Method for separating and screening fungi on surface of koji-covered straw

Abstract

The invention relates to a method for separating and screening fungi on the surface of koji-covered straw in the technical field of biological separation, which comprises the following steps: S1, taking a koji-covered straw sample, 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, separation and purification: coating a rose-bengal culture medium with the bacterial suspensions with different concentrations, carrying out inverted culture, picking single colonies, and inoculating the single colonies to a new rose-bengal flat plate for purification; and S3, molecular identification: extracting the purified strain genome DNA, carrying out PCR amplification by using fungus universal primers ITS1 and ITS4, sequencing a PCR product, carrying out BLAST comparison with an NCBI database, constructing a phylogenetic tree, and determining the strain species. According to the scheme, the fungus strain with specific functional characteristics for white spirit processing is obtained.

Description

Technical Field

[0001] This invention relates to the field of microbial isolation technology, specifically to a method for isolating and screening fungi on the surface of rice straw. Background Technology

[0002] As a key saccharification and fermentation agent and flavor enhancer in the baijiu brewing process, the quality of baijiu daqu (a type of starter culture) directly affects the flavor and taste of baijiu. High-temperature daqu for sauce-aroma baijiu is produced using traditional techniques, relying on a natural fermentation environment to achieve the enrichment and succession of microorganisms, accumulating abundant enzymes and microbial metabolites. These components are an important foundation for the formation of the sauce-aroma flavor. During the daqu production process, the raw materials and open environment provide diverse sources of microorganisms. Wheat is the main source of bacteria, while fungi largely originate from the daqu-making environment.

[0003] Rice straw, as an important auxiliary material in koji (fermentation starter) production, plays a crucial role in preventing sticking and maintaining a stable fermentation environment by wrapping koji blocks during the cultivation process, thanks to its excellent heat retention, moisture retention, and air permeability. Simultaneously, the surface of rice straw carries a rich variety of microorganisms. Fresh rice straw can enrich the microbial diversity in the early stages of koji cultivation, while used rice straw, due to the presence of brewing-related microorganisms, can be reused to achieve an "inoculation" effect, significantly influencing the microbial community structure of koji and the quality of baijiu (Chinese liquor). However, current research on the systematic isolation, screening, and functional characteristics of fungi on the surface of rice straw used for koji wrapping is still insufficient, and their potential application value in baijiu fermentation has not been fully explored. Therefore, conducting research on the isolation, screening, and functional characteristics of fungi on the surface of rice straw used for koji wrapping is of great significance for enriching baijiu brewing microbial resources, optimizing koji production processes, and improving baijiu quality. Summary of the Invention

[0004] The present invention aims to provide a method for isolating and screening fungi on the surface of rice straw to obtain fungal strains with specific functional characteristics for use in the processing of baijiu (Chinese liquor).

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a method for isolating and screening fungi on the surface of rice straw, comprising the following steps: S1. Take a sample of rice straw, add sterile physiological saline, shake to fully disperse the microorganisms, prepare a bacterial suspension mother liquor, and then serially dilute to obtain bacterial suspensions of different concentrations. S2. Isolation and purification: Bacterial suspensions of different concentrations were spread on Bengal red agar, incubated upside down, and single colonies were picked and inoculated onto new Bengal red plates for purification. S3. Molecular identification: Genomic DNA of the strain was extracted and purified, and PCR amplification was performed using universal fungal primers ITS1 and ITS4. The PCR products were sequenced and compared with the NCBI database using BLAST to construct a phylogenetic tree and determine the species of the strain.

[0006] Preferably, as an improvement, the sterile saline in step S1 is 1 mol / L NaCl sterile saline, the mass-to-volume ratio of the wrapped rice straw sample to the saline is 1 g: 9 mL, the shaking conditions are room temperature, 120 r / min, and shaking time is 2 h; the gradient dilution concentration is 10 -1 10 -2 10 -3 .

[0007] Preferably, as an improvement, in step S2, the formula of the Bengal Red culture medium is: 20g agar, 5g yeast extract, 10g tryptone, 1g glucose, 5g sodium chloride, 0.01g Bengal Red, and 1000mL distilled water; the culture conditions are 30℃ and the culture time is 2-3 days.

[0008] Preferably, as an improvement, the genomic DNA of the strain is extracted in step S3 using the sodium lauryl ester method; the PCR reaction conditions are: pre-denaturation at 94℃ for 4 min, denaturation at 94℃ for 45 s, annealing at 55℃ for 45 s, extension at 72℃ for 1 min, 35 cycles, final extension at 72℃ for 10 min, and holding at 4℃; a phylogenetic tree is constructed using MEGA7 software via the nearest neighbor method.

