CN112111416A

CN112111416A - Issatchenkia orientalis strain for whole-process green production of fruit wine and application thereof

Info

Publication number: CN112111416A
Application number: CN202010525940.8A
Authority: CN
Inventors: 王友升
Original assignee: Nanjing Wantuo Biotechnology Co ltd
Current assignee: Nanjing Wantuo Biotechnology Co ltd
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2020-12-22
Anticipated expiration: 2040-06-10
Also published as: CN112111416B

Abstract

The invention relates to Issatchenkia orientalis (Issatchenkia orientalis), which is preserved in China general microbiological culture Collection center (CGMCC) 24 in 24.04.2020, has the preservation number of CGMCC No.19724 and is named as Issatchenkia orientalis WTB 20042304. The strain can effectively utilize citric acid and tartaric acid, has the degradation capability on citric acid, lactic acid, formic acid and succinic acid in fruit wine, and has particularly remarkable degradation capability on citric acid; can effectively improve the content of active substances such as total phenols, total flavonoids and anthocyanidin, and improve the antioxidation level of the fruit wine. In addition, the strain has a certain inhibiting effect on botrytis cinerea. Therefore, the strain can be applied to storage and preservation of picked fruits or acid reduction and fermentation of fruit wine, and is an optimal strain for the whole process of green production of fruit wine.

Description

Issatchenkia orientalis strain for whole-process green production of fruit wine and application thereof

Technical Field

The invention relates to a functional yeast strain, in particular to Issatchenkia orientalis for green production of fruit wine in the whole process and application thereof.

Background

The fruit wine is wine which is prepared by fermenting saccharomycetes into alcohol by utilizing sugar of fruits, contains specific flavor and nutrient components of the fruits, and is brewed by the earliest human society. The fruit of the fruit wine can be made from the fruit of the Ficus eight-flower phyla, and the fruit is more ideal from kiwi fruit, waxberry, orange, grape, blueberry, red date, cherry, lychee, honey peach, persimmon, strawberry and the like. In fruit wine, most famous wines are popular in recent years because blueberries contain abundant anthocyanin which can resist free radicals and have the function of delaying senescence.

The brewing process of the fruit wine comprises the following steps: picking fresh fruit → sorting → crushing, removing stem → pulp → separating and extracting juice → clarifying → clear juice → fermenting → pouring barrel → storing wine → filtering → cold processing → mixing → filtering → finished product. The variety of the raw materials is one of the important factors for ensuring the quality of the fruit wine product, and the raw materials directly influence the sensory characteristics of the brewed fruit wine. Wherein, the fruits selected from the picked fresh fruits require the ripeness of the fruits to reach full ripeness, high sugar content of the fruit juice, no mildew, rot, deterioration and no plant diseases and insect pests. The brewed finished fruit wine has mellow, harmonious and palatable taste if the acid content is proper. Conversely, the palatability is poor, the sourness is too heavy, the wine juice is cloudy and is not bright, and the purchase desire of consumers is reduced. The acid in the fruit wine is partially brought by raw materials, such as tartaric acid in grapes, malic acid in apples, citric acid in waxberries and the like; there are also yeast metabolites produced during fermentation, such as acetic acid, butyric acid, lactic acid, succinic acid, and the like. L-malic and tartaric acids are among the most prominent organic acids in wine and play a crucial role in wine brewing, including the organoleptic quality and physical, biochemical and microbiological stability of wine. One of the quality indexes of fruit wine is to control the total amount of organic acids in the fruit wine.

In conclusion, in the brewing process of the fruit wine, the preservation of fresh fruits and the control of the total amount of organic acids in finished wine are two important links influencing the cost and the quality of the fruit wine.

According to analysis, the quality deterioration of fresh fruits and vegetables is influenced by a plurality of factors, but diseases are the most main reasons. Among them, rotting and deterioration caused by fungal diseases are the most serious factors in postharvest loss of fruits, main diseases of different fruits are different, grapes mainly cause powdery mildew due to Botrytis cinerea (Botrytis cinerea), and apples mainly cause Penicillium expansum (Penicillium expansum). At present, the common and main methods for preventing fresh fruits from being preserved are physical prevention and chemical agent prevention, the use of chemical pesticides not only causes pathogenic bacteria to generate drug resistance to reduce the sterilization effect, but also causes chemical agent residues to influence the health of people due to unclean treatment. Physical control methods (such as low-temperature storage) require special equipment, often consume energy, and are not favorable for retaining nutrient components in fresh fruits. In the prior art, acid reducing agent (edible alkali), anion exchange resin column acid reduction, low-temperature freezing acid reduction and the like are usually added into fruit wine, the former acid reducing mode influences the flavor and taste of the fruit wine due to the introduction of alkali, the latter acid reducing mode only influences the control of organic acid in the production process, the cost is high, the efficiency is low, and the control of the organic acid in the fruit wine cannot be realized after filling is finished.

