CN108130282B - Novel delbruospora strain and application thereof - Google Patents
Novel delbruospora strain and application thereof Download PDFInfo
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Abstract
The invention provides a novel strain of saccharomyces delbrueckii and application thereof. The collection number of the delbrueckia frutescens is CGMCC NO. 13125. The Debrella yeasts of the invention can be used for the production of a fragrance material, in particular one or more of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate.
Description
Technical Field
The invention relates to a new delbruospora strain and application thereof.
Background
The Derlburgh spore yeast is non-saccharomyces cerevisiae, and the colony morphology is spherical protrusion, smooth surface, opaque and creamy. The yeast has high osmotic pressure resistance, can survive in grape juice with high sugar content, does not produce a large amount of acetic acid, and has strong ester production capacity, thereby enhancing fragrance of the grape wine, such as flower fragrance, fruit fragrance and the like. Such as the noble rot wine and the ice wine in the brewing process.
The acetic acid-2-phenethyl ester is also called phenethyl acetate, phenethyl acetate and benzyl methyl acetate, and is colorless liquid with sweet bottom and rose fragrance. Can be used as substitute for methyl heptynoate in preparation of soap and daily cosmetic essence. It is commonly used for blending essence such as rose, orange flower, violet, tuberose, wild rose, etc., and fruity essence, and has peach fragrance.
The propionic acid-2-phenethyl ester is colorless to yellowish approximately oily liquid, is fragrant and sweet red rose-like fragrance, has fruit bottom fragrance and is similar to the flavor of thick sweet honey and strawberries. The boiling point is 245 ℃ and the flash point is more than 100 ℃. The relative density (d2525) is 1.010 to 1.014, and the refractive index (nD20) is 1.493 to 1.496. Insoluble in water, soluble in propylene glycol and dilute ethanol (1: 4, 70%). Natural products exist in peanut kernels and the like. GB 2760-.
The butyric acid-beta-phenethyl ester is colorless to light yellow liquid. Has fruit and rose fragrance, and has honey sweet fragrance. The boiling point is 238 ℃, and the flash point is more than or equal to 100 ℃. Is insoluble in water and soluble in ethanol. Natural products are found in grapes, wine, and the like. GB 2760-. The preparation method is mainly used for preparing strawberry, raspberry, peach, apple, pineapple, cream, honey and other types of essence and wine essence.
No aromatic substances such as acetic acid-2 phenylethyl ester, propionic acid-2 phenylethyl ester and butyric acid-beta phenylethyl ester have been reported to be produced by the yeast delbrueckii. Laura Canonico et al (Laura Canonico, Alice Agarbati, Torulaspora delbrueckii in the brewing process: A new approach to enhance bio-flavor and to reduce ethanol content [ J ]. Food Microbiology, 2016, 56:45-51) report that the use of Dermasporea yeast during beer brewing can enhance flavor and reduce ethanol content. In this report, beer was fermented with only Debrella yeast, but no flavor substances such as phenylethyl acetate, phenylethyl propionate and β -phenylethyl butyrate were detected, and in the most preferred treatment, a maximum of 0.008mg/L of phenylethyl acetate was detected in the sample when Saccharomyces cerevisiae and Debrella yeast were used in a ratio of 1: 1.
While other reports relating to Dermatophorus are not related to flavor, such as CN 105431539A, mainly mentions that new organic acids can be synthesized by introducing protein nucleic acids of Dermatophorus into host cells. CN 101982546A proposes the preparation of dipeptides from a culture or a bacterial cell of Saccharomyces delbrueckii. US20050129808A1 refers to a high stability liquid yeast composition comprising Dermospora delbrueckii. Andrew king et al, 2000 reported the bioconversion of monoterpene ethanol using Saccharomyces cerevisiae, Kluyveromyces delbrueckii and Kluyveromyces lactis.
Disclosure of Invention
The first aspect of the present invention provides Saccharomyces delbrueckii (Torulaspora delbrueckii) with a collection number of CGMCC NO. 13125.
