Composite microecological preparation for aquatic products as well as preparation method and application of composite microecological preparation

Abstract

The invention discloses a compound micro-ecological preparation for aquatic products as well as a preparation method and application thereof, and belongs to the technical field of micro-ecological preparations. The microecological preparation disclosed by the invention comprises clostridium butyricum, bacillus licheniformis, enterococcus faecalis and lactobacillus plantarum. The microecological preparation is combined with concentrated cottonseed protein for use, can replace fish meal, is added into feed, can remarkably improve the immunocompetence, oxidation resistance and digestive enzyme activity of animals, and can improve the relative abundance of beneficial bacteria in intestinal tracts, inhibit the growth of pathogenic bacteria and regulate the microecological balance of the intestinal tracts after entering the intestinal tracts of the animals. According to the invention, aerobic bacteria and anaerobic bacteria are cooperatively used, oxygen in the surrounding environment is greatly consumed by bacillus licheniformis and enterococcus faecalis, the proliferation of beneficial bacteria such as clostridium butyricum and lactobacillus plantarum is promoted, and the probiotic effect is fully played.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of micro-ecological preparations, and particularly relates to a compound micro-ecological preparation for aquatic products and a preparation method and application thereof. BACKGROUND

[0002] Fish meal has been an important protein source in aquatic feed for a long time due to its rich nutrition, balanced amino acid ratio, good palatability, rich vitamins and minerals, and easy digestion and absorption. In recent years, with the increase of aquaculture species and the expansion of aquaculture scale, and the rapid development of industrialized aquaculture, the demand for fish meal has increased. Under this background, people are constantly looking for other protein ingredients to reduce the demand for fish meal in feed in order to achieve partial or complete replacement of fish meal.

[0003] Plant protein sources are considered to be ideal protein sources for replacing fish meal due to their low price, stable yield, and wide sources. Although plant protein sources have been widely used in aquatic feed and their safety has been fully proven, their large-scale application in feed is limited due to their poor palatability, unbalanced amino acid ratio, low available phosphorus content, and the presence of various anti-nutritional factors. Concentrated cottonseed protein is of great concern due to its high-quality protein, but its use in feed is limited due to the presence of anti-nutritional factors such as gossypol, phytic acid, and polysaccharides. Therefore, high-level plant protein replacement of fish meal will inevitably affect the absorption and utilization of nutrients by fish and even directly harm the health of aquatic animals, causing negative effects on growth, antioxidant capacity, immunity, and intestinal flora structure.

[0004] To further alleviate the negative effects of high-level plant protein replacement of fish meal on farmed animals, researchers have adopted many strategies. One notable method is to add green additives (micro-ecological preparations) to feed. Probiotics as feed additives can bring benefits to farmed animals in many aspects, including improving digestive system health, enhancing the immune system, promoting nutrient absorption, reducing stress response, and improving feed quality. In addition, as living microorganisms, probiotics can regulate the intestinal flora of farmed animals, maintain the balance and stability of the intestinal microbiota through competitive exclusion, production of antibacterial substances, regulation of intestinal environment, enhancement of immune regulation, and promotion of nutrient metabolism. Currently, many studies have added probiotics to feed with high-level plant protein replacement of fish meal to alleviate the negative effects of plant protein on farmed animals.

[0005] Currently, the addition of probiotics in feed is mainly single-strain, which only has specific efficacy and limited inhibitory effect on certain harmful bacteria. In addition, single-strain may be inhibited by the intestinal environment and other microorganisms of farmed animals in some cases, resulting in limited survival and reproduction. SUMMARY

[0006] In view of the above prior art, the present application provides a compound micro-ecological preparation for aquatic products and a preparation method and application thereof, so as to solve the technical problems of limited efficacy of the existing micro-ecological preparation and difficulty in growth and reproduction of single bacterial species.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is to provide a compound micro-ecological preparation, which comprises Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum.

[0008] The beneficial effects of the present application are as follows: Clostridium butyricum, Enterococcus faecalis, Bacillus licheniformis and Lactobacillus plantarum are probiotics from different species, which have different flora components and metabolic functions. Clostridium butyricum is a strict anaerobic gram-positive bacterium with good tolerance, which can produce short-chain fatty acids, digestive enzymes and vitamins and other beneficial substances; Bacillus licheniformis can promote the growth and reproduction of beneficial bacteria and maintain the balance of intestinal flora, which helps to improve the intestinal micro-ecological environment through competitive exclusion of harmful bacteria, production of beneficial metabolites and other ways; Enterococcus faecalis and Lactobacillus plantarum, as representatives of lactic acid bacteria, have many important functions such as regulating intestinal micro-ecological balance, promoting food fermentation, enhancing immunity, antibacterial and antioxidant effects.

