CN116769618A - Functional microorganism for inhibiting ethyl acetate production - Google Patents
Functional microorganism for inhibiting ethyl acetate production Download PDFInfo
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 title claims abstract description 206
- 244000005700 microbiome Species 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- 230000002401 inhibitory effect Effects 0.000 title abstract description 12
- 235000021107 fermented food Nutrition 0.000 claims abstract description 16
- 241000222178 Candida tropicalis Species 0.000 claims description 43
- 239000007788 liquid Substances 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 15
- 230000000813 microbial effect Effects 0.000 claims description 15
- 235000014101 wine Nutrition 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 238000011081 inoculation Methods 0.000 claims description 7
- 239000002068 microbial inoculum Substances 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 5
- 235000013305 food Nutrition 0.000 claims description 4
- 241000894007 species Species 0.000 claims description 4
- 235000019990 fruit wine Nutrition 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 235000020097 white wine Nutrition 0.000 claims description 3
- 238000013124 brewing process Methods 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 abstract description 24
- 230000000593 degrading effect Effects 0.000 abstract description 4
- 238000011217 control strategy Methods 0.000 abstract description 2
- 235000013339 cereals Nutrition 0.000 description 15
- 238000000855 fermentation Methods 0.000 description 13
- 230000004151 fermentation Effects 0.000 description 13
- 244000286779 Hansenula anomala Species 0.000 description 12
- 235000014683 Hansenula anomala Nutrition 0.000 description 12
- 239000000523 sample Substances 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 9
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 9
- 241000436311 Candida orthopsilosis Species 0.000 description 7
- 241000143603 Nakaseomyces Species 0.000 description 7
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 7
- 241000192327 [Candida] membranifaciens Species 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 6
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 6
- 239000001257 hydrogen Substances 0.000 description 6
- 229910052739 hydrogen Inorganic materials 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 5
- 235000013379 molasses Nutrition 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 240000006394 Sorghum bicolor Species 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- -1 ethyl hexyl Chemical group 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 4
- 241000209072 Sorghum Species 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000012159 carrier gas Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 235000009754 Vitis X bourquina Nutrition 0.000 description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 description 2
- 240000006365 Vitis vinifera Species 0.000 description 2
- 235000014787 Vitis vinifera Nutrition 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 235000013405 beer Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000015096 spirit Nutrition 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000011787 zinc oxide Substances 0.000 description 2
- 235000014692 zinc oxide Nutrition 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 244000253911 Saccharomyces fragilis Species 0.000 description 1
- 235000018368 Saccharomyces fragilis Nutrition 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 229940031154 kluyveromyces marxianus Drugs 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
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- 238000002791 soaking Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a functional microorganism for inhibiting ethyl acetate production, and belongs to the technical field of microorganisms. The invention obtains a microorganism which can inhibit other microorganisms from producing ethyl acetate and has the function of degrading ethyl acetate, and after the functional microorganism is additionally added into fermented grains, the ethyl acetate content in the white spirit can be reduced, and the ethyl caproate content can be increased, so that the ethyl caproate ratio is reduced. The invention also provides a control strategy of ethyl acetate in fermented food.
Description
Technical Field
The invention relates to a functional microorganism for inhibiting ethyl acetate production, and belongs to the technical field of microorganisms.
Background
Ethyl acetate is one of the tetraesters that make up the typical style of Luzhou-flavor liquor. The quality and style of the Luzhou-flavor liquor are determined by the relation of the amount ratio of ethyl acetate to other ethyl esters. For example, the ratio of ethyl acetate to ethyl caproate (ethyl hexyl ratio) is preferably less than 1, and the ethyl hexyl ratio of the high-quality strong-flavor base wine is usually about 0.5. For various reasons, the strong aromatic Chinese spirits have the phenomenon that the content of ethyl acetate is higher, even the content of ethyl caproate is far higher (the original liquor can reach 4 g.L) -1 The fermented grains can reach 300 mg.kg -1 ) The method not only affects the harmony and the representativeness of the wine body, but also is not beneficial to later-stage blending, thereby increasing the production cost and reducing the market competitiveness of enterprises. Therefore, only the problem of higher ethyl acetate is solved, the large-scale production of the stability of the Chinese white spirit can be promoted, and the healthy and rapid development of the Chinese white spirit industry is promoted.