Compared with physical and chemical deacidification and biological deacidification, the biological deacidification method can reduce the acidity of the fruit wine, more importantly can increase the stability of the fruit wine and improve the quality of the wine, has small side effect, and is mostly developed by malic acid-lactic acid fermentation (MLF) which is fermented by lactic acid bacteria at present. Lactic acid bacteria produce malic acid-lactase, L-malic acid is changed into L-lactic acid and carbon dioxide under the catalysis of the malic acid, and compared with malic acid, lactic acid is softer, so that the acid reduction effect is achieved. However, lactic acid is also an organic acid, so that the method cannot effectively reduce the organic acid in the fruit wine and improve the mouthfeel of the fruit wine.

Disclosure of Invention

Technical problem to be solved

In order to solve the problems in the prior art, the invention provides Issatchenkia orientalis for green production of fruit wine in the whole process and application thereof. The strain not only has the biocontrol effect, but also has the inhibition effect on the postharvest pathogenic fungi of the fruits, can replace chemical bactericides to prevent and control postharvest diseases of the fruits, and keeps the fruits fresh to prevent the fruits from rotting and deteriorating; meanwhile, the strain has an excellent function of reducing organic acids such as citric acid, lactic acid, formic acid, acetic acid and the like in fruit wine, and can be used for fermenting and reducing the acid of the fruit wine. Especially when the fruit wine is blueberry fruit wine, the strain can also obviously improve the anthocyanin content in the blueberry fruit wine and almost completely degrade citric acid in the blueberry fruit wine.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

the applicant selects and purifies microorganisms in naturally fermented blueberry fruit wine by using a WL nutrient agar culture medium to obtain a strain which can effectively utilize citric acid and tartaric acid and has insensitive acidity reducing capability to alcohol concentration, confirms that the strain is Issatchenkia orientalis (I.orientalis) through identification, classifies and names the strain as I.orientalis WTB20042304, and reserves the strain in China general microbiological culture Collection center at 24/04 in 2020 and with the preservation number of CGMCC No. 19724.

Based on the yeast strains screened by the invention, the invention also provides the following technical scheme:

the invention also relates to application of the Issatchenkia orientalis strain I.orientalis WTB20042304 in storage and preservation of picked fruits or fruit wine deacidification. The method specifically comprises the following steps:

the first scheme is as follows: a fresh fruit preservation method is characterized in that Issatchenkia orientalis I.orientalis WTB20042304 is prepared into bacterial suspension, and the bacterial suspension is used for spraying or dip-coating fresh fruits.

The specific method comprises the following steps: activating Issatchenkia orientalis I.orientalis WTB20042304, fermenting and culturing with YPD liquid culture medium, centrifuging to obtain thallus, cleaning thallus with sterile water to remove culture medium, and making into 1 × 10⁶CFU/mL～1×10⁸CFU/mL of bacterial suspension; putting fruits into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a fresh-keeping box, sealing, and storing at room temperature.

Preferably, the fresh fruit is grape or blueberry.

Preferably, the activating step is: taking single colony on solid culture medium, culturing in YPD liquid culture medium at 26 deg.C under 200r/min for 24 hr, centrifuging at 4000rpm for 5min, collecting thallus, and washing with sterile water for 3 times.

Scheme II: a fruit wine deacidification fermentation method is characterized in that before saccharomyces cerevisiae is inoculated in fruit juice, issatchenkia orientalis I.orientalis WTB20042304 is inoculated, and then fermentation treatment is carried out.

Preferably, the fruit wine is blueberry fruit wine, and the fruit juice is blueberry fruit juice.

