CN112522122B - Fruit wine deacidification strain and application thereof - Google Patents
Fruit wine deacidification strain and application thereof Download PDFInfo
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Abstract
The invention discloses a fruit wine acidity reducing strain and application thereof, and belongs to the technical field of fruit wine brewing. The invention provides a Candida Candida divaricata, which is classified and named as Candida divaricata Z3, is preserved in China center for type culture Collection in Wuhan and has the preservation number of CCTCC No. M2015400. The Candida divaricata Z3 provided by the invention has high alcoholic strength, low pH value and high SO 2 The fruit wine has good fermentation environment adaptation, can quickly degrade citric acid in the fruit wine, can increase fruit wine fragrance, effectively improves the sensory quality of the fruit wine, and has good application prospect.
Description
Technical Field
The invention relates to a fruit wine acidity reducing strain and application thereof, belonging to the technical field of fruit wine brewing.
Background
China has abundant fruit resources, and the production of fruit fermented wine is one of the important ways of developing and utilizing the fruit resources and improving the added value of fruit products. However, many fruits have high acid content, so the fruit wine prepared by taking the fruits as raw materials also has high acid content, and mainly contains citric acid (accounting for about 90 percent of the total acid content), such as waxberries, raspberries, blueberries and the like. In the process of fruit wine fermentation, organic acid with a certain content can make the fruit wine fragrant and mellow and make people feel a refreshing feeling, and can inhibit the growth activity of a certain amount of bacteria, but too high content of the organic acid can cause the fruit fermented wine to have high acidity, uncomfortable taste, rough and uncoordinated wine body and strong sour and astringent feeling. Therefore, the total acid content in the wine needs to be reduced, so that the fruit wine is more mellow and refreshing and meets the requirements of consumers.
The problem of over-high acidity of fruit wine has attracted attention of many scholars long ago, and researchers also try and research various methods aiming at the problem. At present, the most applied acid reduction methods mainly comprise physical acid reduction and chemical acid reduction. The physical deacidification method mainly refers to a freezing deacidification method and an ion exchange resin deacidification method. The physical deacidification has the advantages of effectively reducing the deacidification, having great influence on the flavor of the wine body and being operatedIs complicated and tedious. Chemical deacidification method mainly comprises CaCO 3 Deacidification of KHCO 3 Deacidification, double salt method, double calcium salt method, chitosan deacidification and the like. Although the chemical acid-reducing method is simple and easy to operate and has obvious acid-reducing effect, chemical reactions often occur along with side reactions, which affect the color and taste of the wine, and a large amount of metal ions and other substances are introduced, which may cause unstable factors such as light loss, turbidity and the like of the wine.
The research and development direction of the modern deacidification process is a biological deacidification method, which is typically malic acid-lactic acid fermentation (MLF) in wine, i.e. malic acid is decomposed to form lactic acid with soft acidity. However, MLF has poor degradation effect on citric acid, and cannot be applied to deacidification of fruit wine rich in citric acid. Therefore, screening of excellent strains which can adapt to the fermentation environment of fruit wine and efficiently degrade citric acid is a key problem which needs to be solved for producing high-grade fruit wine.
Disclosure of Invention
The invention mainly aims at the problems that the existing fruit wine with high organic acid content, particularly the fruit wine with high citric acid content is not harmonious in wine body and poor in taste, the existing physical and chemical acid reduction effects are poor, no proper bacterial strain is used for biological acid reduction, and the like, and the bacterial strain capable of rapidly degrading the citric acid in the fruit wine is bred and applied to brewing of the fruit wine.
The invention provides a Candida (Candida diversa) Z3 strain, wherein the Candida (Candida diversa) Z3 is preserved in China Center for Type Culture Collection (CCTCC) 24 months 06 in 2015, the preservation address is Wuhan, Wuhan university, the preservation number is as follows: CCTCC NO: m2015400.
The Candida (Candida diversa) Z3 strain separation source is waxberry orchard soil, waxberry branches and leaves and waxberry fruits, and the yeast enrichment culture medium, the WL culture medium and the deacidification culture medium are sequentially adopted for screening and purifying; inoculating the activated bacterial liquid into a deacidification culture medium containing 10% (v/v) absolute ethyl alcoholScreening ethanol-resistant strains; inoculating the activated bacteria liquid with 100ppm SO 2 In acid reducing medium for SO tolerance 2 Screening strains; the screened SO resistance 2 The activated bacteria liquid is inoculated in an acid reduction culture medium, and strains with high acid rate are screened by measuring the change of acid content in unit time.
Extracting a whole genome from the obtained strain, carrying out PCR amplification, sending the processed gene fragment to a Shanghai gene data research institute for sequencing, carrying out DNA man and NCBI database data comparison on the returned sequencing result, wherein the NCBI data comparison result shows that the screened yeast is Candida CEC Y111 type, the homology of the gene is 99 percent, and the screened yeast can be determined to be Candida and named as Candida (Candida subversia) Z3.