The production of natural ethyl acetate by microorganisms (e.g., yeast and lactic acid bacteria) is well established and has historically been applied to food production. Volatile ethyl acetate is one of the most important aroma compounds in fermented foods such as beer, wine and dairy products. At low concentrations, esters produce a sweet fruity note, but are also considered off-flavors when present in large amounts. The concentration of the ethyl acetate in the dairy product ranges from 50 to 100 mg.L -1 The concentration in beer and wine is 8-64mg.L -1 The concentration range in Chinese strong aromatic Chinese liquor is 1-2 g.L -1 . The yeast can synthesize a large amount of ethyl acetate by using ethanol or glucose as a substrate, such as Kluyveromyces marxianus, wilkham's yeast and Fei Bien Siberian yeast.
If the original strain in the white spirit brewing system can be utilized, the strain for inhibiting the currently known high-yield ethyl acetate can be beneficial to promoting the large-scale production of high-quality white spirit, and further realizing high-quality, high-efficiency and safe production.
Disclosure of Invention
In order to solve the problems, the invention screens out a strain of microorganism which inhibits other microorganisms from producing ethyl acetate and has the function of degrading ethyl acetate from the distilled grains of the white spirit and the environmental samples, namely Candida tropicalis LBM101.
The Candida tropicalis LBM is preserved in China Center for Type Culture Collection (CCTCC) at 2022, 12 and 23, and the preservation number is CCTCC NO: m20222053.
The strain is obtained by screening from strong aromatic Chinese spirits fermented grains and environmental samples.
The strain of the invention has the following characteristics:
(1) Ethyl acetate is not produced;
(2) Can degrade exogenously added ethyl acetate;
(3) After the additional addition of the fermented grains, the ethyl acetate produced by other microorganisms can be inhibited, and meanwhile, the ethyl caproate content can be increased, and the ethyl-hexyl ratio can be reduced.
The second object of the present invention is to provide a microbial agent containing Candida tropicalis LBM101.
The microbial agent is used for controlling the content of ethyl acetate in fermented grains and maintaining the normal ethyl acetate ratio, and Candida tropicalis LBM101 is taken as a main microorganism.
In one embodiment of the present invention, the viable count of Candida tropicalis LBM101 in the microbial inoculum is 10 5 ～10 8 CFU/mL。
In one embodiment of the present invention, the microbial inoculum has a microbial concentration of Candida tropicalis LBM101 in the range of 10 5 ～10 8 CFU/mL。
In one embodiment of the present invention, the microbial inoculum is obtained by preparing a seed solution of Candida tropicalis LBM101 and then expanding culture.
In one embodiment of the present invention, the microbial agent comprises live cells of strain Candida tropicalis LBM of the present invention, freeze-dried dry cells of strain Candida tropicalis LBM of the present invention obtained, immobilized cells of strain Candida tropicalis LBM of the present invention, liquid microbial agent of strain Candida tropicalis LBM of the present invention, solid microbial agent of strain Candida tropicalis LBM of the present invention, or strain Candida tropicalis LBM101 of the present invention in any other form.
In one embodiment of the invention, the microbial inoculum also contains any strain of any species that can be used in fermented foods or in the preparation of fermented foods, such as bacillus licheniformis, saccharomyces cerevisiae, bacillus subtilis, and the like.
In one embodiment of the invention, the microbial inoculum further comprises any carrier that can be used to ferment food.
The third object of the present invention is to provide a method for controlling ethyl acetate in a fermented food, which comprises inoculating Candida tropicalis LBM101 or Candida tropicalis LBM-containing microbial agent of the present invention into a process for producing a fermented food, and controlling ethyl acetate in a fermented food by inhibiting other microorganisms.
In one embodiment of the invention, the fermented food is white spirit.
In one embodiment of the present invention, the method comprises culturing a bacterial solution of a bacterial agent to 10 8 After CFU/mL, the initial inoculation concentration after inoculation to fermented grains is not lower than 10 5 CFU/g, thereby brewing white spirit.
In one embodiment of the present invention, the control method is to inoculate Candida tropicalis LBM strain bacteria liquid prepared by the method of preparing Candida tropicalis LBM strain bacteria liquid into fermented grains for fermentation.
In one embodiment of the present invention, candida tropicalis LBM101 is a control method according to 10 8 CFU/mL is used for preparing bacterial strain bacterial liquid.
In one embodiment of the invention, the medium is a molasses medium.
In one embodiment of the invention, the method is specifically: will Candida tropicalis LBM101Single colonies were inoculated in YPD liquid medium and cultured at 30℃for 24 hours. Then transferring it into molasses culture medium for culturing to 10 8 CFU/mL expanded culture bacterial liquid, and then inoculating the bacterial liquid to fermented grains, wherein the initial inoculation concentration is not lower than 10 5 CFU/g, thereby brewing white spirit.
A fourth object of the present invention is to provide an application of the functional strain Candida tropicalis LBM101 for inhibiting ethyl acetate production, which is applied to the technical field of food, especially the technical field of fermented food.
