CN111789054A

CN111789054A - Method for applying bacillus liquid to stichopus japonicus seedling culture

Info

Publication number: CN111789054A
Application number: CN202010742378.4A
Authority: CN
Inventors: 李石磊; 王旭达; 董颖; 周遵春; 关晓燕; 叶博; 王笑月; 姜苹哲; 孙红娟
Original assignee: LIAONING OCEAN AND FISHERIES SCIENCE RESEARCH INSTITUTE
Current assignee: LIAONING OCEAN AND FISHERIES SCIENCE RESEARCH INSTITUTE
Priority date: 2020-07-29
Filing date: 2020-07-29
Publication date: 2020-10-20

Abstract

The invention discloses a method for applying bacillus liquid to stichopus japonicus offspring seed culture; the mass of the stichopus japonicus seed monomer is 1-10g, and the feeding density is 100-³The bacillus liquid cultured by the method of the invention is prepared by the turbidimetry method according to the ratio of 10⁷Or 10⁹The cfu/g concentration ratio is mixed in the feed for feeding the stichopus japonicus. The daily feed dosage is 3-10% of the weight of the stichopus japonicus. The use of the bacterial liquid can effectively improve the culture water quality, improve the intestinal tracts of aquatic animals, enhance the immunity, promote the growth, reduce the occurrence of diseases and improve the quality of aquatic products. Fundamentally reduces the breeding cost and increases the breeding benefit.

Description

Method for applying bacillus liquid to stichopus japonicus seedling culture

Technical Field

The invention relates to the field of microbial fermentation, in particular to a method for applying bacillus liquid to stichopus japonicus breeding.

Background

Probiotics play a great role in aquaculture at present. The probiotics contain rich nutrient substances, and can directly provide available amino acid, vitamins and the like for aquatic animals. Moreover, probiotics produce a variety of organic acids that promote the absorption of calcium, iron ions, and vitamin D by animals with water coldness. The probiotics can maintain the flora balance in the stomach and intestinal tracts of aquatic animals, improve the gastrointestinal tract function, improve the feed quality and improve the digestion utilization rate, and can synthesize various amino acids, vitamins and the like required by the host animals for maintaining life activities in the intestinal tracts for the host animals to utilize. The probiotics can generate various enzymes in the metabolic process, the enzymes interact with digestive enzymes in aquatic animals and can promote the activity of the digestive enzymes, the growth and development of immune organs of the aquatic animals can be promoted, the immune reaction of organisms can be activated, the humoral immunity and the cellular immunity of the aquatic animals can be stimulated, and the activity of various enzymes in the body fluid of the aquatic animals can be improved. The probiotics have good disease-resistant effect, and on one hand, the pathogenic bacteria are rejected and limited by competition of colonization parts, living and breeding spaces and nutrient substances between the probiotics and the pathogenic bacteria. On the other hand, probiotics produce active substances such as antibiotics through secretion, so that pathogenic bacteria are inhibited. In aquaculture, the probiotics not only have the functions, but also can survive in aquaculture water, degrade organic matters, decompose and convert harmful substances, stabilize the pH value of the water, effectively improve the aquaculture ecological environment and maintain ecological balance.

CN101028007A relates to a special aquaculture probiotics disinfection and bacteriostasis biological preparation, which is mainly prepared by mixing bacillus subtilis, bacillus licheniformis, bifidobacterium, lactein and bait according to a proportion and preparing into powder by a special biotechnology process. The bacillus subtilis, the bacillus licheniformis, the bifidobacterium and the lactobacillus are cultures of purified species, and the probiotics participate in the living and breeding space, time, colonization part and nutrient competition between beneficial flora and pathogenic bacteria in the breeding environment and the digestive tract of animals, and stress the living, breeding, colonization and attachment of pathogenic flora. The beneficial flora generates volatile fatty acid and lactic acid in the metabolic process, reduces ph and Eh in the growth environment, generates hydrogen peroxide and inhibits pathogeny; the growth metabolin is antibiotic and bacteriocin, and can kill pathogenic bacteria. The bait is various nutrient components of algae and is a carrier of probiotics. The invention is suitable for the antibacterial disinfection of various culture species in various aquaculture industries; good effect, no toxicity to the disinfected human body and cultured species, no pollution to water quality and environment; the operation and use method is simple; the probiotics prepared by the special process is a new generation disinfection and sterilization product in aquaculture industry. Especially in inhibiting harmful microbe and preventing vibriosis of aquatic product; improving organism metabolism of cultured species, and supplementing organism nutrient components; stimulating the immune system of the organism and improving the immunity of the organism; the biological degradation is participated in, and the organic pollutant is eliminated.

CN105209596A relates to a process and medium for the production of probiotic micro-organisms of abalone using a novel bioprocessing technology. There is currently no commercially viable bioprocess for the production of abalone probiotics. The invention promotes the biological production of the abalone probiotics as follows: the probiotic product is produced from a small volume, low temperature culture of the bacterial probiotic Vibrio midae, or the yeast probiotic Debaryomyces hansenii, or a combination thereof. The invention describes an inoculation procedure, a fermentation process, biomass collection and the production of biomass into a liquid or dry product that can be incorporated into abalone feed.

CN108208464A relates to a preparation method of a probiotic feed for aquaculture. The method takes the agricultural product processing leftovers and/or marine product processing leftovers as raw materials, the leftovers are subjected to primary fermentation curing and bacteria removal, and then the secondary fermentation is carried out by utilizing a biological fermentation technology to prepare the probiotic feed rich in three different habits of probiotics, so that the leftovers from the agricultural and marine product processing are recycled, the environment is protected, the problem of raw material shortage for preparing the feed in China is greatly relieved, and in addition, the probiotic feed for aquaculture prepared by the method also has the effect of improving the physique of culture species such as fishes, shellfishes, shrimps and crabs and the like, and the culture output is effectively improved.