Preferably, the method is: crushing fresh blueberries, adjusting the components to obtain blueberry juice, adding Issatchenkia orientalis WTB20042304 into the blueberry juice, adding saccharomyces cerevisiae after 2-4 days, and performing sugar degree adjustment, primary fermentation, filtration and post-fermentation to obtain the blueberry juice. The delayed addition of Saccharomyces cerevisiae is intended to allow the first-added Issatchenkia orientalis I.orientalis WTB20042304 to survive in large quantities, and to degrade the organic acids, such as citric acid, malic acid, etc., in the juice. The sugar is added later, so that the problem that the acid reducing effect is lost because the Issatchenkia orientalis WTB20042304 preferentially utilizes the sugar is avoided.

Preferably, the addition amount of the Issatchenkia orientalis I.orientalis WTB20042304 is1 × 10⁶CFU/mL。

Preferably, the bacterial adding amount of the saccharomyces cerevisiae is1 × 10⁶CFU/mL, wherein the sugar adding amount for adjusting the sugar degree is 120g/L, fermenting at the room temperature of 22-26 ℃, stirring for 2 times every day, and filtering after the fermentation is finished, wherein the filtrate is the blueberry fruit wine.

The Issatchenkia orientalis I.orientalis WTB20042304 comprises an activation treatment before use: taking single colony on solid culture medium, culturing in YPD liquid culture medium at 26 deg.C under 200r/min for 24 hr, centrifuging at 4000rpm for 5min, collecting thallus, and washing with sterile water for 3 times.

(III) advantageous effects

The invention has the beneficial effects that:

(1) according to the Issatchenkia orientalis WTB20042304, after the Issatchenkia orientalis I.orientalis WTB20042304 is cultured on a citric acid (citric acid is a unique carbon source) screening culture medium for 96 hours, the content of citric acid in the culture medium is reduced by 92.39 percent, so that the Issatchenkia orientalis can utilize the citric acid; after the culture medium is cultured for 96 hours on a tartaric acid (tartaric acid is a unique carbon source) screening culture medium, the content of tartaric acid in the culture medium is reduced by 38.84%, which indicates that the tartaric acid can be utilized. The strain is added into blueberry juice for fermentation, and no citric acid is detected at the end point of fermentation, which indicates that the strain has strong citric acid degradation capability. In addition, if the blueberry juice fermentation broth only added with saccharomyces cerevisiae is taken as a reference, and the blueberry juice fermentation broth simultaneously added with saccharomyces cerevisiae and issatchenkia orientalis i.orientalis WTB20042304, the content of lactic acid is reduced by 36.17%, the content of formic acid is reduced by 24.39%, the content of acetic acid is reduced by 9.20%, and the content of succinic acid is reduced by 13.43% at the end point of fermentation. Therefore, the Issatchenkia orientalis WTB20042304 screened by the invention has the capacity of degrading citric acid, lactic acid, formic acid, acetic acid and succinic acid in fruit wine, wherein the capacity of degrading the citric acid is particularly remarkable.

The Issatchenkia orientalis WTB20042304 is used as the acid-reducing yeast, can degrade most of organic acids in fruit wine, and more importantly can increase the stability of the fruit wine (can also stabilize the acidity after bottling), and improve the quality of the wine.

(2) The selected Issatchenkia orientalis WTB20042304 is added into blueberry juice fermentation liquor, and if the blueberry juice fermentation liquor only added with saccharomyces cerevisiae is used as a reference, and the blueberry juice fermentation liquor added with saccharomyces cerevisiae and Issatchenkia orientalis WTB20042304 is added at the end of fermentation, the total phenols in the blueberry fruit wine are increased by 7.08%, the total flavonoids are increased by 2.93%, and the content of anthocyanin is increased by 7.91%. Phenols, flavonoids and anthocyanins are important active substances in blueberry fruit wine, and have activities of resisting oxidation, scavenging free radicals, resisting variation and preventing canceration. DPPH free radical scavenging capacity is improved by 5.82%, FRAP total antioxidant capacity is improved by 9.58%, ABTS free radical scavenging capacity is improved by 5.53%, total reducing power is improved by 4.09%, and the improvement of fruit wine antioxidant capacity effectively prevents aging and pathological changes of organisms caused by aging. Therefore, if the Issatchenkia orientalis WTB20042304 screened by the method is applied to brewing of blueberry wine, the quality of the blueberry wine can be greatly improved.