In a second aspect, the present invention provides the use of Saccharomyces delbrueckii (Torulaspora delbrueckii) with a collection number of CGMCC NO.13125 for the production of ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol, phenethyl alcohol and/or fragrance materials.
In one or more embodiments, the fragrance material is selected from: one or more of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate.
In one or more embodiments, the Debrella yeast is used to produce 2-phenylethyl acetate.
In one or more embodiments, the Debrella yeast is used to produce 2-phenylethyl propionate.
In one or more embodiments, the delbrueckia sp.
In one or more embodiments, the Debrella yeast is used to produce any two or all three of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate.
In one or more embodiments, the Debrella yeast is used to produce 3-methyl-1-butanol and/or phenethyl alcohol.
In a third aspect, the present invention provides a fermentation method comprising a step of fermenting Saccharomyces delbrueckii (Torulaspora delbrueckii) with a collection number of CGMCC NO. 13125.
In one or more embodiments, fermentation is performed in a YPD liquid medium containing yeast extract, tryptone and glucose.
In one or more embodiments, the concentration of yeast extract in the YPD liquid medium is 5 to 20 g/L.
In one or more embodiments, the concentration of tryptone in the YPD liquid medium is 10-30 g/L.
In one or more embodiments, the concentration of glucose in the YPD liquid medium is 10 to 30 g/L.
In one or more embodiments, the ambospora delbrueckii is inoculated at a concentration of 0.5-5%.
In one or more embodiments, the fermentation temperature is from 25 to 32 ℃.
In one or more embodiments, the stirring rate during fermentation is 150 to 300 rpm.
In one or more embodiments, the method further comprises the step of activating the bacterial species.
In one or more embodiments, the Debrella yeast is inoculated onto YPD solid plate culture medium, and cultured at 25-32 ℃ for 0.5-3 days to activate the Debrella yeast.
In one or more embodiments, the YPD solid plate medium contains yeast extract, tryptone, glucose, and agar.
In one or more embodiments, the method further comprises the step of preparing a seed liquid.
In one or more embodiments, a single colony of Debrella yeast is selected and cultured in a YPD liquid medium at a constant temperature of 25-32 ℃ and a rotation speed of 150-300 rpm for 18-28 hours, thereby preparing the seed solution.
In one or more embodiments, the YPD liquid medium contains yeast extract, tryptone and glucose.
In one or more embodiments, the concentration of bacteria in the seed solution is 1 to 3X 108CFU/ml。
In one or more embodiments, the fermentation is a batch fermentation, a continuous fermentation, or a fed-batch fermentation.
In a fourth aspect, the present invention provides a process for producing ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol, phenethyl alcohol and/or a fragrance material, which comprises the step of performing fermentation by the fermentation process of the present invention.
In one or more embodiments, the fragrance material is selected from: one, any two or all three of acetic acid-2-phenylethyl ester, propionic acid-2-phenylethyl ester and butyric acid-beta-phenylethyl ester.
In one or more embodiments, the method is used to produce a fragrance material.
In a fifth aspect, the present invention provides a culture comprising Saccharomyces delbrueckii (Torulaspora delbrueckii) with a collection number of CGMCC NO.13125 and a culture medium.
In one or more embodiments, the medium is a YPD medium.
In a sixth aspect, the invention provides a fermentation broth comprising one or more of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate.
In one or more embodiments, the concentration of 2-phenylethyl acetate in the fermentation broth is at least 2.0 mg/kg.
In one or more embodiments, the concentration of 2-phenylethyl propionate in the fermentation broth is at least 4.0 mg/kg.
In one or more embodiments, the concentration of butyric acid- β -phenylethyl ester in the fermentation broth is at least 1.5 mg/kg.
In one or more embodiments, the fermentation broth further comprises Saccharomyces delbrueckii (Torulaspora delbrueckii) with a collection number of CGMCC NO.13125 and a culture medium.
In one or more embodiments, the fermentation broth further comprises one or more of ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol, and phenylethyl alcohol.
Drawings
FIG. 1: colony morphology photographs of delbrueckia pastoris WBRD 1.16083001.