[0009] Bacillus licheniformis and Enterococcus faecalis, as aerobic bacteria, have a unique mechanism of action in biological oxygen deprivation, which can create a low-oxygen environment by consuming oxygen, thereby inhibiting pathogenic bacteria that need oxygen to grow and reproduce, promoting the growth of Clostridium butyricum and Lactobacillus plantarum, two anaerobic bacteria, and improving their survival rate in the intestine, thereby playing a role in the intestine; Clostridium butyricum and Enterococcus faecalis produce butyric acid, which is beneficial to intestinal health and can maintain the integrity of intestinal mucosa and anti-inflammatory capacity, while Bacillus licheniformis and Lactobacillus plantarum can promote the production of short-chain fatty acids, effectively increase the yield of butyric acid and improve the intestinal environment; the compound preparation prepared by combining Clostridium butyricum, Enterococcus faecalis, Bacillus licheniformis and Lactobacillus plantarum comprehensively utilizes the characteristics of different species of probiotics and presents a complementary effect between multiple probiotic species, thereby improving the overall effect and making it superior to single probiotic preparation, achieving more comprehensive intestinal micro-ecological regulation and promoting overall health.

[0010] Clostridium butyricum, Enterococcus faecalis, Bacillus licheniformis and Lactobacillus plantarum maintain the intestinal health of aquatic animals through metabolic complementation, immune regulation, synergistic antibacterial and environmental regulation mechanisms. In addition, they further enhance their comprehensive probiotic effect through signal molecule interaction, intestinal barrier function promotion and symbiotic relationship. Clostridium butyricum and Lactobacillus plantarum produce butyric acid and lactic acid respectively, reduce the pH value of the intestinal tract, inhibit harmful bacteria, and provide a favorable growth environment for each other and other probiotics such as Enterococcus faecalis and Bacillus licheniformis. The nutrients generated by the digestive enzymes secreted by Bacillus licheniformis further promote the growth and metabolism of other probiotics. Enterococcus faecalis and Lactobacillus plantarum activate the intestinal immune system, enhance the host's resistance to pathogenic bacteria, indirectly protect other probiotics, and maintain the balance of intestinal microecology. The antibacterial substances (such as antibacterial peptides and lipopeptide antibiotics) produced by the four probiotics work together to form a broad-spectrum antibacterial effect, effectively inhibit pathogenic bacteria, and promote the growth and colonization of beneficial bacteria.

[0011] The application further discloses a preparation method of the compound microecological preparation.

[0012] (1) Culturing of probiotics:

[0013] S11: inoculating Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum into culture media respectively for culturing, wherein Clostridium butyricum and Lactobacillus plantarum are cultured anaerobically, and Bacillus licheniformis and Enterococcus faecalis are cultured aerobically, and bacterial liquid is obtained respectively;

[0014] S12: centrifuging the bacterial liquid respectively, removing supernatants and collecting bacterial precipitates to obtain bacterial precipitates;

[0015] S13: washing the bacterial precipitates with PBS, mixing the bacterial precipitates of Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum according to a volume ratio of 1-3:1-2:1-2:1-3 to obtain mixed bacterial precipitates;

[0016] (2) Preparation of freeze-drying protective solution:

[0017] S21: dissolving trehalose and sodium glutamate in PBS, adding 4-12 g of trehalose and 0.3-1.5 g of sodium glutamate to 100 mL of PBS to obtain a mixed solution, and sterilizing the mixed solution;

[0018] S22: sterilizing β-cyclodextrin, and adding 6-15 g of sterilized β-cyclodextrin to 100 mL of the mixed solution to obtain a freeze-drying protective solution;

[0019] (3) Preparation of compound freeze-dried bacterial powder:

[0020] S31: The hydroxypropyl starch is sterilized, and then mixed with the mixed bacteria body precipitate at a mass ratio of 0.5-2:1 to obtain a mixture;

[0021] S32: The mixture is added to a freeze-drying protective solution, stirred, frozen, and then dried to obtain a compound microbial agent.

