CN111671001B

CN111671001B - Microecological composite premixed feed containing pediococcus acidilactici and application thereof

Info

Publication number: CN111671001B
Application number: CN202010512546.0A
Authority: CN
Inventors: 郭本月; 刘广; 倪梦丽; 魏万权; 周怡; 王磊
Original assignee: Qingdao Guan Tai Biotechnology Co ltd; Qingdao Master Biological Technology Co ltd
Current assignee: Qingdao Guan Tai Biotechnology Co ltd; Qingdao Master Biological Technology Co ltd
Priority date: 2020-06-08
Filing date: 2020-06-08
Publication date: 2022-04-08
Anticipated expiration: 2040-06-08
Also published as: CN111671001A

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a microecological compound premix feed and application thereof in aquaculture. The microecological compound premix feed comprises pediococcus acidilactici (A)Pediococcus acidilactici) MST02 with the preservation number of CCTCC NO: M2020094 can obviously improve the intestinal health of aquatic animals, improve the immunity and disease resistance of the aquatic animals, improve the digestion and utilization rate of feed, promote the growth of the aquatic animals, and can be widely applied to the field of aquatic feeds.

Description

Microecological composite premixed feed containing pediococcus acidilactici and application thereof

Technical Field

The invention relates to the technical field of functional microorganism screening, in particular to a micro-ecological composite premixed feed containing pediococcus acidilactici and application thereof.

Background

The health of aquaculture animals is largely influenced by their intestinal health, which plays an important role in the healthy growth of the aquatic animals, since the animal intestines are the main site for the digestion and absorption of nutrients. In the present stage, the aquaculture industry pursues high benefits, accompanied by high-density cultivation and the pursuit of growth speed, which requires that the cultivated animals face high-load feed nutrition and the intestinal tract bears great burden. The intestinal mucosa of aquatic animals is damaged due to long-term high nutrition load, and further intestinal diseases are caused. The high incidence of intestinal diseases of aquatic animals becomes an important factor restricting the development of the aquaculture industry at present, and people gradually realize that the good intestinal tracts of the aquatic animals are an important guarantee for the success of the aquaculture in view of the important role of the intestinal tract health on the immunity and growth of organisms.

The addition of antibiotics in the feed is a traditional way for solving the intestinal health problem of aquatic animals, but the use of antibiotics can cause the generation of drug resistance of pathogenic bacteria and finally harm human health. The probiotics can participate in the microbial balance in the body of animals, can not generate drug resistance or residue in the body of aquatic organisms, and a plurality of scholars add active probiotics into feed as additives to improve the immunity of the animals by maintaining the balance of intestinal flora so as to achieve the purposes of disease prevention and disease resistance. Researches show that the probiotics are added into the feed, so that the aquatic animals can be promoted to grow rapidly, the survival rate is improved, the incidence rate of diseases can be reduced by enhancing the immunity of the aquatic animals, and the water quality of the surrounding environment can be improved. The probiotics protect the immune system of the organism by establishing a microbial flora in the intestinal tract of the organism, thereby improving the immunity of the organism and enhancing the digestion and absorption capacity of the organism. The probiotics can directly enhance the inhibition effect of the animals on intestinal harmful microbial communities or enhance the nonspecific immunity function to prevent diseases, thereby indirectly playing a role in promoting the growth of the animals and improving the conversion rate of animal feed. Therefore, excellent probiotics are screened out, and the probiotics and other nutrient elements are reasonably compatible according to the physiological characteristics and nutritional requirements of aquatic animals, and are correctly used in practice, so that the effect of the probiotics can be better exerted, and the method has important significance for improving the formula of the aquatic feed.

Disclosure of Invention

The invention aims to provide a microecological compound premix feed and application thereof in aquaculture. The microecological compound premix feed contains Pediococcus acidilactici (Pediococcus acidilactici), can obviously improve the intestinal health of aquatic animals, improve the immunity and disease resistance of the aquatic animals, improve the digestion and utilization of the feed, promote the growth of the aquatic animals, and can be widely applied to the field of aquatic feeds.

A microecological compound premix feed comprises probiotics, compound trace elements, compound acidifier and carrier.

