Enterococcus citreum capable of simultaneously producing geraniol and linalool

Abstract

In the prior art, the production of linalool mainly relies on artificial extraction or biotechnological expression. Engineered strains constructed with Saccharomyces cerevisiae or Escherichia coli synthesize linalool by expressing the linalool synthase gene, but there are problems with unstable expression in the process of multiple passages of these engineered strains. The present invention provides a Enterococcus casseliflavus that can simultaneously produce geraniol and linalool, and the Enterococcus casseliflavus is screened from high-quality Luzhou-flavor old cellar mud. The Enterococcus casseliflavus can stably grow for multiple generations in the natural environment. The Enterococcus casseliflavus was deposited on June 21, 2022 at the China General Microbiological Culture Collection Center, deposit number: CGMCC NO. 25150.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Enterococcus fuscae that can simultaneously produce geraniol and linalool. Background Art

[0002] Linalool is a non-cyclic monoterpene alcohol with a special fragrance that occurs naturally in plants. It is a chemical substance with a wide range of applications. Linalool is slightly soluble in water, easily soluble in organic solvents, and volatile. Linalool has the aroma of lily of the valley, but the aroma varies depending on the source. Therefore, linalool has important application value in the fields of food, fragrance, medicine, daily chemicals, etc. Due to its aroma, linalool has become a frequently used fragrance in perfumes and household cleaning and care products. Linalool is also used in the food industry as a food flavor. Linalool is also an important intermediate in industrial production. Existing studies have shown that linalool has pharmacological activities such as analgesia, anti-anxiety, sedation and hypnosis, anti-inflammatory, anti-tumor, and antibacterial, and has broad prospects for medicinal use.

[0003] Geraniol is an oxygenated monoterpene. It is widely used across various industries for its aroma. Its rose-like, slightly sweet aroma is widely used in the food and cosmetics industries. It is also used as an additive in advanced fuels, pharmaceuticals, and fungicides.

[0004] In the prior art, the synthesis of linalool and geraniol mainly uses plant extraction and chemical synthesis. Chinese patent publication number CN102397788A discloses a method for selectively hydrogenating dehydrolinalool to produce linalool, which comprises mixing dehydrolinalool with a solvent and then hydrogenating it in a liquid phase over a fixed bed catalyst in hydrogen. The solvent is a C2-C4 fatty alcohol, the solvent ratio is 1:(0.5-1.2), and the liquid hourly space velocity is 2.0-2.5 hr -1 Chemical synthesis methods are usually expensive and produce large amounts of waste gas, waste liquid and waste residue.

[0005] Chinese patent publication number CN101619013 discloses a method for extracting natural linalool from the leaves of Cinnamomum camphora. The method involves selecting high-quality individual Cinnamomum camphora plants to obtain high-quality mother Cinnamomum camphora plants with an oil yield of at least 1.5% from fresh leaves, a leaf oil content of at least 97% linalool, no more than 0.2% camphor, and no more than 0.2% eucalyptol. The selected mother plants are then propagated to extract linalool from the leaves. Based on the obtained high-quality mother plants, pure individual plants are then asexually propagated, such as by tissue culture or cuttings. Monoterpenes extracted from natural aromatic plants are generally expensive due to their low yield and extraction efficiency, as well as their susceptibility to various natural factors.

[0006] Based on this, the method of synthesizing monoterpenes by microbial cells has become a hot research direction to replace plant extraction and chemical synthesis because of its wide source of raw materials, no spatial and environmental restrictions, low cost, single product and short production cycle.

[0007] Existing research has shown that microbial linalool synthesis can be achieved by constructing engineered strains using Saccharomyces cerevisiae or Escherichia coli to express the linalool synthase gene. Chinese Patent Publication No. CN111411101B discloses a linalool synthase mutant containing one or a combination of two of the following amino acid mutations: E352H or E343D. However, due to the demand for the production of multiple alcohols during winemaking, the prior art involves adding multiple engineered strains to the fermentation mixture to achieve wines that meet quality standards. However, the increased number and variety of engineered strains means more demanding fermentation conditions and increases the risk of competition between different fungi. Currently, Enterococcus fuscae is not known to have the ability to simultaneously produce geraniol and linalool. The present invention provides a strain of Enterococcus fuscae that can simultaneously produce geraniol and linalool during its growth.

