A screening method and application of bacteria in yellow water

Abstract

The application discloses a screening method of bacteria in yellow water, and 8 strains of bacteria with excellent performance are screened from the yellow water by using the method. The 8 strains of bacteria have excellent comprehensive tolerance, multi-enzyme activity and broad-spectrum aroma-producing capacity, and can be applied to the fields of composite enzyme preparation, liquor flavoring fermentation and yellow water resource treatment. The application provides an efficient screening strategy for mining functions of microorganisms in extreme environments, and the obtained strains have wide application prospects in the brewing industry and biological conversion.

Description

Technical Field

[0001] This invention relates to the field of microbial screening technology, specifically to a method and application for screening bacteria in yellow water. Background Technology

[0002] Yellow water is a major byproduct of solid-state fermentation of baijiu. During fermentation in the fermentation pits, as microorganisms in the mash metabolize, macromolecules such as starch and protein in the raw materials are decomposed, and free water gradually precipitates out, carrying a large amount of metabolic products and unused components to settle at the bottom of the pit, eventually forming a brownish-yellow or brownish viscous liquid, i.e., yellow water. The composition of yellow water is extremely complex, with extremely high chemical oxygen demand (COD) and biochemical oxygen demand (BOD), and direct discharge would cause serious environmental pollution.

[0003] In terms of composition, yellow water is rich in sugars (accounting for about one-third of the total organic matter), alcohols (mainly ethanol, with an alcohol content of 3.2% to 5.3%), organic acids (mainly lactic acid, accounting for more than 80%, with pH values ​​typically fluctuating between 2.5 and 4.2), esters, and nitrogen-containing compounds. This extreme environment of high acidity, high alcohol content, and high osmotic pressure makes yellow water a unique microbial resource pool, enriched with a large number of microbial communities that have been domesticated over a long period and adapted to extreme environments, possessing enormous potential for functional exploitation.

[0004] Most existing technologies focus on screening strains with single functions (such as acid production or production of specific flavor compounds), with relatively simple screening indicators and a lack of systematic evaluation of strains' multiple tolerances under extreme environments (such as simultaneous tolerance to high temperatures, high ethanol, and high lactic acid). Furthermore, microorganisms in yellow water often possess multiple metabolic potentials due to long-term domestication, but existing screening methods often neglect comprehensive consideration of strains' multi-enzyme activity and broad-spectrum aroma-producing capabilities, resulting in a large number of excellent microbial resources remaining untapped. Summary of the Invention

[0005] The present invention aims to provide a method and application for screening bacteria in yellow water, which can systematically evaluate the environmental tolerance, enzymatic activity and aroma-producing characteristics of the strains, thereby obtaining high-performance microbial resources.

[0006] To achieve the above objectives, the first aspect of this application provides the following technical solution: A method for screening bacteria in yellow water, S1, sampling and isolation: Yellow water is collected from the bottom of a soy sauce-flavored liquor cellar, serially diluted and spread on LB solid medium, and after cultivation, single colonies are picked for purification to obtain pure culture strains; S2. Molecular identification: Genomic DNA of the strain was extracted and purified, its 16S rDNA sequence was amplified and sequenced, and the species of the strain was preliminarily determined by homology comparison. S3. Tolerance re-screening: The strains identified in step 2 were inoculated into LB liquid medium with different temperatures, pH values, ethanol concentrations and lactic acid concentrations and cultured to screen out strains with strong growth ability under stress factors. S4. Enzyme activity screening: The strains screened in step 3 are inoculated onto cellulase, saccharifying enzyme, pectinase, α-amylase, protease and tanninase detection media to screen for strains that produce at least one target enzyme in high quantities. S5. Aroma production capacity screening: The strains screened in step 4 are inoculated into LB liquid medium for fermentation, and the metabolites in the fermentation broth are extracted. GC-MS analysis is used to screen strains that can produce volatile flavor substances, which include at least one of aldehydes, ketones, alcohols, acids, esters and pyrazines.

[0007] Furthermore, the temperature gradient in step 3 is 30℃, 40℃, 50℃, and 60℃; the pH gradient is 2, 3, 4, 5, 6, and 7; the ethanol concentration gradient is 2%, 4%, 6%, and 8% (v / v); and the lactic acid concentration gradient is 5 g / L, 10 g / L, 15 g / L, 20 g / L, and 25 g / L.