The probiotic bacteria are Pediococcus acidilactici MST02(Pediococcus acidilactici MST02), which are preserved in China center for type culture Collection of the university of Wuhan, China in No. 4-29 of 2020, and the preservation number is CCTCC NO: M2020094.

The compound microelements comprise zinc sulfate and sodium selenite.

The compound acidifier is prepared from citric acid, potassium sorbate and tributyrin.

The carrier is rice hull powder.

Further preferably, the microecological compound premix feed comprises the following components in percentage by weight: 38-50g/kg of pediococcus acidilactici powder, 13.0-14.5g/kg of zinc sulfate, 1.0-1.5g/kg of sodium selenite, 20-28g/kg of citric acid, 15-22g/kg of potassium sorbate, 32-55g/kg of tributyrin and 860g/kg of rice hull powder 850-.

Further preferably, the microecological compound premix feed comprises the following components in percentage by weight: 50g/kg of pediococcus acidilactici powder, 13.5g/kg of zinc sulfate, 1.2g/kg of sodium selenite, 28g/kg of citric acid, 22g/kg of potassium sorbate, 32g/kg of glycerol tributyrate and 853.3g/kg of rice hull powder.

The viable bacteria amount in the pediococcus lactis powder is 10¹⁰CFU/g。

The invention also provides the application of the microecological compound premix feed in aquatic feeds.

Has the advantages that:

the pediococcus acidilactici MST02 provided by the invention can obviously improve the water quality of the surrounding environment of aquatic animals, can effectively degrade nitrite nitrogen in the water body, and has a removal rate of the nitrite nitrogen in the water body of the aquaculture pond up to 86.86% after 48 hours of treatment. .

The pediococcus acidilactici is good in acid resistance, can resist the digestive tract environment, is particularly beneficial to the pediococcus acidilactici to play a probiotic role in the bodies of the gastric aquatic animals, the survival rate of the pediococcus acidilactici MST02 is as high as 95% after being treated for 2 hours under the condition of pH2.5, the survival rate still exceeds 80% after being treated for 2 hours under the condition of pH1.5, and the effect is remarkable.

The pediococcus acidilactici can effectively inhibit pathogenic bacteria such as vibrio parahaemolyticus, vibrio splendidus and the like, reduce the occurrence probability of diseases of cultured animals, and meanwhile, can be used as a feed additive, remarkably improve the utilization rate of the cultured animals on the feed, promote the growth of the animals and enhance the immunity. The result of the Micropterus salmoides feeding experiment shows that the specific growth rate of the probiotic group Micropterus salmoides is improved by 36.0 percent and the bait coefficient is reduced by 19.7 percent compared with the control group by adding pediococcus lactici MST02 into the feed; the indexes representing immunity are obviously improved, wherein the total antioxidant capacity and the activities of superoxide dismutase, alkaline phosphatase and acid phosphatase in serum are respectively improved by 148.7%, 45.8%, 107.3% and 13.0%; in addition, 2 weeks after the injection of pathogenic bacteria, namely vibrio splendidus, the cumulative mortality of the probiotic group, namely micropterus salmoides, is reduced by 56.3 percent, and the effect is very obvious.

The composite premixed feed containing pediococcus acidilactici MST02 provided by the invention can effectively improve the micro-environment of the digestive tract of cultured animals, improve the utilization rate of the feed, promote the survival and growth of the cultured animals, and simultaneously can effectively inhibit pathogenic bacteria and enhance the immunity and disease resistance of the cultured animals. Compared with a control group, the survival rate and the weight gain rate of the juvenile Micropterus salmoides of an experimental group added with the microecological compound premix feed are respectively improved by 15.3-26.6 percent and 33.2-67.2 percent, and the total serum antioxidant capacity, the activities of superoxide dismutase and acid phosphatase are respectively improved by 63.2-118.4 percent, 34.4-71.0 percent and 50.0-72.5 percent; compared with a control group, the grass carp intestinal protease activity, the amylase activity and the lipase activity are respectively improved by 56.1-97.2%, 62.2-94.9% and 76.6-106.1%, intestinal villi of young micropterus salmoides and grass carp in an experimental group are remarkably increased, intestinal muscle layers are remarkably thickened, the accumulated death rate is greatly reduced, and unexpected effects are achieved.