The colony morphology of the Candida (Candida divversa) Z3 on the culture medium is milky white, large and flat (see fig. 1 in particular).
In one embodiment of the invention, the Candida (Candida divversa) Z3 is capable of growing normally in wine having one or more of the following conditions: SO (SO) 2 The content is 80 + -20 mg/L, the alcoholic strength (v/v) is 12 + -3%, the pH value is 3.2 + -0.2, and the culture temperature is 23 + -5 ℃.
In one embodiment of the present invention, the fermentation conditions of the Candida (Candida divversa) Z3 strain are as follows:
the additive contains 20g of citric acid, 1g of urea and (NH) 4 ) 2 SO 4 1g，KH 2 PO 4 2.5g，Na 2 HPO 4 0.5g，MgSO 4 ·7H 2 O1g，FeSO 4 ·7H 2 Culturing in a culture medium containing 0.1g of O and 0.5g of yeast powder; the culture was carried out at 30 ℃ for 24h at initial pH3.2 of the fermentation.
The invention also provides a product containing the Candida (Candida divversa) Z3.
The invention also provides the Candida (Candida divversasa) Z3 or the application of the product in reducing the acidity of fruit wine.
In one embodiment of the invention, the application is that the Candida (Candida divversa) Z3 or the product is added in a fruit wine brewing process.
In one embodiment of the present invention, the Candida (Candida diversa) Z3 is added in an amount of at least 0.2% (v/v).
In one embodiment of the invention, the wine is a wine rich in citric acid.
In one embodiment of the invention, the fruit wine is waxberry, blueberry, raspberry, sea buckthorn, blueberry.
The invention also provides a brewing method of the fruit wine, which comprises the following steps:
inoculating the candida to crushed fruits, performing primary fermentation, enzyme treatment, inoculating saccharomyces cerevisiae for secondary fermentation, and ageing to obtain the fruit wine.
In one embodiment of the invention, the fruit wine is obtained by cleaning, crushing and uniformly mixing the raw materials, inoculating the candida, performing primary fermentation, performing enzyme treatment, separating peel and residue after fermentation, inoculating saccharomyces cerevisiae, performing secondary fermentation, separating peel and residue after secondary fermentation, aging, clarifying, filtering and sterilizing.
In one embodiment of the present invention, the saccharomyces cerevisiae is a common saccharomyces cerevisiae, specifically saccharomyces cerevisiae D254, angel yeast RW, angel yeast SY, angel yeast K1; the function is as follows: converting glucose to alcohol.
In one embodiment of the invention, the method comprises the steps of:
(1) cleaning: cleaning with sterile water to remove soil and dust, sieving to remove mildewed and rotten fruits, and air drying;
(2) crushing and uniformly mixing: crushing the cleaned fruits, adding sterile water, and uniformly mixing to obtain a crushing liquid; directly adding sterile water into the raw materials of the fruit residues and the fruit powder without cleaning and crushing, and uniformly mixing;
(3) primary fermentation: inoculating activated Candida (Candida divversasa) Z3 into the crushed liquid obtained in the step (2) for primary fermentation, wherein the inoculation amount of the Candida (Candida divversasa) Z3 is at least 0.2% (v/v);the inoculation amount of the Candida (Candida divarsia) Z3 is at least 1 x 10 6 cfu/mL；
(4) Enzyme treatment: putting the fermentation liquor obtained in the step (3) into a sterilized fermentation tank, adding pectinase, adding white sugar till the total sugar content is 200-210 g/L, standing for 3-4 h, and performing enzyme treatment to obtain crude fruit juice;
(5) and (3) separating skin and residue after fermentation: filtering the crude fruit juice obtained in the step (4) to obtain a filtrate;
(6) and (3) secondary fermentation: adding saccharomyces cerevisiae into the filtrate obtained in the step (5) for secondary fermentation, wherein the fermentation temperature is 23-27 ℃, the fermentation time is 7-14 days, so as to obtain a secondary fermentation mixture, and the inoculation amount of the saccharomyces cerevisiae is at least 1 × 10 6 cfu/mL；
(7) Aging, clarifying, filtering and sterilizing: and (4) filtering the secondary fermentation mixture obtained in the step (6) to obtain a filtrate, standing and ageing the filtrate at the temperature of 20 ℃, clarifying, filtering and sterilizing to obtain the fruit wine.
In one embodiment of the invention, the Candida (Candida diversa) Z3 and Saccharomyces cerevisiae are added in the form of a bacterial suspension.
In one embodiment of the invention, the pectinase is added in an amount of at least 2% by weight of the juice.
In one embodiment of the invention, the final concentration of pectinase is at least 30 mg/L.
The invention also provides a method for degrading citric acid, which is to culture the Candida (Candida divversasa) Z3 in a system containing citric acid.
The invention also provides application of the Candida (Candida diversa) Z3 in preparing products for degrading acidity of fruit wine or citric acid.