[0009] Preferably, as an improvement, it also includes environmental tolerance analysis, specifically including: A1. Temperature tolerance analysis: After the species of the strain was determined, each strain was inoculated onto PDA medium plates and placed in incubators at 30℃, 40℃, 50℃ and 60℃ for 3-5 days. The growth of the strains was observed and the highest growth temperature of the strains was recorded. A2. Lactic acid tolerance analysis: After sterilizing lactic acid separately, it was added to PDA medium cooled to about 50°C in proportion to prepare gradient medium with final lactic acid concentrations of 20 g / L, 40 g / L, 60 g / L, 80 g / L, and 100 g / L; each strain was inoculated into the above medium and cultured at 30°C for 3-5 days. The growth status was observed to determine the highest lactic acid tolerance concentration of the strain. A3. pH tolerance analysis: Prepare YPD medium, add 1 mol / L hydrochloric acid to adjust the pH of the medium to 5, 6, and 7, then add 2% agar powder, sterilize at 121℃ for 20 min, and pour into plates; after inoculating the strain, incubate at 30℃ for 3-5 days and observe the growth of the strain. A4. Ethanol tolerance analysis: Different volume fractions of ethanol were added to PDA medium to prepare gradient medium with ethanol volume fractions of 2%, 4%, 6%, and 8%. After inoculation, the strains were cultured at 30℃ for 3-5 days, and the growth status was observed. The highest ethanol tolerance concentration of the strains was recorded. A5. Enzyme activity assay of fungal strains: The ability of strains to produce cellulase, saccharifying enzyme, pectinase, α-amylase, protease, esterase, and tanninase was determined using the clear zone method. The strains were inoculated onto selection medium plates containing the corresponding substrates and cultured at 30°C for 3-5 days. The presence and size of the clear zone were used to determine whether the strain had the ability to produce the enzyme and the relative strength of the enzyme activity. A6. Analysis of fungal strains' metabolites: Each strain was inoculated into glucose fermentation medium and cultured at 30℃ and 150 r / min for 48 h with shaking. After the culture, the cells were removed by centrifugation at 8000 r / min for 10 min. The supernatant was extracted with an equal volume of ethyl acetate, mixed thoroughly by shaking, and allowed to stand for separation. The organic phase was collected. The organic phase was concentrated using a rotary evaporator, and the concentrated organic phase was analyzed by GC-MS to identify the types of metabolites.

[0010] The advantages of this solution are: 1. This invention establishes a highly efficient method for isolating, screening, and identifying fungi on the surface of rice straw, successfully isolating 8 fungal strains, enriching the microbial resource library related to Baijiu brewing, and clarifying the species classification of each strain through molecular identification, providing a foundation for subsequent functional research and application.

[0011] 2. The selected fungal strains exhibit diverse environmental tolerances. Some strains can tolerate high temperatures of 50℃, high lactic acid concentrations of 80g / L, or high ethanol concentrations of 8%, enabling them to adapt to the complex environmental conditions during the fermentation process of baijiu and providing environmental adaptability assurance for their application in baijiu fermentation.

[0012] 3. Some of the strains screened in this scheme have the ability to produce multiple enzymes such as cellulase, saccharifying enzyme, and α-amylase. These enzymes play an important role in the degradation of raw materials, saccharification and fermentation, and can promote the conversion and utilization of raw materials in the brewing process of liquor and improve fermentation efficiency. Attached Figure Description

[0013] Figure 1 This is a phylogenetic tree diagram constructed based on the ITS sequence of the strain in an embodiment of the present invention.

[0014] Figure 2 This is a table showing the environmental tolerance results of the strains in the embodiments of the present invention.

[0015] Figure 3 This is a table showing the enzyme activity production results of the strains in the embodiments of the present invention.

[0016] Figure 4 This is a table showing the metabolite results of the strains in the embodiments of the present invention. Detailed Implementation

[0017] The following detailed description illustrates the specific implementation method: Example A method for isolating and screening fungi on the surface of rice straw, comprising the following steps: S1. Weigh 10g of the treated rice straw sample and add it to an Erlenmeyer flask containing 90mL of 1mol / L NaCl sterile physiological saline. Shake at 120r / min for 2h at room temperature to prepare a bacterial suspension stock solution. Add 1mL of the stock solution to 9mL of sterile water and mix thoroughly to obtain 10g of the bacterial suspension. -1 Diluents were prepared sequentially using the same method, 10 -2 10 -3 Diluent.

