Thai enterococci capable of producing multiple aldehyde aroma substances and application thereof

Abstract

The present invention provides a Thai Enterococcus derived from the mud of liquor brewing pit and capable of producing a variety of aldehyde aroma substances and its application. The strain is named Enterococcus thailandicus.zqw28, and the preservation number is CGMCC No.25138. The Thai Enterococcus zqw28 provided by the present invention can produce a variety of aldehyde aroma substances such as furfural, decanal, benzaldehyde, phenylacetaldehyde, etc. The Thai Enterococcus zqw28 provided by the present invention can be used in the field of wine fermentation, especially in the field of liquor brewing, including pit mud production, improving the flavor of liquor, etc. The Thai Enterococcus that can produce a variety of aldehyde aroma substances provided by the present invention and its application make up for the blank in the prior art that there is no relevant research report on the metabolites of specific enterococci strains, which is of great significance to the study of microorganisms and the source of flavor substances in the liquor brewing process, and has important industrial and economic value.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to Thai enterococci and the production of multiple aldehyde aroma substances using the Thai enterococci and other applications. Background Art

[0002] The brewing of Chinese liquor uses grains such as sorghum as raw materials, and uses Daqu, Xiaoqu or Fuqu as saccharification and fermentation agents. It undergoes a complex fermentation process including enzyme chemical reactions, biochemical reactions, organic chemical reactions and microbial metabolic activities. The aroma substances in the raw materials themselves, the metabolites of koji microorganisms, the aroma substances produced by the distillation of fermented grains and the aroma substances produced by the metabolism of cellar mud microorganisms together form the complex and diverse flavor components of liquor. The trace components that determine the style and characteristics of liquor account for about 2%. At present, more than 2,000 trace components have been detected, including aldehydes, esters, alcohols, heterocyclics, terpenes, aromatics, ketones, nitrogen-containing compounds, acids, sulfurs, acetals and lactones.

[0003] Functional bacteria usually refer to microorganisms that can directly or indirectly produce beneficial effects on the flavor of liquor during the liquor brewing process. Liquor brewing is an environment where multiple bacteria coexist. In order to improve their own competitiveness, various microorganisms will release their own metabolites into the environment, inhibiting the growth and metabolism of other microorganisms or making it impossible for other microorganisms to use the substances, thereby improving their own competitiveness. When the amount of these metabolites reaches a certain amount and the raw materials are consumed to a certain extent, the growth of various microorganisms will remain relatively stable in quantity, metabolism will almost stop, and the flavor substances will basically take shape.

[0004] Different microbial colony compositions will lead to differences in the production of flavor substances, which in turn creates different types of liquor. The raw materials used for brewing, the type of koji, the brewing process, and the production environment play a vital role in the quality and flavor of liquor, affecting the growth and metabolism of different functional bacteria, forming a special colony environment, and thus forming a unique style of liquor.

[0005] Enterococcus, cells are spherical or oval, in pairs or short chains in liquid culture medium. It does not produce spores and is Gram-positive. Sometimes it moves with flagella and has no obvious capsule. It is facultatively anaerobic, chemoheterotrophic, and metabolizes by fermentation. It can ferment a wide range of carbohydrates, mainly producing L(+)-lactic acid, but no gas, with a final pH of 4.2-4.6, and complex nutritional requirements. It can usually grow at 10°C to 45°C (optimum 37°C), and can also grow at pH 9.6, 6.5% NaCl and 40% bile salts. According to the utilization of sugars by enterococci and their molecular biological characteristics, enterococci can be divided into Enterococcus faecalis, Enterococcus faecium, Enterococcus avium and Enterococcus tenacious.

[0006] Enterococci are members of the normal intestinal flora of humans and animals, and are also part of the normal flora in food. They are widely used in various fermented foods due to their ability to produce acid, hydrolyze protein or hydrolyze fat. In recent years, studies on liquor brewing microorganisms have found that Enterococci have made an important contribution to liquor brewing.