FIG. 2: microscopic morphology of delbrueckia sp WBRD1.16083001 (1000 ×).
Detailed Description
The invention obtains a strain of non-engineering bacteria such as Torulaspora delbrueckii WBRD1.16083001 which can directly utilize pure soybean molasses by strain separation and culture from the soybean molasses, the bacteria are preserved in China general microbiological culture Collection center (CGMCC, No. 3 of West Lu No.1 of the sunward area of Beijing, the institute of microbiology, 100101) in 10.21 days of 2016, and the preservation numbers are as follows: CGMCC NO. 13125.
The Debrella yeast of the present invention can be cultured in a conventional YPD medium. Typically, YPD medium contains yeast extract, tryptone and glucose. The concentration of the yeast extract powder is usually 5-20 g per liter of the culture medium, for example 5-10 g/L. The concentration of tryptone is usually 10-30 g/L, for example 15-25 g/L. The concentration of glucose is usually 10-30 g/L, for example 15-25 g/L.
The YPD medium may be a liquid medium or a solid medium. When preparing the solid culture medium, agar (powder) can be added into a mixture containing yeast extract powder, tryptone and glucose, and water is added for preparation, so that the solid culture medium can be obtained. The dosage of the agar is the conventional dosage in the field, such as 10-30 g/L or 15-25 g/L.
YPD medium can be prepared by a conventional method, for example, in the case of 1L medium, yeast extract, trypsin and glucose are mixed, and the volume is adjusted to 1L with water to obtain YPD medium. When preparing the solid culture medium, a proper amount of agar powder can be added. When necessary, YPD medium can be sterilized by a conventional method.
The Dermatophorus strain of the present invention can be activated by a conventional YPD medium, or a seed liquid thereof can be prepared. For example, in the activation, the Debrella yeast of the invention can be diluted by sterile water, coated on a YPD solid plate culture medium, and cultured at a constant temperature of 25-32 ℃ for 0.5-3 days, preferably until bacterial colonies on the plate are clear.
YPD solid plate medium contained yeast extract powder, tryptone, glucose and agar powder as described above.
When preparing the seed solution, a single colony of the Debrella yeast is selected and inoculated in a YPD liquid culture medium, and is cultured for 18-30 hours at a constant temperature of 25-32 ℃ and 150-300 rpm. Usually, the concentration of the delbrueckia in the seed liquid can reach 1-3 x 108CFU/ml。
YPD liquid seed medium contained yeast extract, tryptone and glucose as described above.
The Debrella yeasts of the invention can be fermented in conventional YPD medium. The medium may be the same as YPD liquid seed medium. The fermentation is usually carried out at 25 to 32 ℃ and 150 to 300rpm, and preferably at a constant temperature. The fermentation may be a batch fermentation, a continuous fermentation or a fed-batch fermentation.
Therefore, the invention provides a fermentation method of the delbrueckia, which comprises the step of fermenting the delbrueckia with the preservation number of CGMCC NO. 13125. Preferably, the fermentation is carried out in a YPD medium as described herein, with the fermentation conditions as described hereinbefore.
In certain embodiments, the fermentation further comprises the steps of strain activation and seed liquid preparation as described above.
A variety of flavor substances can be produced using the fermentation of the present invention, including but not limited to 2-phenylethyl acetate, 2-phenylethyl propionate, β -phenylethyl butyrate, and the like. Accordingly, the present invention also provides a process for the production of a fragrance material, in particular one, any two or all three of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate, which process comprises the step of fermentation using a fermentation process as described herein. Specifically, the method comprises the step of fermenting the delbrueckia delbrueckii with the preservation number of CGMCC NO. 13125.
After the fermentation was completed, the aroma substances were extracted and purified by the method described in CN 104561135 a. For example, the fermentation broth may be subjected to distillation under reduced pressure using a rotary evaporator to distill off volatile components. The specific method comprises the following steps: pouring the fermentation liquor into a round-bottom flask, pouring liquid nitrogen into a primary cold trap for condensation, and extracting at 40 ℃ under the vacuum degree of 20mbar until no liquid is left at a condensation end. And (4) after borrowing and lodging extraction, combining the condensate in the cold trap to obtain total volatile components, and distilling the obtained volatile component liquid.