In conclusion, probiotics have been widely used in aquaculture. However, the probiotic industry in the aquaculture industry still has many problems, such as less strains developed for aquaculture characteristics, easy inactivation of live bacteria preparations during storage and transportation, irregular product quality of aquaculture probiotics in the market, insufficient ability of aquaculture personnel in quality identification of probiotics, and the like, and the most important contradictions are that: the contradiction between the demand of farmers for high-quality probiotic products and the low cost-performance of the aquatic probiotic products. The prior commercial probiotics for aquaculture has good quality, high selling price and large using cost for farmers on large water surface; the product with low price has less viable bacteria and more mixed bacteria, and the product quality can not be ensured. The probiotic products are from manufacturers to the first line of cultivation, the production cost of the manufacturers is eliminated, the costs of packaging, logistics, difference price of intermediate merchants and the like are also added, so that the cost of the probiotic products purchased by cultivation enterprises and cultivation user terminals is high, the timeliness is poor, and the number of live bacteria is reduced in the transportation and storage processes. In recent years, some farmers purchase strains and culture media to perform fermentation operation by themselves by using the existing conditions, but the operation flow is not standard, the number of bacteria contained in the fermented bacteria liquid is small, the amount of mixed bacteria is large, and the using effect is influenced, so that a simple probiotic culture method suitable for the first line of culture is urgently needed, and the breeding enterprises and the farmers can obtain high-quality bacteria liquid meeting the requirement of aquaculture by using the method without using fermentation tanks, so that the breeding cost is reduced while the feeding effect of the probiotics is ensured.

Disclosure of Invention

In order to solve the problems, the invention provides a method for applying a bacillus liquid to stichopus japonicus breeding, wherein the mass of stichopus japonicus seed monomers is 1-10g, and the culture density is 100-³During the period, aeration is kept, the temperature of the seawater is 15-25 ℃, the pH value is 7.8-8.2, and the salinity is 28-32. And feeding the stichopus japonicus after changing water every day, wherein the daily feeding amount of the feed is 3-10% of the weight of the stichopus japonicus. The bacillus liquid cultured by the method is prepared by the turbidimetric method according to the ratio of 10⁷Or 10⁹The cfu/g concentration ratio is mixed in the feed for feeding the stichopus japonicus.

The bacillus cultured by the method has obvious probiotic effect on promoting the growth of stichopus japonicus, improving the activity of digestive enzyme and enhancing the immune defense function, is dose-dependent, and has the addition concentration of 10 percent when the bacillus is added into the feed⁷、10⁹The growth capacity, the digestive enzyme activity, the immunoenzyme activity and the resistance to vibrio splendidus of the stichopus japonicus are obviously improved when cfu/g is adopted, which shows that the bacillus cultured by the method can be used as a safe and effective probiotic additive to be applied to stichopus japonicus culture.

Furthermore, the invention also discloses a simple probiotic culture method suitable for aquaculture;

a simple culture method of probiotics suitable for aquaculture comprises the following steps:

the method comprises the following steps: the temperature in the probiotic culture chamber is required to be controllable, and an ultraviolet lamp is installed according to the room space, wherein the average volume per cubic meter is 2-5 w;

step two: an open plastic barrel is used as a probiotic culture container, transparent plastic cloth is cut according to the size of a barrel opening and is used for sealing, the plastic cloth covers the barrel opening, and the periphery of the plastic cloth is fastened by a rope. Perforating the center of the plastic cloth to facilitate the passage of the inflation tube, fastening the plastic cloth and the contact part of the air tube by using a rope, and irradiating all articles by using an ultraviolet lamp for 1-3 hours for disinfection before inoculation;

step three: placing tools such as a beaker and a measuring cylinder used in the expanding culture process into water, boiling for 10-30 minutes and disinfecting;

step four: filtering the gas-filled pipeline by a precise filtering system;

step five: adding the seawater or the fresh water which is subjected to precipitation and sand filtration into the plastic barrel in the step 1, wherein the water adding amount is 1/2-3/4 of the volume of the barrel. Adding 5-10 ml of sodium hypochlorite (10-20% of available chlorine) into each liter of seawater or fresh water, sealing and inflating according to the step 1, adding 0.45-0.9 g of sodium thiosulfate into each liter of seawater or fresh water for neutralization after 12-16 hours, adding the sodium thiosulfate into a barrel after the sodium thiosulfate is boiled in the fresh water, and testing the existence of residual chlorine by using starch potassium iodide after inflating for half an hour. Adjusting the pH value to 6.5-7.5 with sodium hydroxide or hydrochloric acid solution.

Step six: adding a culture medium into clear water, boiling and melting, pouring into the plastic barrel in the step 5, then adding probiotic strains, inoculating 2-5%, sealing according to the step 1, preserving heat and inflating.

Step seven: after 3-5 days of culture, the number of beneficial bacteria in the bucket is 15 multiplied by 10⁸～40×10⁸And (5) the seeds are directly thrown into a culture pond per ml.

The three-stage precise filter is prepared by using a nano-grade air filtering material, and the preparation method comprises the following steps:

the method comprises the following steps: according to the mass parts, 50-65 parts of long fiber wood pulp, 28-36 parts of basswood pulp fiber, 18-25 parts of mercerized softwood pulp fiber and 10-18 parts of para-aramid pulp fiber are uniformly mixed and then pulped, then 300-400 parts of sulfuric acid with the mass percentage concentration of 2-5% is added, 3-7 parts of allyl dimethoxysilane is added, the temperature is controlled to be 60-72 ℃, after continuous pulping and mixing is carried out for 1-5 hours, 0.5-2 parts of ammonium persulfate and 0.3-2.4 parts of D-allyl glycine are added, the temperature is controlled to be 50-68 ℃, after continuous pulping and mixing is carried out for 0.5-2.5 hours, fluffing, dewatering and drying are carried out to obtain air filter base paper, and the quantitative amount of the air filter base paper is 85-105g/m 2;

step two: then adding 0.1-0.5 part of carboxylated carbon nano tube into 100-200 parts of acrylic acid emulsion with the solid content of 35-45%, mixing and stirring for 10-30min, then carrying out ultrasonic treatment for 30-60min, uniformly dispersing, then coating on the surface of air filter base paper, wherein the coating weight is 4-10g/m2, and then drying at 90-110 ℃ for 120-180s to obtain the nano-scale air filter material.

And introducing allyl into the long fiber wood pulp, the basswood pulp fiber and the mercerized softwood pulp fiber, and copolymerizing the allyl with D-allyl glycine to prepare the silane modified wood pulp fiber. The wood pulp fiber component is mainly cellulose, and partial reaction is shown as follows:

further, the silane modified wood pulp fiber and D-allyl glycine are subjected to graft copolymerization, and a part of reaction is as follows:

according to the simple probiotic culture method suitable for aquaculture, high-quality bacteria liquid for aquaculture can be obtained by the method without using a fermentation tank for aquaculture enterprises and farmers.

