CN110982710B - Wine cellar verruca umbilicalis leveling and application thereof in aging of new wine - Google Patents

Wine cellar verruca umbilicalis leveling and application thereof in aging of new wine Download PDF

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CN110982710B
CN110982710B CN201911381981.8A CN201911381981A CN110982710B CN 110982710 B CN110982710 B CN 110982710B CN 201911381981 A CN201911381981 A CN 201911381981A CN 110982710 B CN110982710 B CN 110982710B
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张晓娟
彭铭烨
孟连君
王俊红
许正宏
史劲松
陆震鸣
柴丽娟
钱建瑛
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Jiangnan University
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Abstract

The invention discloses a cellar verruca plana and application thereof in aging of new wine. The wine cellar flat verruca umbilicalis P-3 is classified and named as Zasmicium cellare, is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, and has the preservation date: year 2019, month 6, day 25, deposit address: the preservation number of No. 3 Xilu Beijing, Chaoyang, and the institute of microbiology, China academy of sciences is CGMCC No. 18126. The verruca umbilicalis P-3 in the cellar is used for accelerating the aging of new wine, can shorten the aging time, has strong controllability and simple and convenient operation, and has important significance for the innovation of the wine brewing process.

Description

Wine cellar verruca umbilicalis leveling and application thereof in aging of new wine
Technical Field
The invention relates to a cellar verruca plana and application thereof in aging of new wine, belonging to the technical field of microbial fermentation.
Background
Generally, newly brewed wine is often characterized by pungent taste and pungent smell, light aroma, poor taste, inelegance and the like. Therefore, the new wine needs to be stored for a certain time to eliminate the foreign flavor, the fragrance is increased, the wine body is mellow and soft, and the taste is coordinated, and the change is generally called aging. The natural aging is a traditional method for producing high-quality wine, and mainly because the reactions such as oxidation, esterification and the like occur in the storage process, the fragrant substances are continuously generated; the new wine contains low boiling point aldehydes, hydrogen sulfide and other irritative and odorous substances, most of which are volatilized after storage, and alcohol molecules are associated with water molecules, so that the irritative taste and the spicy taste of the new wine are greatly reduced. Therefore, the wine is said to have a stale flavor.
The aging time of the new wine is required to be different from several years to several decades according to different raw materials and fermentation processes. In addition, the natural aging of the new wine is not only a very long process, but also occupies a large number of storage containers and storerooms or wine storage holes, so that the problems of long production period, large occupied area of an aging workshop and the like are caused, and thus, the capital overstock is inevitably caused, and the requirement of modern production is not facilitated. For this reason, the wine industry has adopted modern scientific techniques to accelerate the aging process of wine. These techniques can be divided into three major categories: physical, chemical and biological methods. The physical aging method comprises a plurality of methods of external factors such as light, electricity, magnetism, microwave, ultrasonic wave, ions, high pressure and the like, and although the methods have certain functions, the methods of the external factors change the natural aging mechanism of the new wine to a great extent, so that the method is considered to change one kind of wine into another kind of wine. Chemical ageing is carried out by adding some aging marker substances or precursor substances of the substances into the wine, but the safety of the exogenous additive substances is always lower than that of the exogenous additive substances generated naturally, and the content of harmful substances in the fresh wine is not reduced. At present, microorganisms, particularly lactic acid bacteria, are known to play a crucial role in the formation of flavor substances and organic acids in the process of brewing new wines, wherein the more common microorganisms are lactobacillus casei, lactobacillus reuteri and lactobacillus brevis which can promote the flavor maturation of the new wines and increase the flavor of the new wines, but the microorganisms only play a role in the fermentation process, and the existence of the microorganisms is difficult to find in the aging process.
At present, Chinese patent application No. 201210376137.8 discloses a method for accelerating red wine aging by an enzyme method, which utilizes polyphenol oxidase to promote the polymerization of procyanidine in red wine to form polymer precipitates, thereby accelerating the aging process of red wine, but the method can not reduce the content of aldehydes, hydrogen sulfide and other substances, is not beneficial to the synthesis of ester substances, and the red wine obtained after aging has insufficient fragrance and heavy spicy taste. Chinese patent application No. 201810597371.0 discloses a method for rapidly aging yellow wine, which takes potassium hypochlorite as an oxidant to promote the oxidation-reduction reaction and the esterification reaction to be accelerated under the synergistic action of ultraviolet rays and catalyst silicon dioxide, but in the method, the potassium hypochlorite is reduced to generate oxygen, and the generated oxygen can cause the generation of a mycoderm, thereby easily causing the rancidity of the wine. The Chinese patent application No. 201810458720.0 discloses an efficient aging device for new wine simulating natural alcoholization and an application method thereof, the method designs an aging device, and uses nano functional materials as fillers at the lower parts of a grid rod and a shell thereof to shorten the aging time of the wine.