[0007] CN111748504A discloses a technical method for producing Enterococcus faecium by using white wine lees, comprising taking freeze-dried powder of Enterococcus faecium and placing it in a modified MRS culture medium, streaking and culturing, picking 3-5 typical single colonies of Enterococcus faecium and purifying and rejuvenating them continuously for 3 times, selecting the best single colony of Enterococcus faecium as a primary seed liquid strain; inoculating the selected single colony of Enterococcus faecium by aseptic operation into the primary seed culture medium, and culturing on a shaking table to obtain the primary seed liquid; then further inoculating into the secondary seed culture medium to obtain the secondary seed liquid; inoculating the prepared secondary seed liquid into a solid fermentation culture medium containing pretreated white wine lees, and fermenting and culturing; after the fermentation is completed, drying and crushing the fermentation product to obtain the solid fermentation product of Enterococcus faecium.

[0008] CN114317344A discloses an Enterococcus faecium and its application and composition. The Enterococcus faecium can be used in the preparation of feed additives, animal feed, food additives, food or probiotics, etc. The fermentation and culture method of the Enterococcus faecium comprises inoculating the seed liquid of the Enterococcus faecium into the Enterococcus faecium fermentation medium, and fermenting and culturing for 10 hours to 20 hours under the conditions of 35°C to 40°C and pH 5.8 to 7.0.

[0009] CN107541407B discloses a method for brewing red yeast rice wine rich in γ-aminobutyric acid, which includes adding durable enterococcus fermentation liquid to the pre-fermentation mash for post-fermentation to obtain mature red yeast rice wine mash, wherein the preparation of the durable enterococcus fermentation mash includes the preparation of durable enterococcus seed liquid, carrier adsorption of durable enterococci, activation and proliferation of durable enterococci and fermentation of durable enterococci.

[0010] Hu Xiaolong et al. (Hu Xiaolong, Wang Kangli, Niu Guangjie, et al. Analysis of the diversity of microbial communities in medium-temperature Daqu based on high-throughput sequencing technology [J]. Journal of Light Industry, 2019, 34(4): 9) used high-throughput sequencing technology to study the diversity and composition of fungal and bacterial communities in medium-temperature Daqu of a wine company in Henan Province. The results showed that a total of 28 genera were detected in the core of medium-temperature Daqu, among which Enterobacter, Lactobacillus, Lactococcus, Brucella, Enterococcus and Erwinia were the dominant bacterial genera. Except for Brucella, the contents of other dominant bacterial genera in the skin and core samples were quite different.

[0011] Ma Yingyao et al. (Ma Yingyao, Hu Ping, Sun Lilin, et al. Metagenomics analysis of the correlation between dominant bacteria and metabolic functions in the fermentation cellar of Maotai-flavor liquor [J]. Modern Food Science and Technology, 2020, 36(6)9) used metagenomics technology to analyze the changes in the dominant bacterial flora, metabolic function differences and their correlation in the fourth round of Maotai-flavor liquor mash in the cellar. The results showed that the dominant bacteria at 30 days of fermentation were Lactococcus (33.83%), Enterococcus (32.4%), Bacillus (14.07%) and Mycobacterium (8.62%).

[0012] Liu Zheng et al. (Liu Zheng, Qin Peijun, Lu Yanxiang, Wu Zhifei, Zhang Changxia, Li Xifen, Liang Huizhen. Research progress on microbial diversity and metabolic processes in the brewing process of Maotai-flavor liquor [J]. China Brewing, 2022, 41(06): 6-11.) studied the bacterial diversity and its metabolic processes, yeast diversity and its metabolic processes, mold diversity and its metabolic processes, and actinomycete diversity and its metabolic processes in the brewing process of Maotai-flavor liquor. The results showed that yeast cannot survive in a high temperature environment, so its content in high-temperature Daqu is very low. 13 mold genera such as Alternaria, Aspergillus flavus, and Aspergillus niger are the dominant molds in the brewing environment of Maotai-flavor liquor. The metabolites of actinomycetes have a metabolic regulatory effect on the functional microorganisms of brewing, thereby affecting the production of aroma substances.

[0013] Jiang Dongcai et al. (Jiang Dongcai, Zhu Heqin, Luo Zhi, Peng Liqun. Research on substances and microbial flora during high-temperature Daqu fermentation [J]. Brewing, 2022, 49(04): 53-56.) analyzed the substances and microbial flora at each stage of the high-temperature Daqu in the production process of soft and elegant liquor. The results showed that the changes in substances during the fermentation process were closely related to the reproduction of microorganisms. The representative PCR-DGGE band sequencing was compared for similarity. ＞ 95% of the bacteria mainly include 8 genera and uncultivable microorganisms, namely Lactobacillus, Bacillus, Weissella, Staphylococcus, Pediococcus, Leuconostoc, Enterobacter and Enterococcus, among which the dominant bacteria are lactic acid bacteria and Bacillus.