[0022] Further, the culture medium in S11 is cultured for 24-48 h at 37 DEG C.

[0023] Further, the centrifugation condition parameters in S12 are that the rotation speed is 6000 r / min, and the centrifugation time is 10 min.

[0024] Further, the sterilization mode in S21, S22 and S31 is 121 DEG C sterilization for 15 min.

[0025] Further, the freezing mode in S32 is-80 DEG C freezing for 2 h, and the equipment for drying into a powder is a freeze dryer.

[0026] The application further discloses an application of the compound microbial agent in aquaculture.

[0027] Further, the components of the aquatic feed further include concentrated cottonseed protein.

[0028] Further, the adding amount of the compound microbial agent is 0.25%-1% of the mass of the aquatic feed.

[0029] Further, the feeding object of the aquatic feed is Litopenaeus vannamei.

[0030] The application has the following beneficial effects:

[0031] 1. The Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum used in the compound microbial agent prepared in the application are probiotic species that exist in the intestinal tract of Litopenaeus vannamei, and have no influence on the health of Litopenaeus vannamei, and have a promoting effect on immunity, oxidation resistance and digestive enzyme indexes, improve the relative abundance of beneficial bacteria such as Firmicutes, Lactobacillus and Clostridium butyricum, reduce the relative abundance of potential pathogenic bacteria such as Vibrio and Maribacter, and regulate the intestinal microecological balance.

[0032] 2. The aerobic bacteria and anaerobic bacteria are applied in cooperation, the oxygen in the surrounding environment is consumed by the Bacillus licheniformis and Enterococcus faecalis, the growth of beneficial bacteria such as Clostridium butyricum and Lactobacillus is promoted, and the probiotic effect is fully exerted.

[0033] 3. The use of the compound microecological preparation of the present application in combination with concentrated cottonseed protein realizes a fish meal replacement mixture, which is beneficial to improve the economic benefits of breeders, BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The morphological diagram of the compound microecological preparation prepared for Example 2;

[0035] Figure 2 The figure for analyzing the antioxidant and immune enzyme activities of the experimental shrimp in different treatment groups in Experimental Example 2;

[0036] Figure 3 The figure for analyzing the intestinal digestive enzyme activities of the experimental shrimp in different treatment groups in Experimental Example 2;

[0037] Figure 4 The figure for analyzing the intestinal flora alpha diversity of the experimental shrimp in different treatment groups in Experimental Example 2;

[0038] Figure 5 The figure for analyzing the intestinal flora principal coordinates of the experimental shrimp in different treatment groups in Experimental Example 2;

[0039] Figure 6 The figure for analyzing the top 15 bacteria in terms of relative abundance at the level of microbial door in the intestinal tract of the experimental shrimp in different treatment groups in Experimental Example 2;

[0040] Figure 7 The figure for analyzing the top 15 bacteria in terms of relative abundance at the level of microbial genus in the intestinal tract of the experimental shrimp in different treatment groups in Experimental Example 2;

[0041] Figure 8 The figure for analyzing the top 15 bacteria in terms of relative abundance at the level of microbial species in the intestinal tract of the experimental shrimp in different treatment groups in Experimental Example 2;

[0042] Figure 9 The figure for analyzing the LEfSe difference test of the intestinal flora of the experimental shrimp in different treatment groups in Experimental Example 2 (LDA≥3, P<0.05);

[0043] Different lowercase letters on the column chart represent significant differences (P<0.05), and the same or no letters represent no significant differences (P>0.05). DETAILED DESCRIPTION

[0044] The Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum selected in the present application are common bacterial species, which are provided by Biwofeng Biotechnology (Guangdong) Co., Ltd.; the culture medium used for culturing the bacterial species is a Clostridium culture medium, a nutrient broth culture medium and an MRS broth culture medium, which are purchased from Guangdong Huan Kai Microbial Technology Co., Ltd., and the above bacterial species and culture medium can be obtained by the public channel by ordinary technical personnel in the field.