[0018] S2. Separation and purification: Take the mother liquor and 10 ml of the purified liquor separately. -1 10 -2 10 -3 100 μL of each dilution solution was evenly spread onto Bengal red agar plates (Bengal red agar is prepared from the following materials: agar, 20 g; yeast extract, 5 g; tryptone, 10 g; glucose, 1 g; sodium chloride, 5 g; Bengal red, 0.01 g; distilled water, 1000 mL), with three replicates for each concentration. The plates were inverted and incubated at 30°C for 3 days, and the morphology, color, size, and other characteristics of the colonies on the plates were observed.

[0019] S3. Molecular Identification: Genomic DNA of the strain was extracted and purified using a modified sodium lauryl ester method. The ITS fragment of the strain was amplified using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction conditions were as follows: pre-denaturation, 94℃, 4 min; denaturation, 94℃, 45 s; annealing, 55℃, 45 s; extension, 72℃, 1 min, cycle number 35; final extension, 72℃, 10 min; incubation, 4℃. The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequenced sequences were submitted to the NCBI database for BLAST alignment. ITS sequences of known strains with high homology to the strain were downloaded. The MEGA7 algorithm was used to compare homologous sequences, and a phylogenetic tree was constructed using the nearest neighbor method to determine the strain species.

[0020] Eight fungal strains were isolated and screened from the surface of rice straw, and were numbered DC-9, DC-16, DC-21, DC-23, DC-27, DC-28, DC-36, and DC-39, respectively. Figure 1As shown, these 8 strains belong to 5 genera: Aspergillus, Cladosporium, Paecilomyces, Penicillium, and Saccharomycopsis.

[0021] As attached Figure 1 As shown in Figure A, these 8 strains belong to 6 species. Among them, DC-28 and... C. xanthochromaticum MEFC077 clustered together, DC-28 belonged to... C. xanthochromaticum DC-39 and C. cladosporioides D41B5 clusters together, DC39 belongs to... C. cladosporioides DC-27 and S. fibuligera AUMCs clustered together, with DC-27 belonging to... S. fibuligera .like Figure 1 As shown in Figure B, DC-16 and DC-36 are... A. spinulosporus NRRL 2395 clustered together, with DC-16 and DC-36 belonging to... A. spinulosporus DC-23 and Pen. citrinum NRRL 1841 clustered together, DC-23 belonged to Pen. citrinum DC-9 and DC-21 with Pae. variotii CBS 101075 clustered together, with DC-9 and DC-21 belonging to... Pae. variotii .

[0022] Results of environmental tolerance analysis of the strains are as follows Figure 2 As shown, 1. Temperature tolerance analysis: After the species were determined, each strain was inoculated into potato dextrose agar (PDA) medium (components include: potato, 200g; sucrose, 20g; distilled water, 1000mL; agar, 20g) and placed in incubators at 30℃, 40℃, 50℃, and 60℃ for 3-5 days. The growth of the strains was observed, and the highest growth temperature of the strains was recorded.

[0023] As the temperature increases, the growth ability of the strains gradually weakens. The maximum growth temperature of DC-23, DC-28 and DC-39 is 30℃, the maximum growth temperature of DC-9, DC-16, DC-21 and DC-27 is 40℃, and the maximum growth temperature of DC-36 is 50℃.

[0024] 2. Lactic acid tolerance analysis: After sterilizing lactic acid separately, it was added to PDA medium cooled to about 50°C in proportion to prepare gradient medium with final lactic acid concentrations of 20 g / L, 40 g / L, 60 g / L, 80 g / L, and 100 g / L. Each strain was inoculated into the above medium and cultured at 30°C for 3-5 days. The growth status was observed to determine the highest lactic acid tolerance concentration of the strain.

[0025] As lactic acid concentration increased, the growth ability of the strains gradually weakened. The highest lactic acid tolerance concentration (DAC) for DC-28 was 10 g / L, for DC-23 and DC-39 it was 15 g / L, for DC-16, DC-27, and DC-36 it was 20 g / L, and for DC-9 and DC-21 it was 80 g / L. Among these eight strains, DC-9 and DC-21 showed the strongest lactic acid tolerance, while DC-28 showed the weakest.