In one embodiment of the invention, the application is in brewing wine, distilled wine and the like, such as white wine, grape wine, yellow wine and fruit wine.
In one embodiment of the invention, the application is to add the strain for inhibiting the production of ethyl acetate into white wine, grape wine, yellow wine or fruit wine brewing process.
In one embodiment of the invention, the use is to add the strain of the invention inhibiting the production of ethyl acetate to fermented grains.
The invention has the beneficial effects that:
the invention obtains a microorganism which can inhibit other microorganisms from producing ethyl acetate and has the function of degrading ethyl acetate, and after the functional microorganism is additionally added into fermented grains, the functional microorganism can inhibit other strains from producing ethyl acetate, has little influence on the microbial structures of other genera, can maintain normal fermentation of white spirit, can reduce the ethyl acetate content in the white spirit, and can also increase the ethyl caproate content and reduce the ethyl caproate ratio. The invention also provides a control strategy of ethyl acetate in fermented food.
Preservation of biological materials
Candida tropicalis Candida tropicalis, taxonomic designation Candida tropicalis LBM, was deposited in the chinese collection of typical cultures at 2022, 12, 23, with a deposit number of cctccc NO: m20222053, the preservation address is university of Wuhan in Wuhan, china.
Drawings
Fig. 1: different yeasts were fermented in combination with Wickerhamomyces anomalus for 3 days (a) and 6 days (b) respectively for ethyl acetate production; wherein n.g. represents Nakaseomyces glabratus LBM, c.m. represents Candida membranifaciens LBM137, c.o. represents Candida orthopsilosis LBM225,225, c.t. represents Candida tropicalis LBM101,101, w.a. represents Wickerhamomyces anomalus.
Fig. 2: different yeasts are added with 2g.L -1 Fermenting in jowar juice culture medium for 3 days (a) and 6 days (b) to degrade ethyl acetate; wherein n.g. represents Nakaseomyces glabratus LBM, c.m. represents Candida membranifaciens LBM137, c.o. represents Candida orthopsilosis LBM225,225, c.t. represents Candida tropicalis LBM101,101, w.a. represents Wickerhamomyces anomalus.
Detailed Description
Culture medium:
YPD liquid Medium (g.L) -1 ): peptone 20, glucose 20, yeast extract 10.
Sorghum juice medium: crushing sorghum properly, and mixing the crushed sorghum with water according to a feed liquid ratio of 1:4 (w/v), soaking overnight, adding appropriate amount of high temperature resistant alpha-amylase (30U.g) -1 Sorghum), stirring uniformly, sterilizing at 105 ℃ for 1h in a high-temperature sterilizing pot, adding a proper amount of high-temperature resistant alpha-amylase, adding deionized water with 1-2 times of volume, heating and boiling for 2h, cooling to 60 ℃, and adding a proper amount of saccharifying enzyme (300 U.g) -1 Sorghum), saccharifying for 4h at 60 ℃ after stirring uniformly, squeezing juice with 2 layers of gauze, and filtering, wherein 6000 r.min -1 Centrifuging for 10min, collecting supernatant, and freezing at-20deg.C. Before use, the sugar degree was diluted to 7℃bx and the mixture was dispensed into 250mL Erlenmeyer flasks.
Molasses culture medium (g.L) -1 ): molasses 80, magnesium sulfate 0.2, ammonium sulfate 3 and monopotassium phosphate 3.
Example 1: isolation and identification of functional microorganisms inhibiting ethyl acetate production
(1) Isolation of strains: 10g of fresh fermented grains are suspended in 100mL of 0.9% NaCl solution, coated with a solid culture medium and placed in a 30 ℃ incubator for static culture for 3 days; after streaking to obtain single colony, selecting single colony in YPD liquid culture medium, selecting 3 parallel plates, and heating at 30deg.C for 200r.min -1 Is cultured in a shaker for 1 day.
(2) Inoculating the cultured strains into jowar juice culture medium to obtain initial inoculation concentration of 2×10 5 CFU·mL -1 3 strains of bacteria are prepared in parallel, and fermentation is carried out for 6 days at 30 ℃.
(3) Detection of ethyl acetate in fermentation broth after strain culture: sample and anhydrous diethyl ether according to 1:1, mixing and extracting ethyl acetate in proportion, taking the upper liquid, filtering by an organic filter head with the thickness of 0.22 mu m, and detecting by adopting a gas chromatography.
