type Chinese herbal medicine preparation for prawn feed.
Background
In recent years, diseases are always bottleneck problems restricting the healthy development of the prawn breeding industry, and a large amount of antibiotics are used as important technical measures for preventing and treating the diseases, so that a plurality of negative effects are generated, on one hand, pathogenic bacteria are easy to generate drug resistance due to long-term use, and on the other hand, the antibiotic drug residue exceeds the standard and seriously harms human health. Along with the development of green aquaculture technology, the prohibition of antibiotics is imperative, so the development of green, safe and efficient Chinese herbal medicine preparations becomes an important measure for replacing antibiotics.
The Chinese herbal medicine is a pure natural product, has extremely rich Chinese herbal medicine resources in China, and is widely applied due to the advantages of small toxic and side effects, difficulty in generating drug resistance, improvement of animal physiological health and the like. At present, most of Chinese herbal medicine additives are crude products such as powder or powder, the processing depth is not enough, active ingredients in the Chinese herbal medicine are difficult to be fully absorbed and utilized, and the utilization rate of the active ingredients in the Chinese herbal medicine is low, so how to effectively improve the active ingredients and the physiological effect of the Chinese herbal medicine becomes the focus of the current research.
Disclosure of Invention
The invention aims to provide bacillus for prawn feed and a fermentation type Chinese herbal medicine preparation for prawn feed, the preparation uses the compound bacillus to ferment four Chinese herbal medicines of rehmanniae vaporata, angelica, white peony root and rhizoma ligustici wallichii, and the preparation is safe, reliable, green and environment-friendly, and can obviously improve the physiological health level of aquatic animals; the preparation method of the feed is simple and feasible, can improve the drug effect of the Chinese herbal medicine, improves the utilization rate of the Chinese herbal medicine, saves the cost, and can be implemented on a large scale for industrial production. Can improve physiological and biochemical indexes of prawn, improve immunity of prawn, enhance resistance to pathogenic bacteria, reduce culture risk, and increase culture benefit.
In order to achieve the purpose, the invention adopts the following specific technical scheme: bacillus used for prawn feed, characterized by including Bacillus safensis (Bacillus safensis) and Bacillus stratosphericus (Bacillus stratosphericus);
the Saufusbacillus (Bacillus safensis) collection number is as follows: CGMCC number 22059, preservation date: 23/3/2021, depository: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North;
the Bacillus stratosphericus (Bacillus stratosphericus) has the following preservation number: CGMCC number 22060, preservation date: 23/3/2021, depository: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
The bacillus is applied to the preparation of a fermentation type Chinese herbal medicine preparation for prawn feed.
A fermented Chinese herbal medicine preparation for prawn feed is characterized by being prepared by the following steps:
(1) pretreatment of Chinese herbal medicines: drying four Chinese herbal medicines of radix rehmanniae preparata, angelica sinensis, radix paeoniae alba and ligusticum wallichii for 24 hours in an oven, respectively putting the dried four Chinese herbal medicines into a high-speed knife type multistage grinder for grinding, sieving the ground four Chinese herbal medicines by a sieve of 60-100 meshes, accurately weighing the Chinese herbal medicines by using an electronic balance, mixing the Chinese herbal medicines according to a proportion, bagging and sealing the mixture for later use; the mixture in proportion is prepared from 12 to 20 parts by mass of prepared rehmannia root, angelica, white paeony root and szechuan lovage rhizome: 9-25 parts of: 9-12 parts of: mixing 6-10 parts;