In addition, the composite premixed feed containing pediococcus acidilactici MST02 provided by the invention can effectively degrade organic matters in the aquaculture water body, reduce the content of ammonia nitrogen and nitrite nitrogen, improve the aquaculture water body, and is beneficial to reducing the occurrence of aquaculture diseases and maintaining the health of aquaculture animals. Compared with a control group, the COD in the culture water body of the Micropterus salmoides of the experimental group added with the microecological compound premix feed is reduced by 72.8-80.0%, while the contents of ammonia nitrogen and nitrite nitrogen are respectively reduced by 64.2-72.4% and 60.0-70.0%, and the effect is obvious.

Drawings

FIG. 1 is a graph comparing the cumulative mortality of micropterus salmoides in control and experimental groups.

Detailed Description

The equipment and reagents used in the examples of the present invention may be selected from any commercially available ones. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.

The culture medium selected in the examples comprises the following specific formula:

DM culture medium: 1g of sodium nitrite, 1g of sodium carbonate, 0.5g of dipotassium phosphate, 0.03g of magnesium sulfate heptahydrate, 0.4g of ferrous sulfate heptahydrate, 1000ml of distilled water, 12g of agar powder and 7.6-7.8 of ph.

MRS liquid medium: 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast powder, 80lml of tween-tween, 2g of monopotassium phosphate, 2g of diammonium citrate, 5g of sodium acetate, 0.58g of magnesium sulfate, 0.15g of manganese sulfate, 1000ml of distilled water, pH6.2-6.4, and sterilizing at 121 ℃ for 20 min.

MRS culture medium: 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast powder, tween-80 lml, 2g of monopotassium phosphate, 2g of diammonium citrate, 5g of sodium acetate, 0.58g of magnesium sulfate, 0.15g of manganese sulfate, 1000ml of distilled water, pH6.2-6.4, adding agar of-15-20 g to prepare a solid culture medium, and sterilizing at 121 ℃ for 20 min.

The preparation method of pediococcus acidilactici MST02 bacterial powder related in the embodiment of the invention comprises the following steps: activating Pediococcus acidilactici MST02, performing amplification culture, performing liquid fermentation, centrifuging, concentrating, and lyophilizing to obtain product with viable bacteria content of about 10¹⁰CFU/g of bacterial powder.

The invention is further illustrated by the following specific examples.

Example 1: isolation, screening and identification of strains

1. Sample preparation:

collecting live micropterus salmoides intestinal tracts purchased in a certain market in Qingdao Guzhou city, aseptically picking up healthy and strong-activity micropterus salmoides intestinal tracts, and waiting for subsequent separation.

2. Screening method

(1) Enrichment culture

And (3) putting the intestinal tract sample into a grinder, putting the intestinal tract sample into normal saline, homogenizing, centrifuging at a low speed to fully disperse bacteria in the sample into the upper layer of normal saline, and diluting by 10 times in a gradient manner. Transferring the bacterial suspension to a liquid MRS culture medium according to the inoculation amount of 10%, and culturing at 30 ℃ for 24-48 h to obtain the enriched bacterial suspension.

(2) Separation of

Coating 0.2ml of bacterial suspension in a DM solid culture medium plate by adopting a 10-fold dilution method, and culturing in a constant temperature box at 30 ℃; and (5) selecting a single colony with good growth after the plate grows out, and repeatedly streaking on a DM plate to obtain a purified single colony.

(3) Preliminary screening

And selecting single colonies which are uniformly, clearly and well grown after separation and purification, and sequentially naming the single colonies as A1, A2, A3 … … and A30. And carrying out amplification culture on the single colony in an MRS liquid culture medium for 48 h.

(4) Double sieve

And inoculating the preliminarily screened suspected activated strain into an improved MRS (added with 30mg/L sodium nitrite) liquid culture medium, culturing at 30 ℃, sampling for 12 hours at regular time, and continuously sampling for 72 hours. And (4) measuring the concentration of nitrite in the fermentation solution at different time points. The applicant co-screened 5 strains which all had different degrees of degradation effect on nitrite, and the concentrations of nitrite in the fermentation liquor of the 5 strains at different time points are detailed in table 1.