(4) In the process of preparing the blueberry wine by fermentation, the sugar content at the fermentation end point proves that the fermentation process of saccharomyces cerevisiae to the blueberry wine is not influenced after the issatchenkia orientalis I.orientalis WTB20042304 is added. Therefore, the blueberry juice can be normally fermented after the Issatchenkia orientalis I.orientalis WTB20042304 is added, the alcoholic strength is 11.73% at the end point of fermentation, and SSC is 6.81 degrees Bx. If the blueberry juice fermentation broth only added with the saccharomyces cerevisiae is taken as a control (the pH at the end of fermentation is 3.61), and the pH at the end of fermentation is 3.92 after the issatchenkia orientalis I.orientalis WTB20042304 and the saccharomyces cerevisiae are added, the fact that the issatchenkia orientalis I.orientalis WTB20042304 has obvious acid reduction effect is further proved.

(5) The incidence of the grapes inoculated with botrytis cinerea in advance is counted by taking the issatchenkia orientalis WTB20042304 as a biocontrol bacterium, and the result is 42.22%; under the condition of the same concentration of the biocontrol bacteria, the morbidity of the existing biocontrol bacteria (the existing strain with the preservation number of CGMCC No.14909) is 71.11 percent, thereby showing that the yeast strain has better inhibiting effect on the botrytis cinerea compared with the existing biocontrol bacteria.

Therefore, the Issatchenkia orientalis I.orientalis WTB20042304 can prevent the fresh fruits from being rotted and deteriorated after the fresh fruits are picked to ensure the freshness of the fresh fruits, can play a role in reducing acid when the fruit juice is used for fermentation and brewing in the later period, is not influenced by the alcohol concentration, does not influence the normal fermentation process of the saccharomyces cerevisiae, can play a role in reducing acid of most kinds of organic acids in fruit wine, improves the taste and the appearance of the fruit wine (the fruit wine is turbid in wine juice with high acidity), is a preferable strain applicable to the whole process of the fruit wine preparation process, and can still play the role in reducing acid in the fermentation process even if part of biocontrol bacterium suspension sprayed during the fresh-keeping storage of the fresh fruits is remained. In addition, the content of active substances such as phenols, flavonoids, anthocyanin and the like in the fruit wine can be increased, and the antioxidation and total reducing power are improved, so that the quality of the fruit wine is further improved.

Drawings

FIG. 1 is a photograph showing morphological characteristics of Issatchenkia orientalis I.orientalis WTB20042304 screened in the present invention.

FIG. 2 is a PCR electrophoresis detection map of total DNA of Issatchenkia orientalis I.orientalis WTB20042304 screened by the present invention.

FIG. 3 is a phylogenetic tree analysis of Issatchenkia orientalis I.orientalis WTB20042304 screened in accordance with the present invention.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

Screening and identification of strains

Isolation of the Strain

Sampling the naturally fermented blueberry fruit wine at 0d, 3d, 6d, 9d, 12d and 15d respectively, diluting the wine sample liquid in a WL nutrient agar culture medium by 1000 times, 10000 times and 100000 times, paving the sample, culturing at the constant temperature of 26 ℃ for 48 hours to obtain a single bacterial colony, and performing two parallel experiments. And (3) selecting a single colony on a WL nutrient agar plate for separation and purification, culturing for 48h at a constant temperature of 26 ℃, selecting a suspected colony according to morphological characteristics of the yeast for microscopic examination, and carrying out streak separation on the WL plate to obtain a purified strain.

Biological characteristics of Issatchenkia orientalis I.orientalis WTB20042304

(1) Morphological characteristics

Referring to FIG. 1A and FIG. 1B, A represents the colony morphology of WL nutrient agar (fungal culture medium, 75gWL nutrient agar medium dissolved in 1000mL water, subpackaged, sterilized at 121 deg.C for 20 min) cultured at 26 deg.C for 48h, with scale of 1 cm; b represents the cell morphology observed under a 40-fold microscope (10 μm scale). The colony was observed to be irregular, white, flat, rough in shape; the cells were observed under a microscope to have an oval shape and a diameter of about 8 μm.

(1) Molecular biological identification

Firstly, extracting the total DNA of the strain by an SDS boiling method, amplifying target fragments of a 26s rDNA D1/D2 region by taking NL-1 and NL-4 as primers, and carrying out agarose gel electrophoresis analysis (electrophoresis conditions: 180V,30min), wherein the result is shown in figure 2; and (4) detecting that the size of the target fragment is correct, and performing DNA sequencing on the sample.