[0045] Clostridium butyricum is cultured in Clostridium enrichment medium, Bacillus licheniformis is cultured in nutrient broth medium, Enterococcus faecalis and Lactobacillus plantarum are cultured in MRS broth medium, and the specific formulations of the above three culture media are as follows:

[0046] Table 1. Formulation of Clostridium enrichment medium

[0047]

[0048] Table 2. Formulation of nutrient broth medium

[0049]

[0050] Table 3. Formulation of MRS broth medium

[0051]

[0052] The microorganisms involved in the present application are represented by Chinese names, and the microorganisms that have not yet been officially named in Chinese are named according to the nomenclature rules of microorganisms in the present application. The following is a table of the Chinese names of the microorganisms in the present application and the original Latin names:

[0053] Table 4. Table of Chinese names of microorganisms in the present application and Latin names

[0054] Chinese name Latin name Phylum Proteobacteria Proteobacteria Phylum Bacteroidota Bacteroidota Phylum Verrucomicrobiota Verrucomicrobiota Phylum Firmicutes Firmicutes Phylum Actinobacteriota Actinobacteriota Phylum Cyanobacteria Cyanobacteria Phylum Patescibacteria Patescibacteria Phylum Fusobacteriota Fusobacteriota Phylum Bdellovibrionota Bdellovibrionota Phylum Deinococcota Deinococcota Phylum Chloraflexi Chloraflexi Phylum Acidobacteriota Acidobacteriota Phylum Desulfobacterota Desulfobacterota Phylum Myxococcota Myxococcota Donghicola Ruegeria Tamlana Vibrio Haloferula Tenacibaculum Pelomonas Ralstonia Hoppeia Pseudoalteromonas Enterococcus Sungkyunkwania Maribacter Pir4_lineage Acinetobacter Tamlana_agariborans Tenacibaculum_mesophilum Ralstonia_insidiosa Mangrovimonas_sp_ST2L15 Flavobacterium_sp_MS-3 Haloferula_rosea Acinetobacter_schindleri Demequina_globuliformis Ilumatobacter_fluminis Planctomycete_DDSe3016 Maribacter_sp Tamlana_crocina Clostridium_butyricum Pluralibacter_gergoviae Rubritalea Hypnocyclicus Gimesiaceae Pir4_lineage Planctomycete_DDSe3016 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Pir4_lineage.planctomycete_DDse3016 Gillisia Tenacibaculum Tenacibaculum mesophilum Ruegeria Alphaproteobacteria Rhidobacterales Rhodobacteraceae Demequina Demequina globuliformis Pirellulaceae Pirellulales Planctomycetes Planctomycetota Psychrobacter Myxococcata Psychrobacter maritimus Saccharimonadales Aaccharimonadia Micavibrionales Microbacteriacceae Mesorhizobium Bacillales Haloferula rosea Rubritalcaccae Donggicola Weeksellaceae Lactobacillus Lactobacillaceae Clostridia Lactobacillales Firmnicutes Persicirhabdus Kurthia Escherichia_Shigella Boseongicola Planococcaceae Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0055] The specific embodiments of the present application will be described in detail below in conjunction with the examples.

[0056] Example 1

[0057] Clostridium butyricum and Lactobacillus plantarum need to be cultured anaerobically, and Bacillus licheniformis and Enterococcus faecalis need to be cultured aerobically, and the corresponding culture media need to be prepared by different operations, and the specific operation steps are as follows:

[0058] Clostridium enrichment medium (Clostridium butyricum): The prepared liquid medium is placed in a 500 ml anaerobic flask, nitrogen is used to discharge the oxygen in the liquid medium, and then the anaerobic flask is sealed and sterilized, 121 ℃ for 15 min.

[0059] Nutrient broth medium (Bacillus licheniformis): The prepared liquid medium is placed in a 500 ml conical flask, and the conical flask is sterilized at 121 ℃ for 15 min.

[0060] MRS broth medium (Lactobacillus plantarum): The prepared liquid medium is placed in a 500 ml anaerobic flask, nitrogen is used to discharge the oxygen in the liquid medium, and then the anaerobic flask is sealed and sterilized, 121 ℃ for 15 min.

[0061] MRS broth medium (Enterococcus faecalis): the prepared liquid medium was placed in a 500 ml conical flask, and the conical flask was sterilized at 121℃ for 15 min.