In certain embodiments, the present invention also provides a method for the production of any one or more of ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenethyl alcohol, the method comprising the step of fermenting ambospora delbrueckii with a accession number of CGMCC No.13125 as described herein. The invention relates in particular to a method for producing 3-methyl-1-butanol and/or phenethyl alcohol. In the fermentation liquor obtained by the method, the 3-methyl-1-butanol can be more than 10mg/kg, preferably more than 14 mg/kg; the concentration of phenethyl alcohol may be 20mg/kg or more, preferably 25mg/kg or more.
Thus, the present invention also includes a culture which is a culture of the present invention when the strain of Saccharomyces delbrueckii is being cultured, or a product after the culture or fermentation is completed. For example, the culture may be a medium containing Dermatophorus delbrueckii of the present invention, including but not limited to a culture containing Dermatophorus delbrueckii obtained by activating the Dermatophorus delbrueckii of the present invention as described above, or a seed solution obtained during or after the preparation of the seed solution. The culture will generally contain the B.delbrueckii of the invention and the culture medium.
The invention also comprises a fermentation liquid, which can be a liquid substance in the fermentation process or a liquid substance after the fermentation is finished. Typically, the fermentation broth comprises delbrueckia delbrueckii according to the invention and a culture medium. More preferably, the fermentation broth contains a flavouring substance, especially one or more of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate. More preferably, the concentration of 2-phenylethyl acetate in the fermentation broth of the present invention is at least 2.0 mg/kg; and/or the concentration of 2-phenylethyl propionate is at least 4.0 mg/kg; and/or the concentration of butyric acid-beta-phenylethyl ester is at least 1.5 mg/kg.
In certain embodiments, the fermentation broth of the present invention further comprises any one or any plurality of ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol, and phenylethyl alcohol. Preferably, the concentration of ethanol in the fermentation broth can be above 1.5mg/kg, preferably above 2.0 mg/kg; the concentration of 3-methyl-1-butanol may be 10mg/kg or more, preferably 14mg/kg or more; the concentration of 2-methyl-1-butanol may be 1.5mg/kg or more, preferably 2.0mg/kg or more; and/or the concentration of phenethyl alcohol may be 20mg/kg or more, preferably 25mg/kg or more.
The biological synthesis method for preparing the perfume material meets the requirement of green chemistry, and is simple in process and convenient to operate. And the delbrueckia delbrueckii is widely applied to wine and is a safe and reliable strain.
The present invention will be further described with reference to specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.
Example 1: isolation of the Strain
The strain of a soybean molasses sample collected and stored by the soybean industry limited company of Yi Hai (urban harbor defense) is separated, and a bacterial colony obtained by screening is spherical protrusion, smooth in surface, opaque and creamy (see figure 1). Observing the circular thallus and spore-contained delbrueckia yeast (see figure 2) under a microscope, wherein the 18S rDNA base sequence of the delbrueckia yeast is shown as SEQ: ID NO: 1. Reference is made to a screening method (screening and growth conditions [ J ] of soybean molasses high-yield single-cell protein strains reported by Gaoyrong, Liuyang et al, brewing in China, 2010, 8: 33-36).
The separated non-engineering bacteria wild Deerhua delbrueckii WBRD1.16083001 has been deposited in the China general microbiological culture Collection center (CGMCC, Beijing university Hokko No.1 North West Lu, institute of microbiology, China academy of sciences, 100101) at 2016, 10.21.2016, with the following preservation numbers: CGMCC NO. 13125.
Example 2: activation of strains and seed liquid preparation
A. Activation of strains:
diluting and spreading separated Deerhua strain WBRD1.16083001 on YPD solid plate culture medium with sterile water, and culturing at 28 deg.C for 1-2 days until colony on plate is clear.