[0008] Furthermore, the target enzyme in step 4 includes at least one of glucoamylase, protease, and tanninase.

[0009] The second aspect of this application provides the following technical solution: An application of yellow water source bacteria screened according to the method described in the first aspect in the fermentation production of flavor substances in baijiu (Chinese liquor), wherein the bacteria ferment the substrate to produce at least one flavor substance comprising acetaldehyde, cyclohexanone, 3-methyl-1-butanol, phenylethanol, hexanoic acid, ethyl propionate, butyl acetate, and 2,5-dimethylpyrazine.

[0010] Furthermore, the bacteria are used to ferment the substrate to produce at least one flavor compound selected from acetaldehyde, cyclohexanone, 3-methyl-1-butanol, phenylethyl alcohol, hexanoic acid, ethyl propionate, butyl acetate, and 2,5-dimethylpyrazine.

[0011] Furthermore, the application includes fermentation under high temperature, high ethanol, high lactic acid, or low pH conditions.

[0012] Working principle and beneficial effects of the present invention: This invention isolated and screened eight bacterial strains from the yellow water of a soy sauce-flavored fermentation pit. These eight bacterial strains belong to... Bacillus from Velez , Bacillus licheniformis , Bacillus toyonensis , Providence vermiculite and Oceanobacillus profundusThe strains comprise 3 genera and 5 species. Environmental tolerance tests showed that HS-5 and HS-16 exhibited the strongest tolerance to temperature and ethanol, while HS-1 and HS-5 showed the strongest tolerance to lactic acid. HS-5 also showed the strongest tolerance to acidic pH. Overall, strain HS-5 demonstrated the strongest environmental tolerance. These 8 strains produced 6 enzymes: HS-10 produced the strongest cellulase, HS-5 produced the strongest saccharifying enzyme, HS-4 produced the strongest α-amylase, HS-18 produced the strongest protease, HS-16 produced pectinase, and HS-5 produced tanninase. Fermentation using LB liquid medium produced 51 volatile flavor compounds, including aldehydes, ketones, alcohols, acids, and esters, characteristic of baijiu (Chinese liquor). Attached Figure Description

[0013] Figure 1 Phylogenetic tree constructed based on the 16S rDNA sequence of the strain; Figure 2 Temperature tolerance test of the strain; Figure 3 Ethanol tolerance test of the strain; Figure 4 pH tolerance test of the strain; Figure 5 Lactic acid tolerance assay of the strain. Detailed Implementation

[0014] The following detailed description illustrates the specific implementation method: I. Isolation and Identification of Bacteria in Yellow Water 1. Isolation and purification of bacteria in yellow water Take 10 mL of the yellow water and add it to 90 mL of sterile physiological saline with a concentration of 1 mol / L NaCl to prepare 10 -1 Diluent; prepare 10 using the same method. -2 10 -3 Diluent. From 10... -1 10 -2 and 10 -3 Spread 100 μL of the diluted solution onto LB solid medium (agar, 20 g; yeast extract, 5 g; tryptone, 10 g; NaCl, 10 g; distilled water, 1000 mL), and incubate upside down in a 30 ℃ incubator for 2–3 days. Pick a single colony and inoculate it onto a new Bengal red plate for purification.

[0015] 2. Molecular identification Genomic DNA was extracted from the strain using a modified sodium lauryl sulfate method. The 16S rDNA fragment of the strain was amplified using universal bacterial primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR conditions were as follows: pre-denaturation, 95 °C, 5 min; denaturation, 95 °C, 30 s; annealing, 58 °C, 30 s; extension, 72 °C, 1 min, cycle number 35; final extension, 72 °C, 10 min; incubation, 4 °C. The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequenced sequences were submitted to the NCBI GenBank database for BLAST alignment. The 16S rDNA sequences of type strains with high homology to the strain were downloaded. MEGA7 was used to compare homologous sequences, and a phylogenetic tree was constructed using the nearest neighbor method to determine the strain species.

[0016] II. Environmental tolerance of the strain 1. Temperature tolerance analysis The strain was inoculated into LB liquid medium, and the temperature of the shaker was set to 30 ℃, 40 ℃, 50 ℃, and 60 ℃, and the rotation speed was set to 150 r / min, respectively, and the culture was carried out for 48 h. With blank medium as a control, the absorbance of the bacterial suspension was measured at λ=600 nm.