TABLE 1 potential target strains degradation of nitrite nitrogen

As can be seen from the data in Table 1, A12 has the strongest capacity of degrading nitrite nitrogen in 5 strains screened by the invention. After 48 hours of fermentation, the content of nitrite nitrogen in the fermentation liquor of the A12 strain is reduced by 93%, and after 72 hours, nitrite nitrogen in the fermentation liquor is almost completely degraded, thereby achieving unexpected technical effects.

3. Identification of strains

(1) And (3) colony morphology characteristics: the A12 strain has small colony, is white, gram-positive and spherical, is alternatively divided into tetrads on two planes at right angles, and can be preliminarily determined to be pediococcus acidilactici;

(2) extracting genome DNA of the A12 strain, amplifying a 16SrRNA sequence by utilizing a PCR technology, comparing and analyzing by sequencing BLAST, wherein the similarity of the sequence and the 16S rRNA sequence of the published multiple strains of Pediococcus acidilactici is up to 96 percent, and the identification proves that the A12 strain is Pediococcus acidilactici and is consistent with the biochemical identification result.

(3) The A12 strain is named as Pediococcus acidilactici MST02(Pediococcus acidilactici MST02), which is preserved in China center for type culture Collection of Wuhan university, Wuhan, China at 29 months 4 in 2020, with the preservation number of CCTCC NO: M2020094.

Example 2 application of Pediococcus acidilactici MST02 in purifying aquaculture water

1. Preparation of fungal powder

The pediococcus acidilactici MST02 is subjected to amplification culture to prepare 100 hundred million/g of bacterial powder.

2. Site of experiment

A culture plant of a bass in the city of Jiangsu salt.

3. Procedure of experiment

Selecting 6 ponds with similar size and water quality, and respectively detecting the nitrite nitrogen content in each water body by 3 ponds in parallel in a control group and an experimental group. Then 10ppm of pediococcus acidilactici powder was added to the water of the experimental group, and not added to the control group. The experimental water temperature is 28-30 ℃, and the same equipment is used for continuous aeration in the water body during the experiment. And respectively measuring the content of nitrite nitrogen in the water body after 48 hours, averaging the detection values of the 3 parallel groups, and calculating the removal rate of the nitrite nitrogen, wherein the specific results are shown in table 2.

The removal rate (%) is (initial nitrite nitrogen content-ending nitrite nitrogen content/initial nitrite nitrogen content × 100%.

TABLE 2 removal of nitrite nitrogen from water in aquaculture ponds after 48h treatment

Group of	Initial nitrous nitrogen content (mg/L)	Termination of nitrous Nitrogen content (mg/L)	Removal rate%
				Control group	1.38±0.05	1.30±0.04	5.79％
Experimental group	1.37±0.03	0.18±0.02	86.86％

As can be seen from the data in Table 2, the pediococcus acidilactici MST02 provided by the invention can effectively degrade nitrite nitrogen in the water body, and the removal rate of the nitrite nitrogen in the water body of the aquaculture pond is as high as 86.86% after 48 hours of treatment. Because nitrite nitrogen can seriously affect the physiological metabolism of aquatic animals, the degradation problem of nitrite nitrogen becomes a problem in aquaculture. The pediococcus acidilactici MST02 has a remarkable degradation effect on nitrite nitrogen in a water body, can be used for solving the problem of overhigh nitrite nitrogen in an aquaculture water body, and has a wide application prospect.

Example 3 Pediococcus acidilactici MST02 gastric acid resistance test

The simulated gastric juice comprises the following components: 8.5g of tryptone, 3.5g of glucose, 2.05g of sodium chloride, 0.13g of calcium chloride, 0.42g of potassium chloride, 0.55g of monopotassium phosphate, 0.05g of bovine bile salt, 0.11g of lysozyme, 13.5g of pepsin, 1000ml of distilled water, adjusting the pH to 1.5 and 2.5 respectively by hydrochloric acid, and sterilizing at 115 ℃ for 20 minutes.

The experimental control group was another strain of Pediococcus acidilactici AS1.2696, which was obtained from the institute of microbiology, national academy of sciences.