Drawings

The invention is further illustrated with reference to the following figures and examples:

FIG. 1 is a photograph showing the PCR amplification result;

FIG. 2 is an electrophoretogram of bacterial 16S amplification products;

FIG. 3 is a fluorescent quantitative PCR amplification curve of a bacterial 16S gene sample;

FIG. 4 is a fluorescent quantitative PCR melting curve of a bacterial 16S gene sample;

FIG. 5 is a fluorescent quantitative PCR amplification curve of the bacterial 16S gene standard;

FIG. 6 is a flow chart of Illumina PE250 sequencing experiments;

FIG. 7 is a graph of the effect of Bacillus on Apostichopus japonicus trypsin activity;

FIG. 8 is a graph of the effect of Bacillus on the lipase activity of Stichopus japonicus;

FIG. 9 is a graph of the effect of Bacillus on Apostichopus japonicus amylase activity;

FIG. 10 is a graph of the effect of Bacillus on acid phosphatase activity of Stichopus japonicus;

FIG. 11 is a graph showing the effect of Bacillus on the alkaline phosphatase activity of Stichopus japonicus;

FIG. 12 is a graph of the effect of Bacillus on Apostichopus japonicus superoxide dismutase activity;

FIG. 13 is a graph of the effect of Bacillus on the lysozyme activity of Stichopus japonicus;

FIG. 14 shows the bacteriostatic action of Bacillus on attacking Vibrio splendidus of Apostichopus japonicus.

Detailed Description

The following detection methods were as follows:

PCR preliminary experiment

1.1 primer design

1.2 PCR amplification conditions

PCR was performed using a 30. mu.l reaction:

qPCR Mix......................15μl

Mg²⁺(25mM).....................2μl

Forward Primer(10μM)..........0.5μl

Reverse Primer(10μM)..........0.5μl

Template DNA..................2μl

_____________________________________________________

complement ddH₂.

Bacterial 16S gene PCR reaction parameters:

a.1×(3min at 95℃)

b.30×(30s at 94℃；30s at*****℃；30s at 79℃)

1.3 PCR amplification result identification gel Pattern

1.3.1 electrophoretogram of bacterial 16S amplification product

1.4 PCR amplification results

After probing the PCR amplification conditions, the PCR amplification results were counted as follows:

1.5 the results show

1.6. Preparation of standards

After linearization treatment, the constructed plasmid is purified and quantified, and is converted into copy number (copies/mu l) through a formula, the constructed standard product is diluted by 10 times of gradient, 90 mu l of diluent and 10 mu l of plasmid are generally made into 4-6 points, and 10-2-10-6 diluents of the standard product are respectively selected through a pre-experiment to prepare a standard curve.

Injecting: plasmid initial copy number conversion formula (copies/. mu.l) ═ concentration (ng/. mu.l). 10-9. 6.02. 1023/(molecular weight. 660)

1.7. Fluorescent quantitative PCR detection

PCR was performed using a 30. mu.l reaction:

qPCR Mix......................15μl

Mg2+(25mM)....................2μl

Forward Primer(10μM)..........0.5μl

Reverse Primer(10μM)..........0.5μl

Template DNA..................2μl

.

_____________________________________________________

Complement ddH₂.

PCR reaction parameters of bacterial 16S rDNA gene:

a.1×(3min)at 95℃)

b.30×(30s)at 94℃；30s at 60℃；30s at 79℃)

2 bacteria liquid high-throughput sequencing analysis experimental method cultured by using method

2.1 Illumina PE250 sequencing Experimental flow

2.1.1 genomic DNA extraction

After completion of the extraction of the genomic DNA, the extracted genomic DNA was examined by electrophoresis on 1% agarose gel.

2.1.2 PCR amplification

Specific primers with barcode were synthesized according to the designated sequencing region.

In order to ensure the accuracy and reliability of subsequent data analysis, two conditions need to be met, 1) low-cycle amplification is used as far as possible; 2) ensure that the amplification cycles of each sample are consistent. Representative samples were randomly selected for pre-experiments to ensure that the majority of samples were able to amplify the appropriate concentration of product at the lowest cycle number. PCR was performed using a TransGen AP 221-02: TransStartFastpfu DNA Polymerase; a PCR instrument: ABI

Model 9700; all areThe samples are carried out according to formal experimental conditions, each sample is repeated for 3 times, PCR products of the same sample are mixed and detected by 2% agarose gel electrophoresis, the PCR products are recovered by cutting gel by using an AxyPrepDNA gel recovery kit (AXYGEN company), and Tris-HCl is eluted; and (5) detecting by 2% agarose electrophoresis.

2.1.3 fluorescence quantitation

Referring to the preliminary quantification result of electrophoresis, the PCR product was quantified using QuantiFluor^TMThe quantitative determination of ST blue fluorescence system (Promega corporation) followed by mixing in the corresponding proportions according to the sequencing requirements of each sample.

2.1.4 Illumina PE250 library construction

1) Connecting a Y-shaped joint;

2) removing the adaptor self-connecting fragment by magnetic bead screening;

3) enriching the library template by utilizing PCR amplification;

4) sodium hydroxide denaturation produces single-stranded DNA fragments.

2.1.5 Illumina PE250 sequencing

1) One end of the DNA fragment is complementary with the basic group of the primer and is fixed on the chip;

2) the other end is randomly complementary to another primer nearby and is also fixed to form a bridge;

3) performing PCR amplification to generate a DNA cluster;

4) the DNA amplicon is linearized into a single strand.

5) Adding modified DNA polymerase and 4 kinds of fluorescence labeled dNTPs, and synthesizing only one base in each cycle; 6) Scanning the surface of the reaction plate by laser, and reading the nucleotide species polymerized by the first round of reaction of each template sequence;

7) chemically cleaving the "fluorophore" and the "stop group" to restore the 3' terminal viscosity and continuing to polymerize a second nucleotide;

8) and counting the fluorescent signal result collected in each round to obtain the sequence of the template DNA fragment.

2.2 bioinformation analysis procedure

Firstly, splicing PE reads obtained by Illumina PE250 sequencing according to an overlap relation, simultaneously performing quality control and filtration on sequence quality, performing OTU (optical transport unit) clustering analysis and species taxonomy analysis after distinguishing samples, and performing various diversity index analyses on OTU (optical transport unit) and detection on sequencing depth based on OTU clustering analysis results; based on the taxonomic information, statistical analysis of community structure can be performed at various taxonomic levels.