[0062] Example 2

[0063] A composite microecological preparation includes Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum; the composite microecological preparation in the embodiment is prepared through the following steps:

[0064] (1) Cultivation of probiotics

[0065] S11: Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum were inoculated (1% inoculation amount) into the corresponding liquid medium prepared in Example 1 respectively, and cultured at 37℃ for 36 h, to obtain bacterial liquid of Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum respectively;

[0066] S12: The cultured bacterial liquid was centrifuged at a speed of 6000 r / min for 10 min, and the supernatant was removed to collect the bacterial precipitate;

[0067] S13: The bacterial precipitate was washed with PBS for 2 times, and the bacterial precipitates of the four probiotics were mixed according to the volume ratio of the four bacterial liquids in S11, and the volume ratio of Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum was 2:1.5:1.5:2, to obtain a mixed bacterial precipitate;

[0068] (2) Preparation of freeze-drying protective solution

[0069] S21: Trehalose and sodium glutamate were dissolved in PBS, 8 g of trehalose and 0.9 g of sodium glutamate were added to 100 mL of PBS, to obtain a mixed solution, and the mixed solution was sterilized at 121℃ for 15 min;

[0070] S22: β-cyclodextrin was sterilized at 121℃ for 15 min in dry powder state, 10 g of sterilized β-cyclodextrin was added to 100 mL of the mixed solution, and stirred uniformly to obtain a freeze-drying protective solution;

[0071] (3) Preparation of composite freeze-dried bacterial powder

[0072] S31: Hydroxypropyl starch was sterilized at 121℃ for 15 min in dry powder state, and then mixed with the mixed bacterial precipitate at a mass ratio of 1.3:1 to obtain a mixture;

[0073] S32: Take 8% of the total volume of the four kinds of bacteria liquid in S11 as the freeze-drying protective solution, add the mixture to it, then perform thorough stirring, after stirring uniformly, freeze at -80℃ for 2 h, after complete freezing, place in a freeze dryer until it becomes dry powder, to obtain the compound microbial agent.

[0074] Example 3

[0075] A compound microecological preparation, comprising Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum; the compound microecological preparation in the embodiment is prepared through the following steps:

[0076] (1) Culturing of probiotics

[0077] S11: inoculate (1% inoculation amount) Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum into the corresponding liquid culture medium prepared in Example 1 respectively, and culture at 37℃ for 36 h, to obtain the bacteria liquid of Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum respectively;

[0078] S12: centrifuge the cultured bacteria liquid respectively, centrifuge at 6000 r / min for 10 min, remove the supernatant and collect the precipitate to obtain the bacteria precipitate;

[0079] S13: wash the bacteria precipitate once with PBS, mix the bacteria precipitate of the four kinds of probiotics according to the volume ratio of the four kinds of bacteria liquid in S11, the volume ratio of Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum is 1:1:1:1, to obtain the mixed bacteria precipitate;

[0080] (2) Preparation of freeze-drying protective solution

[0081] S21: dissolve trehalose and sodium glutamate in PBS, add 4 g of trehalose and 0.3 g of sodium glutamate to 100 mL of PBS, to obtain a mixed solution, sterilize the mixed solution at 121℃ for 15 min;

[0082] S22: sterilize β-cyclodextrin at 121℃ for 15 min in dry powder state, add 6 g of sterilized β-cyclodextrin to 100 mL of the mixed solution, stir uniformly, to obtain the freeze-drying protective solution;

[0083] (3) Preparation of compound freeze-dried bacteria powder

[0084] S31: sterilize hydroxypropyl starch at 121℃ for 15 min in dry powder state, then mix with the mixed bacteria precipitate at a mass ratio of 0.5:1, to obtain a mixture;

[0085] S32: Take 5% of the total volume of the four kinds of bacteria liquid in S11 as the freeze-drying protective solution, add the mixture to it, then stir thoroughly, freeze at -80℃ for 2 h after uniform stirring, and then place it in a freeze dryer until it becomes dry powder to obtain the compound microbial agent.

[0086] Example 4

[0087] A compound microecological preparation includes Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum; the compound microecological preparation in the embodiment is prepared through the following steps:

[0088] (1) Cultivation of probiotics

[0089] S11: inoculate (1% inoculation amount) Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum into the corresponding liquid culture medium prepared in Example 1, respectively, and cultivate at 37℃ for 36 h to obtain Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum liquid, respectively;

[0090] S12: centrifuge the cultured bacteria liquid, centrifuge at 6000 r / min for 10 min, remove the supernatant and collect the precipitate to obtain the bacteria precipitate;

[0091] S13: wash the bacteria precipitate with PBS for 3 times, mix the bacteria precipitate of the four kinds of probiotics according to the volume ratio of the four kinds of bacteria liquid in S11, the volume ratio of Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum is 3:2:2:3, to obtain the mixed bacteria precipitate;