Chromatographic conditions:
hydrogen flame ionization detector (flame ionization detector, FID), carrier gas is high purity nitrogen (N) 2 ) CP-WAX 57CB chromatographic column (50 m×0.25mm×0.25 μm), sample inlet temperature 220 ℃, detector temperature 220 ℃, hydrogen flow 40mL min -1 Air flow rate 400 mL/min -1 Column flow 2.0mL min -1 Split ratio 5:1, sample injection amount is 1 mu L. The temperature rise program at the time of detection is: kept at 60℃for 5min.
(4) Identification of Strain ITS: and (3) centrifuging the fermentation liquor to obtain cell sediment, extracting genome, determining the ITS sequence of a conserved region to identify species, wherein the primer sequences for PCR are ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', the sequence is shown as SEQ ID NO: 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', the sequence is shown as SEQ ID NO: 2).
The fungi in the fermented grains are separated, 31 strains of yeasts are obtained through preliminary colony morphology judgment and are compared with a Genbank database after ITS gene sequences are determined, the species are determined, and several strains which do not produce or produce ethyl acetate with low yield are obtained, and the results are shown in Table 1.
TABLE 1 isolation and identification of microorganisms
Note that: ND represents below the detection limit
Candida tropicalis LBM101, which has been preserved in China Center for Type Culture Collection (CCTCC) at 2022, 12 and 23, has a preservation number of CCTCC NO: m20222053.
Example 2: application of functional microorganism Candida tropicalis LBM101 in inhibiting ethyl acetate production by other microorganisms
(1) Separating several strains Candida tropicalis LBM (CCTCC NO: M20222053), candida orthopsilosis LBM225, candida membranifaciens LBM137, nakaseomyces glabratus LBM64, and one strain with high ethyl acetate yield (6 g.L for 7 days) -1 The strain (Wickerhamomyces anomalus) above) was activated in YPD liquid medium to give a seed liquid concentration of 10 7 CFU·mL -1 。
(2) The strains which do not produce ethyl acetate or produce ethyl acetate with low yield are respectively inoculated into a sorghum juice culture medium with the same proportion as Wickerhamomyces anomalus, the inoculum size is 3 percent, and the strains are subjected to static culture in a 30 ℃ incubator for 6 days, wherein each strain is 3 times in parallel.
(3) Detection of ethyl acetate in fermentation broth after strain culture: sample and anhydrous diethyl ether according to 1:1, mixing and extracting ethyl acetate in proportion, taking the upper liquid, filtering by an organic filter head with the thickness of 0.22 mu m, and detecting by adopting a gas chromatography.
Chromatographic conditions:
hydrogen flame ionization detector (flame ionization detector, FID), carrier gas is high purity nitrogen (N) 2 ) CP-WAX 57CB chromatographic column (50 m×0.25mm×0.25 μm), sample inlet temperature 220 ℃, detector temperature 220 ℃, hydrogen flow 40mL min -1 Air flow rate 400 mL/min -1 Column flow 2.0mL min -1 Split ratio 5:1, sample injection amount is 1 mu L. The temperature rise program at the time of detection is: kept at 60℃for 5min.
The results are shown in FIG. 1.
These four yeasts produce substantially no ethyl acetate. Compared with Wickerhamomyces anomalus pure culture ethyl acetate production, nakaseomyces glabratus LBM, candida membranifaciens LBM137, candida orthopsilosis LBM and CCTCC NO: m20222053 combined fermentation with Wickerhamomyces anomalus reduced ethyl acetate production by 44.11%, 22.10%, 37.43% and 63.09%, respectively, on day 3; at the end of the fermentation (6 days), the combined fermentation of Nakaseomyces glabratus LBM and Wickerhamomyces anomalus increased ethyl acetate by 5.82%, candida membranifaciens LBM137, candida orthopsilosis LBM225 and CCTCC NO: the combined fermentation of M20222053 and Wickerhamomyces anomalus reduced ethyl acetate by 0.59%, 5.77% and 48.83%, respectively.
To sum up, CCTCC NO: m20222053 has the strongest effect of inhibiting Wickerhamomyces anomalus ethyl acetate production and lasting inhibition effect.
Example 3: application of functional microorganism Candida tropicalis LBM101 in degradation of ethyl acetate
(1) Activating non-or low-ethyl acetate-producing strains Candida tropicalis LBM (i.e. CCTCC NO: M20222053), candida orthopsilosis LBM225, candida membranifaciens LBM137, nakaseomyces glabratus LBM64 and high-ethyl acetate-producing strain (Wickerhamomyces anomalus) separated from fermented grains in YPD liquid culture medium to obtain seed solution with concentration of 10 7 CFU·mL -1 。
(2) Inoculating seed solution to seed solution containing 2g.L -1 In the sorghum juice culture medium of ethyl acetate, the inoculation amount is 3%, and the culture is carried out for 6 days in a 30 ℃ incubator, wherein each strain is 3 times in parallel.