(2) recovering strains: placing the Bacillus safensis in an ultra-low temperature refrigerator at-80 ℃: (Bacillus safensis) And Bacillus stratosphericus (Bacillus stratosphericus) Inoculating the strain into 2216E liquid medium according to the volume ratio of 1-5% for culture for 20-28 h under the conditions of 24-28 ℃ and 160-220 rpm; counting the number of live bacteria by using a plate colony counting method, and when the number of live bacteria in the strain recovery culture medium reaches 3.2 multiplied by 108~5.2×108Stopping the culture of the live bacteria when cfu/ml is reached;
(3) preparing a Chinese herbal medicine liquid fermentation culture medium: firstly, taking a certain amount of the Chinese herbal medicines mixed in proportion obtained in the step 1, adding pure water which is 4-6 times of the mass of the Chinese herbal medicines to obtain Chinese herbal medicine solutions, and respectively adding NaCl with the mass fraction of 4-6% and MgSO 3-5% of the solution to the solutions4·7H20.22-0.63% KH O2PO4Putting 2-4% of cassava flour into a steam sterilization pot, sterilizing at 121 ℃ for 35 minutes, and cooling in an ultra-clean workbench; then adding 0.12-0.34% of L-glutamic acid and 0.2-1.2% of VC (both subjected to ultraviolet sterilization and disinfection) by mass into the cooled Chinese herbal medicine solution, adjusting the pH to 6.8-7.4, preparing a solution, namely a Chinese herbal medicine liquid fermentation culture medium, and storing the solution for later use under an aseptic condition;
(4) preparing Chinese herbal medicine fermentation liquor: the bacillus safensis of the step 2 (A), (B), (C)Bacillus safensis) And Bacillus stratosphericus (Bacillus stratosphericus) Mixing the bacillus subtilis and the bacillus subtilis into composite bacillus according to the volume ratio of 1: 1-1: 4, inoculating 3-11% of the bacillus subtilis and the bacillus subtilis into the Chinese herbal medicine liquid fermentation medium obtained in the step (3), and culturing at the speed of 160-210 rpm and the temperature of 26-32 ℃ for 28-36 hours to obtain Chinese herbal medicine fermentation liquor;
(5) extracting effective components: adding pure water with the mass of 6-10 times of that of the obtained Chinese herbal medicine fermentation liquor, leaching for 2.2-2.8 hours at the low temperature of 45 ℃, leaching for three times at the low temperature, and combining the filtrates of the three times; then adding pure water with the mass of 2 times of that of the extracted medicine residues into the extracted medicine residues, and extracting twice; mixing the filtrates for 5 times;
(6) preparing a fermented Chinese herbal medicine preparation: and (4) carrying out rotary evaporation on the filtrate obtained in the step (5) at the temperature of 45-55 ℃, concentrating to be viscous, and carrying out freeze drying for 24-32 h to obtain the fermented Chinese herbal medicine preparation.
The content of 5-hydroxymethylfurfural, paeoniflorin, ferulic acid and total polysaccharide of the four-ingredient decoction in the Chinese herbal medicines before fermentation is 0.2387, 2.3091, 0.2531 and 69.6607 mg/g respectively; after fermentation, the content of the active substances is 0.2862, 3.1111, 0.3363 and 109.7467 mg/g respectively, and compared with the content before fermentation, the content of the active substances is respectively increased by 19.86%, 34.74%, 32.88% and 57.54% due to fermentation.
A prawn feed based on a fermented Chinese herbal medicine preparation is characterized in that the fermented Chinese herbal medicine preparation is added into a basal feed, and the fermented Chinese herbal medicine preparation added into each kilogram of the basal feed accounts for 0.2-0.8 percent by mass.
The prawn feed is characterized in that fermented Chinese herbal medicine preparations added in each kilogram of basal feed are 0.2%, 0.5% and 0.8% in mass fraction.
The prawn feed is characterized in that the basic feed comprises the following components: by mass, 34% of fish meal, 20% of soybean meal, 16.4% of peanut meal, 22% of wheat flour, 5% of soybean lecithin, 0.6% of vitamin complex and 2% of compound mineral.
The Chinese herbal medicine fermentation preparation for the prawns can obviously improve the immunity of the prawns, improve the survival rate of the prawns, improve the levels of protease and lipase in bodies of the prawns, improve the gene expression level of prawn intestinal tract prawn peptides and comprehensively improve the physiological health level of the prawns.
