Drawings
FIG. 1 is a graph showing the comparison of caproic acid yield of Daqu culture solution. Wherein, the lower case letters are different to show that the difference is obvious (P < 0.05), and the numerical values in the graph are repeated for more than 3 times.
FIG. 2 is a chart showing the color reaction of copper sulfate.
FIG. 3 is a graph of caproic acid yield of caproic acid bacteria. Wherein, the lower case letters are different to show that the difference is obvious (P < 0.05), and the numerical values in the graph are repeated for more than 3 times.
FIG. 4 is a graph showing the comparison of caproic acid production by caproic acid bacteria at various temperatures. Wherein, the lower case letters are different to show that the difference is obvious (P < 0.05), and the numerical values in the graph are repeated for more than 3 times.
FIG. 5 is a graph comparing caproic acid production of each caproic acid bacteria at different ethanol concentrations. Wherein, the lower case letters are different to show that the difference is obvious (P < 0.05), and the numerical values in the graph are repeated for more than 3 times.
FIG. 6 is a graph comparing caproic acid production of each caproic acid bacteria at different pH. Wherein, the lower case letters are different to show that the difference is obvious (P < 0.05), and the numerical values in the graph are repeated for more than 3 times.
FIG. 7 shows agarose gel electrophoresis of PCR products of rDNA of strain DJ001 16S, wherein M, marker 5000, negative control, A, DJ001.
FIG. 8 is a phylogenetic tree of strain DJ001 based on the 16S rDNA sequence.
FIG. 9 is a graph of caproic acid yield of simulated broth culture strain DJ 001. Wherein ", P < 0.0001" and the values in the figures are repeated more than 3 times.
FIG. 10 is a graph showing the temperature change during the culture of Daqu.
FIG. 11 is a graph showing caproic acid content of yeast powder culture broth to which strain DJ001 was added during the course of making Daqu. Wherein "/x", P <0.001, and the values in the figures are repeated more than 3 times.
FIG. 12 is a saccharification force diagram of Daqu. Wherein, ", P <0.05, and the numerical values in the figure are repeated for more than 3 times.
FIG. 13 is a graph showing the yield of white spirit. Wherein, ", P < 0.0001" ", P < 0.05", and the values in the figures are repeated more than 3 times.
FIG. 14 is a graph showing the caproic acid and ethyl caproate content of white spirit. Wherein, ", P <0.05; ns, no SIGNIFICANT DIFFERENCE, and the values in the figure are repeated for more than 3 times.
FIG. 15 is a mass chart of caproic acid and ethyl caproate of Chinese liquor. Wherein, ", P <0.0001; ns, no SIGNIFICANT DIFFERENCE, and the values in the figures are repeated more than 3 times.
Detailed Description
The invention will be further described with reference to the drawings and examples.
Preparation method of sodium ethylacetate medium in the following examples
Sodium ethylacetate culture medium, anhydrous sodium acetate 5g, yeast Extract 1g, magnesium sulfate heptahydrate 0.2g, anhydrous dipotassium hydrogen phosphate 0.4 g, ammonium sulfate 0.5 g and calcium carbonate 10 g, and after weighing and dissolving, the solution is fixed to volume of 1L and the natural pH value. Sterilizing at 121deg.C to 20min, and adding anhydrous ethanol 2% (volume fraction) when cooling. The solid medium should be supplemented with 2% agar powder prior to sterilization.
The method for extracting organic matters such as caproic acid and ethyl caproate in the following examples
Filtering the obtained culture solution with a 0.22 μm microporous filter membrane, taking 500 μl of filtrate in a 5mL volumetric flask, adding anhydrous diethyl ether to a constant volume of 5mL, sufficiently oscillating, standing for 1 min, and sucking the upper layer extract to obtain the solution to be tested.
Gas chromatography detection method in the following examples
The caproic acid and ethyl caproate content in the diethyl ether extract was detected by gas chromatograph. The chromatographic conditions were column flow rate 2.15 mL/min, column temperature set at 50 ℃, detector temperature set at 240 ℃, initial temperature set at 60 ℃ and hold at 1 min, temperature rise to 220 ℃ and hold at 2 min at 10 ℃/min, sample volume set at 1 μl, split sample, split ratio 20:1.
Example 1
The embodiment discloses a separation method of bacillus amyloliquefaciens producing caproic acid, which comprises the following steps:
step 1, selecting high-quality Daqu.