[0026] 3. pH tolerance analysis: Prepare YPD medium (components include: yeast extract, 10g; tryptone, 20g; glucose, 20g; distilled water, 1000mL), add 1mol / L hydrochloric acid to adjust the pH of the medium to 5, 6, and 7, then add 2% agar powder, sterilize at 121℃ for 20min and pour into plates; after inoculating the strain, incubate at 30℃ for 3-5 days and observe the growth of the strain.

[0027] As the pH decreased, the growth ability of the strains gradually weakened. These 8 strains grew best in a medium with pH=7, and could also grow in a medium with pH=5, but the growth was weaker.

[0028] 4. Ethanol tolerance analysis: Different volume fractions of ethanol were added to PDA medium to prepare gradient media with ethanol volume fractions of 2%, 4%, 6%, and 8%. After inoculation, the strains were cultured at 30℃ for 3-5 days, and their growth status was observed. The highest ethanol tolerance concentration of the strains was recorded.

[0029] As the ethanol concentration increased, the growth ability of the strains gradually weakened. The highest ethanol tolerance of DC-39 was 4%, while that of DC-9, DC-23, DC-28, and DC-36 was 6%, and that of DC-16, DC-21, and DC-27 was 8%. Among these eight strains, DC-16, DC-21, and DC-27 showed the strongest ethanol tolerance, while DC-39 showed the weakest.

[0030] 5. Enzyme activity assay of fungal strains: The ability of the strains to produce cellulase, saccharifying enzyme, pectinase, α-amylase, protease, esterase and tanninase was determined by the clear zone method. The strains were inoculated onto selection medium plates containing the corresponding substrates and cultured at 30°C for 3-5 days. The presence and size of the clear zone were used to determine whether the strain had the ability to produce the enzyme and the relative strength of the enzyme activity.

[0031] The results are as follows Figure 3 As shown, these eight strains can produce five enzymes: cellulase, saccharifying enzyme, α-amylase, protease, and tanninase. Two strains (DC-28 and DC-39) are capable of producing cellulase. Two strains (DC-16 and DC-27) are capable of producing saccharifying enzyme. Three strains (DC-9, DC-36, and DC-39) are capable of producing α-amylase. Only one strain (DC-28) is capable of producing tanninase. Except for DC-21 and DC-23, which do not produce any of the seven enzymes, the other strains are capable of producing at least one enzyme. DC-9, DC-16, DC-27, and DC-36 are capable of producing only one enzyme; DC-9 and DC-36 produce α-amylase, and DC-16 and DC-27 produce saccharifying enzyme. DC-28 and DC-39 have the ability to produce two enzymes: DC-28 has the ability to produce cellulase and tanninase, and DC-39 has the ability to produce cellulase and α-amylase.

[0032] 6. Analysis of fungal strains' metabolites: Each strain was inoculated into glucose fermentation medium (components included: 0.2 g magnesium sulfate heptahydrate, 5 g glucose, 1 g disodium hydrogen phosphate heptahydrate, 5 g sodium chloride, 1 g dipotassium hydrogen phosphate, and 1000 mL water), and cultured at 30℃ and 150 rpm for 48 h with shaking. After culture, the cells were removed by centrifugation at 8000 rpm for 10 min, and the supernatant was extracted with an equal volume of ethyl acetate. After shaking and mixing, the mixture was allowed to stand and separate into layers, and the organic phase was collected. The organic phase was concentrated using a rotary evaporator, and the concentrated organic phase was analyzed by GC-MS to identify the types of metabolites.

[0033] The results are as follows Figure 4As shown, these eight bacterial strains, using glucose fermentation medium, were able to produce 22 metabolites, including one alcohol (2-butoxyethanol), one ketone (cyclohexanone), and ten esters (methyl acetate, dimethyl succinate, dimethyl glutarate, dimethyl adipate, ethyl propionate, n-propyl acetate, butyl acetate, sec-butyl acetate, isobutyl acetate, and 1-methoxy-2-propyl acetate). Among these eight strains, strains DC-27 and DC-28 produced the most metabolites, with 18 each; strains DC-9, DC-36, and DC-39 produced the fewest metabolites, with 14 each. In addition, all strains were able to produce nine substances: methyl aldehyde, n-propyl acetate, sec-butyl acetate, butyl acetate, cyclohexanone, 2-butoxyethanol, dimethyl succinate, dimethyl glutarate, and dimethyl adipate.