[0014] Liang Zhen (Liang Zhen. Research on microorganisms and flavor substances in the fermentation process of soy-flavor liquor [D]. 2020) studied the correlation between microorganisms and flavor substances in the fermentation process of soy-flavor liquor. The results showed that there was a good correlation between microorganisms and flavor substances; the bacteria with a strong correlation with flavor substances were mainly Lactobacillus, Weissella, Rhodococcus, Pediococcus, Enterococcus, Lactococcus, and Leuconostoc, and they were mainly lactic acid bacteria.

[0015] Current research is mostly focused on using high-throughput sequencing technology to study the evolution of bacterial genera such as Enterococcus, Enterobacter and Lactobacillus during the liquor brewing process. However, there are no relevant research reports on the metabolites of specific enterococcal strains. Finding an enterococcus that can produce a variety of aldehyde aromatic substances is of significant significance to the liquor brewing process.

[0016] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0017] In view of the shortcomings of the prior art, the present invention provides a Thai Enterococcus that can produce a variety of aldehyde aroma substances. The Thai Enterococcus can specifically produce furfural, decanal, benzaldehyde, phenylacetaldehyde and other aldehyde aroma substances, which can be used in the brewing of various types of liquor.

[0018] Preferably, to achieve the above purpose, the present invention provides a Thai Enterococcus that can produce multiple aldehyde aroma substances, its name is Thai Enterococcus zqw28 (Enterococcus thailandicus), which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit number is: CGMCC NO.25138.

[0019] Preferably, the nucleotide sequence of Thai Enterococcus zqw28 based on 16S rDNA is shown as SEQ ID NO.1.

[0020] Further, the amplification primer sequences are:

[0021]

[0022] Preferably, the biological characteristics of the Thai Enterococcus of the present invention are as follows: on the MRS agar medium, the colonies are slightly convex and round, milky white and opaque, with a smooth and moist surface and neat edges.

[0023] Preferably, the present invention provides a method for screening and identifying the aforementioned Thai Enterococcus, the screening and identification method comprising the following steps: breaking up and separating the Luzhou Laojiao pit mud sample, purifying by streaking, and preserving the strain, and then using the ability to produce aldehyde flavor substances as a screening criterion to obtain Thai Enterococcus that produces aldehyde flavor substances.

[0024] Preferably, the breaking up and separation comprises: taking a suspension of the Luzhou Laojiao pit mud sample and breaking it up by shaking, and spreading a plurality of gradient dilutions on a solid culture medium plate for anaerobic culture.

[0025] Preferably, the streak purification comprises: picking single colonies with different colony morphologies on the plate where the colonies grow, and purifying all the single colonies by secondary streaking.

[0026] Preferably, the bacterial strain preservation comprises: anaerobic cultivation of each purified strain in a liquid culture medium in the form of a single colony.

[0027] Preferably, the present invention provides a screening method for testing the ability of each strain to produce a target compound after separation and purification, so as to screen out strains with high production capacity. Specifically, the screening method comprises dissolving the glycerol storage tube of the strain obtained after separation and purification, inoculating it in MRS liquid culture medium respectively, and culturing it under anaerobic conditions at 37°C for 20 hours, activating it for three generations, inoculating it in 50mL of MRS liquid culture medium at a 2% inoculation amount and culturing it for 20 hours, and obtaining the fermentation liquid to be tested after the fermentation is completed. Thereafter, the target compound is extracted and enriched by headspace solid phase microextraction, and then chromatographic detection and analysis are performed by GC-MS, so as to screen Thai Enterococcus with high production capacity of aldehyde flavor substances by comparing the content of volatile substances.

[0028] Preferably, the present invention provides a molecular identification method for bacteria producing organic acids, organic alcohols and ester compounds, which specifically comprises amplifying the target strain, taking fresh bacterial liquid in the logarithmic growth phase, collecting the bacteria by centrifugation, and extracting genomic DNA using a bacterial genome extraction kit. In particular, in the present invention, the universal primers 27F / 1492R for lactic acid bacteria are used to amplify the full-length 16SrDNA sequence of the target strain.

[0029] Preferably, the present invention also provides a microbial agent, which contains Enterococcus thailandensis.

