Compound microbial agent for enhancing production of beta-phenethyl alcohol in liquor brewing process as well as preparation method and application of compound microbial agent

Abstract

The invention discloses a compound microbial agent for enhancing production of beta-phenethyl alcohol in a white spirit brewing process as well as a preparation method and application of the compound microbial agent, and belongs to the technical field of microorganisms. The compound microbial agent is prepared from aspergillus niger, pichia membranaefaciens, Kazakstan monospora, saccharomyces cerevisiae and sporospora delbrueckii; in the compound microbial agent, the final concentration OD600 of the aspergillus niger, the final concentration OD600 of the pichia membranaefaciens, the final concentration OD600 of the Kazakstein monospora, the final concentration OD600 of the saccharomyces cerevisiae and the final concentration OD600 of the sporospora delbrueckii are 0.8-1.5, 0.8-1.5, 0.5-1, 0.8-1.5 and 0.5-1 respectively. After the compound microbial agent provided by the invention is applied to a solid-state fermentation system of Luzhou-flavor liquor, the yield of the aroma substance beta-phenethyl alcohol can be obviously increased by adjusting the eukaryotic microbial community structure of the fermentation system; the method can effectively solve the problem that the stability of the method for producing beta-phenethyl alcohol in the process of intensifying Baijiu fermentation by a single strain is poor.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a composite microbial agent for strengthening the production of beta-phenylethanol in a liquor brewing process, and a preparation method and application thereof. Background Art

[0002] Liquor occupies a vital position in China's food industry. Luzhou-flavor liquor is one of the four basic types of liquor and is loved by consumers for its pleasant flavor. The trace components in Luzhou-flavor liquor have an important influence on the flavor profile of liquor. Among them, β-phenylethanol is a volatile aroma substance with a rose scent. Studies have shown that eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus niger, and Geotrichum candidum can convert substrates into β-phenylethanol during the fermentation of liquor. Therefore, β-phenylethanol is considered to be one of the important aroma skeleton components of liquor. A higher content of β-phenylethanol can make the wine mellow and sweet and the aroma full.

[0003] Most of the previous studies focused on the study of aroma substances such as esters in liquor, and paid less attention to β-phenylethanol. Only a few studies have found that the use of a single Pichia pastoris or abnormal Wickham yeast can enhance the β-phenylethanol content in liquor production.

[0004] CN111961599A discloses yeast with floral and fruity flavor produced by solid-state fermentation and its application. By inoculating yeast with a preservation number of CGMCC 19814 into mash for fermentation, the β-phenylethanol in the mash is increased from 0.009 g / kg to 0.456 g / kg.

[0005] CN107177520A discloses a high-yield β-phenylethanol yeast strain and its application. By inoculating the β-phenylethanol yeast into a liquor fermentation system, the β-phenylethanol content in the distilled liquor is 110 mg / L.

[0006] The above methods for increasing β-phenylethanol in the fermentation process of liquor are all completed through a single strain, but the solid-state fermentation process of liquor is usually completed by the collaboration of a complex microbial community. It is difficult for a single enhanced strain to maintain high efficiency and stability in actual production. Therefore, the development of composite microbial agents derived from cellars and targeted enhancement of the production of liquor flavor substances have been research hotspots and difficulties in recent years. Summary of the invention

[0007] The technical problem to be solved by the present invention is that the method for strengthening the production of β-phenylethanol in the fermentation process of liquor with a single strain has poor stability. The present invention aims to provide a composite microbial agent, comprising five eukaryotic core strains in the brewing process of Luzhou-flavor liquor. The composite agent is applied to the solid-state fermentation of liquor by artificial strengthening, which can significantly increase the yield of the aroma component β-phenylethanol and help improve the flavor characteristics of liquor.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] In a first aspect, the present invention provides a composite microbial agent for enhancing the production of β-phenylethanol in a liquor brewing process, wherein the composite microbial agent is composed of Aspergillus niger, Pichia membranifaciens, Kazachstania unispora, Saccharomyces cerevisiae and Torulaspora delbrueckii.

[0010] In the above-mentioned composite microbial agent, the final concentration of Aspergillus niger OD 600 The final concentration of Pichia membranifaciens is OD 0.8 to 1.5. 600 The final concentration of Kazachstania unispora is OD 600 The final concentration of Saccharomyces cerevisiae is 0.5-1. 600 The final concentration of Torulaspora delbrueckii was 0.8-1.5. 600 It is 0.5~1.