Advantageous effects
(1) The strain capable of obviously reducing the acidity of the fruit wine is obtained by screening, and particularly, the strain can effectively reduce the content of citric acid, improve the softness and balance of the wine body, modify the flavor of the fruit wine, improve the quality of the fruit wine and avoid the damage of physical or chemical deacidification to the quality of the fruit wine.
(2) Taking a waxberry wine as an example, the waxberry wine brewed by adding Candida albicans (Candida divansa) Z3 provided by the invention has the total acid content and the citric acid content which are both obviously reduced compared with the waxberry fruit juice before fermentation, wherein the total acid content is reduced from 15.50g/L to 6.02g/L, the citric acid content is reduced from 10.85g/L to 1.13g/L, and the degradation rate reaches 89.59%; the total acid of the waxberry wine brewed without adding the Candida (Candida divversas) Z3 provided by the invention is 15.92g/L, which is higher than that of the waxberry fruit juice before fermentation, and the citric acid content is basically not different from that before fermentation and is still as high as 10.81 g/L; the total amount of volatile aroma substances in the waxberry wine brewed by adding the Candida (Candida divversasa) Z3 provided by the invention is increased to a certain extent, and reaches 180.35mg/L, so that the aroma and the flavor of the waxberry wine can be improved.
(3) Taking blueberry wine as an example, the total acid of the blueberry wine brewed by adding Candida (Candida diversa) Z3 provided by the invention is reduced from 9.10g/L to 5.63g/L, and the citric acid content is reduced from 5.23g/L to 0.76 g/L; the total acid of blueberry fruit wine brewed by Candida divaricata Z3 without adding is 10.29g/L, the citric acid content is basically not different from that before fermentation, and still reaches 5.21 g/L; the total amount of volatile aroma substances in blueberry wine brewed by adding Candida (Candida divversasa) Z3 provided by the invention is increased to a certain extent, and reaches 190.41mg/L, so that the aroma and the flavor of the blueberry wine can be improved.
(4) The Candida (Candida diversa) Z3 strain obtained by screening has ethanol resistance and sulfur dioxide resistance, and can also obviously improve the aroma of fruit wine and the flavor and quality of the fruit wine; the deacidification method is simple and convenient to operate, low in cost, suitable for large-scale popularization and application of fruit wine and obvious in economic benefit.
Biological material preservation
A Candida albicans strain Candida divansa Z3, which is taxonomically named as Candida divansa Z3, is deposited in China Center for Type Culture Collection (CCTCC) 24.06.2015, and has a preservation number of CCTCC NO: m2015400, the preservation address is Wuhan, Wuhan university in China.
Drawings
FIG. 1: candida divansa Z3 colony morphology.
FIG. 2: and (3) comparing the gene data with the Candida CEC Y111 base in DNA man.
FIG. 3: two different waxberry wine flavor profile maps.
FIG. 4: two different blueberry wine flavor profile maps.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The Saccharomyces cerevisiae referred to in the examples below was Saccharomyces cerevisiae D254, available from Angel Yeast, Inc.
The media involved in the following examples are as follows:
yeast enrichment medium: weighing 50g of anhydrous glucose, 1g of urea and (NH) 4 ) 2 SO 4 1g，KH 2 PO 4 2.5g，Na 2 HPO 4 0.5g，MgSO 4 ·7H 2 O 1g，FeSO 4 ·7H 2 0.1g of O and 0.5g of yeast powder, dissolving with deionized water, and fixing the volume to 1000 mL. Sterilizing at 121 deg.C for 20 min.
Acid reduction screening culture medium: the anhydrous glucose in the yeast enrichment medium is replaced by citric acid, and other components and a sterilization method are the same as those of the yeast enrichment medium.
WL medium: accurately weighing 4g of yeast powder, 5g of peptone, 50g of anhydrous glucose and 20g of agar. Stock solution A (40 mL) and stock solution B1mL (78) were added, the pH was adjusted to about 6.5, and the mixture was dissolved in deionized water to obtain a volume of 1000 mL. Sterilizing at 121 deg.C for 20 min. Stock C1mL was added after sterilization was complete. Wherein the stock solution A, B, C consists of: stock solution a: accurately weighing KH 2 PO 4 5.5g, KCl 4.25g, anhydrous CaCl 2 1.25g, anhydrous MgSO 4 1.25g, dissolving to a constant volume of 400mL, and storing in a refrigerator at 4 ℃ for later use. And (4) liquid storage B: accurately weighing FeCl 3 ·6H 2 O 0.25g，MnSO 4 ·H 2 Dissolving 0.25g of O, fixing the volume to 100mL, and storing in a refrigerator at 4 ℃ for later use. Stock solution C: accurately weighing bromomethyl0.44g of phenol green is dissolved in a mixed solution of 10mL of deionized water and 10mL of absolute ethyl alcohol, and is stored in a refrigerator at 4 ℃ for later use.