[0030] Preferably, the microbial agent is a liquid agent or a solid agent containing one or more of live cells of Enterococcus thailandensis, freeze-dried dry cells of Enterococcus thailandensis, and immobilized cells of Enterococcus thailandensis.

[0031] Preferably, the present invention provides a freeze-dried bacterial powder, which is prepared by the following method: after activating Thai Enterococcus, inoculate it into a culture medium for enrichment culture, collect the bacteria by centrifugation after the culture is completed, add trehalose, sorbitol, and skim milk powder as freeze-drying protective agents, mix them evenly, and then perform vacuum freeze-drying to obtain freeze-dried bacterial powder.

[0032] Preferably, the present invention provides a use of Enterococcus thailandensis, or a microbial agent containing Enterococcus thailandensis, or a freeze-dried bacterial powder containing Enterococcus thailandensis in the fermentation production of aldehyde flavor substances.

[0033] Preferably, the application of Enterococcus thailandensis in the fermentation production of aldehyde aroma substances can be to culture Enterococcus thailandensis, or a microbial agent containing Enterococcus thailandensis, or a freeze-dried bacterial powder containing Enterococcus thailandensis in a fermentation medium. Specifically, the culture medium includes MRS liquid culture medium, MRS solid culture medium and fermentation medium.

[0034] Preferably, in the present invention, the composition of the fermentation medium is 16g glucose, 10g tryptone, 2.5g yeast powder, 5g beef extract, 3.85g bovine brain, 4.9g bovine heart, 0.5g Tween 80, 1g ammonium citrate, 2.5g sodium chloride, 2.5g anhydrous sodium acetate, 0.05g magnesium sulfate, 0.025g manganese sulfate, 1.25g disodium hydrogen phosphate and 1g dipotassium hydrogen phosphate per liter of culture medium.

[0035] Preferably, the use of Enterococcus thailandensis in the production of a variety of aldehyde aroma substances can be specifically to culture the Enterococcus thailandensis in a fermentation medium at 37°C for 24 hours with a pH value of 7.0, wherein the inoculation amount of the Enterococcus thailandensis is 2% (×10 7 cfu / mL).

[0036] More specifically, the Thai Enterococcus strain can produce furfural, decanal, benzaldehyde, phenylacetaldehyde, 3,4-dimethylbenzaldehyde and 2,3-dihydro-2,2,6-trimethylbenzaldehyde, among which the contents of furfural, decanal, benzaldehyde, phenylacetaldehyde, 3,4-dimethylbenzaldehyde and 2,3-dihydro-2,2,6-trimethylbenzaldehyde are 0.356, 0.322, 0.352, 0.161, 0.321 and 1.196 mg / L respectively.

[0037] Preferably, in the present invention, the aldehyde aroma substances are selected from the group consisting of furfural, decanal, benzaldehyde, phenylacetaldehyde, 3,4-dimethylbenzaldehyde, and 2,3-dihydro-2,2,6-trimethylbenzaldehyde. In particular, among the aldehyde aroma substances produced by Enterococcus thailandensis, the concentration / content of 2,3-dihydro-2,2,6-trimethylbenzaldehyde is the highest, and the concentration / content of phenylacetaldehyde is the lowest. That is, the characteristics of woody, spicy, medicinal, and powdery aromas are more prominent.

[0038] Preferably, the present invention provides a use of Enterococcus thailandensis, or a microbial agent containing Enterococcus thailandensis, or a freeze-dried bacterial powder containing Enterococcus thailandensis in wine fermentation.

[0039] Preferably, the application of Enterococcus thailandensis in wine fermentation can be to add Enterococcus thailandensis, or a microbial agent containing Enterococcus thailandensis, or a freeze-dried bacterial powder containing Enterococcus thailandensis to wine cellar mud. In particular, Enterococcus thailandensis, or a microbial agent containing Enterococcus thailandensis, or a freeze-dried bacterial powder containing Enterococcus thailandensis is added to wine cellar mud for fermentation, so that the wine sample contains aldehyde aroma substances, thereby improving the flavor of the wine.

[0040] Preferably, the present invention provides a use of Enterococcus thailandensis, or a microbial agent containing Enterococcus thailandensis, or a freeze-dried bacterial powder containing Enterococcus thailandensis in liquor brewing.

[0041] Preferably, the application of Enterococcus thailandensis in liquor brewing can be to add Enterococcus thailandensis, or a microbial agent containing Enterococcus thailandensis, or a freeze-dried bacterial powder containing Enterococcus thailandensis during the liquor fermentation process.