[0011] Furthermore, in the above-mentioned composite microbial agent, the final concentration OD of Aspergillus niger 600 The final concentration of Pichia membranifaciens was OD 1. 600 The final concentration of Kazachstania unispora was OD 1. 600 The final concentration of Saccharomyces cerevisiae was 1 OD 600 The final concentration of Torulaspora delbrueckii was 1, and the OD 600 is 1.

[0012] Furthermore, Aspergillus niger has a deposit number of CGMCC 3.17612.

[0013] Furthermore, Pichia membranifaciens has a deposit number of CGMCC 2.5139.

[0014] Furthermore, the Kazakh unispora yeast Kazachstania unispora has a deposit number of CGMCC2.251.

[0015] Furthermore, Saccharomyces cerevisiae has a deposit number of CGMCC 2.3854.

[0016] Furthermore, the Delbrueck spore yeast Torulaspora delbrueckii has a deposit number of CGMCC2.4064.

[0017] In a second aspect, the present invention provides a method for preparing the above-mentioned composite microbial agent: culturing and preparing Aspergillus niger solution, Pichia membranaceus solution, Kazakhstan unisporic yeast solution, Saccharomyces cerevisiae solution and Delbur's spore yeast solution respectively, diluting the obtained bacterial solution according to the final concentration of each bacterial body in the above-mentioned composite microbial agent, and then mixing them, shaking and mixing them thoroughly, to obtain a composite microbial agent for enhancing the production of β-phenylethanol in the liquor brewing process.

[0018] Furthermore, the preparation method of the above-mentioned Aspergillus niger liquid is: inoculate the Aspergillus niger species on a malt extract agar medium plate and activate it at 30° C. for 48 to 72 hours, scrape the Aspergillus niger spores into physiological saline, and obtain the Aspergillus niger liquid.

[0019] Furthermore, the preparation method of the above-mentioned Pichia pastoris bacterial solution is: inoculate the Pichia pastoris strain on a YPD agar medium plate and activate it at 30°C for 48 hours, take the activated Pichia pastoris and inoculate it into a YPD liquid culture medium, and culture it at 30°C for 24 hours to prepare the Pichia pastoris bacterial solution.

[0020] Furthermore, the preparation method of the above-mentioned single-sporic Kazakhstan yeast liquid is: inoculate the single-sporic Kazakhstan yeast strain on a YPD agar medium plate and activate it at 30°C for 48 hours, take the activated single-sporic Kazakhstan yeast and inoculate it into a YPD liquid culture medium, and culture it at 30°C for 24 hours to prepare the single-sporic Kazakhstan yeast liquid.

[0021] Furthermore, the preparation method of the above-mentioned brewer's yeast liquid is: inoculate the brewer's yeast strain on a YPD agar medium plate and activate it at 30°C for 48 hours, take the activated brewer's yeast and inoculate it into a YPD liquid culture medium, and culture it at 30°C for 24 hours to prepare the brewer's yeast liquid.

[0022] Furthermore, the preparation method of the above-mentioned Delb's spore yeast liquid is: inoculate the Delb's spore yeast strain on a YPD agar medium plate and activate it at 30°C for 48 hours, take the activated Delb's spore yeast and inoculate it into a YPD liquid culture medium, and culture it at 30°C for 24 hours to prepare the Delb's spore yeast liquid.

[0023] Furthermore, the composition of the malt extract agar medium is: 130.0 g / L malt extract powder, 15 g / L agar, pH value 6.0±0.2, and high-pressure sterilization at 121° C. for 15 min.

[0024] Furthermore, the composition of the above-mentioned malt extract liquid culture medium is: 130.0 g / L malt extract powder, pH value 6.0±0.2, and high-pressure sterilization at 121° C. for 15 minutes.

[0025] Furthermore, the composition of the above YPD agar medium is: yeast extract 10g / L, peptone 20g / L, glucose 10g / L, agar 15g / L, pH 7.0±0.2, and high-pressure sterilization at 121°C for 15min.

[0026] Furthermore, the composition of the above YPD liquid culture medium is: yeast extract 10g / L, peptone 20g / L, glucose 10g / L, pH 7.0±0.2, and high-pressure sterilization at 121°C for 15min.

[0027] In a third aspect, the present invention provides an application of the composite microbial agent or the composite microbial agent prepared by the above preparation method: adding 1% (v / v) of the composite microbial agent per kilogram of mash for mixed fermentation to enhance the production of β-phenylethanol in the solid-state fermentation process of Luzhou-flavor liquor.

