CN-122012281-A - Composite microbial inoculum for water quality purification and vibrio prevention and control and preparation method thereof

CN122012281ACN 122012281 ACN122012281 ACN 122012281ACN-122012281-A

Abstract

The application relates to the technical field of aquaculture, and particularly discloses a composite microbial inoculum for water quality purification and vibrio prevention and control and a preparation method thereof. The composite microbial inoculum for water quality purification and vibrio prevention and control comprises bacillus subtilis, bacillus natto and bacillus pumilus with the viable count ratio of (1-3): 1-2, wherein the bacillus subtilis is a strain capable of producing protease and amylase, the bacillus natto is a strain capable of producing nattokinase and protease, and the bacillus pumilus is a strain capable of producing antibacterial small peptide for inhibiting vibrio. The application can increase the total viable count of the composite microbial inoculum, increase the COD degradation rate of the composite microbial inoculum in a short time and the vibrio inhibition rate in a long time, and is beneficial to improving the rapid water quality purification performance and the durable vibrio prevention and control performance of the composite microbial inoculum.

Inventors

ZHU XINYING
CHENG CHUNMING
CHENG MING
ZHU XINTAO
LIU GANG

Assignees

连云港荣盛生物科技有限公司

Dates

Publication Date: 20260512
Application Date: 20260226

Claims (5)

1. The preparation method for water quality purification and vibrio prevention and control is characterized by comprising the following steps: The composite microbial inoculum for water quality purification and vibrio prevention and control comprises bacillus subtilis, bacillus natto and bacillus pumilus with the viable count ratio of (1-3): 1-2, wherein the bacillus subtilis is a strain capable of producing protease and amylase, the bacillus natto is a strain capable of producing nattokinase and protease, the bacillus pumilus is a strain capable of producing antibacterial small peptide for inhibiting vibrio, and the total viable count of the composite microbial inoculum for water quality purification and vibrio prevention and control is more than or equal to 2.0x10 10 CFU/g; S1, performing liquid seed expansion culture on single strains of bacillus subtilis, bacillus natto and bacillus pumilus respectively to obtain bacillus subtilis liquid seed liquid, bacillus natto liquid seed liquid and bacillus pumilus liquid seed liquid; s2, mixing the bacillus subtilis liquid seed liquid, the bacillus natto liquid seed liquid and the bacillus pumilus liquid seed liquid according to the ratio of the viable count of (1-3): (1-2): (1-2), and then inoculating the mixture into a solid fermentation medium for common solid fermentation, and obtaining a fermentation product after the fermentation is finished; s3, drying and crushing the fermentation product at low temperature to obtain the composite microbial inoculum for water quality purification and vibrio prevention and control.
2. The method for preparing the composite microbial inoculum for water quality purification and vibrio prevention and control according to claim 1, wherein the solid fermentation medium comprises bran, bean pulp and corn flour in a mass ratio of (4-6): 2.5-3.5): 2.
3. The method for preparing the composite microbial inoculum for water quality purification and vibrio prevention and control according to claim 1 is characterized in that the step S2 is that the bacillus subtilis liquid seed liquid, the bacillus natto liquid seed liquid and the bacillus pumilus liquid seed liquid are mixed according to the ratio of viable bacteria number (1-3): (1-2): (1-2), inoculated into a solid fermentation culture medium, subjected to common solid fermentation at the fermentation temperature of 35-37 ℃ for 36-72 hours, and then fermented to obtain a fermentation product.
4. The method for preparing a composite microbial inoculant for water quality purification and vibrio prevention and control according to claim 1, wherein the bacillus subtilis is subjected to strain screening according to the following steps: collecting humus soil, adding the humus soil into normal saline, shaking in a shaking table at a constant temperature of 30 ℃ until the humus soil is uniformly dispersed, diluting supernatant, respectively coating the diluted supernatant on a casein-containing culture medium and a starch-containing culture medium, and inversely culturing the culture medium at a temperature of 30-37 ℃ for 24-48 hours; Observing whether transparent rings appear around colonies on a culture medium containing casein, measuring the ratio of the diameter of the transparent rings to the diameter of the colonies, collecting bacterial strains of the colonies with the ratio of the diameter of the transparent rings to the diameter of the colonies being more than or equal to 2.0, obtaining the bacillus subtilis producing protease, pouring lugol's iodine solution on the culture medium containing starch, standing, observing whether the transparent rings appear around the colonies on the culture medium containing starch, measuring the ratio of the diameter of the transparent rings to the diameter of the colonies, collecting bacterial strains of the colonies with the ratio of the diameter of the transparent rings to the diameter of the colonies being more than or equal to 1.5, obtaining the bacillus subtilis producing amylase, and identifying the bacillus subtilis producing protease and the bacillus subtilis producing amylase, and then preserving the identified qualified bacterial strains to obtain the bacillus subtilis.
5. The method for preparing a composite microbial inoculant for water quality purification and vibrio prevention and control according to claim 1, wherein the bacillus pumilus is subjected to strain screening according to the following steps: taking out the content in the intestinal canal of the prawn, dispersing in normal saline, shaking in a 30 ℃ constant temperature shaking table until the content is dispersed uniformly, diluting the supernatant, coating the diluted supernatant on a nutrient agar plate, culturing for 24-48 hours at 30-37 ℃, and selecting single colony with different forms, colors and sizes for purification to obtain 23 pure cultures; Inoculating vibrio parahaemolyticus on a liquid culture medium, culturing in a 30 ℃ constant temperature shaking table to a logarithmic mid-term to obtain an indicator bacteria liquid, uniformly mixing the indicator bacteria liquid with soft agar at 45-50 ℃, pouring the mixture on a bottom plate formed by solidifying nutrient agar, after solidification, placing sterile oxford cups on the bottom plate at equal intervals, respectively dispersing 23 pure cultures, adding the 23 pure cultures into sterile oxford cup holes, pre-dispersing for 2-4 hours at 4 ℃, transferring to 30 ℃ for forward culture for 18-24 hours, observing whether a bacteria inhibition zone appears around the bacteria colony, measuring the diameter of the bacteria inhibition zone, collecting bacterial strains of the bacteria colony with the diameter of more than or equal to 15mm, and carrying out identification on the bacterial strains to obtain the bacterial strains qualified by identification.