Results of homology alignment and evolutionary tree

And (3) comparing the sequencing Gene sequence with the corresponding sequence of the known strain in the Gene Bank, finding the corresponding model strain in strainafo, and constructing a phylogenetic evolution tree according to the homology comparison result. As shown in fig. 3. The results show that: the strain and I.orientalis (EU585754.1) have 100 percent of confidence on the same branch and have close affinity (distance is 0), so that the strain is determined to be the Issatchenkia orientalis (I.orientalis).

Second, acid-reducing property of Issatchenkia orientalis I.orientalis WTB20042304

(one) the acid reducing capability of Issatchenkia orientalis I.orientalis WTB20042304 is tested by using a culture medium with citric acid and tartaric acid as unique carbon sources

(1) Experimental Material

Firstly, strains: issatchenkia orientalis I.orientalis WTB20042304, I.orientalis WTB2 (blueberry wine fermented mash), I.orientalis WTB 3 (grape wine fermented mash), I.orientalis WTB 3 (blueberry wine fermented mash), I.orientalis WTB 4 (blueberry wine fermented mash), I.orientalis WTB 5 (hawthorn wine fermented mash) P.cactophia (preservation strain number CGMCC No.14909)

② drugs and reagents

TABLE 1 drugs and reagents

(iii) laboratory instruments

TABLE 2 Main Instrument of the experiment

Fourthly, culture medium

Citric acid medium: 1g of citric acid, 1g of yeast extract, 2g of peptone and 0.1g of potassium dihydrogen phosphate were dissolved in 100mL of water and sterilized by autoclaving at 121 ℃ for 20min for further use.

Tartaric acid culture medium: 1g of tartaric acid, 1g of yeast extract, 2g of peptone and 0.1g of potassium dihydrogen phosphate were dissolved in 100mL of water and sterilized by autoclaving at 121 ℃ for 20min for further use.

(2) Experimental methods

Firstly, respectively picking single strains of cultured Issatchenkia orientalis I.orientalis WTB20042304, I.orientalis WTB2 (blueberry fruit wine fermented mash), I.orientalis WTB 3 (grape fruit wine fermented mash), I.orientalis WTB 3 (blueberry fruit wine fermented mash), I.orientalis WTB 4 (blueberry fruit wine fermented mash), I.orientalis WTB 5 (hawthorn fruit wine fermented mash) P.cactophila (preservation strain number is CGMCC No.14909), dropping the single strains into a test tube filled with 5ml of YPD culture medium, shaking the test tube on a shaking table for 16-24h to ensure the purity and the activity of the YPD culture medium, taking out, removing supernatant, adding water, cleaning, centrifuging for 3 times, and finally adding 1ml of water to prepare a strain suspension. Diluting the prepared bacterial suspension by 10 times, 100 times and 1000 times in gradient, selecting 1000 times, counting cells with a blood counting plate under a microscope, calculating the bacterial suspension concentration by a formula, and diluting to 2 × 10⁶CFU/mL。

② respectively adding 100 mul of the acid-adding culture medium (the citric acid culture medium and the tartaric acid culture medium) and 100 mul of bacterial suspension into a 96-well plate, and making three strains of bacteria in parallel. Scanning the absorbance at 590nm at 0h, 24h, 48h and 72h respectively, calculating the difference value of the absorbance with 0h, reflecting the growth condition of the strains through the difference value of the absorbance, and further judging whether the strains can grow by using corresponding organic acid.

(3) Single acid reduction effect analysis

3 portions of citric acid medium and tartaric acid medium, 50mL each per bottle of medium, were prepared and sterilized for use. Respectively selecting 3 cultured strains, respectively dropping into test tube containing 5ml LYPD culture medium, shaking on shaking table for 16-24 hr to ensure purity and activity, taking out, removing supernatant, centrifuging for 3 times, adding 1ml water to desired volume, and making into final productForming bacterial suspension and counting blood cells. Adding 3 strains of yeast into corresponding triangular flasks respectively to make the final concentration of yeast be 10⁶And (3) placing the CFU/mL into a constant-temperature shaking table for shaking, sampling at 26 ℃ and 200rpm for 0h, 48h and 96h respectively, taking 2X 4mL each time, and measuring the content of the organic acid at the sampling time point respectively.