[0028] The beneficial effect of the present invention is that after the composite microbial agent provided by the present invention is applied to the solid-state fermentation system of Luzhou-flavor liquor, the yield of the aroma substance β-phenylethanol can be significantly increased by adjusting the eukaryotic microbial community structure of the fermentation system, which is beneficial to improving the flavor characteristics of the liquor. The composite microbial agent of the present invention has a promoting effect and important significance on improving the quality of Luzhou-flavor liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The changes of the relative content of β-phenylethanol during the solid-state fermentation of liquor (CK: control group; T: enhanced group);

[0030] Figure 2 for the comparison of the diversity of eukaryotic microbial communities (Shannon index);

[0031] Figure 3 The species composition of the eukaryotic microbial community. DETAILED DESCRIPTION

[0032] The technical solution of the present invention can be specifically implemented in the following manner.

[0033] The invention discloses a composite microbial agent for strengthening the production of beta-phenylethanol in the process of liquor brewing. The composite microbial agent consists of Aspergillus niger, Pichia membranifaciens, Kazachstania unispora, Saccharomyces cerevisiae and Torulaspora delbrueckii.

[0034] In the embodiments, strains preserved by China General Microbiological Culture Collection Center (CGMCC) are preferred for carrying out the experiments in the embodiments, and the specific strain numbers are as follows:

[0035] Aspergillus niger, the deposit number is CGMCC 3.17612.

[0036] Pichia membranifaciens, the deposit number is CGMCC 2.5139.

[0037] The single-spore Kazakh yeast Kazachstania unispora has a deposit number of CGMCC 2.251.

[0038] Saccharomyces cerevisiae, the deposit number is CGMCC 2.3854.

[0039] Delbrueck's spore yeast Torulaspora delbrueckii, the deposit number is CGMCC 2.4064.

[0040] The culture medium and its components involved in the embodiment are as follows:

[0041] Malt extract agar medium: malt extract powder 130.0 g / L, agar 15 g / L, pH 6.0±0.2, high pressure sterilization at 121℃ for 15 min.

[0042] Malt extract liquid culture medium: malt extract powder 130.0 g / L, pH 6.0±0.2, high pressure sterilization at 121℃ for 15 min.

[0043] YPD agar medium: yeast extract 10 g / L, peptone 20 g / L, glucose 10 g / L, agar 15 g / L, pH 7.0±0.2, high pressure sterilization at 121℃ for 15 min.

[0044] YPD liquid medium: yeast extract 10 g / L, peptone 20 g / L, glucose 10 g / L, pH 7.0±0.2, high pressure sterilization at 121℃ for 15 min.

[0045] The technical solution and effects of the present invention are further illustrated below through practical examples.

[0046] Example

[0047] Example 1 Preparation of composite microbial agent

[0048] (1) Preparation of seed solution of each strain

[0049] The preparation method of the seed liquid of Aspergillus niger CGMCC 3.17612 is as follows: the strain is inoculated on a malt extract agar medium plate for activation (30°C, 48h), the Aspergillus niger spores are scraped into physiological saline, and a spore suspension is prepared as the seed liquid, wherein the bacterial cell concentration is OD 600 =24, continue to dilute the Aspergillus niger seed solution with physiological saline to a final concentration of OD 600 =1, standby.

[0050] The preparation method of the seed solution of Pichia pastoris CGMCC 2.5139 is as follows: the strain is inoculated on a YPD agar medium plate for activation (30°C, 48h), the activated Pichia pastoris is inoculated into a YPD liquid medium, and cultured at 30°C for 24h to prepare the seed solution; wherein the bacterial cell concentration is OD 600 =13.61, and then continue to dilute the Pichia pastoris seed solution with physiological saline to a final concentration of OD 600 =1, standby.

[0051] The preparation method of the seed solution of the monosporic Kazakh yeast CGMCC 2.251 is as follows: the strain is inoculated on a YPD agar medium plate for activation (30°C, 48h), the activated monosporic Kazakh yeast is inoculated into a YPD liquid medium, and cultured at 30°C for 24h to prepare the seed solution; wherein the bacterial cell concentration is OD 600 =7.28, continue to use physiological saline to dilute the final concentration of the monosporic Kazakhstan yeast seed solution OD 600 =1, standby.

[0052] The preparation method of the seed solution of Saccharomyces cerevisiae CGMCC 2.3854 is as follows: the strain is inoculated on a YPD agar medium plate for activation (30°C, 48h), the activated Saccharomyces cerevisiae is inoculated into a YPD liquid medium, and cultured at 30°C for 24h to prepare the seed solution; wherein the bacterial cell concentration is OD 600 =12.1, continue to dilute the brewer's yeast seed solution with physiological saline to a final concentration of OD 600=1, standby.