The Chinese herbal medicine fermentation of the invention is characterized in that under proper conditions, one or more beneficial bacteria are utilized, a complex containing active ingredients of the Chinese herbal medicine, thallus and metabolites thereof is prepared by biological conversion of the Chinese herbal medicine, and the Chinese herbal medicine fermentation has the advantages of improving the efficacy of the Chinese herbal medicine, improving the utilization rate of the Chinese herbal medicine, generating new active substances and the like by virtue of an enzyme system rich in microorganisms, thereby having extremely wide application prospect.
The invention has the advantages and beneficial effects that:
(1) the drug effect is improved. The bacillus contains rich cellulase, phytase and other enzyme systems, has strong decomposition capacity under mild conditions, and can quickly break cell walls of Chinese herbal medicines and release more effective components, so that the drug effect is improved, the dosage of traditional Chinese medicines is saved, and the absorption of the effective components of the traditional Chinese medicines by organisms is better promoted. According to the method, four Chinese herbal medicines are fermented, and the fermentation improvement rates of active substance components of the four Chinese herbal medicines, namely 5-hydroxymethylfurfural, paeoniflorin, ferulic acid and total polysaccharide of four-ingredient decoction are respectively as follows: 19.86%, 34.74%, 32.88% and 57.54%.
(2) The synergistic effect of Chinese herbal medicine and bacillus. The bacillus is screened from prawn intestinal tracts, is easy to adapt to the environment in the intestinal tracts of aquatic animals, and can improve the immunity of prawn organisms and promote the growth and development of aquatic animals. The Chinese herbal medicine can promote the growth and proliferation of probiotics, generate more enzyme system wall breaking, release more traditional Chinese medicine active substances, promote the absorption and utilization of traditional Chinese medicine components, supplement each other and accord with the concept of 'green cultivation, ecological cultivation and healthy cultivation' in China.
(3) Can improve the phagocytic activity of blood cells of litopenaeus vannamei. When the external pathogen invades the prawn body and the diameter is less than 10 mu m, phagocytosis of blood cells can be induced, and the phagocytosis of the blood cells is an important barrier for resisting the external invasion of the prawn. The prawn phagocytic activity of the test group is obviously higher than that of the control group (P < 0.05) within 21 days of feeding the fermented Chinese herbal medicine feed additive, and the control group has no obvious difference.
(4) Can improve the antibacterial activity of the blood plasma of the litopenaeus vannamei. The antibacterial activity reflects the resistance of the prawn body to external microorganisms, and is an important expression for reflecting the strength of the immunity of the prawn body. Within 21 days of feeding the fermented Chinese herbal medicine feed additive, the antibacterial and bacteriolytic activities of the hemolymph of the prawns in the test group are both obviously higher than those of the control group (P < 0.05), and the control group has no obvious difference.
(5) Can improve the oxidation resistance of the litopenaeus vannamei. In order to prevent damage to the body from excess reactive oxygen species, crustaceans have a complex and effective antioxidant defense system to cope with oxidative stress. The total oxidation resistance (T-AOC) of the prawns in the test group is obviously higher than that of the prawns in the control group (P < 0.05) within 21 days after the prawns are fed with the fermented Chinese herbal medicine feed additive.
(6) Can improve the activity of the digestive enzyme of the litopenaeus vannamei. The activity of the digestive enzyme is an important index for evaluating the digestive ability of the cultured animals to nutrient substances. Within 21 days of feeding the fermented Chinese herbal medicine feed additive, the digestive enzyme activity in the hepatopancreas of the test group is obviously higher than that of the control group (P is less than 0.05), and the control group has no obvious difference.
(7) Can improve the gene expression level of the litopenaeus vannamei intestinal tract prawn peptide. Prawn peptides are an important component of the innate immune system. The gene expression level of the prawn Peptide (PEN) of the test group is obviously higher than that of the control group (P < 0.05) within 21 days of feeding the fermented Chinese herbal medicine feed additive.