[0092] (2) Preparation of freeze-drying protective solution

[0093] S21: dissolve trehalose and sodium glutamate in PBS, add 12 g of trehalose and 1.5 g of sodium glutamate to 100 mL of PBS to obtain a mixed solution, and sterilize the mixed solution at 121℃ for 15 min;

[0094] S22: sterilize β-cyclodextrin at 121℃ for 15 min in dry powder state, add 15 g of sterilized β-cyclodextrin to 100 mL of the mixed solution, stir uniformly to obtain the freeze-drying protective solution;

[0095] (3) Preparation of compound freeze-dried bacteria powder

[0096] S31: sterilize hydroxypropyl starch at 121℃ for 15 min in dry powder state, then mix with the mixed bacteria precipitate at a mass ratio of 2:1 to obtain a mixture;

[0097] S32: 10% of the total volume of the four kinds of bacteria solution in S11 was taken as the freeze-drying protective solution, the mixture was added, then fully stirred, and after uniform stirring, frozen at-80℃ for 2h, and after complete freezing, placed in a freeze dryer until dry powder was obtained, to obtain the compound microbial agent.

[0098] Comparative Example 1

[0099] Preparation of Clostridium butyricum microecological preparation

[0100] (1) Cultivation of probiotics

[0101] S11: Clostridium butyricum was inoculated (1% inoculation amount) into the corresponding liquid medium prepared in Example 1, and cultured at 37℃ for 36h to obtain a Clostridium butyricum bacterial solution;

[0102] S12: The cultured bacterial solution was centrifuged at a speed of 6000r / min for 10min, the supernatant was removed, and the bacterial sediment was collected;

[0103] S13: The bacterial sediment was washed with PBS for 2 times;

[0104] (2) Preparation of freeze-drying protective solution

[0105] This step is the same as step (2) of preparing the freeze-drying protective solution in Example 2;

[0106] (3) Preparation of compound freeze-dried bacterial powder

[0107] S31: Hydroxypropyl starch was sterilized at 121℃ for 15min in dry powder state, then mixed with the bacterial mixed sediment at a mass ratio of 7:1 to obtain a mixture;

[0108] S32: 8% of the volume of the bacterial solution in S11 was taken as the freeze-drying protective solution, the mixture was added, then fully stirred, and after uniform stirring, frozen at-80℃ for 2h, and after complete freezing, placed in a freeze dryer until dry powder was obtained, to obtain the Clostridium butyricum bacterial agent.

[0109] Experimental Example 1

[0110] Application of compound microecological preparation in Litopenaeus vannamei

[0111] This experiment is divided into a control group, an experimental group and a comparison group, because the components and effects of the compound microecological preparation prepared in Examples 2-4 of the application are similar, the application effect of the compound microecological preparation prepared in Example 2 is taken as an example to illustrate, ​ is a morphological diagram of the compound microecological preparation.

[0112] Penaeus vannamei for experiment were purchased from a local farm in Donghai Island, Zhanjiang, and were placed in 0.5 cubic meter aquaculture barrels for feeding. Fish meal, concentrated cottonseed protein, the compound microecological preparation prepared in the above examples, and Clostridium butyricum microecological preparation were mixed with the basal feed as additives to feed Penaeus vannamei. The basal feed was divided into a control group (FM) with 25% fish meal protein and an experimental group (CPC) with 40% fish meal replaced by concentrated cottonseed protein. The composition of the basal feed in the FM group and the CPC group is shown in Table 5:

[0113] Table 5 Composition and ingredient content of basal feed

[0114] Control group (FM) feed: The control group feed contains 25% fish meal, which is referred to as the FM group.

[0115] Experimental group feed: The experimental group uses concentrated cottonseed protein to replace 40% of the fish meal in the control group (FM) and adds 0%, 0.25%, and 0.50% of the compound microecological preparation prepared in Example 1 to the basal feed, which is referred to as the CPC, 0.25%, and 0.50% groups. The remaining ingredients and composition are the same as those of the control group feed.

[0116] Comparison group (CB 0.50%) feed: The comparison group uses concentrated cottonseed protein to replace 40% of the fish meal in the control group (FM) and adds 0.50% of the Clostridium butyricum microecological preparation prepared in Comparative Example 1 to the basal feed, which is referred to as the CB 0.50% group. The remaining ingredients and composition are the same as those of the control group feed.