Adopting a liquid chromatograph to detect conditions:

a chromatographic column: agilent 5 TC-C18 (250X 4.6mm), using diammonium hydrogen phosphate with pH of 2.4 as mobile phase, column temperature of 45 deg.C, flow rate of 0.8ml/min, and sample amount of 20 μ L, respectively centrifuging and diluting samples containing citric acid and tartaric acid culture medium and 3 strains of acid-reducing yeast added organic acid culture medium for 48h and 96h, and detecting with high performance liquid chromatography. Determining the retention time of the organic acid through the chromatogram results of the citric acid and tartaric acid standard substances, and comparing the retention time with the chromatographic peak area of each organic acid in the 0h culture medium to detect the acid reduction effect of each strain. Before testing, samples of 0h citric acid and 0h tartaric acid medium were first diluted 20-fold with ultrapure water, loaded for testing, and then diluted and tested for 48h, 96h media, respectively.

Establishment of a standard curve: weighing a certain amount of organic acid standard substance, putting the organic acid standard substance into a volumetric flask, diluting with ultrapure water to prepare a mother solution with the concentration of 10mg/ml, diluting with a mobile phase to the required concentration with the concentration gradient of 2.5mg/ml, 2mg/ml, 1mg/ml, 0.4mg/ml, 0.2mg/ml and 0.1mg/ml respectively, carrying out sample injection in sequence to obtain corresponding peak areas, and obtaining a corresponding linear regression equation by using a least square method.

(4) Results of the experiment

The deacidification effect by taking citric acid and tartaric acid as unique carbon sources is shown in the table 3-table 5:

TABLE 3 OD in citric acid relative to the start of the experiment₅₉₀Increase in value

TABLE 4 relative OD in tartaric acid at the beginning of the experiment₅₉₀Increase in value

TABLE 5 contents of citric acid and tartaric acid for 72h

As is clear from table 3, when the i.orientalis WTB20042304 strain was added to the citric acid medium (citric acid was used as the sole carbon source), the absorbance increased by 1.6 or more at 48h and 72h of culture, as is clear from table 3. Higher than other strains. As can be seen from table 4, when the i.orientalis WTB20042304 strain was added to the tartaric acid medium (tartaric acid was the sole carbon source), the absorbance increased by 1.1 or more at 48h and 72h of culture. Higher than other strains. From table 5, it can be seen that the degradation rate of i.orientalis WTB20042304 on citric acid reaches 92.39%, and other strains are all lower than 90%; the degradation rate of tartaric acid reaches 41.04%, and other strains are lower than 40%.

In conclusion, the strains are most potent in utilizing citric acid and tartaric acid.

Effect of Issatchenkia orientalis I.orientalis WTB20042304 on organic acids and active substances in blueberry mash

(1) Experimental Material

Blueberry: provided by the sunshine gift winery 2019, 1 month and 6 days, and the variety is Bei Lu

② drugs and reagents

TABLE 6 drugs and reagents

(iii) laboratory instruments

TABLE 7 main instruments of experiment

(2) Experimental methods

The blueberries are crushed and then distributed into 3 fermentation tanks, and 3.5 kg of the blueberries are placed in each fermentation tank. Respectively adding activated Issatchenkia orientalis WTB20042304 and Pichia capraphila BY35 (the existing strain and the preserved strain are CGMCC No.14909) into 2 fermentation tanks, wherein the addition amount is1 × 10⁶CFU/mL, control without any acid reducing yeast. Adding Saccharomyces cerevisiae at day 3, the concentration is also 1 × 10⁶CFU/mL, sugar addition 120 g/L. The fermenter was placed at 22 ℃ for fermentation with 2 stirring times per day. Filtering after fermentation is finished, and sealing and storing the fruit wine filtrate.

(3) Blueberry fruit wine index determination

a. Measurement of physical and chemical indexes

And (3) pH value measurement: measured with a pH meter. SSC determination: measured with a glucometer. Alcohol content determination: measured with an alcohol meter. And (3) total sugar determination: phenol-sulfuric acid method, 1mL sample +0.5mL 5% phenol solution +2.5mL concentrated sulfuric acid, mixing well for 5min, taking out and cooling to room temperature, and measuring absorbance at 490nm of spectrophotometer. And (3) total acid determination: measured according to the national standard method.

b. Active substance assay

The determination of the total phenol content is carried out by the method of Singleton [ Singleton V.L., Rossi J.A.colorimetry of total phenolics with phospho-molar phosphoric acid reagents [ J ]. American Journal of Enlogy and Viticulture,1965,16: 144-. The total flavone content is determined according to methods of Wang Youyan, Wang Youyang, Zhao Rubia and the like, the extraction condition research of antioxidant and free radical scavenging active substances in sophora flower [ J ] food industry science and technology, 2009(12): 130-. The anthocyanin content is determined by referring to the method of the production process research of dry blueberry wine [ D ]. Anhui university 2014 ].