YPD solid plate medium composition included (unit 1L): 10g of yeast extract powder (OXOID, Inc.), 20g of tryptone (OXOID, Inc.), 20g of glucose (national drug group chemical reagent, Inc.), and 10-20g of agar powder (Aladdin industries, Inc.), wherein the volume is constant to 1L by using distilled water, and the pH value is natural.
B. Preparing a seed solution:
a large single colony is picked from the plate and inoculated in a liquid YPD medium, and is cultured for 20-24h at the constant temperature of 220r/min at the temperature of 28 ℃. The concentration of the bacteria can reach 1-3 multiplied by 108CFU/ml。
YPD liquid seed medium composition comprises, in units of 1L: 10g of yeast extract powder (OXOID, Inc.), 20g of tryptone (OXOID, Inc.), and 20g of glucose (national drug group chemical reagent, Inc.), wherein the volume is adjusted to 1L by using distilled water, and the pH value is natural.
Example 3: non-inoculated YPD fermentation medium volatile flavor substance analysis
The YPD fermentation medium was the same as that described for the YPD liquid seed medium in example 2.
And (3) flavor component analysis: YPD was directly analyzed without inoculating the wild-type Dryospora delbrueckii of the present invention. The flavor components were analyzed by HS-SPME GC-MS. GC-MAS detection method: GC, Agilent 6890N/5973Agilent (USA Corp.); and (3) meteorological chromatographic conditions: HP-5MS (30X 0.25mm X0.25 μm film thickness), temperature programmed: the initial temperature is 60 ℃, the temperature is kept for 2min, then the temperature is raised to 150 ℃ at the rate of 5 ℃/min, the temperature is kept for 1min, then the temperature is raised to 300 ℃ at the rate of 5 ℃/min, the temperature is kept for 10min, the flow rate of a high-purity He column is 1.0ml/min, the temperature of a sample inlet is 250 ℃, the sample injection amount is 1 mu l of split flow 50:1, wherein, the mass spectrum condition is as follows: interface temperature 280 ℃, EI source, ionization voltage 70eV, ion source temperature 230 ℃, and scanning range 29-400 amu.
And searching the mass-to-charge ratio and the wind degree of fragments corresponding to each peak through a built-in NIST spectral library to obtain corresponding compounds.
The results of the tests show that the volatile flavour of the product obtained in this example relates to 13, and that the key two flavour enhancing substances, 2-phenylethyl acetate and 2-phenylethyl propionate, were not detected.
The flavor profile is shown in table 1 below.
Table 1: analysis table of volatile flavor component of uninoculated YPD medium
| Retention time | Peak area | Content (mg/kg) | Name of Compound | |
| 1 | 6.367 | 94132 | 0.098 | 3-methyln-butanol |
| 2 | 10.691 | 26527 | 0.028 | Hexanal |
| 3 | 14.803 | 17284 | 0.018 | Styrene (meth) acrylic acid ester |
| 4 | 15.241 | 25835 | 0.027 | Heptanal |
| 5 | 16.378 | 28358 | 0.029 | Hexanal methyl ester |
| 6 | 16.985 | 112418 | 0.117 | 2-methyl-3-heptanone |
| 7 | 18.265 | 389721 | 0.405 | Benzaldehyde |
| 8 | 18.863 | 31949 | 0.033 | Hexanoic acid |
| 9 | 21.651 | 55053 | 0.057 | 2-Ethyl hexanol |
| 10 | 21.997 | 92982 | 0.097 | Benzyl alcohol |
| 11 | 22.619 | 141141 | 0.147 | Phenylacetaldehyde |
| 12 | 26.033 | 22223 | 0.023 | Nonanal |
| 13 | 46.565 | 53303 | 0.055 | Quinoline carboxaldehyde |
Example 4: fermentation broth inoculated with Delbecco strain WBRD1.16083001, and volatile flavor substance analysis.
The fermentation method comprises the following steps: 1% of the seed liquid obtained in example 1 was inoculated into YPD fermentation medium and subjected to shake-flask culture at a speed of 220r/min at 28 ℃ for 24 hours. The YPD fermentation medium was the same as that described for the YPD liquid seed medium in example 2.
Taking the fermentation liquor to analyze volatile flavor substances. The analytical procedure was as in example 3.