[0017] 2. Ethanol Tolerance Analysis Ethanol was added to LB liquid medium as needed, adjusting the volume fraction of ethanol to 2%, 4%, 6%, and 8%. The bacterial strain was inoculated into the ethanol gradient medium and incubated in a shaker at 30 °C and 150 r / min for 48 h. Using blank medium as a control, the absorbance of the bacterial suspension was measured at λ=600 nm.

[0018] 3. pH tolerance analysis Prepare LB liquid medium and adjust the pH of the medium to 2, 3, 4, 5, 6, and 7 by adding 1 mol / L hydrochloric acid. Inoculate the bacterial strain into the pH gradient medium and incubate in a shaker at 30 °C and 150 r / min for 48 h. Using blank medium as a control, measure the absorbance of the bacterial suspension at λ=600 nm.

[0019] 4. Lactic acid tolerance analysis Lactic acid was sterilized separately (115 °C, 15 min) and then added to LB liquid medium as needed, adjusting the final concentrations to 5 g / L, 10 g / L, 15 g / L, 20 g / L, and 25 g / L. The strain was inoculated into lactic acid gradient media and incubated in a shaker at 30 °C and 150 r / min for 48 h. Using a blank medium as a control, the absorbance of the bacterial suspension was measured at λ=600 nm.

[0020] III. Enzyme Activity Assay of Strains The strains were spotted onto the culture media for detecting cellulase activity, saccharifying enzyme activity, pectinase activity, α-amylase activity, protease activity, esterification enzyme activity, and tanninase activity. The presence of enzyme-producing activity was determined by whether a clear zone could be formed around the colony, and the ratio of the diameter of the clear zone (D) to the diameter of the colony (d) (D / d) was calculated.

[0021] IV. Analysis of the strain's metabolites The bacterial strain was inoculated into LB liquid medium and cultured at 30 °C and 150 r / min for 48 h. The cells were then removed by centrifugation at 8000 r / min for 10 min. An equal volume of dichloromethane was added to the supernatant for extraction. After thorough mixing, the mixture was allowed to stand and separate into layers to obtain the organic phase. The organic phase was concentrated using a rotary evaporator, and the substances in the organic phase were detected using GC-MS.

[0022] V. Results and Analysis 1. Isolation and identification of bacteria in yellow water Eight bacterial strains were isolated from yellow water and numbered as follows: HS-1, HS-4, HS-5, HS-10, HS-12, HS-14, HS-16, and HS-18. The 16S rDNA sequences of these eight strains were compared with sequences in the NCBI GenBank database. Sequences with high homology to the 16S rDNA sequences of the target strains were downloaded, and a phylogenetic tree was constructed, as shown in the attached figure. Figure 1 As shown. HS-1 and Bacillus belye ( Bacillus velezensis CBMB205 clustered together, and HS-1 was named Bacillus velezensis HS-1; HS-4, HS-10 and HS-18 are related to Bacillus licheniformis ( Bacillus lichen-like ATCC 14580 is grouped together, with HS-4, HS-10, and HS-18 named respectively. Bacillus lichen-like HS-4 Bacillus licheniformis HS-10 and Bacillus licheniformis HS-18; HS-14 and Bacillus tonkinensis ( Bacillus toyonensis BCT-7112 clustered together, and HS-14 was named... Bacillus toyonensis HS-14; HS-5 and Providencia larvae ( Providence vermiculite OP1 clustered together, and HS-5 was named Providence vermiculite HS-5; HS-12 and HS-16 with deep-sea Bacillus ( Oceanobacillus profundus CL-MP28 clustered together, with HS-12 and HS-16 named respectively. Oceanobacillus profundus HS-12 and Oceanobacillus profundus HS-16.

[0023] These eight bacteria belong to three genera: Bacillus, Providencia, and others. Providence ) and Bacillus spp. ( Oceanobacillus Five strains of Bacillus (HS-1, HS-4, HS-10, HS-14 and HS-18) and two strains of Bacillus aquaticus (HS-12 and HS-16) belong to Firmicutes at the phylum level, while one strain of Providencia (HS-5) belongs to Proteobacteria at the phylum level.