c. Detection of acid reducing effect of blueberry fruit wine

Preparing a mixed standard product: taking 10mg of solid standard substance and 100 μ L of liquid standard substance mother liquor, metering to 10ml with mobile phase, mixing, and storing in refrigerator at 4 deg.C in dark place. In use, 1ml of 45. mu.L microporous membrane was taken out by syringe and tested in a liquid phase vial under optimum conditions.

Sample pretreatment: the samples were first diluted 20 times with ultrapure water, tested by loading, and then diluted and tested for 48h, 96h of culture medium, respectively.

Liquid phase conditions: a chromatographic column: agilent 5 TC-C18 (250X 4.6mm), using diammonium hydrogen phosphate with pH of 2.4 as mobile phase, column temperature of 45 deg.C, flow rate of 0.8ml/min, and sample amount of 20 μ L, respectively centrifuging and diluting 20 times samples of culture medium containing citric acid and tartaric acid and organic acid culture medium after adding 2 strains of acid-lowering yeast, fermenting for 48h and 96h, and detecting with high performance liquid chromatography. Determining the retention time of the organic acid through the chromatogram results of the citric acid and tartaric acid standard substances, and comparing the retention time with the chromatographic peak area of each organic acid in the 0h culture medium to detect the acid reduction effect of each strain.

(4) Results of the experiment

Variation of citric acid content

The citric acid content in the blueberry juice was high at 6.81g/L at the beginning of fermentation, while no citric acid was detected in the remaining 2 groups at the end of fermentation, except for the control group (without the addition of acid-reducing yeast).

Therefore, the I.orientalis WTB20042304 yeast has strong degradation capability on citric acid in the blueberry fruit wine.

Variation of lactic acid content

The lactic acid content at the beginning of the fermentation was very low, only 0.08 g/L. As can be seen by comparing the lactic acid content at the end of fermentation with that at the beginning of fermentation, the Saccharomyces cerevisiae produces lactic acid during the fermentation process, so that the lactic acid content at the end of fermentation in the control group is 0.94 g/L. Specific results are shown in table 8:

TABLE 8

In addition, when the screened yeast strains are added, the content of lactic acid at the fermentation end point is reduced by 36.17 percent compared with that of a control group, and the lactic acid reducing capability of the screened yeast strains is obviously superior to that of the existing yeast strains and is also superior to that of other experimental groups.

Variation of formic acid content

Formic acid detected in the blueberry juice at the beginning of fermentation is only 0.07g/L, and saccharomyces cerevisiae can generate more formic acid in the fermentation process, and the formic acid content of the contrast is 0.41g/L at the end of fermentation. By comparing the formic acid content at the end of the fermentation with that at the beginning of the fermentation, it can be seen that the saccharomyces cerevisiae produces formic acid during the fermentation. Specific results are shown in table 9:

TABLE 9

After the calcium of BY35 was added to the blueberry mash, the formic acid content was increased, presumably BY decomposing other organic acids to form part of formic acid. The I.orientalis WTB20042304 screened by the invention has the capability of degrading the formic acid, the content of the formic acid is 0.31g/L at the fermentation end point, and is reduced by 24.39 percent compared with a control group, and the degradation of the formic acid by the I.orientalis of other experimental groups is less than 10 percent.

Variation of acetic acid content

The blueberry juice contains a very small amount of acetic acid which is only 0.15g/L at the beginning of fermentation, a certain amount of acetic acid can be generated by saccharomyces cerevisiae in the fermentation process, and the acetic acid content of a control group reaches 14.79g/L at the end of fermentation. The saccharomyces cerevisiae is easy to produce acetic acid in the fermentation process. The results are shown in Table 11.

Watch 10

After the I.orientalis WTB20042304 is added, the content of acetic acid is reduced by 9.20 percent compared with the control group, the I.orientalis WTB 5 has equivalent degradation capability on the acetic acid with the I.orientalis WTB20042304, the I.orientalis WTB2 is reduced by less than 5 percent compared with the control group, and other strains accumulate more acetic acid compared with the control group.