The laboratory has 5 kinds of strong aromatic Daqus, which are respectively numbered DQ1, DQ2, DQ3, DQ4 and DQ5, the Daqus are fully ground into yeast powder and evenly added into an ethanol sodium acetate culture medium, and the caproic acid yield is detected after 7 d is cultured, and the result is shown in figure 1. The caproic acid yield of Daqu DQ2 and DQ3 is higher and is respectively 0.64 mg/mL and 0.52 mg/mL, so that the high-yield caproic acid bacteria are separated and screened from Daqu DQ2 and DQ3 in the follow-up process.
And 2, selecting a strain with caproic acid production capacity from the Daqu based on a copper sulfate chromogenic method.
Placing Daqu culture solution 200 uL in 2 mL centrifuge tube, dipping small amount of culture solution, streaking and coating to separate and purify strain, selecting single colony, placing in sodium ethylacetate culture medium, culturing 7 d, placing 2 mL culture solution in 10 mL centrifuge tube, adding 1 mL of 20% copper sulfate solution, and observing whether blue-green precipitate is generated. As a result, as shown in FIG. 2, a part of the strain produced a bluish-green precipitate, and the corresponding strain was kept.
And step 3, detecting caproic acid production capacity of the strain obtained in the step 2 by combining a gas chromatograph, and screening to obtain the strain with high caproic acid production according to a detection result.
Specifically, the strain isolated in the step 2 was cultured in a sodium ethylacetate medium, and after culturing at 37℃to 7 d, the caproic acid yield was detected by a gas chromatograph. Finally, 3 caproic acid bacteria with stronger caproic acid production capacity are obtained, wherein the yield of the caproic acid of DJ001 is highest. As shown in FIG. 3, the yield of hexanoic acid of DJ001 was 1.56 mg/mL.
And 4, testing the tolerance of the candidate strain DJ001 obtained in the step 3. The process is as follows:
(4.1) detection of temperature tolerance of Strain
200 Mu L of DJ001, 1.39-1 and 4.39-1 bacterial suspensions of the seed-retaining species are respectively transferred into a 37 ℃ anaerobic culture 7 d in a sodium ethylacetate liquid culture medium to activate the strain. DJ001, 1.39-1, 4.39-1 bacterial suspensions were inoculated into sodium ethylacetate liquid medium at an inoculum size of 2%, and the liquid loading amount was 90%, and the culture temperatures were set to 22℃C, 27℃C, 32℃C, 37℃C, 42℃C, 47℃C, 52℃C, respectively. Anaerobic culture 7 d. The caproic acid yield of each group was measured and the results are shown in FIG. 4. The yields of DJ001, 1.39-1 and 4.39-1 caproic acid are increased and then reduced along with the increase of the culture temperature, the DJ001 has the strongest resistance to the temperature, and when the temperature reaches 52 ℃, the DJ001 still has higher caproic acid yield.
(4.2), Detection of ethanol tolerance of Strain
200 Mu L of DJ001, 1.39-1 and 4.39-1 bacterial suspensions of the seed-retaining species are respectively transferred into ethanol sodium acetate liquid culture medium, 7 d is anaerobically cultured at 37 ℃, and the strain is activated. DJ001, 1.39-1, 4.39-1 bacterial suspensions were inoculated into sodium ethylacetate liquid culture media with ethanol concentrations of 0%, 2%, 4%, 6%, 8%, 10%, 12% vol, respectively, at a liquid loading of 90%. Anaerobic culture at 37℃7 d. The caproic acid yield of each group was measured and the results are shown in FIG. 5. The yields of DJ001, 1.39-1 and 4.39-1 caproic acid increase and decrease with increasing ethanol concentration, and DJ001 still had higher caproic acid yields when ethanol concentration was 12% vol.
(4.3), Detection of Strain pH tolerance
200 Mu L of DJ001, 1.39-1 and 4.39-1 bacterial suspensions of the seed-retaining species are respectively transferred into ethanol sodium acetate liquid culture medium, 7 d is anaerobically cultured at 37 ℃, and the strain is activated. The DJ001, 1.39-1 and 4.39-1 bacterial suspensions were inoculated into sodium ethylacetate liquid culture media with pH values of 1, 3, 5, 7, 9 and 11 respectively according to an inoculum size of 2%, and the liquid loading amount was 90%. Anaerobic culture at 37℃7 d. The caproic acid yield of each group was measured and the results are shown in FIG. 6. The yields of DJ001, 1.39-1 and 4.39-1 caproic acid increase and decrease with increasing pH, and DJ001 has the best adaptability to pH and the highest yield of caproic acid when the pH is 1-11.