Disclosure of Invention
In order to solve the technical problems, the invention provides the wine cellar verruca plana and the application thereof in aging new wine, which can shorten the aging time, have strong controllability and simple and convenient operation and have important significance for the innovation of the wine brewing process.
The invention aims to provide a cellar flat verruca acuminata P-3, which is classified and named as Zasamium cellare, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation date: year 2019, month 6, day 25, deposit address: no. 3 of Xilu No.1 of Beijing Korean Yang district, the preservation number is CGMCC No. 18126.
The verruca umbilicalis P-3 of the wine cellar is separated from the cave wall of a pure yang cave in a Luzhou old cellar tourism area, and has the biological characteristics that: the growth temperature range is 8-25 ℃, and the optimal growth temperature is 20 ℃; the growth pH range is 4-7, and the optimal growth pH is 5; the ethanol tolerance range (measured by ethanol solubility in air) is 0-8%. On a PDA culture medium, the bacterial colony is spherical, the surface structure is granular, the spore is black or gray black, the conidium is spherical, the hyphae are nearly colorless, no diaphragm, few branches and no locked combination.
The second purpose of the invention is to provide the application of the drugchin peaceful wart P-3 in the aging of new wine.
Further, the new wine is new white wine, new wine or new yellow wine.
Further, the application specifically comprises the following steps:
(1) culturing the drugchin planifolia P-3 in a slant culture medium at 18-22 ℃ until dense spores grow, adding a spore suspension to suspend the spores, and filtering to remove mycelia to obtain a spore solution;
(2) inoculating the spore liquid obtained in the step (1) into a seed culture medium, and performing static culture at 18-22 ℃ for 36-48 h to obtain a strain seed liquid of the verruca acuminata drugstore P-3;
(3) inoculating the P-3 seed liquid of the drugchin peacockia wine cellar obtained in the step (2) into a growth culture medium, standing and culturing for 36-48 h at 18-22 ℃, centrifuging to obtain P-3 cells of the drugchin peacockia wine cellar, adding water to prepare a P-3 cell suspension of the drugchin peacockia wine cellar, wherein the number of the cells in the cell suspension is 107～109Per mL;
(4) and (4) spraying the cell suspension of the verruca acuminata P-3 prepared in the step (3) onto the surface of a wine storage container.
Further, the wine storage container is a wine storage hole, an oak barrel or a pottery jar, and the cell number of the wall of the wine storage hole is 1010～1012Per m2The number of cells on the surface of the pottery jar was 106～108Per dm2The number of cells on the surface of oak barrel is 105～107Per dm2。
Further, the spore suspension is 0.01-0.02 g/L Tween80 and 0.08-0.10 g/L NaH2PO4、0.50～0.55g/L Na2HPO4And 0.08-0.10 g/L NaCl.
Further, the slant culture medium is a PDA culture medium, the seed culture medium is a PDB culture medium, and the growth culture medium is a medium containing 3 to up to one5mg/L MgSO4And 6-10 mg/L CaSO4The PDB medium of (1).
The third purpose of the invention is to provide a microbial agent containing the drugcojiangpingya verrucosa P-3.
Further, the microbial agent is a solid microbial agent or a liquid microbial agent.
The invention has the beneficial effects that:
the invention provides a cellar flat verruca acuminata P-3, which is classified and named as Zasmicium cellare, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has the preservation date: year 2019, month 6, day 25, deposit address: the preservation number of No. 3 Xilu Beijing, Chaoyang, and the institute of microbiology, China academy of sciences is CGMCC No. 18126. The verruca umbilicalis P-3 in the cellar is used for accelerating the aging of new wine, can shorten the aging time, has strong controllability and simple and convenient operation, and has important significance for the innovation of the wine brewing process.
Microbiological material preservation information: the cellar flat verruca umbilicalis P-3 is classified and named as Zasmicium cellare, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation date: year 2019, month 6, day 25, deposit address: the preservation number of No. 3 Xilu Beijing, Chaoyang, and the institute of microbiology, China academy of sciences is CGMCC No. 18126.