Drawings
FIG. 1 is a graph showing the result of the phagocytosis rate of the hemocytes of the prawns in example 2.
FIG. 2 is a graph showing the results of the antibacterial activity of the plasma of the prawns in example 2.
FIG. 3 is a graph showing the results of the total antioxidant capacity of the prawns of example 2.
FIG. 4 is a graph showing the results of gene expression of prawn intestinal prawn peptide 3 (PEN 3) in example 2.
FIG. 5 is a graph showing the results of the prawn hepatopancreatin protease in example 2.
FIG. 6 is a graph showing the results of the hepatopancreas lipase of the prawn in example 2.
Detailed Description
The following are specific examples of the present invention and further description of the technical solutions of the present invention, but the scope of the present invention is not limited to the examples, and those skilled in the art can modify and change the present invention according to the spirit of the present invention, and all changes or equivalent substitutions that do not depart from the spirit of the present invention are included in the scope of the present invention. Wherein the prepared rhizome of rehmannia, angelica, white peony root and rhizoma ligustici wallichii are all sold in the market, bacillus safensis (A), (B) and (B)Bacillus safensis) And Bacillus stratosphericus (acillusstratosphericus) The prawn intestinal tract screening agent is obtained by screening researchers in aquatic animal environment physiology laboratories of aquatic institute of China oceanic university, wherein the bacillus safensis is preserved in China general microbiological culture Collection center (Beijing) with the preservation number of 22059; the stratosphericus bacillus is preserved in China general microbiological culture Collection center (Beijing) with the preservation number of 22060.
Example 1:
the preparation of the prawn fermentation Chinese herbal medicine microecological preparation and the breeding feed comprises the following steps:
firstly, preparing effective components of fermented Chinese herbal medicines. The fermented Chinese herbal medicine preparation is prepared by mixing Bacillus safensis (Bacillus safensis) and Bacillus stratosphericus (Bacillus stratosphericus) screened from intestinal tracts of litopenaeus vannamei in a ratio of 1: 1-1: 4 (V: V) to form composite Bacillus, and inoculating 3-11% of the composite Bacillus into a Chinese herbal medicine fermentation culture medium.
The Chinese herbal medicine components in the Chinese herbal medicine fermentation medium are 12-20 parts of prepared rhizome of rehmannia, 9-25 parts of angelica, 9-12 parts of white peony root and 6-10 parts of ligusticum wallichii according to parts by weight.
The preparation method comprises the following steps:
(1) the preparation method comprises the steps of drying the four Chinese herbal medicines of the radix rehmanniae preparata, the angelica, the white paeony root and the ligusticum wallichii for 24 hours in an oven, respectively placing the dried Chinese herbal medicines into a high-speed knife type multistage grinder for grinding, sieving the ground Chinese herbal medicines by a sieve of 60-100 meshes, accurately weighing the Chinese herbal medicines by using an electronic balance, mixing the Chinese herbal medicines according to a proportion, bagging and sealing for later use.
(2) Recovering strains: inoculating bacillus safensis and bacillus stratosphericus which are placed in an ultra-low temperature refrigerator at minus 80 ℃ into a 2216E liquid medium according to the inoculation amount of 1-5% (volume ratio) for culturing, and culturing for 20-28 h under the conditions of 24-28 ℃ and 160-220 rpm; counting the number of live bacteria by using a plate colony counting method, and when the number of live bacteria in the strain recovery culture medium reaches 3.2 multiplied by 108~5.2×108When cfu/ml, the culture of viable bacteria was stopped.