The invention is further illustrated by the following specific examples:

example 1

The method comprises the following steps: the temperature in the lactobacillus culture room is required to be controllable, and an ultraviolet lamp is arranged according to the room space, wherein the average volume is 2w per cubic meter;

step two: an open plastic barrel is used as a lactobacillus culture container, transparent plastic cloth is cut according to the size of a barrel opening and is used for sealing, the plastic cloth covers the barrel opening, and the periphery of the plastic cloth is fastened by a rope. Perforating the center of the plastic cloth to facilitate the passage of the inflation tube, fastening the contact part of the plastic cloth and the air tube by using a rope, and irradiating all articles by using an ultraviolet lamp for 1 hour for disinfection before inoculation;

step three: placing tools such as a beaker and a measuring cylinder in water for boiling for 10 minutes for disinfection in the expanding culture process;

step four: filtering the gas-filled pipeline by a precise filtering system;

step five: adding the seawater or the fresh water which is subjected to precipitation and sand filtration into the plastic bucket in the step 1, wherein the water adding amount is 3/4 of the volume of the bucket. Adding 5ml of sodium hypochlorite (effective chlorine 10) into each liter of seawater or fresh water, sealing and inflating according to the step 1, adding 0.45g of sodium thiosulfate into each liter of seawater or fresh water for neutralization after 12 hours, adding the sodium thiosulfate into a barrel after the sodium thiosulfate is boiled in clear water, and testing whether residual chlorine exists by using starch potassium iodide after inflating for half an hour. The pH is adjusted to 7.5 with sodium hydroxide or hydrochloric acid solution.

Step six: adding the culture medium into clear water, boiling to melt, pouring into the plastic barrel in the step 5, then adding the lactic acid bacteria strain, inoculating 5%, sealing according to the step 1, preserving heat and inflating.

Step seven: after 5 days of culture, the number of the beneficial bacteria in the bucket is 40 multiplied by 108/ml, and the beneficial bacteria are directly thrown into a culture pond.

Taking 1ml of the bacterial liquid in the barrel, diluting by 100 times, counting by using a blood counting chamber, quantifying 22.6 multiplied by 108 lactic acid bacteria/ml, and detecting no mixed bacteria in a microscopic examination.

the method comprises the following steps: according to the mass parts, 50 parts of long fiber wood pulp, 28 parts of basswood pulp fiber, 18 parts of mercerized softwood pulp fiber and 10 parts of para-aramid pulp fiber are uniformly mixed and then pulped, then 300 parts of sulfuric acid with the mass percentage concentration of 2% is added, 3 parts of allyl dimethoxysilane is added, the temperature is controlled at 60 ℃, after pulping and mixing are continued for 1h, 0.5 part of ammonium persulfate and 0.3 part of D-allyl glycine are added, the temperature is controlled at 50 ℃, after pulping and mixing are continued for 0.5h, fluffing, dewatering and drying are carried out to obtain air filter base paper, and the ration of the air filter base paper is 85-105g/m 2;

step two: then adding 0.1 part of carboxylated carbon nano tube into 100 parts of acrylic emulsion with the solid content of 35%, mixing and stirring for 10min, then carrying out ultrasonic treatment for 30min, uniformly dispersing, and then coating on the surface of air filter base paper, wherein the coating weight is 4g/m²And then drying at 90 ℃ for 120s to obtain the nano-scale air filter material.

Carrying out fluorescent quantitative PCR experiment on the lactobacillus cultured by the method

Table 1: PCR preliminary experiment sample detection result

The target band size of the PCR product is correct, the concentration is proper, the melting curve peak diagram is consistent with the target peak, the quantitative values are all more than 500, the quantitative values are accurate and reliable, and the subsequent experiment can be carried out.

Table 2: lactic acid bacteria liquid diversity high-throughput sequencing result

Name of the genus	Number of
		Acidovorax	2
Acinetobacter	14
		Aerococcus	232
Bacillus	2
		Bradyrhizobium	7
Brevundimonas	19
		Chloroplast_norank	2
Enterococcus	21
		Flavobacterium	9
Halolactibacillus	1
		Lactobacillus	29337
Lactococcus	23
		Leuconostoc	12
Macrococcus	40
		Pseudonocardia	7
Staphylococcus	3
		Vagococcus	3
Vibrio	5
		Vibrionimonas	1
Weissella	3

On the genus level, the Lactobacillus liquid obtained by the method has the highest Lactobacillus content, the ratio of the Lactobacillus to the total bacteria is up to more than 99%, and the ratio of other bacteria is less than 1%.

Example 2

the method comprises the following steps: the temperature in the bacillus subtilis culture room is required to be controllable, and an ultraviolet lamp is arranged according to room space, wherein the average volume is 3w per cubic meter;

step two: an open plastic bucket is used as a bacillus subtilis culturing container, transparent plastic cloth is cut according to the size of a bucket opening and is used for sealing, the plastic cloth covers the bucket opening, and the periphery of the plastic cloth is fastened by a rope. Perforating the center of the plastic cloth to facilitate the passage of the inflation tube, fastening the contact part of the plastic cloth and the air tube by using a rope, and irradiating all articles by using an ultraviolet lamp for 2 hours for disinfection before inoculation;

step three: placing tools such as a beaker and a measuring cylinder in water for boiling for 15 minutes for disinfection in the expanding culture process;

step four: filtering the gas-filled pipeline by a precise filtering system;

step five: adding the seawater or the fresh water which is subjected to precipitation and sand filtration into the plastic bucket in the step 1, wherein the water adding amount is 3/5 of the volume of the bucket. Adding 7ml of sodium hypochlorite (14% of available chlorine) into each liter of seawater or fresh water, sealing and inflating according to the step 1, adding 0.6g of sodium thiosulfate into each liter of seawater or fresh water for neutralization after 14 hours, adding the sodium thiosulfate into a barrel after the sodium thiosulfate is boiled in clear water, and testing whether residual chlorine exists by using starch potassium iodide after inflating for half an hour. The pH is adjusted to 7 with sodium hydroxide or hydrochloric acid solution.

Step six: adding the culture medium into clear water, boiling to melt, pouring into the plastic barrel in the step 5, then adding the bacillus licheniformis with the inoculation amount of 4%, sealing according to the step 1, preserving heat and inflating.

Step seven: after 4 days of culture, the number of the bacillus subtilis in the bucket is 22 multiplied by 10⁸And (5) the seeds are directly thrown into a culture pond per ml.

the method comprises the following steps: 55 parts of long fiber wood pulp, 31 parts of basswood wood pulp fiber and 19 parts of mercerized softwood pulp in parts by massUniformly mixing pulp fibers and 15 parts of para-aramid pulp fibers, pulping, adding 380 parts of sulfuric acid with the mass percentage concentration of 3%, adding 5 parts of allyl dimethoxysilane, controlling the temperature to 65 ℃, continuously pulping and mixing for 2 hours, then adding 1 part of ammonium persulfate and 1.3 parts of D-allyl glycine, controlling the temperature to 55 ℃, continuously pulping and mixing for 0.8 hour, defibering, dehydrating and drying to obtain air filter base paper, wherein the ration of the air filter base paper is 95g/m²；

Step two: and then adding 0.3 part of carboxylated carbon nanotubes into 120 parts of acrylic emulsion with the solid content of 38%, mixing and stirring for 15min, then carrying out ultrasonic treatment for 50min, uniformly dispersing, coating on the surface of base paper for air filtration, wherein the coating weight is 6g/m2, and then drying for 150s at 90-110 ℃ to obtain the nano-scale air filtration material.