The detection result shows that the volatile flavor of the fermented product prepared in the embodiment relates to 20 types, wherein the content of key flavor substances, namely, the 2-phenylethyl acetate can reach 2.83mg/kg, the content of the 2-phenylethyl propionate can reach 4.895mg/kg, and the content of the butyric acid-beta-phenylethyl ester can reach 2.087 mg/kg. The three kinds of perfume materials are good perfume materials in food and chemical industries.
The flavour profile is shown in table 2.
TABLE 2
| Number of | Retention time | Peak area | Content (mg/kg) | Name of Compound |
| 1 | 4.036 | 175428 | 0.131 | Acetaldehyde |
| 2 | 4.234 | 2799580 | 2.084 | Ethanol |
| 3 | 5.649 | 165667 | 0.123 | Ethyl acetate |
| 4 | 5.827 | 496852 | 0.370 | 2-methyl-1-propanol |
| 5 | 6.367 | 122020 | 0.091 | 3-methyl-1-butanol |
| 6 | 7.701 | 39561 | 0.029 | Propionic acid ethyl ester |
| 7 | 8.351 | 20012374 | 14.896 | 3-methyl-1-butanol |
| 8 | 8.452 | 2708343 | 2.016 | 2-methyl-1-butanol |
| 9 | 9.863 | 69932 | 0.052 | 2, 3-butanediol |
| 10 | 18.256 | 1040373 | 0.774 | Benzaldehyde |
| 11 | 20.35 | 65188 | 0.049 | Octanal |
| 12 | 21.983 | 426355 | 0.317 | Benzyl alcohol |
| 13 | 22.609 | 473783 | 0.353 | Phenylacetaldehyde |
| 14 | 26.033 | 154256 | 0.115 | Nonanal |
| 15 | 26.711 | 38738761 | 28.835 | Phenylethanolic acid |
| 16 | 34.878 | 3802066 | 2.830 | Acetic acid 2-phenylethyl ester |
| 17 | 40.05 | 6576647 | 4.895 | Propionic acid-2-phenylethyl ester |
| 18 | 42.246 | 2803998 | 2.087 | Butyric acid-beta-phenylethyl ester |
| 19 | 46.681 | 332105 | 0.247 | Butyric acid 2-methyl-2-phenylethyl ester |
| 20 | 46.868 | 83755 | 0.062 | Butyric acid 3-methyl-2-phenylethyl ester |
Sequence listing
<110> Fengyi (Shanghai) Biotechnology research and development center, Inc
<120> a new strain of Derlichospora delbrueckii and application thereof
<130> 167238
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 617
<212> DNA
<213> Delbert's spore yeast (Torulaspora delbrueckii)
<400> 1
ttttttcggg tccttgtttc aagacgggcg gcatataacc agtatgccag catccttgac 60
gtaaagtcgc agtcctcagt cccagctggc agtatttcta caggatataa cacttaccga 120
ggcaagctac attcctgcag atttatcctg ccgccaaaac tgatgctggc ccagtgagct 180
gcgagattcc cccacccaca aggagcagag ggcgcaaaac accatgtctg atcaaatgcc 240
cttccctttc aacaatttca cgtacttttt cactctcttt tcaaagttct tttcatcttt 300
ccatcactgt acttgttcgc tatcggtctc tcgccaatat ttagctttag atggaattta 360
ccacccactt agagctgcat tcccaaacaa ctcgactctt caaaagcact ttacaaagaa 420
ctgggatcct cgccacacgg gattctcacc ctctatgacg tcctgttcca aggaacatag 480
acaaggacca gccccaaagt taccttctac aaattacaac tcgggcaccg aaggtaccag 540
atttcaaatt tgagcttttg ccgcttcact cgccgttact aaggcaatgc ccggttggtt 600
tcttttcctc cgctttt 617
Claims (28)
1. Deerhella delbrueckii with preservation number CGMCC number 13125 (Torulaspora delbrueckii）。
2. Deerhella delbrueckii with preservation number CGMCC number 13125 (Torulaspora delbrueckii) Use in the production of ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol, phenethyl alcohol and/or a fragrance material;
wherein the perfume material is selected from: one or more of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate.