YPD liquid medium (g/L): 10 parts of yeast powder, 20 parts of peptone and 20 parts of anhydrous glucose, and dissolving to a constant volume of 1000 mL. Sterilizing at 121 deg.C for 20 min.
YPD solid Medium (g/L): agar was added in an amount of 20g/L based on the YPD liquid medium.
The detection methods referred to in the following examples are as follows:
the detection method of total acid comprises the following steps:
according to the national standard GB/T15038-2006, 10mL of sample is sucked into a 100mL beaker, 50mL of water is added, an electrode is inserted, a rotor is arranged on the beaker, the beaker is arranged on an electromagnetic stirrer, stirring is started, and titration is carried out by using a sodium hydroxide standard solution. The volume of sodium hydroxide standard solution consumed was recorded and a blank experiment was performed.
X＝[c×(V 1 -V 0 )×75]/V 2
X represents the total acid content (calculated by tartaric acid) in the sample, g/L; c is the concentration of a sodium hydroxide standard solution, and the unit mol/L; v 0 The volume of the sodium hydroxide standard solution consumed in the blank experiment is expressed in unit mL; v 1 The concentration of the sodium hydroxide standard solution consumed by the sample is unit mL; v2 is the volume of sample aspirated, in mL; and 75 is the molar mass value of tartaric acid, unit g/moL.
The detection method of total sugar comprises the following steps:
firstly, decomposing total sugar into reducing sugar, measuring a certain amount of sample V 1 In a 100mL volumetric flask, the sugar content is adjusted to 0.2-0.4g, 5mL of hydrochloric acid solution is added, water is added to 20mL, and the mixture is shaken up. Hydrolyzing in water bath at 68 + -1 deg.C for 15min, cooling, neutralizing with sodium hydroxide to neutrality, adjusting temperature to 20 deg.C, adding water, and metering to volume 2 And (5) standby. V 2 /V 1 Is the dilution multiple of the wine sample. Taking 0.4mL of diluted sample, adding 0.6mL of LDNS reagent into a test tube, boiling for 5min, cooling, adding 3mL of water, and measuring the light absorption value by using a spectrophotometer at the wavelength of 540 nm. The total sugar concentration in g/L was calculated using a standard curve plotted on glucose.
The detection method of the citric acid content comprises the following steps:
the wine samples for detection need to be filtered through a 0.22 mu m water phase filter membrane, diluted 7 times by mobile phase (20mM sodium dihydrogen phosphate) and placed in a sample injection bottle for sample injection, and the process is repeated three times.
ACQUITY UPLC HSS T3(i.d 2.1X 150mm, 1.7 μm) chromatographic column, detecting wavelength 210nm, column temperature 30 deg.C, flow rate 0.3mL/min, injecting 1 μ L, mobile phase: 20mM NaH 2PO 4 solution.
The detection method of the alcoholic strength comprises the following steps:
firstly, decomposing total sugar into reducing sugar, measuring a certain amount of sample V 1 In a 100mL volumetric flask, the sugar content is adjusted to 0.2-0.4g, 5mL of hydrochloric acid solution is added, water is added to 20mL, and the mixture is shaken up. Hydrolyzing in water bath at 68 + -1 deg.C for 15min, cooling, neutralizing with sodium hydroxide to neutrality, adjusting temperature to 20 deg.C, adding water, and metering to volume 2 And (5) standby. V 2 /V 1 Is the dilution multiple of the wine sample. Taking 0.4mL of diluted sample, adding 0.6mL of LDNS reagent into a test tube, boiling for 5min, cooling, adding 3mL of water, and measuring the light absorption value by using a spectrophotometer at the wavelength of 540 nm. The total sugar concentration in g/L was calculated using a standard curve plotted on glucose.
The detection method of the total aroma comprises the following steps:
5mL of the wine sample and 1.5g of NaCl were placed in a 20mL empty bottle, 10. mu.L of an internal standard (2-methoxy-D3-phenol, final concentration 609.76. mu.g/L) was added, and after sealing with a hollow magnetic metal cap with a PTFE/blue silica gel spacer, HS-SPME operation was performed by a multifunctional autosampler system MPS 2. The sample is balanced for 5min at 50 ℃, extracted for 45min, and the rotating speed is set to be 250 r/min. After extraction, the extraction head with the volatile compounds and semi-volatile compounds adsorbed thereon is desorbed at 250 deg.C for 5min without flow splitting at the GC sample inlet, and subjected to GC × GC-TOFMS analysis. Each sample was injected 3 times under the same conditions.
GC × GC-TOFMS analysis method and mass spectrum conditions:
the GC x GC-TOFMS analysis method and the mass spectrum condition refer to the method established by optimization in the laboratory. GC-MS column oven temperature program: the initial temperature of 45 ℃ was maintained for 2min, and the temperature was raised to 230 ℃ at a rate of 4 ℃/min and maintained for 15 min. The modulator modulation time is 4s, the thermal modulation time is 1s, and the modulation compensation temperature is 20 ℃.