[0034] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for isolating and screening fungi on the surface of rice straw, characterized in that, Includes the following steps: S1. Take a sample of rice straw, add sterile physiological saline, shake to fully disperse the microorganisms, prepare a bacterial suspension mother liquor, and then serially dilute to obtain bacterial suspensions of different concentrations. S2. Isolation and purification: Bacterial suspensions of different concentrations were spread on Bengal red agar, incubated upside down, and single colonies were picked and inoculated onto new Bengal red plates for purification. S3. Molecular identification: Genomic DNA of the strain was extracted and purified, and PCR amplification was performed using universal fungal primers ITS1 and ITS4. The PCR products were sequenced and compared with the NCBI database using BLAST to construct a phylogenetic tree and determine the species of the strain.

2. The method for isolating and screening fungi on the surface of rice straw according to claim 1, characterized in that: The sterile saline solution in step S1 was a 1 mol / L NaCl sterile saline solution. The mass-to-volume ratio of the wrapped rice straw sample to the saline solution was 1 g:9 mL. The shaking conditions were room temperature, 120 r / min, and shaking time of 2 h. The serial dilution concentration was 10... -1 10 -2 10 -3 .

3. The method for isolating and screening fungi on the surface of rice straw according to claim 2, characterized in that: In step S2, the formula for the Bengal Red culture medium is: 20g agar, 5g yeast extract, 10g tryptone, 1g glucose, 5g sodium chloride, 0.01g Bengal Red, and 1000mL distilled water; the culture conditions are 30℃ and the culture time is 2-3 days.

4. The method for isolating and screening fungi on the surface of rice straw according to claim 3, characterized in that: Genomic DNA of the strain was extracted in step S3 using the sodium lauryl laurate method. The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 4 min, denaturation at 94℃ for 45 s, annealing at 55℃ for 45 s, extension at 72℃ for 1 min, 35 cycles, final extension at 72℃ for 10 min, and incubation at 4℃. A phylogenetic tree was constructed using MEGA7 software using the nearest neighbor method.

5. The method for isolating and screening fungi on the surface of rice straw according to claim 4, characterized in that: It also includes environmental tolerance analysis, specifically including: A1. Temperature tolerance analysis: After the species of the strain was determined, each strain was inoculated onto PDA medium plates and placed in incubators at 30℃, 40℃, 50℃ and 60℃ for 3-5 days. The growth of the strains was observed and the highest growth temperature of the strains was recorded. A2. Lactic acid tolerance analysis: After sterilizing lactic acid separately, it was added to PDA medium cooled to about 50°C in proportion to prepare gradient medium with final lactic acid concentrations of 20 g / L, 40 g / L, 60 g / L, 80 g / L, and 100 g / L; each strain was inoculated into the above medium and cultured at 30°C for 3-5 days. The growth status was observed to determine the highest lactic acid tolerance concentration of the strain. A3. pH tolerance analysis: Prepare YPD medium, add 1 mol / L hydrochloric acid to adjust the pH of the medium to 5, 6, and 7, then add 2% agar powder, sterilize at 121℃ for 20 min, and pour into plates; after inoculating the strain, incubate at 30℃ for 3-5 days and observe the growth of the strain. A4. Ethanol tolerance analysis: Different volume fractions of ethanol were added to PDA medium to prepare gradient medium with ethanol volume fractions of 2%, 4%, 6%, and 8%. After inoculation, the strains were cultured at 30℃ for 3-5 days, and the growth status was observed. The highest ethanol tolerance concentration of the strains was recorded. A5. Enzyme activity assay of fungal strains: The ability of strains to produce cellulase, saccharifying enzyme, pectinase, α-amylase, protease, esterase, and tanninase was determined using the clear zone method. The strains were inoculated onto selection medium plates containing the corresponding substrates and cultured at 30°C for 3-5 days. The presence and size of the clear zone were used to determine whether the strain had the ability to produce the enzyme and the relative strength of the enzyme activity. A6. Analysis of fungal strains' metabolites: Each strain was inoculated into glucose fermentation medium and cultured at 30℃ and 150 r / min for 48 h with shaking. After the culture, the cells were removed by centrifugation at 8000 r / min for 10 min. The supernatant was extracted with an equal volume of ethyl acetate, mixed thoroughly by shaking, and allowed to stand for separation. The organic phase was collected. The organic phase was concentrated using a rotary evaporator, and the concentrated organic phase was analyzed by GC-MS to identify the types of metabolites.

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