[0042] Preferably, the present invention also provides a liquor, which can be obtained by adding Enterococcus thailandensis, or a microbial agent containing Enterococcus thailandensis, or a freeze-dried agent containing Enterococcus thailandensis during the fermentation process of the liquor. In particular, Enterococcus thailandensis is added during the liquor brewing process to make the liquor sample rich in aldehyde aroma substances, thereby improving the flavor of the liquor.

[0043] The beneficial technical effects of the present invention include: the Thai Enterococcus zqw28 provided by the present invention can produce a variety of aldehyde aroma substances, which is of great significance for studying the microorganisms and the source of flavor substances in the liquor brewing process. The Thai Enterococcus zqw28 provided by the present invention can provide strain resources for optimizing the liquor brewing process and has a relatively broad market application prospect.

[0044] Biological Deposit Description

[0045] The Thai Enterococcus zqw28 (Enterococcus thailandicus) provided by the present invention is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.25138, a deposit date of June 21, 2022, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0046] The present invention provides a Thai enterococcus that can produce a variety of aldehyde aroma substances, the name of which is Thai enterococcus zqw28 (Enterococcus thailandicus), which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.25138, a deposit date of June 21, 2022, and a deposit address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0047] Example 1

[0048] According to a preferred embodiment, this embodiment provides an application of Enterococcus thailandicus zqw28 in the production of a variety of aldehyde aroma substances. Further, the application specifically includes culturing Enterococcus thailandicus zqw28 in a culture medium at 37°C for 24 hours, with a pH value of 7.0, wherein the inoculation amount of Enterococcus thailandicus zqw28 is 2% (×10 7 cfu / mL). In particular, the culture medium includes MRS liquid culture medium, MRS solid culture medium and fermentation medium.

[0049] Specifically, in this embodiment, the specific composition of the MRS liquid culture medium is 20 g / L glucose, 10 g / L peptone, 4 g / L yeast powder, 5 g / L beef extract powder, 1 g / L Tween 80, 2 g / L triammonium citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate and 1 g / L potassium dihydrogen phosphate. In particular, the pH of the MRS liquid culture medium is 7.0, and the MRS liquid culture medium needs to be sterilized by high pressure steam at 115° C. for 20 minutes.

[0050] According to a preferred embodiment, in this example, the specific composition of the MRS solid culture medium is to add 15 g / L agar to the MRS liquid culture medium. In particular, the MRS solid culture medium needs to be sterilized by high pressure steam at 115° C. for 20 minutes.

[0051] More specifically, the fermentation medium is composed of 16 g / L glucose, 10 g / L tryptone, 2.5 g / L yeast powder, 5 g / L beef extract, 3.85 g / L bovine brain, 4.9 g / L bovine heart, 0.5 g / L Tween 80, 1 g / L ammonium citrate, 2.5 g / L sodium chloride, 2.5 g / L anhydrous sodium acetate, 0.05 g / L magnesium sulfate, 0.025 g / L manganese sulfate, 1.25 g / L disodium hydrogen phosphate, and 1 g / L dipotassium hydrogen phosphate. In particular, the pH of the fermentation medium is 7.0, and the fermentation medium needs to be sterilized by high pressure steam at 115° C. for 20 minutes.

[0052] According to a preferred embodiment, the Thai Enterococcus zqw28 (Enterococcus thailandicus) provided by the present invention can be applied to liquor brewing. Specifically, the Thai Enterococcus zqw28 (Enterococcus thailandicus) of the present invention can be added during the liquor brewing fermentation process, or a liquid bacterial agent or solid bacterial agent containing one or more of Thai Enterococcus zqw28 (Enterococcus thailandicus) living cells, freeze-dried Thai Enterococcus zqw28 (Enterococcus thailandicus) dry bacterial bodies, and immobilized Thai Enterococcus zqw28 (Enterococcus thailandicus) cells can be added.

[0053] According to a preferred embodiment, this embodiment also provides a freeze-dried bacterial powder containing Thai Enterococcus zqw28 (Enterococcus thailandicus). Specifically, the preparation method of the freeze-dried bacterial powder containing Thai Enterococcus zqw28 (Enterococcus thailandicus) is as follows: After activating the Thai Enterococcus zqw28 (Enterococcus thailandicus) described in this embodiment, inoculate it in a culture medium for enrichment culture, collect the bacteria by centrifugation after the culture is completed, add trehalose, sorbitol, and skim milk powder as freeze-drying protective agents, mix them evenly, and then perform vacuum freeze drying to obtain freeze-dried bacterial powder.