Table 3: PCR preliminary experiment sample detection result

Table 4; diversity high-throughput sequencing result of bacillus liquid

On the genus level, the Bacillus liquid obtained by the method has the highest content of Bacillus (Bacillus), the ratio of the Bacillus liquid to the total number of bacteria is up to more than 99%, and the ratio of other mixed bacteria is less than 1%.

Example 3

the method comprises the following steps: the temperature in the lactobacillus culture room is required to be controllable, and an ultraviolet lamp is arranged according to the room space, wherein the average volume is 5w per cubic meter;

step two: an open plastic barrel is used as a probiotic culture container, transparent plastic cloth is cut according to the size of a barrel opening and is used for sealing, the plastic cloth covers the barrel opening, and the periphery of the plastic cloth is fastened by a rope. Perforating the center of the plastic cloth to facilitate the passage of the inflation tube, fastening the plastic cloth and the contact part of the air tube by using a rope, and irradiating all articles by using an ultraviolet lamp for 3 hours for disinfection before inoculation;

step three: placing tools such as a beaker and a measuring cylinder in water for boiling for 30 minutes for disinfection in the expanding culture process;

step four: filtering the gas-filled pipeline by a precise filtering system;

step five: adding the seawater or the fresh water which is subjected to precipitation and sand filtration into the plastic bucket in the step 1, wherein the water adding amount is 3/4 of the volume of the bucket. Adding 10ml of sodium hypochlorite (20% of available chlorine) into each liter of seawater or fresh water, sealing and inflating according to the step 1, adding 0.9g of sodium thiosulfate into each liter of seawater or fresh water for neutralization after 16 hours, adding the sodium thiosulfate into a barrel after the sodium thiosulfate is boiled in clear water, and testing whether residual chlorine exists by using starch potassium iodide after inflating for half an hour. The pH is adjusted to 7.5 with sodium hydroxide or hydrochloric acid solution.

Step seven: after 5 days of culture, the number of lactic acid bacteria in the bucket was 40X 10⁸And (5) the seeds are directly thrown into a culture pond per ml.

the method comprises the following steps: according to the mass portion, 65 portions of long fiber wood pulp, 36 portions of basswood pulp fiber, 25 portions of mercerized softwood pulp fiber and 18 portions of para-aramid pulp fiber are uniformly mixed and then pulped, 400 portions of sulfuric acid with the mass percentage concentration of 5% are added, 7 portions of allyl dimethoxy silane are added, the temperature is controlled to be 72 ℃, after pulping and mixing are continued for 5 hours, 2 portions of ammonium persulfate and 2.4 portions of D-allyl glycine are added, the temperature is controlled to be 68 ℃, after pulping and mixing are continued for 2.5 hours, fluffing, dewatering and drying are carried out to obtain air filter base paper, and the ration of the air filter base paper is 105g/m 2;

step two: and then adding 0.5 part of carboxylated carbon nanotubes into 200 parts of acrylic emulsion with the solid content of 45%, mixing and stirring for 30min, then carrying out ultrasonic treatment for 60min, uniformly dispersing, coating on the surface of base paper for air filtration, wherein the coating weight is 10g/m2, and then drying for 180s at 110 ℃ to obtain the nanoscale air filtration material.

The lactobacillus cultured by the method is used for carrying out fluorescence quantitative PCR experiment.

Table 5: PCR preliminary experiment sample detection result

Table 6: lactic acid bacteria liquid diversity high-throughput sequencing result

Name of the genus	Number of
		Acidovorax
Acinetobacter
		Aerococcus
Bacillus
		Bradyrhizobium
Brevundimonas
		Chloroplast_norank
Enterococcus
		Flavobacterium
Halolactibacillus
		Lactobacillus
Lactococcus
		Leuconostoc
Macrococcus
		Pseudonocardia
Staphylococcus
		Vagococcus
Vibrio
		Vibrionimonas
Weissella

On the genus level, the Lactobacillus liquid obtained by the method has the highest Lactobacillus content, the ratio of the Lactobacillus to the total bacteria is up to more than 99%, and the ratio of other bacteria is less than.

Example 4

A method for applying bacillus liquid to stichopus japonicus breeding comprises the following steps:

1 materials and methods

1.1 materials

The young stichopus japonicus and the feed for the test are provided by a breeding center introduced in mariculture in Liaoning province. The mass of the stichopus japonicus juvenile sea cucumber monomer is 7.17 +/-0.86 g, and a fresh and alive sample is taken back and temporarily cultured for one week under the conventional condition of an artificial seedling pond. During the temporary culture period, aeration is kept, the temperature of seawater is 16 ℃, the pH value is 7.9, and the salinity is 29.3. And feeding the young sea cucumbers with the feed after changing water every day, wherein the daily dosage of the feed is 3-5% of the weight of the young sea cucumbers. The test uses Bacillus for the cultivation of the method. Pathogenic bacteria used in the challenge test are vibrio splendidus which are separated from the focus of the stichopus japonicus suffering from skin rot syndrome.

1.2 Breeding method

Randomly distributing the stichopus japonicus after temporary rearing for 7d into aquariums with the same specification, wherein 200 heads are distributed in each aquarium. Culturing the strain to logarithmic phase, centrifuging at 4 deg.C and 12000r/min for 10min, discarding supernatant, and adding sterile seawater for resuspension. The bacterial liquid is mixed in feed according to the concentration ratio of 105, 107, 109 and 1011cfu/g respectively by adopting a turbidimetric method and is added into different groups of aquaculture water bodies. During the test period, aeration is kept, and the feeding condition is consistent with the temporary rearing period. The experiment was set up in 5 treatments, 3 replicates each of which was repeated 3 times. Samples were taken at 0, 10, 20, 30 and 40d of the test to determine the growth, digestion and immunity indexes of stichopus japonicus.

1.3 measurement of growth index of Stichopus japonicus

The mass of the stichopus japonicus is measured in 0, 10, 20, 30 and 40 days after the bacillus is fed, and the mass increasing rate and the specific growth rate are calculated. The mass increase rate and the specific growth rate are calculated according to the following formula:

the mass increase rate calculation formula (i) WGR/% (Wt-W0)/Wt;

the specific growth rate calculation formula (SGR/%, d-1) ═ lnWt-lnW 0)/t.