Description

Composite microbial inoculum for water quality purification and vibrio prevention and control and preparation method thereof Technical Field The invention relates to the technical field of aquaculture, in particular to a composite microbial inoculum for water quality purification and vibrio prevention and control and a preparation method thereof. Background With the rapid development of intensive aquaculture industry, aquaculture water pollution and disease frequency have become key problems restricting industry sustainable development. Organic pollutants such as residual baits, feces, biological remains and the like generated in the cultivation process are accumulated in a large quantity, so that water eutrophication is caused, ammonia nitrogen and nitrite are out of standard, the substrate is deteriorated, and a hotbed is provided for breeding pathogenic microorganisms. Vibriosis is one of the most common and most harmful bacterial diseases in aquaculture, and often causes large-scale death of economic species such as prawns, fishes and the like, and brings about huge economic loss. Currently, the main methods to deal with the above problems include the use of chemical disinfectants and antibiotics. However, the methods have the defects that the chemical disinfectant can destroy beneficial microecological balance in the water body while killing harmful bacteria, toxic byproducts can be generated, and the abuse of antibiotics can easily cause the problems of drug residues, enhanced bacterial resistance, environmental pollution and the like. Therefore, the development of safe, efficient and environment-friendly microecological preparations has become a research hotspot in the aquaculture industry. In the related art, most of microecologics are single strains or composite bacterial agents with simple functions, and the defects of single function, slow onset of action, short duration of action and the like exist. For example, some water purifying bactericides have strong capability of decomposing organic matters, but have no inhibition effect on pathogenic bacteria, and some antibacterial bactericides can reduce the number of vibrio transiently, but cannot solve the fundamental problem of organic pollution of water, and the antibacterial effect is not durable because bacterial strains cannot effectively colonize in the water. Therefore, a novel compound microbial agent capable of simultaneously realizing dual functions of rapidly purifying water and durably preventing and controlling vibrio is urgently needed in the field. Disclosure of Invention The application provides a composite microbial inoculum for water quality purification and vibrio prevention and control and a preparation method thereof. In a first aspect, the application provides a composite microbial inoculum for water quality purification and vibrio prevention and control, which adopts the following technical scheme: A composite microbial inoculum for water quality purification and vibrio prevention and control comprises bacillus subtilis, bacillus natto and bacillus pumilus with the viable count ratio of (1-3): 1-2. In a specific embodiment, the bacillus subtilis is a strain capable of producing protease and amylase, the bacillus natto is a strain capable of producing nattokinase and protease, and the bacillus pumilus is a strain capable of producing antibacterial small peptides inhibiting vibrio. In a specific embodiment, the total viable count of the composite microbial inoculum for water quality purification and vibrio prevention and control is more than or equal to 2.0X10 10 CFU/g. In a specific embodiment, the viable count ratio of bacillus subtilis, bacillus natto and bacillus pumilus is 2:1:1. In a second aspect, the application provides a preparation method of a composite microbial inoculum for water quality purification and vibrio prevention and control, which adopts the following technical scheme: A preparation method of a composite microbial inoculum for water quality purification and vibrio prevention and control comprises the following steps: S1, performing liquid seed expansion culture on single strains of bacillus subtilis, bacillus natto and bacillus pumilus respectively to obtain bacillus subtilis liquid seed liquid, bacillus natto liquid seed liquid and bacillus pumilus liquid seed liquid; s2, mixing the bacillus subtilis liquid seed liquid, the bacillus natto liquid seed liquid and the bacillus pumilus liquid seed liquid according to the ratio of the viable count of (1-3): (1-2): (1-2), and then inoculating the mixture into a solid fermentation medium for common solid fermentation, and obtaining a fermentation product after the fermentation is finished; s3, drying and crushing the fermentation product at low temperature to obtain the composite microbial inoculum for water quality purification and vibrio prevention and control. In a specific embodiment, the solid fermentation medium comprises bran, soybean meal and corn meal i