Description of the drawings:
FIG. 1 is a graphical representation of the morphological characteristics of P-3 drugchin peaceful spores on PDA medium;
FIG. 2 is a tree diagram and an electrophoresis diagram of drugchin peaceful wart P-3 development based on ITS1 rRNA gene.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
The flavor index detection method comprises the following steps: sample pretreatment: taking 8mL of diluted sample, adding 2g of NaCl into a headspace sample bottle, adding 2 mu L of 2-octanol (1.6g/L) serving as an internal standard, detecting the composition of a volatile compound by adopting a headspace solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method, and carrying out quantitative analysis on the corresponding compound according to the peak area and the concentration of the internal standard 2-octanol. GC an Agilent DB-wax capillary chromatography column (30m 0.25mm 0.25 μm) was used as separation column. The initial temperature of the chromatographic column is 40 ℃, the chromatographic column is maintained for 5min, then the temperature is increased to 60 ℃ at the speed of 5 ℃/min, and then the temperature is increased to 230 ℃ at the speed of 10 ℃/min, and the chromatographic column is maintained for 8 min. Helium was used as the carrier gas, the flow rate was 1.3mL/min, and the split ratio was 10: 1. The ion source temperature of the mass spectrum is 220 ℃, and the mass range is 33-450amu by full spectrum scanning.
Example 1: preparation of drugcharynia planifolia P-3
Scraping 10g of wall sample on the wall of a pure sun cave in a Luzhou Laojiao tourist area, quickly adding the wall sample into a conical flask containing 90mL of sterile water, adding sterilized glass beads, and fully oscillating on a vortex oscillator for 10min to fully disperse the sample. Diluting the supernatant with 10 times gradient to obtain 10 dilution-3、10-4、10-5、10-6、10-7Sucking 0.2mL of sample liquid by using a liquid transfer gun, uniformly coating the sample liquid on a PDA screening culture medium by adopting a flat plate coating method, inverting the prepared flat plate and culturing for 72-120 h in a constant-temperature incubator at 20 ℃, and providing ethanol with different solubilities by using a constant-temperature incubator of a humidifier (an ethanol solubility meter in air). High ethanol tolerant strains were isolated by plate streaking. Selecting a strain which can grow well on a culture medium containing 5-8% of ethanol solubility, separating and culturing the strain by streaking for multiple times to separate out a single colony, then inoculating the single colony on a PDA slant culture medium, and storing the single colony in a refrigerator for later use at 4 ℃.
Example 2: identification of drugstore verrucaria peregrina P-3
Step 1: morphological identification of drugcharynia peacefolicus P-3
The isolated strains were observed for their colony, hyphae, spores and other structures using a PDA plate and a microscope according to the method of the fungal identification Manual (edited by Weijing super, Shanghai science and technology Press, 1979). The result shows that the bacterial strain is cultured for 48-60 h at 20 ℃ on a PDA culture medium, the bacterial colony is spherical, the surface structure is granular, the spores are black or gray black, the conidia are spherical, and the hyphae are nearly colorless, have no diaphragm, have few branches and have no locked combination. Referring to the "fungal identification Manual", the selected strain was preliminarily considered to be drusen (Zamidium cellare)
Step 2: molecular biological identification of drugchin peaceful wart P-3 in cellar
Genomic DNA of drugchin flat Verticillum P-3 (Zamidium cellare P-3) was extracted using a fungal genomic kit (purchased from Takara, Code No. 9765). PCR amplification was performed using the fungal ITS1 rDNA universal primer.
The PCR reaction system is as follows: 10-20 ng of template DNA obtained in step 2, 25 ul of PCR Premix, 0.5 ul of Forward primer (20 pmol/. mu.l), 0.5 ul of Reverse primer (20 pmol/. mu.l), ddH2O make up to a total volume of 50. mu.l.
PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 50-58 ℃ for 1min, extension at 72 ℃ for 1min, and 30 cycles; extending for 5min at 72 ℃, cooling to 4 ℃, and taking out the product.
The sequencing work is carried out by the amplified PCR product sample and sequencing, and the sequencing is completed by Shanghai Biotechnology limited.
According to morphological characteristics, physiological and biochemical indexes and blast comparison results of an ITS1 rDNA gene sequence in NCBI, the similarity of 99.37% with Zamidium cellare shown, so that the strain P-3 can be identified as the Zmidium cellare.