[0054] Example 2

[0055] According to a preferred implementation, this embodiment is a further improvement on Embodiment 1, and repeated contents will not be repeated any more.

[0056] According to a preferred embodiment, the Thai Enterococcus zqw28 (Enterococcusthailandicus) in the present invention can be obtained by the following method:

[0057] S2.1 Separation: After the pit mud sample is broken up by oscillation, the suspension is diluted and multiple gradient dilutions are spread on solid culture medium plates for anaerobic culture.

[0058] Specifically, 20 g of Luzhou Laojiao pit mud sample was placed in a conical flask containing 180 mL of sterile distilled water, and the sample was shaken on a constant temperature shaker for 10 min to fully disperse and mix the sample. 1 mL of the sample suspension was taken and diluted to 10 by the multiple dilution method. -2 ~10 -7, take 100 μL from each gradient dilution and spread it evenly on the MRS solid culture medium plate, prepare two plates in parallel, invert them, and culture them at 37℃ under anaerobic conditions for 36 to 48 hours, and observe them in time.

[0059] S2.2 Purification: Take out the plate with grown colonies, pick out single colonies with different colony morphologies, and perform secondary streaking until all single colonies are purified.

[0060] S2.3 Preservation: Each purified strain was cultured anaerobically in liquid culture medium as a single colony.

[0061] Specifically, a single colony of each strain after purification was picked into 5 mL MRS liquid culture medium, placed in 37°C anaerobic culture for 20 to 24 hours, 1 mL of bacterial solution was pipetted into a bacterial preservation tube, 0.5 mL of 60% sterile glycerol solution was added, resuspended, and stored at -80°C.

[0062] Example 3

[0063] This embodiment is a further improvement of Embodiment 1 and / or Embodiment 2, and the repeated contents will not be repeated here.

[0064] According to a preferred embodiment, this embodiment provides a screening method, which can detect the ability of each strain to produce a target compound after separation and purification and preservation in a glycerol storage tube, so as to screen out strains with high production capacity, wherein the target compound in this embodiment is a variety of aldehyde substances. Specifically, in this embodiment, the aldehyde substances include furfural, decanal, benzaldehyde, and phenylacetaldehyde. Further, the aldehyde substances are selected from the group consisting of furfural, decanal, benzaldehyde, phenylacetaldehyde, 3,4-dimethylbenzaldehyde, and 2,3-dihydro-2,2,6-trimethylbenzaldehyde.

[0065] According to a preferred embodiment, a strain with high aldehyde production capacity can be screened by detecting the content of aldehydes produced by each strain. Specifically, the screening method includes:

[0066] S3.1. Prepare the bacterial solution to be tested

[0067] Specifically, the glycerol storage tube of the isolated and purified strain was dissolved, and the strain was inoculated into MRS liquid culture medium, and cultured at 37°C under anaerobic conditions for 20 hours. After activation for three generations, the strain was inoculated into 50 mL of MRS liquid culture medium at a 2% (volume ratio) inoculation amount and cultured for 20 hours. After the fermentation was completed, the fermentation liquid to be tested was obtained.

[0068] S3.2 Qualitative detection of aldehydes

[0069] Preferably, the fermentation broth to be tested is subjected to headspace solid phase microextraction to extract and enrich the target compound, and then chromatographic detection and analysis is performed by GC-MS.

[0070] Specifically, the bacterial solution was centrifuged, the supernatant was taken, added to the headspace bottle, and a saturated NaCl solution was added. The prepared sample was kept at 60°C for 5 minutes, and then extracted at 60°C for 40 minutes using a 50 / 30μmDVB / CAR / PDMS extraction head. After the extraction, the compound search results were matched with the NIST standard spectral library, and the target compound was confirmed to be more than 80% similar. The culture solution without bacterial fermentation was used as the blank control group, and each volatile substance was qualitatively analyzed.

[0071] In particular, the operating conditions for GC-MS detection chromatography are:

[0072] Gas chromatography conditions: HP-INNOWAX column (60m×0.25mm×0.25μm); temperature program: starting temperature 40°C, hold for 5min, increase to 100°C at 4°C / min, then increase to 230°C at 6°C / min, hold for 10min, carrier gas is high-purity helium (1.0mL / min); injection port temperature 250°C, no split.