[0008] 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

[0009] In response to the deficiencies of the prior art, the present invention provides a strain of Enterococcus casseliflavus. The strain provided by the present invention has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on June 21, 2022, with the deposit number CGMCC NO.25150 and the name: Enterococcus casseliflavus zqw97 (Enterococcus casseliflavus).

[0010] The strain provided by the present invention can simultaneously produce geraniol and linalool that improve the flavor of liquor, and optimizes the fermentation medium without polluting the natural environment, thereby improving the flavor richness of liquor during the production process.

[0011] The strain provided by the present invention was screened from nature. Using a two-phase fermentation process, the strain produced 9.77 mg / L of geraniol and 3.06 mg / L of linalool. While maintaining levels comparable to those produced by two strains, respectively, the strain provided by the present invention is capable of simultaneously producing both flavor compounds and maintaining stable expression across multiple generations.

[0012] According to a preferred embodiment, the strain can be isolated from an alcohol fermentation product. Preferably, the alcohol fermentation product in the present invention can be a substance involved in alcohol brewing, such as cellar mud, mash, fillers, additives, distiller's yeast, and other substances containing microbial components and involved in the brewing process.

[0013] The present invention provides a method for separating and purifying Enterococcus leucoderma, which comprises the following steps:

[0014] The pit mud samples were cultured on MRS solid medium plates to obtain single colonies with different colony morphologies;

[0015] Single colonies with different colony morphologies were inoculated twice into MRS solid culture medium to obtain the purified Enterococcus leucoderma.

[0016] The purified strain was obtained by multi-generation cultivation. Through this purification method, it was confirmed that the strain provided by the present invention can be stably inherited for multiple generations.

[0017] The Enterococcus leucoderma was subjected to molecular biological identification, and its 16S rDNA sequence was shown as SEQ ID NO.1.

[0018] The present invention provides use of the strain in simultaneously producing geraniol and linalool.

[0019] According to a preferred embodiment, the Enterococcus leucoderma is capable of producing geraniol and linalool in a culture environment containing ethanol.

[0020] The present invention provides a culture medium capable of culturing the strain.

[0021] The present invention also relates to compositions comprising the strain of the present invention, a microorganism-free extract, and other aromatic alcohols. The present invention also relates to methods for producing geraniol and linalool based on the strain provided by the present invention, comprising using effective amounts of the strain of the present invention, a habitat for the strain, propagation material for the strain, a microorganism-free extract, a culture medium, and a composition comprising other aromatic alcohols.

[0022] According to a preferred embodiment, the culture medium comprises glucose, peptone, yeast powder, beef extract powder, Tween 80, triammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate and dipotassium hydrogen phosphate. Preferably, the strains involved in the present invention can grow and reproduce in MRS liquid or solid culture medium.

[0023] Linalool has a sweet, woody, green aroma, reminiscent of rosewood and freshly brewed green tea. It combines floral notes of lilac, lily of the valley, and rose with woody and fruity notes. The aroma is soft, light, and penetrating, yet not very persistent. Linalool with a left-handed structure is sweeter and mellower, while linalool with a right-handed structure is lighter and greener. Geraniol, as an aroma aid, contributes to a variety of floral notes, such as citrus and geranium. In terms of the flavor of alcoholic beverages, linalool and geraniol enhance the richness of baijiu (white spirits). Aroma plays a crucial role in human health and function. For example, linalool can induce a sense of well-being. American doctor Sternford and psychologist Reynolds conducted a study on 5,000 people, confirming that frequent exposure to floral fragrance can have a positive impact on mood and health. People's fascination with the aroma of baijiu is partly due to the fact that its aromatic compounds interact with the sensory system, thereby creating a sense of well-being.

[0024] The quality of baijiu (white liquor) is determined by the quality of the pit mud used during the brewing process. In the prior art, the microbial composition of the pit mud is complex and opaque, requiring manual adjustments based on feel for each brewing phase, making it impossible to achieve a consistent taste. For baijiu with excellent taste, the complex microbial composition of the mash and pit mud makes it difficult to explore the factors influencing their production. The present invention provides a strain of Enterococcus fuscae, which is screened from high-quality, aged pit mud of a Luzhou-flavor liquor. By isolating, purifying, and verifying the microbial components present in the pit mud, clues can be provided for exploring the contribution of the pit mud to winemaking and its impact on the quality of the liquor. Based on this, the present invention provides a liquor comprising at least geraniol and linalool produced by the strain. Linalool and geraniol are known to be important flavor enhancers in liquor. Therefore, the inclusion of a strain capable of simultaneously producing geraniol and linalool in microbially fermented liquor plays an important role in improving the quality of the liquor during the fermentation process. The strain provided by the present invention is extracted from pit mud, which also verifies that the strain provided by the present invention can grow and reproduce in an environment with ethanol and produce geraniol and linalool.