(3) Preparing a Chinese herbal medicine liquid fermentation culture medium: firstly, a certain amount of mixed Chinese herbal medicines are taken according to a certain proportion, pure water (g: ml) with the mass 4-6 times of that of the Chinese herbal medicines is added, and 4-6% of NaCl (M: V) and 3-5% of MgSO (MgSO) are respectively added in4·7H2O,，0.22~0.63% KH2PO4Adding 2-4% of cassava powder, placing the cassava powder into a steam sterilization pot, sterilizing the cassava powder for 35 minutes at 121 ℃, and placing the cassava powder in an ultra-clean workbench for cooling; then, 0.12-0.34% of L-glutamic acid and 0.2-1.2% of VC (both of which are subjected to ultraviolet sterilization) are added into the sterilized culture medium, the pH is adjusted to 6.8-7.4, and the prepared liquid culture medium is stored under the aseptic condition for later use.
(4) Mixing the bacillus safensis and the bacillus stratosphericus according to the ratio of 1: 1-1: 4 (V: V) to form composite bacillus, inoculating 3-11% of the composite bacillus into a Chinese herbal medicine liquid fermentation culture medium, and culturing at 160-210 rpm and 26-32 ℃ for 28-36 hours to obtain the mixed Chinese herbal medicine fermentation liquid.
(5) Extracting effective components: adding 6-10 times of pure water into the obtained Chinese herbal medicine fermentation liquor, leaching for 2.2-2.8 hours at a low temperature of 45 ℃, leaching for three times at a low temperature, and combining the filtrates of the three times; then adding 2 times of pure water into the leached dregs of a decoction, and fully soaking and washing twice; and combining the filtrates for 5 times, performing rotary evaporation at 45-55 ℃, concentrating the filtrate to be viscous, and performing freeze drying for 24-32 hours to obtain the effective components in the fermented Chinese herbal medicine.
The three fermented Chinese herbal medicine preparations with different concentrations and the corresponding culture feeds are respectively prepared according to the preparation method, namely, the effective components of the mixed fermentation of the rehmannia glutinosa, the angelica, the white paeony root and the ligusticum wallichii are added into the basic feed to obtain the prawn feeds containing 0.2 percent, 0.5 percent and 0.8 percent by mass respectively.
Example 2:
the influence of the Chinese herbal medicine fermented preparation of the prawns on the physiological health efficacy of the litopenaeus vannamei.
In the experiment, the inventor feeds the litopenaeus vannamei with the fermented Chinese herbal medicine preparation prepared in the example 1, and researches the actual effect of the invention on improving the physiological health efficacy of the litopenaeus vannamei by analyzing the related indexes of the physiological health of the litopenaeus vannamei.
1 materials and methods
1.1 materials
Litopenaeus vannamei for test (A)Litopenaeus vannamei) Purchased from a sunshine market seashell prawn breeding factory, the weight of the seashell prawn is 2.3 plus or minus 0.22g, and the ingredients of the basic feed for the test are as follows: by mass fraction, 34% of fish meal, 20% of soybean meal, 16.4% of peanut meal, 22% of wheat flour, 5% of soybean lecithin, 0.6% of multivitamins and 2% of compound minerals. The experimental calculation shows that the nutrient components in the feed are as follows: 42.4% of crude protein, 7.2% of crude fat and 8.4% of ash in mass fraction.
1.2 methods
1.2.1 test design
After temporarily breeding the litopenaeus vannamei for the test for 7 days, randomly dividing the litopenaeus vannamei into 7 groups, namely a control group, a test group 1, a test group 2, a test group 3, a test group 4, a test group 5 and a test group 6, wherein each group is provided with 3 replicates, and each replicate group is used for stocking 50 shrimps. The control group was fed with the basic feed, the test group 1, the test group 2 and the test group 3 were fed with the basic feed of unfermented Chinese herbal medicines added with 0.2%, 0.5% and 0.8% respectively per kg, and the test group 4, the test group 5 and the test group 6 were fed with the basic feed of prawn added with the fermented Chinese herbal medicines (prepared in example 1) added with 0.2%, 0.5% and 0.8% respectively per kg. After the culture experiment lasts for 21 days, the relevant immunity index of the prawn is determined. During the test period, the feed is fed for 2 times a day, the daily feeding amount is 5 percent of the weight, and during the test period, the water temperature, the salinity and the PH are 22-23 ℃,34 per thousand and 7.8-8.0 respectively.