[0073] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40-450 m / z.

[0074] According to a preferred embodiment, a strain having a stronger ability to produce aldehyde compounds is obtained from the isolated strains by the above method, and the strain is selected for further study.

[0075] In particular, the above screening method can be used as a method for detecting the ability of microorganisms to produce aldehydes.

[0076] According to a preferred embodiment, in the present invention, "headspace solid phase microextraction" can be a solid phase microextraction technology that uses headspace sampling, wherein the headspace is used as a sampling method, and the sample to be tested can be placed in a constant temperature closed container, and the volatile components are evaporated from the sample by heating. After the two phases in the headspace bottle reach thermodynamic equilibrium, the top gas is directly extracted and injected into a gas chromatography-mass spectrometer for separation and analysis, thereby detecting some volatile or odorous substances; and the role of solid phase microextraction technology is to use a fused quartz fiber coated with a stationary phase to adsorb and enrich when extracting gas samples, and then perform analysis and sampling.

[0077] According to a preferred embodiment, in the present invention, "GC-MS" can be a gas chromatography-mass spectrometer, which is a device that combines the characteristics of gas chromatography and mass spectrometry to identify different substances in a sample. When a multi-component mixed sample enters the chromatographic column, due to the different adsorption forces of the adsorbent on each component, after a certain period of time, the running speed of each component in the chromatographic column is also different. Components with weak adsorption forces are easily desorbed and leave the chromatographic column first to enter the detector, while components with the strongest adsorption forces are least likely to be desorbed and therefore leave the chromatographic column last. In this way, the components are separated from each other in the chromatographic column and sequentially enter the detector to be detected and recorded.

[0078] Example 4

[0079] This embodiment is a further improvement of Embodiment 1, Embodiment 2 and / or Embodiment 3, and the repeated contents will not be repeated here.

[0080] According to a preferred embodiment, this embodiment provides a molecular identification method for bacteria producing organic acids, organic alcohols and ester compounds, which can be used to determine the species information of the screened strains. Specifically, the identification method may include:

[0081] The target strain was cultured, fresh bacterial liquid in the logarithmic growth phase was taken, the bacteria were collected by centrifugation, and the genomic DNA was extracted using a bacterial genome extraction kit.

[0082] According to a preferred embodiment, the universal primers 27F / 1492R for lactic acid bacteria are used to amplify the full-length sequence of 16SrDNA, as follows:

[0083]

[0084] ①Reaction system (50μL)

[0085]

[0086] ②Reaction procedure

[0087]

[0088] In particular, the denaturation, annealing and extension steps are preferably repeated for 35 cycles.

[0089] According to a preferred embodiment, the PCR product is separated and tested by 1.0% agarose gel electrophoresis, the voltage is about 11V / cm, and the electrophoresis time is 20min. In particular, the detection method of the PCR product also includes polyacrylamide gel electrophoresis. Further, the commonly used purification methods of PCR products include silica gel chromatography column method, magnetic bead method, permeate combined with alcohol precipitation purification method, direct precipitation purification and gel recovery purification, etc.

[0090] According to a preferred embodiment, the sequencing of PCR products often adopts the cycle sequencing method, that is, dideoxynucleoside triphosphates (ddNTPs) are added to the PCR reaction system at the same time, and primers labeled with radionuclides or fluorescein are used to guide amplification, so that the template amplification and sequencing are carried out simultaneously.

[0091] According to a preferred embodiment, the gene sequence of the 16S rDNA fragment obtained by sequencing is compared with the BLAST (Basic Local Alignment Search Tool) of NCBI (National Center for Biotechnology Information) to determine the species information of the strain, identify it as Thai Enterococcus, and name it Thai Enterococcus zqw28 (Enterococcus thailandicus).

[0092] Example 5

[0093] This embodiment is a further improvement of Embodiment 1, Embodiment 2, Embodiment 3 and / or Embodiment 4, and the repeated contents will not be repeated here.