Wherein Wt is the weight of Stichopus japonicus, W0 is the initial weight of Stichopus japonicus, and t is time (d).

1.4 determination of the Activity of intestinal digestive enzymes of Stichopus japonicus

Randomly taking 5 heads of stichopus japonicus from each test group at 0 th, 10 th, 20 th, 30 th and 40 th days after feeding bacillus, dissecting the stichopus japonicus to obtain intestinal tissues, removing intestinal contents and mesentery on an ice tray, washing with phosphate buffer (pH7.5), drying with filter paper, and homogenizing with 10 times of phosphate buffer. Centrifuging the homogenate at 4 deg.C and 3000r/min for 20min to obtain supernatant as crude enzyme solution.

The trypsin activity is determined according to a trypsin test kit of Nanjing engineering science and technology Limited, and the enzyme activity unit is defined as that the change of the absorbance by 0.003 per minute of trypsin contained in each milligram of protein under the conditions of pH8.0 and 37 ℃.

The lipase activity is measured according to a lipase test box of Nanjing technology Limited, and is defined that each milligram of tissue protein reacts with a substrate in the reaction system for 1min at the temperature of 37 ℃, and each 1umol of the substrate is consumed as one enzyme activity unit.

The amylase activity is measured according to the amylase test box of Nanjing technology Limited, and the activity of each mg of protein in the tissue is defined to act on a substrate for 30min at 37 ℃, and the activity of hydrolyzing 10mg of starch is defined as 1 amylase activity unit.

1.5 determination of immune index in body cavity fluid of Stichopus japonicus

Randomly taking 5 heads of stichopus japonicus from each test group at 0 th, 10 th, 20 th, 30 th and 40 th days of feeding bacillus respectively, draining for 10min, then using a sterile syringe to extract 0.5ml of coelomic fluid from the abdomen of the stichopus japonicus, placing the coelomic fluid in a precooled centrifuge tube, and combining the coelomic fluid. Centrifuging at 4 deg.C and 3000r/min for 20min, and collecting supernatant for measuring acid phosphatase, alkaline phosphatase, superoxide dismutase and lysozyme activity. The kit of Nanjing institute of bioengineering is used for detecting the activity of the immunoenzyme of the stichopus japonicus.

The determination principle of the acid phosphatase and the alkaline phosphatase is that the acid phosphatase and the alkaline phosphatase decompose disodium phenyl phosphate to generate free phenol and phosphoric acid, the phenol reacts with 4-aminoantipyrine in an alkaline solution to generate red quinone derivatives through potassium ferricyanide oxidation, and the enzyme activity can be determined according to the red shade. Acid phosphatase activity was defined as the activity of 1mg phenol produced by the action of 100mL of supernatant on the substrate at 37 ℃ for 30 min. Alkaline phosphatase activity was defined as the activity of 1mg phenol produced by the action of 100mL of supernatant on the substrate at 37 ℃ for 15 min.

The principle of measuring superoxide dismutase is that xanthine and oxidase thereof are generated by a reaction system (O2-), the latter oxidizes hydroxylamine to form nitrite, the nitrite presents purple red under the action of a color developing agent, and the absorbance of the nitrite is measured by a visible light spectrophotometer. When the tested sample contains superoxide dismutase, the control superoxide anion free radical has specific inhibition effect, so that the formed nitrite is reduced, and the absorbance value of the determination tube during color comparison is lower than that of the control tube. The activity of the superoxide dismutase is defined as that the enzyme amount corresponding to the enzyme inhibition rate of 50 percent in each reaction is one superoxide dismutase activity unit.

The principle of lysozyme determination is that in turbid bacterial liquid with a certain concentration, because lysozyme can hydrolyze peptidoglycan on bacterial cell walls to crack bacteria, the concentration is reduced, and the transmittance is enhanced, the content of lysozyme can be presumed according to the transmittance change. The activity of the lysozyme is measured by a turbidimetric method, and the activity of the lysozyme in a sample is calculated according to the transmittance of the sample, a standard substance and distilled water when the sample, the standard substance and the distilled water react with an applied enzyme solution at 37 ℃.

1.6 Effect of Bacillus on the resistance of Stichopus japonicus against Vibrio splendidus infection

In order to verify the immune protection effect of the bacillus on the stichopus japonicus, 20 stichopus japonicus in each group are randomly extracted for the vibrio splendidus challenge test after the feeding test is finished. The second activated vibrio splendidus is inoculated on 2216E culture medium, after 24h of culture at 28 ℃, the vibrio splendidus is diluted to a series of concentrations by sterile physiological saline, and the semi-lethal concentration of the vibrio splendidus on the stichopus japonicus is determined to be 2 x 107 cfu/mL by a preliminary experiment. Injecting 100uL of vibrio splendidus with half-lethal concentration into the abdominal cavity of each stichopus japonicus, recording the physiological reaction and the cumulative death rate of each group of stichopus japonicus after attacking poison for 14 days, and calculating the relative immune protection rate. After 14 days of detoxification, randomly taking 5 stichopus japonicus from each test group, dissecting and cutting intestinal tissues (about 0.5cm multiplied by 0.5 cm) respectively, combining the cut tissues, putting the cut tissues into a 50mL centrifuge tube, adding 30mL PBS buffer solution, putting the centrifuge tube into a tissue homogenizer for treatment for 10min, centrifuging the homogenate at 4 ℃ and 3000r/min for 10min, diluting the supernatant by 10 times with sterile normal saline, coating 200 mu L of the supernatant onto a TCBS vibrio selective culture medium, performing inversion culture at 28 ℃, and counting the number of intestinal vibrios of each group of stichopus japonicus after 2 days. The cumulative mortality and the relative immune protection rate are calculated according to the following formula: the formula of the calculation of the intestinal vomiting rate is (ER/%) < Ne/N0;

cumulative mortality calculation formula (CMR/% ═ N0-Nt)/N0;

the relative immunoprotection rate calculation formula (RPS/%) 1- (Mt/M0).

In the formula, N0 is the initial individual number of the stichopus japonicus test, Ne is the viscus discharging individual number of the stichopus japonicus test, Nt is the terminal survival individual number of the stichopus japonicus test, Mt is the mortality of a challenge group, and M0 is the mortality of a control group.

1.7 data processing

Data were analyzed by SPSS19.0 software for One-way ANOVA and Dunncan multiple comparisons and expressed as mean. + -. standard deviation (X. + -. SD), with differences considered significant when P <0.05 and significant when different lower case letters are indicated in the results.