Activating Pediococcus acidilactici MST02 and Pediococcus acidilactici AS1.2696 under the same condition, inoculating the activated Pediococcus acidilactici MST02 and the Pediococcus acidilactici AS1.2696 into 100ml of MRS liquid culture medium according to the inoculation amount of 0.6%, culturing at 30 ℃ for 24h, respectively adding the two bacterial liquids into prepared sterile simulated gastric juice, detecting the number of viable bacteria by a plate counting method after 0h and 2h respectively, and calculating the survival rate of the bacterial strains, wherein the specific results are shown in Table 3.

The survival rate (%) was 2h viable cell count/0 h viable cell count × 100%.

TABLE 3 survival of Pediococcus acidilactici under different acidic conditions

As can be seen from the data in Table 3, the tolerance of Pediococcus acidilactici MST02 provided by the invention to simulated gastric juice is better than that of Pediococcus acidilactici AS1.2696, the survival rate of Pediococcus acidilactici MST02 is up to 95% after 2h under the condition of pH2.5, and the survival rate still exceeds 80% after 2h under the condition of pH1.5, so that the effect is remarkable. The acid resistance is good, so that the pediococcus acidilactici MST02 can efficiently pass through the stomach of the stomach-containing fish (such as micropterus salmoides), the microenvironment can be better improved in the intestinal tract of the fish, the body growth can be promoted, and the excellent probiotic function can be exerted.

Example 4 antagonistic Effect of Pediococcus acidilactici MST02 on pathogenic bacteria

The antagonistic action of the pediococcus acidilactici MST02 on pathogenic bacteria is detected by a plate antagonistic method by using Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio splendidus (Vibrio spleendidus) as indicator bacteria. The screened bacteria MST02 bacterial liquid is spotted on the plate coated with the indicator bacteria, pure water is spotted on a blank control group, and a pediococcus acidilactici AS1.2696 control group is arranged. Culturing at 28 deg.C, and observing whether a bacteriostatic transparent area or a bacteriostatic covered area appears around the dibbling area within 48 h. Specific results are shown in table 4.

TABLE 4 Pediococcus acidilactici bacteriostasis

As can be seen from the data in Table 4, the pediococcus acidilactici MST02 provided by the invention can effectively inhibit the growth of two pathogenic bacteria of vibrio parahaemolyticus and vibrio splendidus, and the antibacterial effect is obviously superior to that of the pediococcus acidilactici AS 1.2696.

Example 5 Effect of Pediococcus acidilactici MST02 on growth, immune competence and disease resistance of Micropterus salmoides

The experiment is provided with a control group and a probiotic group, wherein each group is respectively provided with three parallels, and each parallels 40 micropterus salmoides. Commercial expanded feed of Micropterus salmoides is used as basic feed. Wherein the control group is fed with basal feed and the probiotic group is fed with feed containing 10⁸CFU/g Pediococcus acidilactici MST02 feed. The culture experiment lasts for 60 days, the micropterus salmoides is fed according to 3-5% of the weight of the micropterus salmoides every day, the feeding amount is adjusted at any time according to the food intake condition, the micropterus salmoides is fed once every morning and evening, and the bottom suction and pollution discharge are performed once. During the experiment, the dissolved oxygen is more than or equal to 7mg/l, the temperature is 28 +/-1 ℃, the salinity is 5-8 per mill, and the pH is 8.0 +/-0.4. And after the culture experiment is finished, counting and weighing the micropterus salmoides, and calculating the specific growth rate of the micropterus salmoides. Blood is taken from tail vein, and indexes such as total antioxidant capacity, serum alkaline phosphatase, serum acid phosphatase, serum superoxide dismutase activity and the like are measured. The vibrio splendidus is taken as a pathogenic bacterium, and the celiac is injected with micropterus salmoides for 2 weeks for observation, so that the cumulative mortality is counted. The specific results are shown in Table 5.

TABLE 5 influence of Pediococcus acidilactici MST02 on growth and immune competence of Micropterus salmoides

	Control group	Probiotic group
			Initial body weight g	35.23±0.06	35.25±0.08
Terminal body weight g	87.25±0.87a	110.27±0.75b
			Coefficient of bait	1.22±0.04^b	0.98±0.03^a
Specific growth Rate%/d	2.86±0.19^a	3.89±0.27^b
			Total antioxidant power (U/ml)	12.92±0.37^a	32.13±0.50^b
Serum superoxide dismutase (U/ml)	125.58±4.64^a	183.14±6.29^b
			Serum alkaline phosphatase (King's unit/100 ml)	3.55±1.37^a	7.36±1.53^b
Serum acid phosphatase Activity U/100ml CLS	3.86±0.21	4.36±0.13
			Cumulative mortality%	67.36±3.01^b	29.46±1.98^a

Note: different letters indicate significant differences (P < 0.05).