[0117] The addition amounts of fish meal, concentrated cottonseed protein, and microecological preparation in each group are shown in Table 6:

[0118] Table 6 Composition and ingredient content of additives

[0119] The addition of the microbial agent is carried out by mixing and feeding. To ensure the activity of probiotics, the experimental feed is prepared daily.

[0120] The experiment includes five groups: FM, CPC, 0.25%, 0.50%, and CB 0.50%, with three replicates in each group and 25 shrimp in each replicate. Feeding is carried out four times a day at 7:00, 11:00, 17:00, and 21:00, and the breeding period is three weeks.

[0121] Experimental Example 2

[0122] Sample collection and analysis

[0123] After 3 weeks of experiment, the serum antioxidant and immune enzyme activity, intestinal digestive enzyme activity and intestinal microbiota of the experimental shrimps were detected. The data were analyzed by one-way ANOVA and Duncan multiple range test using SPSS 23.0 (IBM, USA) statistical software to determine whether there was a significant difference between groups at the level of P<0.05. The data were expressed as mean ± SEM.

[0124] 1. Serum antioxidant and immune enzyme activity detection

[0125] The blood lymph of 6 shrimps randomly selected from each barrel was collected in a 1.5 ml centrifuge tube, and the supernatant was collected after centrifugation for enzyme activity analysis. The serum superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GPX), complement 3 (C3), complement 4 (C4) and alkaline phosphatase (AKP) commercial kits were used for detection.

[0126] The serum antioxidant and immune enzyme activities of different treatment groups are shown in ​ As can be seen from the figure, the serum SOD, GPX and AKP of the FM and CPC groups were significantly lower than those of the 0.25% and 0.50% groups (P<0.05), the MDA activity of the 0.50% group was significantly lower than that of the CPC and FM groups (P<0.05), the C3 and C4 activities of the CPC group were significantly lower than those of the other three groups (P<0.05), and the antioxidant and immune enzyme activities of the CB 0.50% group were significantly lower than those of the 0.50% group (P<0.05).

[0127] 2. Intestinal digestive enzyme activity detection

[0128] The intestinal tract of 4 shrimps randomly selected from each barrel was used for digestive enzyme detection. The digestive enzymes included trypsin, amylase and lipase, which were all detected by commercial kits.

[0129] The intestinal digestive enzyme activities of different treatment groups are shown in ​ As can be seen from the figure, the trypsin and amylase activities of the 0.25% and 0.50% groups were significantly higher than those of the other two groups, and the activity of the 0.50% group was the highest (P<0.05); the lipase activity was the lowest in the CPC group, and the activity of the 0.50% group was the highest (P<0.05); the three digestive enzyme activities of the CB 0.50% group were significantly lower than those of the 0.50% group (P<0.05), the trypsin activity was significantly higher than that of the FM and CPC groups, and the lipase activity was significantly higher than that of the CPC group (P<0.05).

[0130] 3. Intestinal microbiota analysis

[0131] The intestinal tract of 6 shrimps randomly selected from each barrel was stored at -80°C for intestinal microbiota analysis.

[0132] Statistical analysis of alpha diversity index is shown in Table 2 ​ As can be seen from the figure, there is no significant difference in the alpha diversity index of each group; ​ The results of principal coordinate analysis (PCoA) are shown in Figure 2, and it can be seen from the figure that there is no overlap between the samples in the 0.25%, 0.50% and CB 0.50% groups, indicating that the microbial community structure is different among the three groups.

[0133] The top 15 bacteria with high relative abundance at the phylum level are shown in Table 3 ​ As can be seen from the figure, the relative abundance of Firmicutes in the 0.25%, 0.50% and CB 0.50% groups is significantly increased (P<0.05), and the relative abundance of Verrucomicrobia in the 0.25% group is significantly the highest (P<0.05).

[0134] The top 15 bacteria with high relative abundance at the genus level are shown in Table 4 ​ As can be seen from the figure, the relative abundance of Vibrio, Marinobacter and Pir4 lineage in the 0.50% group is significantly reduced (P<0.05).