2. Results and analysis

2.1 measurement of growth index of Stichopus japonicus

Before feeding the bacillus, the average wet weight of young sea cucumber individuals in each test group is not greatly different. By the termination of the test, the mass of the young ginseng bodies of each test group shows a growing trend. Wherein the weight gain rate and specific growth rate of the stichopus japonicus in the test group with the bacillus addition concentration of 109cfu/g are relatively highest. The weight gain rate and specific growth rate of the stichopus japonicus in the test groups with bacillus added concentrations of 105 and 1011cfu/g are lower than those of the control group (Table 1).

Table 7: influence of bacillus on weight gain rate and specific growth rate of stichopus japonicus

Note: data are presented as mean ± standard deviation the same column data are shoulder-labeled with different lower case letters indicating significant difference (P <0.05) and labeled with the same lower case letters indicating no significant difference between groups (P > 0.05).

2.2 Effect of Bacillus on the Activity of Apostichopus japonicus digestive enzymes

The effect of different concentrations of bacillus on the intestinal digestive enzyme activity of stichopus japonicus is shown in fig. 7-9. Wherein the live bacteria addition concentration of the bacillus in A, B, C, D groups of feeds is 10 respectively⁵、10⁷、10⁹、10¹¹cfu/g, CK group as control group without bacillus added in feed. FIG. 7 is a graph showing the effect of Bacillus on the intestinal trypsin activity of Stichopus japonicus. With the increase of the feeding time, the trypsin activity of the test group and the control group gradually increased. Group A and group B had significantly higher trypsin activity than the control group (P) at 10 days of feeding<0.05). After 20 days of feeding, group AThe trypsin activity increased slowly, while the trypsin activity was significantly higher in groups B and C than in the control group (P)<0.05)。

The effect of bacillus on the intestinal lipase activity of stichopus japonicus is shown in fig. 8. The lipase activities of the test group and the control group gradually increased with the increase of the feeding time. The lipase activity of the group B is increased faster than that of the control group and other test groups, and the lipase activity is obviously higher than that of the control group and the test groups at the same time (P < 0.05).

The effect of bacillus on the amylase activity of stichopus japonicus intestinal tract is shown in fig. 9. The amylase activity of the test group and the amylase activity of the control group tend to increase and decrease along with the extension of the feeding time, and the amylase activity of the control group and each test group reaches the highest value at the feeding time of 30 days. Wherein the amylase activity of group D was lower than that of the control group at the same time period, while the amylase activity of group B and C showed significant difference (P <0.05) compared to the control group.

2.3 Effect of Bacillus on Apostichopus japonicus Immunity

The effect of different concentrations of bacillus on the body cavity fluid immunoenzyme activity of stichopus japonicus is shown in fig. 10-fig. 13. Wherein the live bacteria addition concentration of the bacillus in A, B, C, D groups of feeds is 10 respectively⁵、10⁷、10⁹、10¹¹cfu/g, CK group as control group without bacillus added in feed. FIG. 10 is a graph showing the effect of Bacillus on the activity of acid phosphatase in body cavity fluid of Apostichopus japonicus. The acid phosphatase activity of the test group is higher than that of the control group along with the extension of the feeding time, and the acid phosphatase activity of the group B and the group C is obviously higher than that of the control group (P) after the feeding for 30 days<0.05), whereas the activity of acid phosphatase in groups a and D was not significantly different compared to the control group (P)>0.05)。

The effect of bacillus on the alkaline phosphatase activity of the body cavity fluid of stichopus japonicus is shown in fig. 11. As the feeding time increased, the alkaline phosphatase activity increased gradually and higher in each test group than in the contemporary control group. Wherein, the alkaline phosphatase activity of group C is increased most, and the alkaline phosphatase activity of group B is increased next. Both alkaline phosphatase activities were significantly higher than the contemporary control group (P < 0.05).

The effect of bacillus on the activity of superoxide dismutase in the body cavity fluid of stichopus japonicus is shown in fig. 12. The superoxide dismutase activity of the test group and the superoxide dismutase activity of the control group show a trend of increasing firstly and then decreasing, and the superoxide dismutase activity of the control group and each test group reaches the highest when the animals are fed for 30 days. After 30 days of feeding, the activity of superoxide dismutase in the group B and the group C is obviously higher than that of the control group (P <0.05), and the activity of the superoxide dismutase in the group A and the group D is not obviously different from that of the control group (P > 0.05).

The effect of bacillus on the lysozyme activity of the stichopus japonicus coelomic fluid is shown in fig. 13. The lysozyme activity of the test group and the control group is gradually increased along with the extension of the feeding time, the lysozyme activity of the group B and the group D is obviously higher than that of the control group (P <0.05) when the group B and the group D are fed for 20 days, the lysozyme activity of the group D is slowly increased after the group D and the group B and the group C are obviously higher than that of the control group (P < 0.05).

2.4 Effect of Bacillus on the resistance of Stichopus japonicus against Vibrio splendidus infection

After vibrio splendidus is injected, the tested group and the control group of the stichopus japonicus have adverse physiological reactions of mouth swelling, shaking head, intestinal vomiting and the like with different degrees. The concentration of Bacillus is 10¹¹The cumulative mortality rate and the intestinal vomiting rate of the stichopus japonicus are higher and the relative immune protection rate is lower when cfu/g is used. The concentration of Bacillus is 10⁷The cumulative mortality and vomiting rate of apostichopus japonicus was relatively low at cfu/g, while the relative immunoprotection rate was high (table 2). After the challenge test, each group of intestinal tissue diluent of stichopus japonicus is coated on a vibrio selective culture medium, wherein the live bacteria addition concentration of bacillus in A, B, C, D groups of feeds is 10 respectively⁵、10⁷、10⁹、10¹¹cfu/g, CK group as control group without bacillus added in feed. Wherein the number of vibrio splendidus in group B is the least, and the number of vibrio splendidus in group C is the next, and the number of vibrio splendidus in both groups is obviously less than that of the control group and other treatment groups.