Example 3: application of cellar verruca plana P-3 in white spirit aging
(1) Transferring the distilled white spirit into a clean pottery jar, wherein the storage amount of the white spirit is 4/5 of the volume (V/V) of the pottery jar, sealing the opening of the pottery jar by using airtight canvas, sealing the periphery of the canvas by using mud heads, and transporting the pottery jar filled with the white spirit to a wine storage hole by using a trailer for natural aging;
(2) culturing drugchin planiformis P-3 in a PDA (potato dextrose agar) slant culture medium, culturing at 20 ℃ for 96 hours until dense spores grow, adding a spore suspension, scraping off the spores growing on the surface of the PDA slant culture medium, and filtering by using sterile filter paper to remove mycelia;
(3) diluting the spore liquid obtained in step (2) to 1 × 10 with sterile water5Inoculating to PDB seed culture medium at 20 deg.CStanding and culturing for 48h to obtain seed liquid of P-3 strain of drugchin peachblosa;
(4) transferring the seed liquid of the drugcojiangpingueing verruca acuminata P-3 in the step (3) to a liquid containing 4mg/L MgSO4And 8mg/L CaSO4Culturing the PDB growth culture medium at 20 ℃ for 48h, centrifuging the culture medium, removing the supernatant to obtain corresponding P-3 cells of verruca peregrina, adding sterile water to prepare a P-3 cell suspension of verruca peregrina, wherein the number of cells in the suspension can reach 109Per mL;
(5) transferring the cell suspension of the verruca plana P-3 in the cellar obtained in the step (4) into a sprayer, spraying the cell suspension onto the wall of the wine storing hole and the surface of the pottery jar for storing wine, wherein the number of the cells sprayed onto the wall of the hole is 1012Per m2The number of cells on the surface of the pottery jar was 108Per dm2；
(6) And (3) taking out the pottery jar arranged in the wine storage hole in the step (5) after three months, and subpackaging the white wine in the pottery jar into sterilized wine bottles, wherein the white wine in the pottery jar is the aged wine obtained after accelerated aging.
Example 4: application of cellar verruca plana P-3 in wine aging
(1) Transferring the wine into clean oak barrel, wherein the wine storage amount is 4/5 of the volume (V/V) of the oak barrel, sealing the opening of the oak barrel with an oak board, and transporting the oak barrel with the wine to a cellar for natural aging;
(2) culturing drugchin planiformis P-3 in a PDA (potato dextrose agar) slant culture medium, culturing at 20 ℃ for 96 hours until dense spores grow, adding a spore suspension, scraping off the spores growing on the surface of the PDA slant culture medium, and filtering by using sterile filter paper to remove mycelia;
(3) diluting the spore liquid obtained in step (2) to 1 × 10 with sterile water5Inoculating the strain per mL into a PDB seed culture medium, and standing and culturing at 20 ℃ for 36 hours to obtain a strain seed liquid of the drugchin flat verrucaria P-3;
(4) transferring the seed liquid of the drugcojiangpingueing verruca acuminata P-3 in the step (3) to a liquid containing 4mg/L MgSO4And 8mg/L CaSO4Culturing at 20 deg.C for 36 hr in PDB growth medium, centrifuging, and removingClear liquid is obtained to obtain corresponding P-3 cells of the drugchin peacockia, and sterile water is added to prepare a suspension of the P-3 cells of the drugchin peacockia so that the number of the cells in the suspension can reach 107Per mL;
(5) transferring the cell suspension of P-3 of drugchin peachblosa obtained in the step (4) into a sprayer, spraying the cell suspension onto the surface of an oak barrel, wherein the number of the cells sprayed onto the oak barrel is 107Per dm2。
(6) And (3) taking out the oak barrel placed in the cellar in the step (5) after three months, and subpackaging the glucose wine in the oak barrel into sterilized wine bottles, wherein the glucose wine in the bottles is the aged wine obtained after accelerated aging.