[0025] The present invention provides a composition comprising the strain. The strain of the present invention can be applied in the form of a composition to a substance requiring geraniol or linalool as a component.

[0026] According to a preferred embodiment, the strain provided by the present invention can be combined with other strains to form a composition for use in the preparation of edible wine. Beneficial strains present in edible wine include Aspergillus, Rhizopus, Actinomycetes, Thermotolerant Bacillus, yeast, and bacteria. Specifically, bacteria include lactic acid bacteria, caproic acid bacteria, acetic acid bacteria, butyric acid bacteria, methanogens, propionic acid bacteria, and butyric acid bacteria. Lactic acid bacteria include lactococci and lactobacilli. Yeast includes cerevisiae and ester-producing yeast. The strain provided by the present invention can be combined with one or more of the above-mentioned strains to form a composition. The composition can play a role in the alcohol fermentation process.

[0027] Preferably, the composition provided by the present invention comprises:

[0028] Enterococcus leucoderma, Aspergillus niger, Rhizopus spp., Saccharomyces cerevisiae;

[0029] Enterococcus leucoderma, Monascus purpureus, Rhizopus spp., Saccharomyces cerevisiae;

[0030] Enterococcus fulvum, Aspergillus niger, Monascus purpureus, Rhizopus niger, Saccharomyces cerevisiae;

[0031] Enterococcus pyogenes, Aspergillus niger, Monascus purpureus, Rhizopus niger, Saccharomyces cerevisiae, Hexanoic acid bacteria;

[0032] Enterococcus pyogenes, Aspergillus niger, Monascus purpureus, Rhizopus niger, ester-producing yeast, hexanoic acid bacteria.

[0033] Enterococcus fulvidraco, Lactococcus, Aspergillus niger, Monascus purpureus, Rhizopus niger, Saccharomyces cerevisiae;

[0034] Enterococcus pyogenes, Lactobacillus, Aspergillus niger, Rhizopus niger, Saccharomyces cerevisiae, Hexanoic acid bacteria, Acetic acid bacteria, Butyric acid bacteria, Methanogens, Propionibacterium.

[0035] The present invention provides an application of Enterococcus pyogenes in food, medicine, and antibacterial agents. In the food field, geraniol or linalool is widely used. Specifically, linalool is a key component of Earl Grey tea, and the amount added to tea leaves or powder is as high as 0.3%. The strain for producing linalool provided by the present invention can be used in the production of tea substances. Geraniol or linalool is an important flavor raw material in perfumery. The strain for producing linalool and geraniol provided by the present invention can be used in the field of flavor purification.

[0036] Based on the above product requirements, in industrial production, the strain provided by the present invention is used to produce geraniol and linalool, and geraniol and linalool are obtained by separation and purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the GC-MS graph of linalool production by the strain;

[0038] Figure 2 This is the GC-MS molecular fragment mass spectrum of linalool produced by the strain;

[0039] Figure 3 This is the GC-MS molecular fragmentation mass spectrum of linalool standard;

[0040] Figure 4 This is the GC-MS graph of geraniol production by the strain;

[0041] Figure 5 This is the GC-MS molecular fragment mass spectrum of geraniol produced by the strain;

[0042] Figure 6 This is the GC-MS molecular fragment mass spectrum of geraniol standard. DETAILED DESCRIPTION

[0043] The following is a detailed description with reference to the accompanying drawings.

[0044] The strain species and / or strains described herein include vegetative and non-sporulating forms of the strain.

[0045] According to a preferred embodiment, the composition may comprise an amount of about 1×10 3 CFU / g is about 1×10 13 CFU / g of one or more microorganisms. Preferably, the one or more microorganisms are present at about 1×10 5 CFU / g is about 1×10 11 CFU / g. Preferably, the one or more microorganisms are present in an amount of about 1×10 6 CFU / g is about 1×10 10 CFU / g. Preferably, the one or more microorganisms are present in an amount of about 1×10 8 CFU / g is about 1×10 10 CFU / g are present in the composition. Preferably, the composition comprises about 1×10 CFU / g, about 1×10 2 CFU / g, about 1×10 3 CFU / g, about 1×10 4 CFU / g, about 1×10 5 CFU / g, about 1×10 6 CFU / g, about 1×10 7 CFU / g, about 1×10 8 CFU / g, about 1×10 9 CFU / g, about 1×10 10 CFU / g, about 1×10 11 CFU / g, about 1×10 12 CFU / g, about 1×10 13 CFU / g, about 1×10 14 CFU / g or about 1×10 15 One or more microorganisms are present in an amount of CFU / g.