From the data in table 5, it can be seen that compared with the control group, the survival growth and the feed utilization rate of the probiotic group of micropterus salmoides added with pediococcus acidilactici MST02 are significantly improved, wherein the specific growth rate is improved by 36.0%, and the feed coefficient is reduced by 19.7%; the indexes representing immunity are obviously improved, wherein the total antioxidant capacity and the activities of superoxide dismutase, alkaline phosphatase and acid phosphatase in serum are respectively improved by 148.7%, 45.8%, 107.3% and 13.0%; in addition, 2 weeks after the injection of pathogenic bacteria, namely vibrio splendidus, the cumulative mortality of the probiotic group, namely micropterus salmoides, is reduced by 56.3 percent, and the effect is very obvious. Therefore, the pediococcus acidilactici MST02 provided by the invention can effectively promote the survival and growth of micropterus salmoides, enhance the immunity and disease resistance of micropterus salmoides, further contribute to increase the yield and quality of micropterus salmoides and improve the culture benefit of micropterus salmoides.

In conclusion, the pediococcus acidilactici MST02 provided by the invention can effectively solve the problem of overhigh nitrite nitrogen in aquaculture water, reduce the occurrence probability of diseases of aquaculture animals, and simultaneously can be used as a feed additive, improve the digestive tract microenvironment of the aquaculture animals (including gastric animals), obviously improve the utilization rate of the aquaculture animals on feed, promote the growth of the aquaculture animals, and enhance the immunity and disease resistance of the aquaculture animals, thereby being beneficial to improving the economic benefit of farmers. In addition, pediococcus acidilactici MST02 can effectively inhibit pathogenic bacteria in water, and has wide application prospect.

Example 6

A microecological compound premix feed comprises the following components in each kilogram of compound premix feed: 38g of pediococcus acidilactici powder, 14.5g of zinc sulfate, 1.0g of sodium selenite, 23g of citric acid, 20g of potassium sorbate, 45g of tributyrin and 858.5g of rice hull powder.

Example 7

A microecological compound premix feed comprises the following components in each kilogram of compound premix feed: 45g of pediococcus acidilactici powder, 13.0g of zinc sulfate, 1.5g of sodium selenite, 20g of citric acid, 15g of potassium sorbate, 55g of glycerol tributyrate and 850.5g of rice hull powder.

Example 8

A microecological compound premix feed comprises the following components in each kilogram of compound premix feed: 50g of pediococcus acidilactici powder, 13.5g of zinc sulfate, 1.2g of sodium selenite, 28g of citric acid, 22g of potassium sorbate, 32g of tributyrin and 853.3g of rice hull powder.

Example 9 Effect of Microecological Compound premix feed on growth, immunization and Water quality of Micropterus salmoides

The experiment is carried out in a certain scale California perch culture base in Zhejiang, wherein the California perch is used for the experiment, young California perches are hatched in the same pool in the base, and the size of the fry is that the initial weight of the young California perches is 28.62 +/-0.85 g. The control group T0 is fed with basal feed with 43 percent of crude protein and 14 percent of crude fat, the experimental group is added with the microecological compound premix feed provided by the invention according to the mass ratio of 2 per thousand in the basal feed, the T1 is added with the microecological compound premix feed described in the example 6, the T2 is added with the microecological compound premix feed described in the example 7, and the T3 is added with the microecological compound premix feed described in the example 8.