[0135] The top 15 bacteria with high relative abundance at the species level are shown in Table 5 ​ As can be seen from the figure, the relative abundance of Ralstonia eurydice and Acinetobacter schindleri in the 0.50% group is significantly increased (P<0.05); it is worth noting that the relative abundance of Clostridium butyricum in the 0.25% and 0.50% groups is significantly increased, and the highest in the 0.50% group (P<0.05), while the CB 0.50% group basically does not detect the existence of Clostridium butyricum, because Enterococcus faecalis and Bacillus licheniformis in the complex microecological preparation are aerobic bacteria that can create a low-oxygen environment by consuming oxygen, promoting the growth and reproduction of Clostridium butyricum.

[0136] LEfSe analysis further shows the bacterial categories with significant differences in each group, as shown in Figure 3 ​ As can be seen from the figure, the indicator species in the 0.50% group are mostly beneficial bacteria, such as Firmicutes, Clostridia and Lactobacillus.

[0137] In summary, after replacing fish meal with concentrated cottonseed protein, the addition of complex microecological preparation and Clostridium butyricum microecological preparation can promote the immune, antioxidant and digestive enzyme indicators of Litopenaeus vannamei, and increase the abundance of beneficial bacteria in the intestine. By further comparing the complex microecological preparation and Clostridium butyricum microecological preparation, it is found that the effect of the complex microecological preparation is better, and the effect of adding 0.50% is the best, and through 16S determination, it is found that the content of Clostridium butyricum in the complex microecological preparation is significantly increased.

[0138] Although the specific embodiments of the present application have been described in detail, it should be understood that the present patent is not limited to the details of the foregoing embodiments. Various modifications and changes can be made to the described embodiments without departing from the scope of the present patent.

Claims

1. A compound microecological preparation, characterized in that: The compound microecological preparation includes Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis, and Lactobacillus plantarum.

2. The preparation method of the compound microecological preparation according to claim 1, characterized in that, Includes the following steps: (1) Cultivation of probiotics: S11: Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum were inoculated into the culture medium and cultured separately. Clostridium butyricum and Lactobacillus plantarum were cultured anaerobically, while Bacillus licheniformis and Enterococcus faecalis were cultured aerobically to obtain bacterial solutions. S12: Centrifuge the bacterial solutions separately, remove the supernatant and collect the precipitate to obtain bacterial cell precipitate; S13: The bacterial precipitate was washed with PBS. The precipitates of Clostridium butyricum, Bacillus licheniformis, Enterococcus faecalis and Lactobacillus plantarum were mixed in a volume ratio of 1~3:1~2:1~2:1~3 to obtain a mixed bacterial precipitate. (2) Preparation of freeze-drying protection solution: S21: Dissolve trehalose and monosodium glutamate in PBS. Add 4-12 g of trehalose and 0.3-1.5 g of monosodium glutamate to every 100 mL of PBS to obtain a mixed solution. Sterilize the mixed solution. S22: Sterilize β-cyclodextrin, add 6~15 g of sterilized β-cyclodextrin to every 100 mL of mixed solution to obtain lyophilized protective solution; (3) Preparation of compound freeze-dried bacterial powder: S31: Sterilize hydroxypropyl starch, then mix it with the mixed bacterial precipitate at a mass ratio of 0.5~2:1 to obtain a mixture; S32: Add the mixture to the freeze-drying protective solution, stir, freeze and make into dry powder to obtain the compound microbial agent.

3. The method for preparing the compound microecological preparation according to claim 2, characterized in that: The culture conditions for the bacteria in the S11 culture medium are 37℃ for 24-48 h.

4. The method for preparing the compound microecological preparation according to claim 2, characterized in that, The centrifugation conditions in S12 are: rotation speed of 6000 r / min and centrifugation time of 10 min.

5. The method for preparing the compound microecological preparation according to claim 2, characterized in that: The sterilization method for S21, S22 and S31 is sterilization at 121℃ for 15 min.

6. The method for preparing the compound microecological preparation according to claim 2, characterized in that: The freezing method in S32 is freezing at -80℃ for 2 hours, and the equipment used to produce dry powder is a freeze dryer.

7. The application of the compound microecological preparation according to claim 1 in aquaculture, characterized in that: The compound microecological preparation is added as an additive to aquatic feed.

8. The application according to claim 7, characterized in that: The aquatic feed also includes concentrated cottonseed protein.

9. The application according to claim 8, characterized in that: The amount of the compound microecological preparation added is 0.25% to 1% of the mass of the aquatic feed.

10. The application according to any one of claims 7-9, characterized in that: The aquatic feed is intended for Litopenaeus vannamei.

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