Two-dimensional column oven temperature rise program: the initial temperature was maintained at 40 ℃ for 2min, and the temperature was raised to 250 ℃ at 5 ℃/min and maintained for 5 min. High purity helium (> 99.995%) as carrier gas, constant flow mode, flow rate: 1 mL/min.
Time-of-flight mass spectrometry conditions: the EI ion source is adopted, the temperature of the ion source is 230 ℃, the temperature of a transmission line is 280 ℃, and the voltage is controlled to be 70 eV. The mass number range of the detector is 35-400 amu, the collection frequency is 100spec-trum/s, and the voltage of the detector is 430V. Data were collected from the LECO company Pega-sus4D workstation.
The detection method of the volatile acid comprises the following steps:
referring to the national standard 15038-2006, low boiling point acids, i.e. volatile acids, in the sample are distilled out in a distillation manner, and the content of the volatile acids in the sample is obtained by combining an alkali standard solution with sulfur dioxide and by calculation and correction.
10mL of sample (V) [ 20 ℃ solution temperature ] was aspirated]Distilling, supplementing water in the distillation process, and collecting 100mL of distillate. Heating the distillate to boiling, adding 2 drops of phenolphthalein indicator solution (10g/L), titrating to pink with sodium hydroxide standard titration solution (cNaOH is 0.05mol/L), keeping the titration end point within 30s, and recording the volume (V) of consumed sodium hydroxide standard titration solution 2 )
Calculating the content of volatile acid in the sample:
X 1 ＝(C×V 1 ×60.0)/V
in the formula:
X 1 -the content of volatile acids in the sample, in g/L, calculated as acetic acid.
C-concentration of sodium hydroxide standard titration solution, unit mol/L.
V 1 Volume unit mL of consumed sodium hydroxide standard titrant.
60.0 molar mass number of acetic acid, unit g/mol.
V-volume of aspirated sample in mL.
Example 1: screening and identification of waxberry wine deacidification strain
The method comprises the following specific steps:
separation, screening and purification of deacidification bacterial strain
(1) Accurately sampling and weighing samples (5.00 g of each soil in an orchard of water chestnut red bayberry and Dongkui red bayberry, 2.00g of each soil in branch and leaf of red bayberry and 10.00g of each fruit of red bayberry) at multiple points, respectively placing the obtained samples into 250mL conical flasks, adding 100mL of physiological saline (0.9% NaCl solution) and a little glass beads, respectively placing the marked samples into a 30-DEG C shaking table at 200r/min, shaking and shaking uniformly for about 2h, and standing. And (3) accurately sucking 5mL of the supernatant respectively, inoculating the supernatant into a yeast enrichment culture medium (50 mL of culture solution is filled in each 250mL conical flask) sterilized in advance, and culturing for 1 day in a shaking table at 30 ℃ and 200r/min to obtain a pretreatment culture solution.
(2) Respectively standing the pretreated culture solution obtained in the step (1) and then performing gradient dilution (10) -1 ～10 -6 ) 3 appropriate dilution gradients (10) were selected -3 、10 -4 、10 -5 ) 0.1mL of each dilution was pipetted and applied to WL medium plates, and each dilution was repeated 3 times, and incubated at a constant temperature in an incubator at 30 ℃ for 3 days. In order to eliminate the interference of bacteria in the screening process, 50-100 mg of chloramphenicol can be added into the sterilized WL culture medium.
(3) And (3) respectively inoculating single colonies with complete colony morphology growth in a WL culture medium into acid reduction screening culture medium test tubes, culturing for 4 days at the temperature of 30 ℃ and the speed of 200r/min, observing the growth condition of thalli in the test tubes every 12 hours, and recording the growth condition after the culture is finished.
(4) And selecting test tubes which become turbid after culture, namely test tubes with bacteria growing, and numbering the test tubes. And (4) streaking and separating on the sterilized deacidified culture medium, and purifying the strains.
Second, screening of bacterial strain with ethanol resistance, sulfur dioxide resistance and high deacidification rate
(1) Inoculating the pure strain obtained by separation into sterilized YPD liquid culture medium with the inoculation amount of 100 μ L, and performing shake bed activation culture at 30 deg.C and 200r/min for 24 h. Inoculating the activated bacterial liquid into an acid reduction culture medium containing 10% of absolute ethyl alcohol, wherein the inoculation amount is 6% (v/v). Shaking culture at 30 deg.C and 200 r/min. After 4 days of culture, 7 test tubes become turbid, and the 7 strains of bacteria can grow under the condition of higher alcohol content, have better alcohol resistance and better accord with the production conditions in the wine brewing process.
(2) Inoculating the 7 strains of bacteria solution after alcohol-resistant activation to a strain containing 100ppm SO 2 The amount of the inoculum in the acid-reducing medium of (4) is 6% (v/v). Shaking culture at 30 deg.C and 200 r/min. After 4 days of culture, 5 test tubes become turbid, and the 5 strains have better SO 2 And (4) tolerance.