[0046] According to a preferred embodiment, the composition of the culture medium is as follows: per liter of culture medium, the composition comprises 10-20 g glucose, 5-15 g tryptone, 1-5 g yeast extract, 5-15 g beef extract, 1-5 g bovine brain, 5-15 g bovine heart, 0.1-5 g Tween 80, 1 g ammonium citrate, 0.1-5 g sodium chloride, 0.1-5 g anhydrous sodium acetate, 0.1-5 g magnesium sulfate, 0.1-5 g manganese sulfate, 0.1-5 g disodium hydrogen phosphate, and 0.1-5 g dipotassium hydrogen phosphate. After sterilization, 1% to 20% ethyl acetate is added.

[0047] According to a preferred embodiment, the liquid culture medium is composed of: 10-30 g / L glucose, 5-15 g / L peptone, 5-15 g / L yeast extract, 5-15 g / L beef extract powder, 0.1-5 g / L Tween 800, 0.1-5 g / L triammonium citrate, 5-15 g / L sodium acetate, 0.01-1 g / L magnesium sulfate, 0.01-1 g / L manganese sulfate, and 0.1-5 g / L potassium hydrogen phosphate. The pH of the liquid culture medium is adjusted to between 7.0 and 7.5. Sterilization is performed by high-pressure steam.

[0048] Example 1

[0049] This embodiment provides a culture medium for Enterococcus fulvicus.

[0050] The culture medium for increasing the number of active Enterococcus fuscae comprises glucose, peptone, yeast powder, beef extract powder, Tween 80, triammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate and dipotassium hydrogen phosphate. The culture medium is a liquid culture medium.

[0051] Specifically, the liquid culture medium contains: 20 g / L glucose, 10 g / L peptone, 4 g / L yeast extract, 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 dipotassium hydrogen phosphate. The pH of the liquid culture medium is adjusted to 7.0. The medium is sterilized by high-pressure steam at 115°C for 20 minutes. The liquid culture medium obtained by the above method is used to cultivate Enterococcus fuscae.

[0052] Enterococcus spp. was placed in the culture medium and cultured at 37°C for 3 days with the pH value maintained at 7.0. The inoculum size of Enterococcus spp. was 2% (×10 6 cfu / mL).

[0053] According to a preferred embodiment, to obtain high yields of linalool and geraniol, Enterococcus fusogenum is fermented in a culture medium containing, per liter, 16g glucose, 10g tryptone, 2.5g yeast extract, 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. After sterilization at 115°C, 10% ethyl acetate is added.

[0054] Example 2

[0055] This embodiment provides a method for separating and purifying Enterococcus fulvicidal strains.

[0056] (1) Separation method: Take 20g of pit mud sample and place it in a conical flask filled with 180mL of sterile distilled water. Oscillate on a constant temperature shaker for 10min to fully break up and mix the sample. Take 1mL of sample suspension and dilute it to 10% by the doubling dilution method. -2 ~10 -7 , aspirate 100 μL from each gradient dilution and evenly spread it on the MRS solid culture medium plate, prepare two plates in parallel, invert them, and culture them in an anaerobic environment at 37°C for 36 to 48 hours, and observe them in time.

[0057] (2) Streak purification: Take out the plate with colonies, pick out single colonies with different colony morphologies, and perform streaking twice until all single colonies are purified.

[0058] (3) Strain preservation: After purification, a single colony of each strain was placed in 5 mL of MRS liquid culture medium and cultured at 37°C under anaerobic conditions for 20–24 h. 1 mL of the bacterial solution was transferred to a preservation tube, and 0.5 mL of 60% sterile glycerol solution was added, the tube was resuspended, and the tube was stored at −80°C.

[0059] Example 3

[0060] This embodiment provides a method for detecting the ability of Enterococcus fulvidraco to produce geraniol and linalool.