1.2.2 measurement indices and methods
Samples were taken on days 0, 21, respectively, after the experimental feeds were fed. Taking 20 litopenaeus vannamei tails per group randomly, directly inserting sterilized No. 5 needle and 1mL syringe into the peripheral heart chamber around the posterior margin of the cephalic and thoracical carapace of the litopenaeus vannamei for blood sampling by about 3mm, and sucking 0.3mL improved precooled litopenaeus vannamei anticoagulant (0.34M NaCl, 0.01M KCl, 0.01M EDTA-Na) into the syringe before blood drawing20.01M HEPES, pH7.45, osmotic pressure 780 mOsm.kg-1) Finally, the ratio of anticoagulant to hemolymph is 1: 1. And (3) measuring the phagocytosis rate of a part of hemolymph samples, centrifuging the rest hemolymph samples at the temperature of 4 ℃ for 10min at 800g, taking blue supernatant, namely a plasma sample, and measuring the antibacterial activity and the total antioxidant capacity.
Cutting prawn hepatopancreas, washing with pre-cooled 8% normal saline to remove foreign matters, drying with absorbent paper, placing in mortar, adding liquid nitrogen, grinding, pulverizing, and storing in-80 deg.C ultra-low temperature refrigerator for determination of prawn hepatopancrease and lipase. Cutting off midgut of prawn, placing in mortar, adding liquid nitrogen, grinding, adding 1ml Trizol per 0.1g of ground intestine powder, shaking thoroughly, storing in-80 deg.C ultra-low temperature refrigerator, and determining PEN (para-shrimp peptide).
The method for measuring phagocytic activity was carried out according to the methods of the prior art references: immune responses and expression of Immune-related genes in a design flaw lattice tubular aggregate to an expressed Immune-N strain, Comparative Biochemistry and Physiology Part A; the antibacterial activity determination method is carried out according to the methods of the prior documents, and can be referred to as follows: an institute of infection, Purification and properties of microorganism induced microorganisms from a hydrolyzed microorganism of amplified pathogen of Hyperophara cecropia, European Journal of Biochemistry; the total antioxidant capacity is measured by using a Nanjing Makeup biological Limited company production kit; the protease activity was measured according to the methods of the prior art references: protein measurement with the Folin-phenol reagent; the lipase activity was measured according to the methods of the prior art references: compatibility and improvement of the thread determination methods for thread activity; the method for measuring the expression level of PEN (prawn peptide) is carried out according to the methods of the prior art references: analysis of relative gene expression data using real-time quantitative PCR and the 2- Δ CT method.
2 results
2.1 phagocytic Activity of blood cells
As can be seen from fig. 1: after 21 days of feeding the fermented Chinese herbal medicine immune preparation for the prawns, the phagocytic activity of blood cells of the prawns is obviously higher than that of a control group (P < 0.05), after 21 days, the phagocytic activity of six phagocytic activities of an experimental group is obviously higher than that of an unfermented Chinese herbal medicine feed group (P < 0.05), and the phagocytic activities of four and five experimental groups are higher than that of the unfermented Chinese herbal medicine feed group, but the difference is not significant (P > 0.05).
2.2 antibacterial Activity
As can be seen from fig. 2: after 21 days of feeding the fermented Chinese herbal medicine preparation for the prawns, the antibacterial activity of the prawns fed with the fermented Chinese herbal medicine feed group (experimental group five and six) is obviously higher than that of the unfermented Chinese herbal medicine feed group (P is less than 0.05). The antibacterial activity of the low-concentration fermented Chinese herbal medicine feed group (experimental group four) is higher than that of the unfermented Chinese herbal medicine feed group (experimental group one, two and three).