Table 8: influence of Bacillus on resistance of Apostichopus japonicus to infection by Vibrio splendidus

Example 5

the mass (4.20 +/-0.51) g of the stichopus japonicus monomer and the temporary culture density of 385g/m³During the period, the aeration is kept, the temperature of seawater is 24 ℃, the pH value is 8, and the salinity is 30.5. And feeding the young sea cucumbers with the feed after changing water every day, wherein the daily dosage of the feed is 8 percent of the weight of the young sea cucumbers. The bacillus subtilis liquid is prepared by a turbidimetric method according to the proportion of 10⁵、10⁷、10⁹、10¹¹The concentration ratios of cfu/g are respectively mixed in feed and added into different groups of aquaculture water bodies. During the test period, aeration is kept, and the feeding condition is consistent with the temporary rearing period. The experiment was set up with 5 treatments, 3 replicates, i.e. 3 replicates per treatment. The mass of the stichopus japonicus is measured at 0, 10, 20, 30 and 40d of the bacillus fed respectively, 5 individuals are taken in each group in parallel, and the mass increase rate and the specific growth rate are calculated. The mass increase rate and the specific growth rate are calculated according to the following formula:

the mass increase rate calculation formula (i) WGR/% (Wt-W0)/Wt;

the specific growth rate calculation formula (SGR/%, d-1) ═ lnWt-lnW 0)/t.

Wherein Wt is the body mass of Stichopus japonicus td, W0 is the initial body mass of Stichopus japonicus, and t is the time (d)

Before feeding the bacillus, the average wet weight of young sea cucumber individuals in each test group is not greatly different. By the termination of the test, the mass of the young ginseng bodies of each test group shows a growing trend. Wherein the bacillus is added at a concentration of 10⁷And 10⁹The weight gain and specific growth rate of the stichopus japonicus of the test group cfu/g were relatively high. (Table 9).

Table 9: influence of bacillus on weight gain rate and specific growth rate of stichopus japonicus

Example 6

the stichopus japonicus monomer mass is 1.14 +/-0.51 g, and the temporary culture density is 200g/m³During the period, the aeration is kept, the temperature of the seawater is 20 ℃, the pH value is 8.2, and the salinity is 28. And feeding the young sea cucumbers with the feed after changing water every day, wherein the daily dosage of the feed is 6 percent of the weight of the young sea cucumbers. The bacillus subtilis liquid is prepared by a turbidimetric method according to the proportion of 10⁵、10⁷、10⁹、10¹¹The concentration ratios of cfu/g are respectively mixed in feed and added into different groups of aquaculture water bodies. During the test period, aeration is kept, and the feeding condition is consistent with the temporary rearing period. The experiment was set up with 5 treatments, 3 replicates, i.e. 3 replicates per treatment. The mass of the stichopus japonicus is measured at 0, 10, 20, 30 and 40d of the bacillus fed respectively, 5 individuals are taken in each group in parallel, and the mass increase rate and the specific growth rate are calculated. The mass increase rate and the specific growth rate are calculated according to the following formula:

the mass increase rate calculation formula (i) WGR/% (Wt-W0)/Wt;

the specific growth rate calculation formula (SGR/%, d-1) ═ lnWt-lnW 0)/t.

Before feeding the bacillus, the average wet weight of young sea cucumber individuals in each test group is not greatly different. By the termination of the test, the mass of the young ginseng bodies of each test group shows a growing trend. Wherein the bacillus is added at a concentration of 10⁷And 10⁹The weight gain and specific growth rate of the stichopus japonicus of the test group cfu/g were relatively highest. (Table 10).

Table 10: influence of bacillus on weight gain rate and specific growth rate of stichopus japonicus

In conclusion, the Bacillus bacteria cultured by the present methodHas obvious probiotic effect on promoting the growth of young stichopus japonicus, improving the activity of digestive enzyme and enhancing the immune defense function, and is dose-dependent. According to various indexes, when the addition concentration of the bacillus in the feed is 10⁷、10⁹The growth capacity, the digestive enzyme activity, the immunoenzyme activity and the resistance to vibrio splendidus of the stichopus japonicus are obviously improved when cfu/g is adopted, which shows that the bacillus cultured by the method can be used as a safe and effective probiotic additive to be applied to stichopus japonicus culture.

Claims

1. A method for applying bacillus liquid to stichopus japonicus breeding comprises the following steps:

the stichopus japonicus seed with the mass of 1-10g of monomer is added with the weight of 100-³The density culture of (3). Culturing bacillus liquid by simple culture method, and turbidimetric treating the bacillus liquid by 10⁷Or 10⁹The cfu/g is mixed in the feed according to the concentration ratio for feeding the stichopus japonicus, and the daily dosage is 3-10% of the weight of the stichopus japonicus.

2. The method for feeding stichopus japonicus according to claim 1, comprising the following steps: the weight range of the stichopus japonicus offspring seeds is 1-10g, and the weight range is 100-³The density culture of (3).

3. The method for feeding stichopus japonicus according to claim 1, comprising the following steps: during the period, aeration is kept, the temperature of the seawater is 15-25 ℃, the pH value is 7.8-8.2, and the salinity is 28-32.

4. The method for feeding stichopus japonicus according to claim 1, comprising the following steps: the daily bait feeding amount is 3-10% of the weight of the stichopus japonicus.

5. The method for feeding stichopus japonicus according to claim 1, comprising the following steps: the addition concentration of the bacillus liquid in the feed is 10⁷Or 10⁹The growth power and the digestive enzyme activity of the stichopus japonicus are improved at cfu/gThe activity of the enzyme and the capability of resisting vibrio splendidus.

6. The method for feeding stichopus japonicus according to claim 1, comprising the following steps: the strain is filtered by a precise filtering system during simple culture, and the precise filtering system uses a nano-grade air filtering material.

7. The method for feeding stichopus japonicus according to claim 6, wherein the method comprises the following steps: a nano-scale air filter material is prepared by the following steps:

the method comprises the following steps: according to the mass portion, 50-65 portions of long fiber wood pulp, 28-36 portions of basswood wood pulp fiber, 18-25 portions of mercerized softwood pulp fiber and 10-18 portions of para-aramid pulp fiber are uniformly mixed and then pulped, then 300 portions of sulfuric acid with the mass percentage concentration of 2-5% is added, 3-7 portions of allyl dimethoxy silane are added, the temperature is controlled to be 60-72 ℃, after continuous pulping and mixing is carried out for 1-5h, 0.5-2 portions of ammonium persulfate and 0.3-2.4 portions of D-allyl glycine are added, the temperature is controlled to be 50-68 ℃, after continuous pulping and mixing is carried out for 0.5-2.5h, fluffing, dewatering and drying are carried out, and then the air filtration base paper is obtained.

8. The method for simply culturing probiotics suitable for aquaculture according to claim 7, wherein the method comprises the following steps: and introducing allyl into the long fiber wood pulp, the basswood pulp fiber and the mercerized softwood pulp fiber to prepare the silane modified wood pulp fiber.

9. The method for simply culturing probiotics suitable for aquaculture according to claim 7, wherein the method comprises the following steps: and (3) carrying out graft copolymerization on the silane modified wood pulp fiber and D-allyl glycine.