CN-121991805-A - Separation and screening method for fungi on surface of covered rice straw

CN121991805ACN 121991805 ACN121991805 ACN 121991805ACN-121991805-A

Abstract

The invention relates to a method for separating and screening fungi on the surface of rice covered with rice straw, which comprises the following steps of S1, taking a rice covered straw sample, adding sterile normal saline, oscillating to enable microorganisms to be fully dispersed, preparing a fungus suspension mother solution, and then carrying out gradient dilution to obtain fungus suspensions with different concentrations, S2, separating and purifying, namely coating the fungus suspensions with different concentrations on a Bengal culture medium, reversely culturing, picking single colonies, inoculating the single colonies on a new Bengal plate for purification, S3, carrying out molecular identification, extracting genome DNA of the purified strain, carrying out PCR amplification by using fungus universal primers ITS1 and ITS4, carrying out BLAST comparison on the PCR products after sequencing and NCBI databases, constructing a phylogenetic tree, and determining the genus of the strain. The scheme obtains the fungus strain with specific functional characteristics for white spirit processing.

Inventors

WANG XINYE
HE MINGYUAN
LIU ZIWEI
ZHANG XIN
XIAO DINGYI
QIU ZIFEI
Luo Xiaolang
ZHAI HANBIN

Assignees

茅台学院

Dates

Publication Date: 20260508
Application Date: 20260211

Claims (5)

1. The method for separating and screening fungi on the surface of the covered rice straw is characterized by comprising the following steps of: s1, taking a sample of rice straw covered with yeast, adding sterile normal saline, oscillating to fully disperse microorganisms, preparing a bacterial suspension mother solution, and carrying out gradient dilution to obtain bacterial suspensions with different concentrations; S2, separating and purifying, namely coating bacterial suspensions with different concentrations on a Bengalia red culture medium, reversely culturing, picking single bacterial colonies, and inoculating the bacterial suspensions on a new Bengalia red plate for purifying; S3, molecular identification, namely extracting genome DNA of the purified strain, performing PCR amplification by using fungus universal primers ITS1 and ITS4, sequencing a PCR product, performing BLAST comparison with NCBI database, constructing a phylogenetic tree, and determining strain species.
2. The method for separating and screening fungi on the surface of covered rice straw according to claim 1, wherein the sterile physiological saline in the step S1 is NaCl sterile physiological saline with the concentration of 1mol/L, the mass-volume ratio of the covered rice straw sample to the physiological saline is 1g to 9mL, the shaking condition is room temperature and 120r/min, the shaking time is 2h, and the gradient dilution concentration is 10 -1 、10 -2 、10 -3 .
3. The method for separating and screening fungi on the surface of covered rice straw according to claim 2, wherein in the step S2, the formula of the Bengalia amara red culture medium is 20g of agar, 5g of yeast extract, 10g of tryptone, 1g of glucose, 5g of sodium chloride, 0.01g of Bengalia amara red and 1000mL of distilled water, and the culture condition is 30 ℃ and the culture time is 2-3d.
4. The method for separating and screening fungi on the surface of covered rice straw according to claim 3, wherein the step S3 is characterized in that the genome DNA of the strain is extracted by adopting a sodium laurate method, the PCR reaction conditions are that the genome DNA is pre-denatured for 94 ℃ for 4min, denatured for 94 ℃ for 45S, annealed for 55 ℃ for 45S, extended for 72 ℃ for 1min, circulated for 35 times, extended for 72 ℃ for 10min and kept at 4 ℃, and a phylogenetic tree is constructed by adopting MEGA7 software through a proximity method.
5. The method for separating and screening fungi on the surface of covered rice straw according to claim 4, wherein the method further comprises the following steps of: A1, temperature tolerance analysis, namely respectively inoculating each strain after determining the strain species into a PDA culture medium plate, respectively placing the strains into a 30 ℃, 40 ℃,50 ℃ and 60 ℃ incubator to be cultured for 3-5 days, observing the growth condition of the strain, and recording the highest growth temperature of the strain; A2, lactic acid tolerance analysis, namely independently sterilizing lactic acid, adding the lactic acid into a PDA culture medium cooled to about 50 ℃ according to a proportion, preparing a gradient culture medium with the final concentration of the lactic acid of 20g/L, 40g/L, 60g/L, 80g/L and 100g/L, inoculating each strain into the culture medium, culturing for 3-5d at 30 ℃, observing the growth state, and determining the highest lactic acid tolerance concentration of the strain; A3, pH tolerance analysis, namely preparing a YPD culture medium, adding 1mol/L hydrochloric acid to adjust the pH of the culture medium to 5, 6 and 7, adding 2% agar powder, sterilizing at 121 ℃ for 20min, pouring the culture medium into a flat plate, inoculating a strain, culturing at 30 ℃ for 3-5d, and observing the growth condition of the strain; A4, ethanol tolerance analysis, namely adding ethanol with different volume fractions into a PDA culture medium, preparing a gradient culture medium with the volume fractions of 2%, 4%, 6% and 8%, culturing for 3-5d at 30 ℃ after inoculating the strain, observing the growth state, and recording the highest ethanol tolerance concentration of the strain; A5, measuring enzyme activity of the fungus strain, namely measuring production capacity of cellulase, saccharifying enzyme, pectase, alpha-amylase, protease, esterifying enzyme and tannase of the strain by adopting a transparent loop method, inoculating the strain to a screening culture medium flat plate containing corresponding substrates, culturing for 3-5 days at 30 ℃, and observing whether a transparent loop is produced or not, and judging whether the strain has the production capacity of the enzyme and the relative strength of the enzyme activity according to the existence and the size of the transparent loop; A6, analyzing metabolites of fungus strains, namely inoculating each strain to a glucose fermentation culture medium, carrying out shaking culture for 48 hours at 30 ℃ and 150r/min, centrifuging for 10min at 8000r/min after the culture is finished, removing thalli, taking supernatant, adding equal volume of ethyl acetate for extraction, standing for layering after shaking and mixing uniformly, collecting an organic phase, concentrating the organic phase by adopting a rotary evaporator, detecting and analyzing the concentrated organic phase by adopting a GC-MS technology, and identifying the types of the metabolites.