2.3 Total Oxidation resistance
As can be seen from fig. 3: after 21 days of feeding the fermented Chinese herbal medicine preparation for the prawns, the total antioxidant capacity of the blood plasma of the prawns is remarkably improved (P is less than 0.05), and the total antioxidant capacity of the prawns fed with the fermented Chinese herbal medicine feed group is remarkably higher than that of the prawns fed with the unfermented Chinese herbal medicine feed group (P is less than 0.05).
2.4 relative expression levels of PEN
As can be seen from fig. 4: after 21 days of feeding the prawn fermentation Chinese herbal medicine preparation, the prawn peptide gene expression level (P < 0.05) of the prawn intestinal tract is obviously improved, the prawn intestinal tract PEN gene expression levels of the experimental groups five and six are obviously higher than that of the unfermented Chinese herbal medicine feed group (P < 0.05), and the prawn intestinal tract PEN gene expression level of the fermented Chinese herbal medicine low-concentration group (the experimental group four) is higher than that of the unfermented Chinese herbal medicine feed group on average.
2.5 protease specific Activity
As can be seen from fig. 5: after 21 days of feeding the prawn fermented Chinese herbal medicine preparation, the specific activity of the prawn hepatopancreas protease of the medium-high dose group is obviously higher than that of the contrast group (P < 0.05), after 21 days, the specific activity of the prawn hepatopancreas protease of the experimental group four is higher than that of the contrast group, and the specific activities of the proteases of all groups added with the fermented Chinese herbal medicine are higher than those of the contrast group and the unfermented Chinese herbal medicine.
2.6 Lipase specific Activity
As can be seen from fig. 6: after 21 days of feeding the fermented Chinese herbal medicine preparation for the prawns and the prawns, the specific activities of the hepatopancreas lipase of the prawns and the prawns are all obviously higher than those of a control group (P < 0.05), after 21 days, the specific activities of the experiment group five and the experiment group six lipase are all obviously higher than those of an unfermented Chinese herbal medicine feed group (P < 0.05), and the specific activities of the lipase of a fermented Chinese herbal medicine low-dose group are higher than those of an unfermented Chinese herbal medicine high-dose group, but the difference is not significant (P > 0.05).
Experiments prove that on one hand, the shrimp fermentation Chinese herbal medicine preparation provided by the invention can improve the drug effect, promote the effective components in the Chinese herbal medicine to be separated out, and improve the utilization rate of the Chinese herbal medicine; on the other hand, the invention can improve the nonspecific immunity of the litopenaeus vannamei, enhance the disease resistance and reduce the loss of farmers to a certain extent. In addition, the Chinese herbal medicine preparation is safe and nontoxic, aquatic animals cannot generate drug dependence, and the concept of 'green cultivation, ecological cultivation and healthy cultivation' in China is met.

Claims (5)

1. The bacillus used for prawn feed is characterized by comprising bacillus safensis (Bacillus safensis)Bacillus safensis) And Bacillus stratosphericus (Bacillus stratosphericus）；
The Bacillus safensis bacterium (A), (B), (C), (B), (C), (B), (C), (B), (C)Bacillus safensis) The preservation number is: CGMCC number 22059, preservation date: 23/3/2021, depository: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North;
bacillus stratosphericus (A), (B) and (C)Bacillus stratosphericus) The preservation number is: CGMCC number 22060, preservation date: 23/3/2021, depository: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
2. A fermented Chinese herbal medicine preparation for prawn feed is characterized by being prepared by the following steps:
(1) pretreatment of Chinese herbal medicines: drying four Chinese herbal medicines of radix rehmanniae preparata, angelica sinensis, radix paeoniae alba and ligusticum wallichii for 24 hours in an oven, respectively putting the dried four Chinese herbal medicines into a high-speed knife type multistage grinder for grinding, sieving the ground four Chinese herbal medicines by a sieve of 60-100 meshes, accurately weighing the Chinese herbal medicines by using an electronic balance, mixing the Chinese herbal medicines according to a proportion, bagging and sealing the mixture for later use; the mixture in proportion is prepared from 12 to 20 parts by mass of prepared rehmannia root, angelica, white paeony root and szechuan lovage rhizome: 9-25 parts of: 9-12 parts of: mixing 6-10 parts;
(2) recovering strains: the Bacillus safensis strain of claim 1 (Bacillus safensis) placed in an ultra-low temperature refrigerator at-80 ℃Bacillus safensis) And Bacillus stratosphericus (Bacillus stratosphericus) Inoculating the strain into 2216E liquid medium according to the volume ratio of 1-5% for culture for 20-28 h under the conditions of 24-28 ℃ and 160-220 rpm; counting the number of live bacteria by using a plate colony counting method, and when the number of live bacteria in the strain recovery culture medium reaches 3.2 multiplied by 108~5.2×108Stopping the culture of the live bacteria when cfu/ml is reached;
(3) preparing a Chinese herbal medicine liquid fermentation culture medium: firstly, taking a certain amount of the Chinese herbal medicines mixed in proportion obtained in the step 1, adding pure water with the mass of 4-6 times of that of the Chinese herbal medicines to obtain a Chinese herbal medicine solution,adding NaCl with the mass fraction of 4-6% and MgSO 3-5% into the solution respectively4·7H2O, 0.22-0.63% KH2PO4Putting 2-4% of cassava flour into a steam sterilization pot, sterilizing at 121 ℃ for 35 minutes, and cooling in an ultra-clean workbench; then adding 0.12-0.34% of L-glutamic acid and 0.2-1.2% of VC by mass into the cooled Chinese herbal medicine solution, adjusting the pH to 6.8-7.4, preparing a solution, namely a Chinese herbal medicine liquid fermentation culture medium, and storing the solution for later use under the aseptic condition;
(4) preparing Chinese herbal medicine fermentation liquor: the bacillus safensis of the step 2 (A), (B), (C)Bacillus safensis) And Bacillus stratosphericus (Bacillus stratosphericus) Mixing the bacillus subtilis and the bacillus subtilis into composite bacillus according to the volume ratio of 1: 1-1: 4, inoculating 3-11% of the bacillus subtilis and the bacillus subtilis into the Chinese herbal medicine liquid fermentation medium obtained in the step (3), and culturing at the speed of 160-210 rpm and the temperature of 26-32 ℃ for 28-36 hours to obtain Chinese herbal medicine fermentation liquor;
(5) extracting effective components: adding pure water with the mass of 6-10 times of that of the obtained Chinese herbal medicine fermentation liquor, leaching for 2.2-2.8 hours at the low temperature of 45 ℃, leaching for three times at the low temperature, and combining the filtrates of the three times; then adding pure water with the mass of 2 times of that of the extracted medicine residues into the extracted medicine residues, and extracting twice; mixing the filtrates for 5 times;
(6) preparing a fermented Chinese herbal medicine preparation: and (4) carrying out rotary evaporation on the filtrate obtained in the step (5) at the temperature of 45-55 ℃, concentrating to be viscous, and carrying out freeze drying for 24-32 h to obtain the fermented Chinese herbal medicine preparation.
3. A prawn feed based on a fermented Chinese herbal medicine preparation is characterized in that the Chinese herbal medicine preparation of claim 2 is added into a basal feed, and the fermented Chinese herbal medicine preparation added into each kilogram of the basal feed accounts for 0.2-0.8 percent by mass.
4. The feed for prawns according to claim 3, characterized in that the fermented Chinese herbal preparation added per kilogram of basal feed is 0.2%, 0.5% or 0.8% by mass.
5. The prawn feed according to claim 3 or 4, wherein the basal feed comprises the following components: by mass, 34% of fish meal, 20% of soybean meal, 16.4% of peanut meal, 22% of wheat flour, 5% of soybean lecithin, 0.6% of vitamin complex and 2% of compound mineral.
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