[0053] The preparation method of the seed solution of Delb's spore yeast CGMCC 2.4064 is as follows: the strain is inoculated on a YPD agar medium plate for activation (30°C, 48h), the activated spore yeast is inoculated into a YPD liquid medium, and cultured at 30°C for 24h to prepare the seed solution; wherein the bacterial cell concentration is OD 600 =20.84, continue to dilute the Delburian spore yeast seed solution with physiological saline to a final concentration of OD 600 =1, standby.

[0054] (2) Preparation of microbial agents

[0055] The Aspergillus niger seed solution, Pichia pastoris seed solution, Kazakhstan yeast seed solution, Saccharomyces cerevisiae seed solution and Delburian spore yeast seed solution prepared in step (1) were mixed and shaken thoroughly to obtain a total volume of 100 ml and a final concentration of OD 600 =1 composite microbial agent.

[0056] Example 2 Intensification of β-phenylethanol production during liquor solid-state fermentation

[0057] The composite microbial agent prepared in Example 1 was used in a liquid form for enhanced application in the solid-state fermentation process of Luzhou-flavor liquor. The differences in β-phenylethanol production and fungal community structure between the enhanced group and the control group were tracked and compared, and the enhanced application effect of the composite agent was comprehensively evaluated.

[0058] Take 24 1L sterilized ground-mouth bottles, add 550g of fermented grains (including koji) from a certain Luzhou-flavor winery and 3% (v / v) yellow water (yellow water from the 60th day of fermentation in the winery) respectively. The enhanced group was connected with 1% (v / v) of composite microbial agent, and the control group was supplemented with the same volume of sterile water, and mixed evenly. There were 12 bottles in the control group and 12 bottles in the enhanced group. The sealed ground-mouth bottles were placed in a biochemical incubator and fermented at a constant temperature of 30°C for 28 days. Samples were taken on the 0th, 7th, 14th, and 28th days of fermentation, and 3 bottles were taken from the control group and the enhanced group each time. The fermented grains in each bottle were fully mixed and then sampled for β-phenylethanol production and fungal community structure analysis.

[0059] (1) Comparison of β-phenylethanol production

[0060] Headspace solid phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to analyze the volatile components in the lees. Figure 1As shown in the figure, as the fermentation progresses, the relative content of β-phenylethanol in the mash of the fortified group with the composite microbial agent is always higher than that of the control group, and increases by 13.1%, 19.8% and 98.1% respectively on days 7, 14 and 28 compared with the control group. This indicates that the composite microbial agent of the present invention can effectively enhance the production of β-phenylethanol in the solid-state fermentation process of liquor.

[0061] (2) Comparison of fungal community structure

[0062] The metagenomic DNA of fermented grains was extracted by CTAB method. The metagenomic DNA that passed the quality inspection was used as a template, and the universal primer pair (5'-GGAAGTAAAAGTCGTAACAAGG-3' and 5'-GCTGCGTTCTTCATCGATGC-3') was used to PCR amplify the ITS1-5F region of eukaryotic microorganisms. A small fragment library was constructed for the amplified products, and the library was sequenced at both ends based on the Illumina NovaSeq sequencing platform. The sequencing results were subjected to Reads splicing and filtering, species annotation and α diversity analysis, and the species differences between the enhanced group and the control group were compared.

[0063] Depend on Figure 2 It can be seen that as the fermentation progressed, the eukaryotic microbial community diversity (Shannon index) of the control group continued to decrease, but the eukaryotic diversity of the enhanced group showed a trend of first decreasing and then increasing. The results showed that the addition of the composite microbial agent significantly changed the eukaryotic diversity of the enhanced group on the 14th and 28th days, especially the Shannon value of the enhanced group at the end of fermentation was significantly higher than that of the control group.

[0064] Relative abundance of species Figure 3 As shown in the figure, it can be seen that as the fermentation progressed, the eukaryotic microbial species composition of the control group changed. The species composition of the enhanced group was similar to that of the control group at each fermentation time, but the relative abundance of each species was significantly different from that of the control group on the 14th and 28th days. For example, the relative proportion of Thermomyces lanuginosus increased from 0.7% in the control group to 10.07% in the enhanced group on the 28th day. Therefore, the composite microbial agent can improve the β-phenylethanol yield of Luzhou-flavor liquor solid-state fermentation by regulating the structure of the eukaryotic microbial community.

Claims

1. A composite microbial agent for strengthening the production of β-phenylethanol in the liquor brewing process, characterized in that: The composite microbial agent is composed of Aspergillus niger, Pichia membranifaciens, Kazachstania unispora, Saccharomyces cerevisiae and Torulaspora delbrueckii; The final concentration OD of Aspergillus niger in the composite microbial inoculant is 600 The final concentration of Pichia membranifaciens is OD 0.8 to 1.