3. The fermentation method is characterized by comprising the step of fermenting the saccharomyces delbrueckii with the preservation number of CGMCC number 13125Torulaspora delbrueckii) The step (2).
4. The method of claim 3, wherein the fermentation is performed in a YPD liquid medium containing yeast extract, tryptone and glucose.
5. The method according to claim 4, wherein the concentration of yeast extract powder in the YPD liquid medium is 5 to 20 g/L.
6. The method according to claim 4, wherein the concentration of tryptone in the YPD liquid medium is 10 to 30 g/L.
7. The method according to claim 4, wherein the concentration of glucose in the YPD liquid medium is 10 to 30 g/L.
8. The method according to claim 3, wherein the Brevibacillus delbrueckii is inoculated at a concentration of 0.5 to 5%.
9. The method of claim 3,
the fermentation temperature is 25-32 ℃, and the stirring speed during fermentation is 150-300 rpm; and/or
The fermentation is batch fermentation, continuous fermentation or fed-batch fermentation.
10. The method of any one of claims 3 to 9, further comprising the steps of activating the bacterial species and preparing the seed solution.
11. The method according to claim 10, wherein the delbrueckia sp.
12. The method of claim 11, wherein said YPD solid plate medium comprises yeast extract, tryptone, glucose and agar.
13. The method according to claim 10, wherein the seed solution is prepared by culturing a single colony of Debrella in a YPD liquid medium at 25-32 ℃ and 150-300 rpm for 18-28 hours.
14. The method of claim 13, wherein the YPD liquid medium comprises yeast extract, tryptone and glucose.
15. The method of claim 14, wherein the concentration of bacteria in the seed solution is 1 to 3 x 108CFU/ml。
16. A method of producing ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol, phenethyl alcohol and/or a fragrance material, the method comprising the step of performing fermentation using the fermentation method of any one of claims 3-15;
wherein the perfume material is selected from: one or more of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate.
17. A culture comprising Saccharomyces delbrueckii (CGMCC NO. 13125)Torulaspora delbrueckii) And a culture medium.
18. The culture of claim 17, wherein the medium is a YPD medium.
19. A fermentation broth comprising one or more of 2-phenylethyl acetate, 2-phenylethyl propionate and β -phenylethyl butyrate; wherein the fermentation liquor also contains the delbrueckia delbrueckii with the preservation number of CGMCC NO.13125Torulaspora delbrueckii) And a culture medium.
20. The fermentation broth of claim 19, wherein the concentration of 2-phenylethyl acetate in the broth is at least 2.0 mg/kg.
21. The fermentation broth of claim 19, wherein the concentration of 2-phenylethyl propionate in the broth is at least 4.0 mg/kg.
22. The fermentation broth of claim 19, wherein the concentration of β -phenylethyl butyrate in the fermentation broth is at least 1.5 mg/kg.
23. The fermentation broth of claim 19, further comprising one or more of ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol, and phenylethyl alcohol.
24. The fermentation broth of claim 23,
the concentration of the ethanol is more than 1.5 mg/kg;
the concentration of the 3-methyl-1-butanol is more than 10 mg/kg;
the concentration of the 2-methyl-1-butanol is more than 1.5 mg/kg; and/or
The concentration of the phenethyl alcohol is more than 20 mg/kg.
25. The fermentation broth of claim 24, wherein the ethanol concentration is greater than 2.0 mg/kg.
26. The fermentation broth of claim 24, wherein the concentration of 3-methyl-1-butanol is above 14 mg/kg.
27. The fermentation broth of claim 24, wherein the concentration of 2-methyl-1-butanol is greater than 2.0 mg/kg.
28. The fermentation broth of claim 24, wherein the concentration of phenylethyl alcohol is above 25 mg/kg.
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| CN111621528B (en) * | 2020-04-24 | 2021-09-17 | 厦门欧米克生物科技有限公司 | Method for biologically synthesizing ethanolamine |
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