(3) Detection of ethyl acetate in fermentation broth after strain culture: sample and anhydrous diethyl ether according to 1:1, mixing and extracting ethyl acetate in proportion, taking the upper liquid, filtering by an organic filter head with the thickness of 0.22 mu m, and detecting by adopting a gas chromatography.
Chromatographic conditions:
hydrogen flame ionization detector (flame ionization detector, FID), carrier gas is high purity nitrogen (N) 2 ) CP-WAX 57CB chromatographic column (50 m×0.25mm×0.25 μm), sample inlet temperature 220 ℃, detector temperature 220 ℃, hydrogen flow 40mL min -1 Air flow rate 400 mL/min -1 Column flow 2.0mL min -1 Split ratio 5:1, sample injection amount is 1 mu L. The temperature rise program at the time of detection is: kept at 60℃for 5min.
The results are shown in FIG. 2.
Wickerhamomyces anomalus mainly plays the role of ester production, and has no obvious phenomenon of degrading ethyl acetate. At 3 days of fermentation, nakaseomyces glabratus LBM, candida membranifaciens LBM, 137, candida orthopsilosis LBM225 and CCTCC NO: m20222053 degraded 8.2%, 5.5%, 21.9% and 18.5% ethyl acetate, respectively, compared to the non-sterile blank; at the end of fermentation (6 days), these four strains degraded 12.5%, 11.2%, 44.0% and 31.6% of ethyl acetate, respectively, compared to the no-added control. Taken together, c.orthosilosis LBM225 and CCTCC NO: m20222053 has a strong ability to degrade ethyl acetate.
Example 4: application of Candida tropicalis LBM101 strain
Cctccc NO: after activation of M20222053, it was transferred to 28L of molasses medium for expansion to 10 8 CFU·mL -1 Then freeze-drying the mixture into bacterial powder of 10 5 CFU·g -1 Inoculating the initial concentration of (2) into fermented grains, fermenting for 4 months, and taking a wine sample to detect the change of ethyl acetate and ethyl caproate.
The control sample (without microbial inoculum) is CCTCC NO: m20222053, obtained as described above. Table 2 shows the ethyl acetate content, ethyl caproate content and ethyl acetate ratio of the functional strain. The result shows that the functional strain is used for intervening the fermented grains, the ethyl acetate content in the wine sample is reduced by 16.94%, the ethyl caproate content is increased by 31.64%, and the ethyl-hexyl ratio is obviously reduced, so that the purpose of regulating and controlling the ethyl acetate is achieved.
TABLE 2 variation of ethyl acetate, ethyl caproate content and ethyl caproate content by addition or absence of functional Strain
It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present invention and their spirit, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the following claims.
Claims (10)
1. A candida tropicalis Candida tropicalis, wherein Candida tropicalis is preserved in the China center for type culture collection (CCTCC NO) at 12-23 of 2022: m20222053.
2. A microbial agent characterized by the preservation number cctccc NO: candida tropicalis of M20222053.
3. The microbial agent of claim 2, wherein the principal microorganism of the microbial agent is CCTCC NO: m20222053.
4. The microbial agent according to claim 2, characterized in that it contains the live cells of the cells of Candida tropicalis of claim 1, dried cells of Candida tropicalis obtained by freeze-drying, cells of immobilized Candida tropicalis strain, liquid microbial agent of Candida tropicalis, solid microbial agent of Candida tropicalis strain, or Candida tropicalis in any other form.
5. The microbial agent according to claim 2, further comprising any strain of any species that can be used in the preparation of fermented food or fermented food, or further comprising any carrier that can be used in fermented food.
6. A method for controlling ethyl acetate in a fermented food product, comprising inoculating Candida tropicalis according to claim 1 or a microbial inoculum comprising Candida tropicalis according to claim 1 into a process for producing a fermented food product.
7. The control method according to claim 6, wherein the fermented food is white spirit; optionally, the method is to culture the bacterial liquid of the bacterial agent to 10 8 After CFU/mL is higher, the initial inoculation concentration after inoculation to fermented grains is not lower than 10 5 CFU/g, thereby enterBrewing Chinese liquor.
8. Use according to claim 1 as Candida tropicalis.
9. The use according to claim 8, characterized in that it is applied in the field of food technology; optionally, the application is applied to brewing wine and distilled wine production.
10. The use according to claim 8, wherein the use is to add strain Candida tropicalis to a white wine, yellow wine or fruit wine brewing process.
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