5. 600 The final concentration of Kazachstania unispora is OD 600 The final concentration of Saccharomyces cerevisiae is 0.5-1. 600 The final concentration of Torulaspora delbrueckii was 0.8-1.

5. 600 It is 0.5~1.

2. The composite microbial agent for strengthening the production of β-phenylethanol in the liquor brewing process according to claim 1, characterized in that: At least one of the following is met: Aspergillus niger, the deposit number is CGMCC 3.17612; Pichia membranifaciens, with the accession number of CGMCC 2.5139; Kazachstania unispora, with the accession number CGMCC 2.251; Saccharomyces cerevisiae, deposit number CGMCC 2.3854; Delbrueck's spore yeast Torulaspora delbrueckii, the deposit number is CGMCC 2.4064.

3. The method for preparing the composite microbial agent according to any one of claims 1 to 2, characterized in that: Aspergillus niger solution, Pichia membranaceus solution, Kazakhstan uni-sporic yeast solution, Saccharomyces cerevisiae solution and Delbur's spore yeast solution are cultured and prepared respectively, the obtained bacterial solutions are diluted according to the final concentration of each bacterial body in the composite microbial agent according to claim 1, and then the mixtures are mixed and fully shaken to obtain the composite microbial agent for enhancing the production of β-phenylethanol in the liquor brewing process.

4. The method for preparing the composite microbial agent according to claim 3, characterized in that: The preparation method of the Aspergillus niger liquid is as follows: inoculate the Aspergillus niger species on a malt extract agar medium plate and activate it at 30° C. for 48 to 72 hours, scrape the Aspergillus niger spores into physiological saline, and obtain the Aspergillus niger liquid.

5. The method for preparing the composite microbial agent according to claim 3, characterized in that: The preparation method of the Pichia pastoris bacterial liquid is as follows: the Pichia pastoris strain is inoculated on a YPD agar medium plate and activated at 30°C for 48 hours, the activated Pichia pastoris is inoculated into a YPD liquid medium, and cultured at 30°C for 24 hours to prepare the Pichia pastoris bacterial liquid.

6. The method for preparing the composite microbial agent according to claim 3, characterized in that: The preparation method of the monosporic Kazakhstan yeast liquid is as follows: the monosporic Kazakhstan yeast strain is inoculated on a YPD agar medium plate and activated at 30°C for 48 hours, the activated monosporic Kazakhstan yeast is inoculated into a YPD liquid medium, and cultured at 30°C for 24 hours to prepare the monosporic Kazakhstan yeast liquid.

7. The method for preparing the composite microbial agent according to claim 3, characterized in that: The preparation method of brewer's yeast liquid is as follows: the brewer's yeast strain is inoculated on a YPD agar medium plate and activated at 30°C for 48 hours, the activated brewer's yeast is inoculated into a YPD liquid medium, and cultured at 30°C for 24 hours to prepare the brewer's yeast liquid.

8. The method for preparing the composite microbial agent according to claim 3, characterized in that: The preparation method of the Delb's spore yeast liquid is as follows: the Delb's spore yeast strain is inoculated on a YPD agar medium plate and activated at 30°C for 48 hours, the activated Delb's spore yeast is inoculated into a YPD liquid medium, and cultured at 30°C for 24 hours to prepare the Delb's spore yeast liquid.

9. The method for preparing the composite microbial agent according to any one of claims 4 to 8, characterized in that: The composition of malt extract agar medium is: malt extract powder 130.0g / L, agar 15g / L, pH 6.0±0.2, autoclaved at 121℃ for 15min; The composition of the malt extract liquid medium is: malt extract powder 130.0 g / L, pH 6.0 ± 0.2, autoclaved at 121 °C for 15 min; The composition of YPD agar medium was as follows: yeast extract 10 g / L, peptone 20 g / L, glucose 10 g / L, agar 15 g / L, pH 7.0 ± 0.2, autoclaved at 121 °C for 15 min; The composition of YPD liquid medium is: yeast extract 10 g / L, peptone 20 g / L, glucose 10 g / L, pH 7.0±0.2, and high-pressure sterilization at 121°C for 15 min.

10. Use of the composite microbial agent according to any one of claims 1 to 2 or the composite microbial agent prepared by the preparation method according to any one of claims 3 to 9, characterized in that: 1% (v / v) composite microbial agent was added to each kilogram of mash for mixed fermentation to enhance the production of β-phenylethanol in the solid-state fermentation process of Luzhou-flavor liquor.

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