The experiment is carried out in a temperature-controlled culture pond, and the temperature among all groups is kept consistent. The water body for cultivating the micropterus salmoides is continuously aerated, the water temperature is kept at 26 +/-0.5 ℃, the pH value is 7.8-8.0, and the bait feeding amount is adjusted according to the ingestion condition of the fry, the water quality condition and the number of the remaining micropterus salmoides fry. The feeding experiment lasted 4 weeks. Counting the number of live micropterus salmoides after the experiment is finished, calculating the survival rate of micropterus salmoides, and randomly sampling to determine the total antioxidant capacity, superoxide dismutase and acid phosphatase activity in the blood serum of micropterus salmoides; intestinal sections were prepared from intestinal tracts of micropterus salmoides, and the intestinal tissue structure was observed and measured, with the results shown in tables 6 and 7. Collecting a water body for micropterus salmoides larva culture, and measuring the content of ammonia nitrogen and nitrite nitrogen in the water body, wherein the specific results are shown in a table 8. After the culture experiment is finished, 25 fishes are randomly selected, the vibrio parahaemolyticus with the semilethal concentration is injected to carry out the challenge experiment, the death rate is observed and calculated, and the result is shown in figure 1.

TABLE 6 influence of the micro-ecological compound premix feed on the growth and immunity of micropterus salmoides

TABLE 7 influence of the micro-ecological composite premix feed on intestinal tissue structure of micropterus salmoides

As can be seen from the data in tables 6 and 7, compared with the control group, the survival rate and the weight gain rate of the experimental group of the micropterus salmoides juvenile fish added with the microecological compound premix feed are respectively improved by 15.3-26.6 percent and 33.2-67.2 percent, the total serum antioxidant capacity, the superoxide dismutase and the acid phosphatase activity are respectively improved by 63.2-118.4 percent, 34.4-71.0 percent and 50.0-72.5 percent, the intestinal villus of the experimental group of micropterus salmoides juvenile fish is remarkably increased, the intestinal muscular layer is remarkably thickened, and unexpected technical effects are achieved. In addition, as shown in fig. 1, the cumulative mortality of the young micropterus salmoides in the control group is up to 83% within 7 days of the challenge, while the cumulative mortality of the experimental group is generally reduced by 28% -65%. Therefore, the compound premix feed containing pediococcus acidilactici MST02 provided by the invention can effectively improve the micro-environment of the digestive tract of micropterus salmoides, improve the utilization rate of the feed, promote the survival and growth of micropterus salmoides, effectively inhibit pathogenic bacteria, enhance the immunity and disease resistance of micropterus salmoides, and has remarkable effect.

TABLE 8 influence of the micro-ecological compound premix feed on the water quality of micropterus salmoides culture water

Group of	COD(mg/L)	Ammonia nitrogen concentration mg/ml	Concentration of nitrous nitrogen mg/ml
				T0	26.30±0.25	5.36±0.12	0.10±0.03
T1	7.16±0.14	1.74±0.10	0.04±0.02
				T2	7.32±0.17	1.92±0.15	0.03±0.05
T3	5.28±0.13	0.58±0.13	0.03±0.06

As can be seen from the data in Table 8, compared with the control group, the COD in the culture water of Micropterus salmoides of the experimental group added with the microecological compound premix feed of the invention is reduced by 72.8-80.0%, and the contents of ammonia nitrogen and nitrite nitrogen are respectively reduced by 64.2-72.4% and 60.0-70.0%, thereby demonstrating that the compound premix feed containing Pediococcus acidilactici MST02 provided by the invention can effectively degrade the organic matters in the culture water, reduce the contents of ammonia nitrogen and nitrite nitrogen, improve the culture water, be beneficial to reducing the occurrence of culture diseases and maintain the health of cultured animals.

Example 10 Effect of the Microecological Compound premix feed on the growth and disease resistance of grass carp

Young grass carp with initial weight of 30.52 +/-0.52 g is selected and randomly distributed into 4 groups, each group is provided with 4 repetitions, and each repetition is 50 fish. Control group (T0): basic feed is fed, and the experimental group is as follows: the microecological compound premix feed is added into a basal feed according to the mass ratio of 1 per thousand, wherein the microecological compound premix feed described in the embodiment 6 is added into T1, the microecological compound premix feed described in the embodiment 7 is added into T2, and the microecological compound premix feed described in the embodiment 8 is added into T3. The test period is 8 weeks, the water temperature is 25 + -1 deg.C, and the dissolved oxygen is maintained at 7.0 mg/L. After the experiment is finished, the grass carp weight is weighed, the weight gain rate and the specific growth rate are calculated, 25 grass carps are randomly selected, the intestinal tract is taken, the intestinal tract slices are made, the intestinal tract tissue structure is observed and measured, and the results are shown in tables 9 and 10. After the culture experiment is finished, 25 fishes are randomly selected, the aeromonas hydrophila with the semilethal concentration is injected for the challenge experiment, and the mortality is observed and calculated.