(3) The 5 plants not only resist alcohol, but also have better SO 2 The strain solution after the activation of the tolerant strain was inoculated into a 250mL Erlenmeyer flask in an amount of 6% (v/v) in a deacidification medium (liquid). Shaking culture at 30 deg.C and 200 r/min. Sampling 1mL every 12 hours, centrifuging for 1min under 10000r/min, taking supernatant, diluting to 10mL, and determining the residual acid amount in the culture solution. Wherein 1 strain has high deacidification rate, can degrade the citric acid from 10.85g/L to 7.65g/L within 12h, and can reduce the citric acid to 1.03g/L after 24 h.
Identification of acid-reducing strains
By subjecting the 1 strain obtained above to resistance to both alcohol and SO 2 Meanwhile, the strain with higher deacidification rate extracts the whole genome, the processed gene fragment is sent to Shanghai gene data research institute for sequencing after PCR amplification, and DNA man and NCBI database data comparison are carried out on the returned sequencing result. The results of the comparison of NCBI data show that the screened yeast is Candida CEC Y111 type, the homology of the gene is 99 percent, and the nucleotide sequence is shown as SEQ ID NO. 1; can be identified as Candida. FIG. 2 shows the alignment of the returned gene data with Candida CEC Y111 bases in DNA man, with the shaded areas being homologous. And finally determining the Candida as the Candida by integrating morphological feature observation, physiological and biochemical tests and molecular biological sequencing results, wherein the Candida is named Candida divarsansa Z3.
Example 2: brewing method of waxberry fruit wine
The red bayberry fruit wine is brewed by taking red bayberries of Zhejiang Xiancheng water chestnuts as raw materials and is used as a starting sample for biological deacidification.
The brewing process of the waxberry wine is as follows:
(1) preparation of Candida (Candida divansa) Z3 and Saccharomyces cerevisiae suspensions
Preparation of a suspension of Candida (Candida diversa) Z3 bacteria: candida albicans (Candida divansa) Z3 obtained in example 1 was inoculated into YPD liquid medium and cultured at 30 ℃ to give a cell concentration of 1X 10 6 cfu/mL of bacterial suspension.
Preparing a saccharomyces cerevisiae suspension: inoculating Saccharomyces cerevisiae D254 into sucrose water with mass fraction of 5%, activating at 37 deg.C to obtain strain concentration of 1 × 10 6 cfu/mL of bacterial suspension;
(2) selecting fresh waxberry fruits, and crushing to prepare waxberry pulp crushing liquid;
(3) primary fermentation: inoculating the Candida (Candida divansa) Z3 bacterial suspension obtained in the step (1) into the waxberry pulp crushing liquid, and fermenting for 24 hours at 20 ℃;
(4) enzyme treatment: adding 30mg/L pectinase solution and SO 2 (60mg/L) and sucrose was added to a total sugar content of 210 g/L; standing and reacting for 12h to obtain crude fruit juice;
(5) and (3) separating skin and residue after fermentation: filtering the crude fruit juice mixture obtained in the step (4) to obtain a filtrate;
(6) and (3) secondary fermentation: inoculating the saccharomyces cerevisiae D254 bacterial suspension obtained in the step (1) into the filtrate obtained in the step (5), and then carrying out alcoholic fermentation at 25 ℃; when the concentration of reducing sugar in the waxberry pulp is less than or equal to 4g/L, cooling the fermented mash to 4 ℃, and adding SO 2 (60mg/L), stopping alcoholic fermentation to obtain a secondary fermentation mixture;
(7) and (4) filtering the secondary fermentation mixture obtained in the step (6) to obtain a filtrate, standing and ageing the filtrate at the temperature of 20 ℃, clarifying, filtering and sterilizing to obtain the waxberry wine 1.
The preparation method of the control fruit wine is the same as that of the waxberry fruit wine 1, except that Candida albicans (Candida diversa) Z3 is not inoculated, namely, the step (3) is to stand the waxberry pulp crushing liquid obtained in the step (2) for 24 hours at 20 ℃ to prepare the waxberry fruit wine 2.
The physical and chemical indexes of the waxberry juice and the two waxberry wines and the contents of the organic acid and the volatile aroma components are measured, and the results are shown in table 1.
TABLE 1 basic physical and chemical indexes of waxberry wine
Note: inoculating Candida utilis Z3 into fructus Myricae Rubrae wine 1, and inoculating Candida utilis Z3 into fructus Myricae Rubrae wine 2
As can be seen from Table 1, the content of total acid and citric acid of the waxberry wine 1 added with the Candida (Candida diversa) Z3 provided by the invention is remarkably reduced compared with that of the waxberry wine before fermentation, wherein the content of the total acid is reduced from 15.50g/L to 6.02g/L, the content of the citric acid is reduced from 10.85g/L to 1.13g/L, and the degradation rate reaches 89.59%.