[0024] 2. Environmental tolerance analysis of the strain The temperature tolerance of these eight strains was determined, as shown in the attached figure. Figure 2 As shown, the concentrations of bacterial suspensions of HS-1, HS-4, HS-5, HS-12, HS-14, HS-16, and HS-18 gradually decreased with increasing temperature, reaching their lowest concentrations at 60 °C. The concentration of bacterial suspension of HS-10 showed a trend of first increasing and then decreasing with increasing temperature, reaching its highest concentration at 40 °C. Under the same temperature gradient, HS-16 exhibited the highest bacterial suspension concentration and the strongest temperature tolerance, followed by HS-5.

[0025] As attached Figure 3 As shown, with increasing ethanol concentration, the bacterial suspension concentrations of HS-1, HS-5, HS-14, HS-16, and HS-18 gradually decreased, reaching their lowest concentrations at 8% ethanol. The bacterial suspension concentrations of HS-4, HS-10, and HS-12 showed a trend of first increasing and then decreasing, reaching their highest concentrations at 4% ethanol. When the ethanol concentration reached 2% or higher, under the same ethanol gradient, HS-5 exhibited the highest bacterial suspension concentration and the strongest tolerance to ethanol, followed by HS-16.

[0026] As attached Figure 4As shown, the growth of these eight bacterial strains was weak when the pH of the culture medium was between 2 and 4. With increasing pH, the concentration of the bacterial suspension showed a continuous upward trend, reaching its highest concentration at pH 5 for HS-1; and its highest concentrations at pH 7 for HS-4, HS-5, HS-10, HS-12, HS-14, HS-16, and HS-18. Under the same pH gradient, HS-5 exhibited the highest bacterial suspension concentration, indicating that HS-5 showed strong tolerance to acidic pH.

[0027] As attached Figure 5 As shown, with increasing lactic acid concentration, the concentrations of bacterial suspensions of HS-1, HS-4, HS-5, HS-14, and HS-18 exhibited a trend of first increasing, then decreasing, and then increasing again, reaching the highest concentration at 25 g / L lactic acid. The concentrations of bacterial suspensions of HS-10 and HS-12 showed a trend of first decreasing and then increasing, reaching the highest concentration at 25 g / L lactic acid. The concentration of bacterial suspension of HS-16 showed a trend of first increasing and then decreasing, reaching the highest concentration at 10 g / L lactic acid. When the lactic acid concentration reached 15 g / L or higher, under the same lactic acid gradient, HS-1 had the highest bacterial suspension concentration and the strongest tolerance to lactic acid, followed by HS-5.

[0028] 3. Determination of enzyme production capacity of the strain These eight strains of bacteria can produce six enzymes: cellulase, saccharifying enzyme, pectinase, α-amylase, protease, and tanninase. As shown in Table 1, two strains, HS-4 and HS-10, possess cellulase production capabilities; HS-4 has a D / d ratio of 1.28, and HS-10 has a D / d ratio of 1.75. Of these two strains, HS-10 exhibits stronger cellulase production. Four strains, HS-4, HS-5, HS-10, and HS-14, possess saccharifying enzyme production capabilities; HS-4 has a D / d ratio of 1.34, HS-5 has a D / d ratio of 1.47, HS-10 has a D / d ratio of 1.31, and HS-14 has a D / d ratio of 1.37. Of these four strains, HS-5 exhibits the strongest saccharifying enzyme production capability. One strain, HS-16, possesses pectinase production capabilities, with a D / d ratio of 1.34. Three strains exhibited α-amylase production capacity: HS-1, HS-4, and HS-16. The D / d ratios were 1.06 for HS-1, 1.28 for HS-4, and 1.19 for HS-16. Among these three strains, HS-4 showed the strongest α-amylase production capacity. Two strains exhibited protease production capacity: HS-5 and HS-12. The D / d ratios were 3.13 for HS-5 and 8.25 for HS-12. Of these two strains, HS-12 showed the stronger protease production capacity. One strain, HS-5, exhibited tannin production capacity, with a D / d ratio of 1.56.

[0029] These eight strains all possess the ability to produce at least one enzyme. HS-14 produces one enzyme, a saccharifying enzyme. HS-1, HS-10, HS-12, and HS-18 produce two enzymes: HS-1 produces both saccharifying and α-amylase; HS-10 produces both cellulase and saccharifying; HS-12 produces both saccharifying and protease; and HS-18 produces both saccharifying and protease. HS-4, HS-5, and HS-16 produce three enzymes: HS-4 produces cellulase, saccharifying, and α-amylase; HS-5 produces saccharifying, protease, and tanninase; and HS-16 produces cellulase, pectinase, and α-amylase.