[0094] According to a preferred embodiment, this example provides a detection method for the production of multiple aldehyde aroma substances by Enterococcus thailandicus zqw28. Specifically, the detection method comprises:

[0095] After dissolving the glycerol storage tube of Enterococcus thailandicus zqw28, it was inoculated into MRS liquid culture medium and cultured at 37°C under anaerobic conditions for 24 hours. After activation for three generations, it was inoculated into 50mL fermentation medium at a 2% (volume ratio) inoculation amount and cultured for 24 hours. Further, after the fermentation, the bacterial liquid was obtained, and the supernatant was taken after centrifugation at 10000rpm for 5 minutes. The detection method was the same as the GC-MS detection method in Example 3, with 0.822mg / mL of 2-octanol as the internal standard and the original culture medium as the blank control, and the content of each volatile substance was calculated.

[0096] According to a preferred embodiment, the detection results of aldehyde aroma substances are shown in Table 1, which can produce furfural, decanal, benzaldehyde, phenylacetaldehyde, 3,4-dimethylbenzaldehyde and 2,3-dihydro-2,2,6-trimethylbenzaldehyde, and the corresponding contents are 0.356, 0.322, 0.352, 0.161, 0.321, and 1.196 mg / L respectively.

[0097] Table 1 Detection results of aldehyde aroma substances in the fermentation broth of Enterococcus thailandensis zqw28

[0098]

[0099] The results showed that in the fermentation broth of Enterococcus thailandicus, 2,3-dihydro-2,2,6-trimethylbenzaldehyde had the highest concentration / content in the fermentation broth of Enterococcus thailandicus, the concentration / content of furfural and benzaldehyde in the fermentation broth of Enterococcus thailandicus was equivalent, the concentration / content of decanal and phenylacetaldehyde in the fermentation broth of Enterococcus thailandicus was equivalent and slightly lower than the concentration of furfural and benzaldehyde, and the concentration / content of phenylacetaldehyde in the fermentation broth of Enterococcus thailandicus was the lowest.

[0100] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the description of the present invention is illustrative and does not constitute a limitation on the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", which means that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept.

Claims

1. A Thai Enterococcus, characterized in that The Thai Enterococcus (Enterococcusthailandicus) zqw28 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.25138 and the deposit date June 21, 2022.

2. A microbial agent, characterized in that: Contains the Enterococcus thailandensis according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The bacterial agent is a liquid bacterial agent or a solid bacterial agent containing one or more of live cells of Enterococcus thailandensis, dry bacterial bodies of Enterococcus thailandensis obtained by freeze drying, and immobilized cells of Enterococcus thailandensis.

4. A freeze-dried bacterial powder, characterized in that: The freeze-dried bacterial powder is prepared by the following method: after activating the Thai Enterococcus described in claim 1, inoculating it in a culture medium for enrichment culture, collecting the bacterial bodies by centrifugation after the culture is completed, adding trehalose, sorbitol, and skim milk powder as freeze-drying protective agents, mixing them evenly, and performing vacuum freeze drying to obtain freeze-dried bacterial powder.

5. Use of the Enterococcus thailandensis according to claim 1, or the microbial agent according to claim 2 or 3, or the freeze-dried bacterial powder according to claim 4 in fermentation production of aldehyde aroma substances, characterized in that: The aldehyde aroma substance is selected from the group consisting of furfural, decanal, benzaldehyde, phenylacetaldehyde, 3,4-dimethylbenzaldehyde, and 2,3-dihydro-2,2,6-trimethylbenzaldehyde.

6. The use according to claim 5, characterized in that The Enterococcus thailandensis according to claim 1, or the microbial agent according to claim 2 or 3, or the freeze-dried bacterial powder according to claim 4 is added to a fermentation system for fermentation, and the fermentation conditions are anaerobic culture at 37° C. for 24 hours.

7. Use of the Enterococcus thailandensis according to claim 1, or the microbial agent according to claim 2 or 3, or the freeze-dried bacterial powder according to claim 4 in wine fermentation or liquor brewing.

8. The use according to claim 7, characterized in that Add the Enterococcus thailandensis described in claim 1, or the microbial agent described in claim 2 or 3, or the freeze-dried bacterial powder described in claim 4 to the pit mud.

9. The use according to claim 7, characterized in that: The Enterococcus thailandensis described in claim 1, or the microbial agent described in claim 2 or 3, or the freeze-dried bacterial powder described in claim 4 is added during the fermentation process of white wine.

10. A method for preparing liquor, characterized in that: The Enterococcus thailandis described in claim 1, or the microbial agent described in claim 2 or 3, or the freeze-dried bacterial powder described in claim 4 is added during the fermentation process of the liquor.

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