The total acid of the waxberry wine 2 fermented by only inoculating saccharomyces cerevisiae is 15.92g/L, but is slightly higher than the waxberry juice before fermentation, probably because a fermentation by-product generated in the fermentation process contains a small amount of organic acid, and the content of citric acid is basically not different from that before fermentation and is still as high as 10.81 g/L.
The volatile aroma components of the waxberry fruit wine are analyzed by GC-MS, and the result shows that the total amount of aroma substances of the waxberry fruit wine after the fermentation is finished is obviously higher than that of the waxberry fruit juice before the fermentation. Meanwhile, the total amount of volatile aroma substances of the waxberry wine 1 prepared by adding the Candida (Candida diversa) Z3 provided by the invention is increased to a certain extent and reaches 180.35mg/L, while the total amount of aroma substances of the waxberry wine 2 prepared by adding the saccharomyces cerevisiae is 152.21mg/L, which proves that the aroma and flavor of the waxberry wine can be improved by adding the Candida (Candida diversa) Z3. Although the content of volatile acid in the waxberry fruit wine 1 slightly rises to reach 0.41g/L, the content is still far lower than the maximum limit standard (1.2g/L) of the national standard (wine GB 15037-2006).
Quantitative descriptive analysis is carried out on two waxberry wines (as shown in figure 3), and the result shows that the content of acid in a control (waxberry wine 2) which is not inoculated with Candida (Candida divversasa) Z3 is obviously higher than that in the waxberry wine 1 inoculated with Candida Z3, and the astringency of the waxberry wine 2 are enhanced due to the high acid, so that the astringency of the waxberry wine 2 is also obviously higher than that of the waxberry wine 1.
In addition, as can be seen from fig. 3, the waxberry wine 1 inoculated with candida Z3 is obviously higher than the waxberry wine 2 not inoculated with candida Z3 in both fruity flavor and flowery flavor, the plant aroma is not obviously different, and in conclusion, the waxberry wine quality is obviously improved after the candida Z3 is inoculated.
Example 3: brewing method of blueberry fruit wine
The blueberry wine is brewed by taking Jiangsu Suzhou rabbit eye blueberries as raw materials and is used as an initial sample for biological deacidification.
The brewing process of the blueberry wine is as follows:
(1) selecting fresh blueberry fruits, and crushing to prepare a blueberry pulp crushing liquid;
(2) primary fermentation: inoculating Candida (Candida divversa) Z3 bacterial suspension obtained in the step (1) of the example 2 into the blueberry pulp crushing liquid, and fermenting for 24 hours at 20 ℃;
(3) enzyme treatment: adding 30mg/L pectase and SO2(60mg/L), and adding sucrose to total sugar content of 200 g/L; standing and reacting for 12h to obtain crude fruit juice;
(4) and (3) separating skin and residue after fermentation: filtering the crude juice mixture obtained in step (3);
(5) and (3) secondary fermentation: inoculating the filtrate obtained in the step (4) with the saccharomyces cerevisiae D254 bacterial suspension obtained in the step (1) of the embodiment 2, and then carrying out alcoholic fermentation at 25 ℃; when the concentration of reducing sugar in blueberry pulp is less than or equal to 4g/L, cooling the fermented mash to 4 ℃, and adding SO 2 (60mg/L), stopping alcoholic fermentation to obtain a secondary fermentation mixture;
(6) and (4) filtering the secondary fermentation mixture obtained in the step (5) to obtain a filtrate, standing and ageing the filtrate at the temperature of 20 ℃, clarifying, filtering and sterilizing to obtain the blueberry fruit wine 1.
The preparation method of the control fruit wine is the same as that of the blueberry fruit wine 1, and is different from that of the blueberry fruit wine 1 in that Candida albicans (Candida diversa) Z3 is not inoculated, namely, the step (2) is that the crushed liquid obtained in the step (1) is kept stand at 20 ℃ for 24 hours to prepare the blueberry fruit wine 2.
The physical and chemical indexes of the blueberry juice and the two blueberry wines and the contents of the organic acid and the volatile aroma components are measured, and the results are shown in table 2.
TABLE 2 basic physical and chemical indexes of blueberry wine
Note: the blueberry wine 1 is inoculated with candida Z3, and the blueberry wine 2 is not inoculated with candida Z3
As can be seen from Table 2, the total acid content of blueberry wine 1 added with Candida (Candida diversa) Z3 provided by the invention is reduced from 9.10g/L to 5.63g/L, and the citric acid content is reduced from 5.23g/L to 0.76 g/L.
The total acid of the blueberry fruit wine 2 obtained by fermenting with the saccharomyces cerevisiae alone is 10.29g/L, the citric acid content is basically not different from that before fermentation and still reaches 5.21g/L, and the effect of reducing the acid can not be achieved by only adding the saccharomyces cerevisiae.