DJ001 was selected as a strain for subsequent study and application based on the results of the caproic acid production and tolerance analysis described above.
Step 5, identification of strains
(5.1), Extraction of genomic DNA of the Strain
Genomic DNA of DJ001 was extracted using SPARKEASY bacterial genomic DNA rapid extraction kit.
(5.2), PCR amplification
The genome DNA is used as a template for PCR amplification, and the universal primer 27F/1492R is used for PCR amplification. The primer sequences were as follows:
27F:5-AGAGTTTGATCCTGGCTCAG-3’;
1492R:5’-GGTTACCTTG TTACGACTT-3’;
The PCR reaction procedure was 98℃pre-denaturation for 2 min, 98℃denaturation for 15 s,55℃annealing for 30s, 72℃extension for 30s cycles, 72℃extension for 5 min.
(5.3) Detection of amplified products by gel electrophoresis
After 3. Mu.L of the amplified product was mixed with 3. Mu. L Loading Buffer, the mixed product of 3.3 uL was aspirated and electrophoresed in a 1% agarose gel. As a result, the amplified bands were single and were about 1500 bp a in size, as shown in FIG. 7.
(5.4), DNA sequencing and sequence alignment
The purified PCR product was sent to Shanghai megagene technologies Inc. for sequencing to obtain the DJ001 strain 16S rDNA sequence, the sequence information is as follows.
ACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTATTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCAAAGGTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGAC
The above sequences were submitted to the NCBI database for Blast alignment and the results are shown as bacillus amyloliquefaciens (b. Phylogenetic trees were constructed based on their sequences, and the results are shown in fig. 8, with DJ001 and b. DJ001 is therefore B.amyloquefaciens, which is designated B. amyloliquefaciens DJ001.
Bacillus amyloliquefaciens B. amyloliquefaciens DJ001,001 obtained in the example is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2025, 1 month and 3 days and a preservation number of CGMCC No. 33299.
Example two
The example discloses simulated fermentation of bacillus amyloliquefaciens B. amyloliquefaciens DJ001,001 obtained in the first example in a laboratory, and the specific description is as follows:
Taking fermented grains 1 kg prepared for brewing by a certain fermented body for later use.
Steaming 1 kg fermented grains 30min with 2L sterile water, filtering, collecting filtrate, placing in beaker, constant volume to 5L, packaging, sterilizing at 115deg.C for 30min, and collecting the simulated fermentation broth.
CK group, namely adding 3g yeast powder, 5g fermented grains and 5mL sterile water into a conical flask containing 270 mL simulated fermentation liquor, and DJ001 group, namely adding 3g yeast powder, 5g fermented grains and 5mL DJ001 bacterial liquid into a conical flask containing 270 mL simulated fermentation liquor. After fermentation of 7 d in both groups, the caproic acid yield was measured. As a result, as shown in FIG. 9, the yield of CK caproic acid was 0.038 mg/mL, the yield of DJ001 caproic acid was 0.73 mg/mL, and the yield of caproic acid was increased by 1821.05%.
Therefore, the addition of B. amyloliquefaciens DJ001 has better caproic acid production effect.
Example III
The embodiment discloses an application of bacillus amyloliquefaciens B. amyloliquefaciens DJ001 obtained in the first embodiment in making Daqu, and the specific description is as follows:
(1) Method for making reinforced Daqu
The bacterial liquid of bacillus amyloliquefaciens B. amyloliquefaciens DJ001,001 obtained in the first embodiment is uniformly added into starter propagation raw materials for preparing medium-high temperature Daqu, the DJ001 bacterial liquid dosage of each room Daqu is 50L, and the conventional Daqu added by a sterile strain is a Control (CK). The total of the Daqu per room is 3840.
(2) Temperature monitoring of Daqu
After the Daqu enters the room and is put, the Daqu product temperature is recorded at 8:00 am every day until the Daqu exits the room after 24 days (after the temperature is stable, the temperature of the Daqu room is not recorded). The curve of the temperature of the Daqu is shown in FIG. 10. The temperature change of the 2-room Daqu was substantially consistent, indicating that the management during the Daqu culture was stable.