Example 5: application of cellar flat verruca acuminata P-3 in yellow wine aging
(1) Transferring the yellow wine which is just brewed into a clean pottery jar, wherein the storage amount of the pottery jar is 4/5 of the volume (V/V) of an oak barrel, sealing the opening of the pottery jar by using airtight canvas, sealing the periphery of the canvas by using mud heads, and transporting the pottery jar filled with the yellow wine to a storage warehouse by using a trailer for natural aging;
(2) culturing drugchin planiformis P-3 in a PDA (potato dextrose agar) slant culture medium, culturing at 20 ℃ for 72h until dense spores grow, adding a spore suspension, scraping off the spores growing on the surface of the PDA slant culture medium, and filtering by using sterile filter paper to remove mycelia;
(3) diluting the spore liquid obtained in step (2) to 1 × 10 with sterile water5Inoculating the strain per mL into a PDB seed culture medium, and standing and culturing at 20 ℃ for 40h to obtain a strain seed liquid of the verruca acuminata P-3 strain of the cellar;
(4) transferring the seed liquid of the drugcojiangpingueing verruca acuminata P-3 in the step (3) to a liquid containing 4mg/L MgSO4And 8mg/L CaSO4Culturing at 20 deg.C for 40h in PDB growth culture medium, centrifuging the culture medium, removing supernatant to obtain corresponding P-3 cells, adding sterile water to prepare P-3 cell suspension, and making the cell number in the suspension reach 108Per mL;
(5) transferring the cell suspension of the verruca plana P-3 obtained in the step (4) into a sprayer, spraying the cell suspension onto the surface of a pottery jar,the number of cells sprayed on the pottery jar is 109Per dm2。
(6) And (3) taking out the pottery jar placed in the storage in the step (5) after three months, and subpackaging the yellow wine in the pottery jar into sterilized wine bottles, wherein the yellow wine in the pottery jar is the aged wine obtained after accelerated aging.
Comparative example 1:
the other steps are the same as example 3, only step (5) is not spraying drugstore verrucaria peregrina P-3 to the cavity wall of the wine storage cavity and the surface of the pottery jar.
Comparative example 2:
the other steps were identical to example 4, except that step (5) was carried out without spraying drugchin applanatum P-3 onto the surface of the oak barrel.
Comparative example 3:
the other steps are identical to example 5, except that step (5) does not spray drugchin applanatum P-3 onto the surface of the pottery jar.
The flavor index of the wines prepared in examples 3-5 and comparative examples 1-3 was analyzed as shown in Table 1:
TABLE 1
Figure BDA0002342498220000081
The sensory index of the wines prepared in examples 3-5 and comparative examples 1-3 is analyzed as shown in Table 2:
TABLE 2
Figure BDA0002342498220000091
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (7)

1. A cellar-smoothing drusen (Verruca umbilicalis)Zasmidiumcellare) P-3, characterized byThe culture medium is collected in the China general microbiological culture Collection center, and the preservation date is as follows: year 2019, month 6, day 25, deposit address: no. 3 of Xilu No.1 of Beijing Korean Yang district, the preservation number is CGMCC number 18126.
2. Use of drugcorona planiformis P-3 according to claim 1 for aging new wine.
3. The use of claim 2, wherein the new wine is new white wine, new wine or new yellow wine.
4. The application according to claim 3, characterized in that it comprises in particular the following steps:
(1) 18-22 percent of drugchin peaceful wart spore P-3 in a slant culture mediumoC, culturing until dense spores grow out, adding the spore suspension to suspend the spores, and filtering to remove mycelia to obtain a spore solution;
(2) inoculating the spore liquid obtained in the step (1) into a seed culture medium, wherein the seed culture medium contains 18-22 percent of spore liquidoC, standing and culturing for 36-48 h to obtain a seed solution of the P-3 strain of the drugchin peaceful wart;
(3) inoculating the P-3 seed liquid of the drugchin peachblosa obtained in the step (2) into a growth culture medium, and inoculating the seed liquid to the growth culture medium for 18-22oC, standing and culturing for 36-48 h, centrifuging to obtain the P-3 cells of the drugchin peachblosoma, adding water to prepare a P-3 cell suspension of the drugchin peachblosoma peachblosum, wherein the number of the cells in the cell suspension is 107~109Per mL;
(4) spraying the cell suspension of the verruca plana P-3 prepared in the step (3) on the surface of a wine storage container;
the spore suspension is 0.01-0.02 g/L Tween80 and 0.08-0.10 g/L NaH2PO4、0.50~0.55 g/L Na2HPO4And 0.08-0.10 g/L NaCl;
the slant culture medium is a PDA culture medium, the seed culture medium is a PDB culture medium, and the growth culture medium is MgSO 3-5 mg/L-MgSO4And 6-10 mg/L CaSO4The PDB medium of (1).
5. The use of claim 4, wherein the wine storage container is a wine storage hole, an oak barrel or a pottery jar, and the number of cells on the wall of the wine storage hole is 1010~1012Per m2The number of cells on the surface of the pottery jar was 106~108Per dm2The number of cells on the surface of oak barrel is 105~107Per dm2。
6. A microbial inoculant comprising the drugchin flat verrucaria P-3 of claim 1.
7. The microbial agent according to claim 6, wherein the microbial agent is a solid microbial agent or a liquid microbial agent.
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