The volatile aroma component analysis result of the blueberry wine shows that the total amount of volatile aroma substances in the blueberry wine 1 added with the Candida (Candida diversa) Z3 provided by the invention is increased to reach 190.41mg/L, while the total amount of aroma of the blueberry wine 2 added with the saccharomyces cerevisiae is 157.68mg/L, and the aroma and the flavor of the blueberry wine can be improved by adding the Candida (Candida diversa) Z3. Although the content of the volatile acid of the blueberry fruit wine 1 is slightly increased to reach 0.38g/L, the content is still far lower than the maximum limit standard (1.2g/L) of the national standard (wine GB 15037-2006).
Quantitative description analysis is carried out on the blueberry wine 1 and the blueberry wine 2 (as shown in figure 4), and the results show that the acid content of the blueberry wine 1 inoculated with Candida (Candida divversa) Z3 is obviously lower than that of the control (blueberry wine 2) not inoculated with Candida Z3, and the astringency is also obviously lower than that of the blueberry wine 2.
In addition, as can be seen from fig. 4, the fruit fragrance of the candida inoculated Z3 blueberry fruit wine 1 is obviously higher than that of the candida unseeded Z3 blueberry fruit wine 2, and the flower fragrance and the costustoot are not very different.
In conclusion, after Candida (Candida divversas) Z3 is inoculated, the wine body balance of the blueberry fruit wine is obviously improved.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> fruit wine acid-reducing strain and application thereof
<130> BAA201321A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 541
<212> DNA
<213> Artificial sequence
<400> 1
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tttcgacgcg ttgtagcgtg gttcagtctt tgagtgacgg atgactaagt cccctggaac 120
ggggtgccat agagggtgag agccccgtga gttgtctttt tagctcttta agtctttacc 180
aaagagtcga gttgtttggg aatgcagctc taagtgggtg gtaaattcca tctaaggcta 240
aataccggcg agagaccgat agcgaacaag tacagtgatg gaaagatgaa aagcactttg 300
aaaagagagt gaaacagtac gtgaaattgt tgaaagggaa gggtatttgg cccgacatgg 360
gttctgtgca ccgttgcctc ttgtaggcgg cgctctgctg gagcctgggc cagcatcagt 420
tttccggcga ggataagaag ttttgaacca cattgtgggg atgagtttga tgctcgcatg 480
ggggactgag gactgccgtt gtaggatgct ggcataacgg ccaaataccg cccgtcttaa 540
a 541
Claims (10)
1. A strain of Candida (Candida subvsa), wherein the Candida is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2015400, and the preservation date is 2015, 06 months and 24 days.
2. A product comprising the Candida of claim 1.
3. Use of candida according to claim 1, or a product according to claim 2, for reducing the acidity of wine.
4. Use according to claim 3, wherein the candida according to claim 1 or the product according to claim 2 is added during wine brewing.
5. Use according to claim 3 or 4, wherein the wine is a wine containing citric acid.
6. A brewing method of fruit wine is characterized by comprising the following steps: inoculating the crushed fruit with the candida as described in claim 1, performing primary fermentation, enzyme treatment, inoculating saccharomyces cerevisiae, performing secondary fermentation, and aging to obtain the fruit wine.
7. The method of claim 6, comprising the steps of:
(1) cleaning, screening out mildewed fruits and rotten fruits, and airing water;
(2) crushing and uniformly mixing: crushing the cleaned fruits, and adding water for mixing to obtain a crushing liquid; directly adding water for mixing when using the fruit residues and the fruit powder as raw materials without cleaning and crushing;
(3) primary fermentation: inoculating activated candida into the crushed liquid obtained in the step (2), and performing primary fermentation, wherein the inoculation amount of the candida is at least 1 x 10 6 cfu/mL；
(4) Enzyme treatment: sterilizing the fermentation liquor obtained in the step (3), adding pectinase, adding white sugar until the total sugar content is 200-210 g/L, standing for 10-12 h, and performing enzyme treatment to obtain crude fruit juice;
(5) and (3) separating skin and residue after fermentation: filtering the crude fruit juice obtained in the step (4) to obtain filtrate;
(6) and (3) secondary fermentation: inoculating saccharomyces cerevisiae into the filtrate obtained in the step (5) for secondary fermentation at the fermentation temperature of 23-27 ℃ for 7-14 days to obtain a secondary fermentation mixture, wherein the inoculation amount of the saccharomyces cerevisiae is at least 1 × 10 6 cfu/mL；
(7) Aging: and (4) filtering the secondary fermentation mixture obtained in the step (6) to obtain a filtrate, standing and ageing the filtrate at the temperature of 20-25 ℃, and clarifying, filtering and sterilizing to obtain the fruit wine.
8. The method of claim 7, wherein in step (4), the pectinase is added in an amount of at least 2% by weight of the juice and at a final concentration of at least 30 mg/L.
9. A method for degrading citric acid, wherein the Candida of claim 1 is cultured in a system containing citric acid.
10. Use of candida according to claim 1 for the preparation of a product for degrading the acidity of fruit wine or degrading citric acid.
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