TABLE 9 Effect of the Microecological Compound premix on the growth behavior of grass carp

TABLE 10 influence of the micro-ecological composite premix feed on the intestinal tissue structure of young grass carp

Group of	T0	T1	T2	T3
					Intestinal villus height/um of foregut	476.47±12.28	526.52±13.56	530.89±12.53	576.88±16.43
Foregut muscularis thickness/um	164.28±6.03	178.71±7.06	174.28±8.89	194.35±6.85
					Villus height/um of midgut intestinal tract	424.34±9.02	464.13±8.57	462.68±10.24	494.97±14.37
Thickness/um of middle intestinal muscularis	128.59±10.45	138.69±15.87	131.88±10.67	168.35±10.28
					Intestinal villus height/um of hindgut	332.84±9.88	362.76±5.69	361.89±6.54	382.99±6.43
Posterior intestinal layer thickness/um	86.87±7.85	96.57±14.25	98.66±5.25	123.56±6.12

From the results in tables 9 and 10, it can be seen that after the grass carp eats the experimental feed added with the microecological compound premix feed, the weight gain rate and the specific growth rate are remarkably improved, the intestinal protease activity, the amylase activity and the lipase activity of the grass carp are respectively improved by 56.1-97.2%, 62.2-94.9% and 76.6-106.1% compared with those of a control group, and intestinal villi of the grass carp in the experimental group are remarkably increased, and intestinal muscle layers are remarkably thickened, so that unexpected effects are achieved. In addition, the cumulative mortality of the control group is up to 72.5 percent and the cumulative mortality of the experimental group is reduced by 38 to 61 percent within 7 days of toxicity attack. Therefore, the microecological compound premix feed containing pediococcus acidilactici MST02 provided by the invention can improve the intestinal tract digestive enzyme activity of the grass carps, promote the absorption and utilization of nutritional ingredients of the feed by the grass carps, thereby promoting the growth and obviously improving the disease resistance of the grass carps.

In conclusion, the microecological compound premix feed containing pediococcus acidilactici MST02 provided by the invention can significantly improve the utilization rate of feed by cultured animals, promote the growth of the cultured animals, effectively solve the problem that the ammonia nitrogen content of aquaculture water exceeds the standard, keep the water quality of the aquaculture water stable, improve the disease resistance of the cultured animals, improve the intestinal health of the cultured animals, improve the survival rate of the cultured animals in the whole period, contribute to improving the economic benefit of farmers, and has a wide application prospect.

Claims

1. The microecological compound premix feed is characterized by comprising probiotics, compound trace elements, a compound acidifier and a carrier; the probiotics is pediococcus acidilactici (A)Pediococcus acidilactici) MST02 with the preservation number of CCTCC NO: M2020094.

2. The microecological composite premix feed of claim 1, wherein the composite trace elements consist of zinc sulfate and sodium selenite.

3. The microecological compound premix feed of claim 2, wherein the compound acidifier comprises citric acid, potassium sorbate and tributyrin.

4. The microecological composite premix feed according to claim 3, wherein the carrier is rice hull powder.

5. The microecological compound premix feed according to claim 4, wherein the microecological compound premix feed comprises the following components in parts by weight: 38-50g/kg of pediococcus acidilactici powder, 13.0-14.5g/kg of zinc sulfate, 1.0-1.5g/kg of sodium selenite, 20-28g/kg of citric acid, 15-22g/kg of potassium sorbate, 32-55g/kg of tributyrin and 860g/kg of rice hull powder 850-.

6. The microecological compound premix feed according to claim 5, wherein the microecological compound premix feed comprises the following components in parts by weight: 50g/kg of pediococcus acidilactici powder, 13.5g/kg of zinc sulfate, 1.2g/kg of sodium selenite, 28g/kg of citric acid, 22g/kg of potassium sorbate, 32g/kg of glycerol tributyrate and 853.3g/kg of rice hull powder.

7. The micro-ecological composite premix feed as claimed in claim 5 or 6, wherein the amount of viable bacteria in the pediococcus acidilactici powder is 10¹⁰CFU/g。