[0030] Table 1 Enzyme production capacity of strains

[0031] 4. Analysis of bacterial metabolites As shown in Table 2, these eight strains of bacteria were able to produce 51 volatile substances using LB liquid medium, including one aldehyde (acetaldehyde), two ketones (cyclohexanone and 2-piperidinone), five alcohols (3-methyl-1-butanol, 2-butoxyethanol, 2-methylthioethanol, benzyl alcohol, and phenethyl alcohol), five acids (2-methylpropionic acid, 3-methylbutyric acid, hexanoic acid, 2-hexylcyclopropaneacetic acid, and n-decanoic acid), 14 esters (ethyl propionate, methyl formate, ethyl propionate, n-propionate, isobutyrate, isobutyl acetate, butyl formate, butyl acetate, isoamyl formate, propylene glycol methyl ether acetate, dimethyl succinate, dimethyl glutarate, dimethyl adipate, and di(2-ethylhexyl) adipate), and one pyrazine (2,5-dimethylpyrazine).

[0032] Table 2 Metabolites of the strain

[0033] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the claims.

Claims

1. A method for screening bacteria in yellow water, characterized in that, Includes the following steps: S1. Sampling and separation: Yellow water was collected from the bottom of the fermentation pit of soy sauce-flavored liquor, serially diluted and spread on LB solid medium. After cultivation, single colonies were picked and purified to obtain pure culture strains. S2. Molecular identification: Genomic DNA of the strain was extracted and purified, its 16S rDNA sequence was amplified and sequenced, and the species of the strain was preliminarily determined by homology comparison. S3. Tolerance re-screening: The strains identified in step 2 were inoculated into LB liquid medium with different temperatures, pH values, ethanol concentrations and lactic acid concentrations and cultured to screen out strains with strong growth ability under stress factors. S4. Enzyme activity screening: The strains screened in step 3 are inoculated onto cellulase, saccharifying enzyme, pectinase, α-amylase, protease and tanninase detection media to screen for strains that produce at least one target enzyme in high quantities. S5. Aroma production capacity screening: The strains screened in step 4 are inoculated into LB liquid medium for fermentation, and the metabolites in the fermentation broth are extracted. GC-MS analysis is used to screen strains that can produce volatile flavor substances, which include at least one of aldehydes, ketones, alcohols, acids, esters and pyrazines.

2. The method for screening bacteria in yellow water according to claim 1, characterized in that, The temperature gradient in step 3 is 30℃, 40℃, 50℃, and 60℃; the pH gradient is 2, 3, 4, 5, 6, and 7; the ethanol concentration gradient is 2%, 4%, 6%, and 8% (v / v); and the lactic acid concentration gradient is 5 g / L, 10 g / L, 15 g / L, 20 g / L, and 25 g / L.

3. The method for screening bacteria in yellow water according to claim 2, characterized in that, The target enzyme in step 4 includes at least one of glucoamylase, protease, and tanninase.

4. The application of yellow water source bacteria screened according to any one of claims 1 to 3 in the fermentation production of flavor substances in Baijiu (Chinese liquor), characterized in that, Using the bacterial fermentation substrate, at least one flavor compound is produced, comprising acetaldehyde, cyclohexanone, 3-methyl-1-butanol, phenylethanol, hexanoic acid, ethyl propionate, butyl acetate, and 2,5-dimethylpyrazine.

5. The application of yellow water source bacteria screened according to the method of claim 5 in the fermentation production of flavor substances in baijiu (Chinese liquor), characterized in that, Using the bacterial fermentation substrate, at least one flavor compound is produced, comprising acetaldehyde, cyclohexanone, 3-methyl-1-butanol, phenylethanol, hexanoic acid, ethyl propionate, butyl acetate, and 2,5-dimethylpyrazine.

6. The application of yellow water source bacteria screened according to any one of claims 1 to 3 in improving the environmental tolerance of fermentation systems, characterized in that, The applications include fermentation under high temperature, high ethanol, high lactic acid, or low pH conditions.

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