[0061] (1) Prepare the bacterial solution to be tested:

[0062] Twenty strains were screened and glycerol tubes were dissolved and inoculated into MRS liquid medium. The culture was incubated at 37°C under anaerobism for 20 hours. After activation for three generations, the culture was inoculated into 50 mL of MRS liquid medium at a 2% (volume ratio) inoculum and cultured for 20 hours. After fermentation, a bacterial suspension was obtained.

[0063] (2) GC-MS detection method:

[0064] Headspace solid-phase microextraction (SPME) was used: the supernatant was transferred to a headspace vial, saturated NaCl solution was added, and the mixture was equilibrated at 60°C for 5 minutes. Extraction was then performed using a 50 / 30 μm DVB / CAR / PDMS tip at 60°C for 40 minutes. Desorption was then performed at 250°C for 5 minutes in the GC inlet. Compound search results were matched against the NIST standard library, and compounds with a similarity of at least 80% were confirmed as the target compounds.

[0065] GC-MS detection chromatographic conditions:

[0066] Gas chromatography conditions: HP-INNOWAX column (60 m × 0.25 mm × 0.25 μm); temperature program: initial temperature 40°C, hold for 5 min, increase to 100°C at 4°C / min, then increase to 230°C at 6°C / min, hold for 10 min, carrier gas: high-purity helium (1.0 mL / min); inlet temperature 250°C, no split.

[0067] 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.

[0068] GC-MS detection showed that only one of the 20 strains screened had the ability to produce both geraniol and linalool. The GC-MS mass spectrum of the strain culture fluid was as follows: Figures 1 to 6 shown.

[0069] According to the GC-MS mass spectrum, it was confirmed that the bacterial solution contained geraniol and linalool.

[0070] Example 4

[0071] This embodiment provides a method for identifying Enterococcus leucoderma.

[0072] 16SrDNA sequence amplification: aspirate 1 mL of the culture medium of the strain and centrifuge at 10,000 rpm for 5 min. Remove the supernatant to obtain bacterial mud, add CTAB solution and phenol-chloroform-isoamyl alcohol (25:24:1) to extract nucleic acid DNA, centrifuge at 12,500 rpm for 5 min to obtain the supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1), mix well, centrifuge at 12,000 rpm for 5 min to obtain the supernatant, rinse twice with ethanol solution, centrifuge at 12,000 rpm for 5 min to remove the supernatant, blow dry the precipitate, add 50 μL of sterile water to resuspend it, and obtain the strain DNA template.

[0073] The primers were 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′.

[0074] After mixing the above systems, PCR amplification was performed.

[0075] The gene sequence of the 16S rDNA fragment was obtained by sequencing, and the species information of the strain was determined by NCBI BLAST comparison. It was identified as Enterococcus casseliflavus and named Enterococcus casseliflavus zqw97.

[0076] Example 5

[0077] This embodiment provides a method for fermenting and culturing Enterococcus fulvicinus and detecting the content of geraniol and linalool.

[0078] The obtained Enterococcus pyogenes was inoculated into a 10mL MRS liquid medium at a 10% inoculum size, and then inoculated into a 400mL fermentation medium containing 40mL ethyl acetate at a 2% inoculum size after standing and culturing for 24h at 37°C. Two-phase fermentation was carried out and cultured continuously for 3 days. After 20mL of the upper organic phase was concentrated and evaporated to dryness, the volume was made up to 5mL with chromatographic grade methanol. The detection method was the same as the volatile component determination method in Example 2, with 0.822mg / mL of 2-octanol as the internal standard, and the content was calculated. After Enterococcus pyogenes was continuously cultivated for 3 days under this process, the geraniol production could reach 9.77mg / L, and the linalool production could reach 3.06mg / L.

[0079]

[0080]

[0081] 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 present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", all of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the features guided by "preferably" are only an optional method and should not be understood as having to be set, so the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A strain of Enterococcus casseliflavus ( Enterococcus casseliflavus ), strain zqw97, characterized in that The preservation number is CGMCC NO. 25150.

2. The Enterococcus casseliflavus according to claim 1, characterized in that, The strain was isolated from an alcohol fermentation product.

3. Use of the strain according to claim 1 in the simultaneous production of geraniol and linalool.

4. The use according to claim 3, characterized in that, The Enterococcus casseliflavus is used for wine brewing fermentation by producing geraniol and linalool.

5. A composition comprising the strain according to claim 1.

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