Description

Separation and screening method for fungi on surface of covered rice straw Technical Field The invention relates to the technical field of microbial separation, in particular to a separation and screening method for fungi on the surface of covered rice straw. Background The Daqu of the white spirit is used as a key saccharification starter and a flavoring agent in the white spirit brewing process, and the quality of the Daqu directly influences the flavor and the taste of the white spirit. The Maotai-flavor high-temperature Daqu is produced by adopting a traditional process, and enrichment and succession of microorganisms are realized by relying on a natural fermentation environment, so that rich enzymes and microorganism metabolites are accumulated, and the components are an important foundation for forming the Maotai-flavor. In the process of producing Daqu, the starter propagation raw material and the open environment provide various sources for microorganisms, wherein wheat is a main source of bacteria, and fungi are mostly sourced from the starter propagation environment. The rice straw is used as an important auxiliary material in starter propagation production, and is used for wrapping the starter block in the process of culturing bacteria by virtue of excellent heat preservation, moisture preservation and air permeability, so that the effects of isolating and preventing adhesion and maintaining the stability of fermentation environment are achieved. Meanwhile, the surfaces of the rice straws are rich in microorganisms, the new rice straws can enrich the microorganism types in the initial period of Daqu bacteria culture, and the used old rice straws can be reused to realize the effect of inoculating bacteria due to adhesion of related microorganisms for brewing wine, so that the new rice straws have important influence on the structure of Daqu microorganism communities and the quality of white spirit. However, at present, the research on systematic separation screening and functional characteristics of fungi on the surface of the covered rice straw is not deep, and the potential application value of the fungi in white spirit fermentation is not fully excavated. Therefore, the separation screening and functional research of the fungi on the surface of the covered rice straw are developed, and the method has important significance for enriching the microbial resources for brewing the white spirit, optimizing the Daqu production process and improving the quality of the white spirit. Disclosure of Invention The invention aims to provide a method for separating and screening fungi on the surface of covered rice straw so as to obtain a fungus strain with specific functional characteristics for white spirit processing. In order to achieve the aim, the invention adopts the following technical scheme that the method for separating and screening fungi on the surface of the covered rice straw comprises the following steps: s1, taking a sample of rice straw covered with yeast, adding sterile normal saline, oscillating to fully disperse microorganisms, preparing a bacterial suspension mother solution, and carrying out gradient dilution to obtain bacterial suspensions with different concentrations; S2, separating and purifying, namely coating bacterial suspensions with different concentrations on a Bengalia red culture medium, reversely culturing, picking single bacterial colonies, and inoculating the bacterial suspensions on a new Bengalia red plate for purifying; S3, molecular identification, namely extracting genome DNA of the purified strain, performing PCR amplification by using fungus universal primers ITS1 and ITS4, sequencing a PCR product, performing BLAST comparison with NCBI database, constructing a phylogenetic tree, and determining strain species. Preferably, as an improvement, the sterile physiological saline in the step S1 is NaCl sterile physiological saline with the concentration of 1mol/L, the mass-volume ratio of the straw covered sample to the physiological saline is 1g to 9mL, the shaking condition is room temperature and 120r/min, the shaking time is 2h, and the gradient dilution concentration is 10 -1、10-2、10-3. Preferably, in the step S2, the formula of the Bengalia culture medium comprises 20g of agar, 5g of yeast extract, 10g of tryptone, 1g of glucose, 5g of sodium chloride, 0.01g of Bengalia and 1000mL of distilled water, wherein the culture condition is 30 ℃ and the culture time is 2-3d. Preferably, as an improvement, the genomic DNA of the strain is extracted in the step S3 by adopting a sodium laurate method, the PCR reaction conditions are that the pre-denaturation is carried out for 4min at 94 ℃, the denaturation is carried out for 45S at 94 ℃, the annealing is carried out for 45S at 55 ℃, the extension is carried out for 72 ℃ for 1min, the cycle is carried out for 35 times, the final extension is carried out for 10min at 72 ℃, the temperature is kept at 4 ℃, and a phylogenetic tre