(3) Selection of Daqu sample
And 5 Daqus are selected in a mode of 5-point sampling for each Daqu, and the obtained same Daqu sample is ground, crushed and uniformly mixed for subsequent experiments.
(4) Detection of caproic acid production capacity of yeast powder culture solution
The yeast powder was added to sodium ethylacetate medium at 1.5% inoculum size, incubated at 37℃for 7d, and the caproic acid yield was examined. As shown in FIG. 11, the caproic acid content of the enriched Daqu powder culture solution prepared by adding DJ001 bacterial solution is improved by 600.00% compared with that of the control Daqu CK added by the aseptic strain.
(5) Daqu saccharification force detection
As shown in FIG. 12, the saccharification force of the reinforced Daqu prepared by adding DJ001 bacterial liquid is improved by 31.11% compared with that of the control Daqu CK added by the aseptic strain. DJ001 is bacillus amyloliquefaciens, and DJ001 is added during the preparation of the Daqu, so that the conversion of starch into glucose is possible, and the saccharification capacity of the Daqu is improved.
In summary, the bacillus amyloliquefaciens B. amyloliquefaciens DJ001,001 in the embodiment I is added into the Daqu, so that the caproic acid production capacity of the culture solution of the yeast powder can be obviously improved, and the saccharification capacity of the Daqu can be obviously improved.
Example IV
The embodiment discloses an application of bacillus amyloliquefaciens B. amyloliquefaciens DJ001 obtained in the embodiment I in white spirit production, and the application is specifically described as follows:
In the production of the strong aromatic white spirit, the strain B, amyloliquefaciens DJ and the strain B are uniformly added into fermented grains according to the inoculation amount of 1 percent, and 70 percent d of the fermented grains are put into a pool for fermentation, and the white spirit with normal grade and excellent grade is obtained after distillation. Weighing to obtain the yield of white spirit of each grade. As shown in FIG. 13, the yields of the added DJ001 group normal grade and premium grade white spirits are respectively improved by 17.76% and 32.69% compared with the yields of the added CK group normal grade and premium grade white spirits. The total white spirit yield of each group is calculated, and the DJ001 group is increased by 23.04% compared with the CK group.
The caproic acid and ethyl caproate content of each wine sample was detected by gas chromatograph, and then the average concentration of caproic acid and ethyl caproate of each group of overall wine samples was calculated in combination with each yield. The results are shown in fig. 14, and the ethyl caproate concentration was increased by 22.11% with no significant difference in the concentration of caproic acid added to the DJ001 group as compared with the CK group.
The combined concentration and the yield of each wine sample are calculated to obtain the mass of the caproic acid and the ethyl caproate in each group of overall wine samples. As a result, as shown in FIG. 15, the mass of caproic acid in the DJ 001-added group was improved by 15.37% (not statistically significant), and the mass of ethyl caproate was improved by 51.71% as compared with the CK group.
Therefore, the addition of the functional bacteria B. amyloliquefaciens DJ001,001 has remarkable effect on improving the yield of white spirit and the content of ethyl caproate. The absence of significant differences in caproic acid concentration between DJ001 and CK group spirits may be related to two factors, (1) dilution of caproic acid concentration after increased liquor yield, and (2) conversion of caproic acid to ethyl caproate.
In summary, the present invention provides a bacillus amyloliquefaciens derived from Daqu and having multiple tolerance and caproic acid producing properties, and a method for isolating the same. The strain can be added into Daqu to improve caproic acid production capacity and saccharification capacity of Daqu, and can be added into fermented grains to improve yield of Chinese liquor and ethyl caproate content. The invention has important value in the aspects of improving the yield and quality of white spirit, saving cost, improving efficiency and the like, and can increase the enterprise and social benefits.
The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings, and the examples described herein are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the spirit and scope of the present invention. The individual technical features described in the above-described embodiments may be combined in any suitable manner without contradiction, and such combination should also be regarded as the disclosure of the present invention as long as it does not deviate from the idea of the present invention. The various possible combinations of the invention are not described in detail in order to avoid unnecessary repetition.
The present invention is not limited to the specific details of the above embodiments, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope of the present invention without departing from the scope of the technical concept of the present invention, and the technical content of the present invention is fully described in the claims.