Fifth change of succinic acid content

The blueberry juice contains a very small amount of succinic acid of only 0.05g/L at the beginning of fermentation, the saccharomyces cerevisiae can generate succinic acid in the fermentation process, and the succinic acid content of a control group at the end of fermentation is 0.67 g/L. It can be seen by comparing the succinic acid content at the end of fermentation with that at the beginning of fermentation that the Saccharomyces cerevisiae produces succinic acid during the fermentation. The results are shown in Table 10.

TABLE 11

The addition of the strain I.orientalis WTB20042304 of the invention reduces the succinic acid by 13.43 percent relative to the control group, and the accumulation of the succinic acid of other strains relative to the control group is larger than that of the control group.

Therefore, I.orientalis WTB20042304 has degradation effects on citric acid, lactic acid, formic acid, acetic acid and succinic acid in blueberry fruit wine fermentation, and particularly has remarkable degradation effect on citric acid.

I.Orientalis WTB20042304 influence on active substances in blueberry fruit wine

The content of total phenols in the control group at the end of fermentation is 1.13mg/g, the content of total flavonoids is 1.05mg/g, and the content of anthocyanidin is 120.29 mg/L.

Compared with a control group, when the strain I.orientalis WTB20042304 is added, the content of total phenols is increased by 10.37 percent and is higher than that of other strains, the content of total flavonoids is increased by 2.93 percent and is higher than that of other strains, and the content of anthocyanin is 7.91 percent.

TABLE 12

Compared with a control group, the antioxidant indexes of the strain I.orientalis WTB20042304 are increased, wherein DPPH is increased by 9.44%, FRAP is increased by 9.58%, ABTS is increased by 6.50%, and the total reducing power is increased by 15.92%. The increase of FRAP is slightly lower than that of strain I.orientalis WTBD4, but other antioxidant indexes are higher than that of strain I.orientalis WTBD 4.

I. Effect of orientalis WTB20042304 on pH of blueberry fruit wine

The blueberry fruit wine added with the acid-reducing yeast can be normally fermented, the alcoholic strength of all treatments at the end point of fermentation is 11.73%, and SSC is 6.87 degrees Bx. The pH of the control at the end of fermentation was 3.61. After addition of Issatchenkia orientalis I.orientalis WTB20042304, the fermentation end point pH was 3.92. The I.orientalis WTB20042304 of the invention has obvious acid reduction effect.

Biological control effect of Issatchenkia orientalis WTB20042304 on fresh fruits

(1) Experimental Material

Firstly, strains: yeast: issatchenkia orientalis I.orientalis19724

Pathogenic bacteria: botrytis cinerea (Botrytis cinerea) for laboratory preservation

Fruit: grape, purchased from Beijing New-onset

② main experimental instruments, refer to Table 2

(2) Experimental methods

Step 1: preparing yeast suspension as biocontrol bacteria suspension, comprising the following steps:

activating the strain: 1 single colony was inoculated to YPD liquid medium on YPD plate and shake-cultured at 26 ℃ for 24 hours.

Collecting thalli: centrifuging, removing culture medium, washing with 1mL sterile water (blowing with pipette or shaking, mixing well), centrifuging at 12000r/min at 4 deg.C for 2 min. This operation was repeated three times. After the water is removed and the thallus is left, 1mL of sterile water is added and mixed evenly, and the mixture is diluted to a proper time.

③ counting the blood corpuscle plate: cover the slide glass first, add 10 uL bacterial suspension in the upper chamber and lower chamber separately, find the square with 10 times mirror first, change 40 times mirror count again. And counting and diluting to obtain suspension with target concentration.

Step 2: preparing a mould bacterial suspension

Adding 2mL sterile water into sterilized 10mL centrifuge tube, clamping mycelia on the surface of the culture medium with sterilized forceps, adding into the centrifuge tube, blowing and mixing with pipette to completely mix spores into water, filtering with filter cloth, counting with blood counting plate, and diluting to 5 × 10⁴Spore suspension at CFU/mL concentration.

And step 3: fruit preparation

Selecting bought grapes, placing damaged grapes separately, cleaning selected good fruits with consistent maturity and size with clear water, treating with sodium hypochlorite aqueous solution (0.5%) for 5min, placing into cleaned boxes, placing 15 grapes per box, drying grape surface water, and pricking with pricking needleThe puncture was started, 1 hole per fruit, and one box per puncture was burned with the inoculating needle. After the grape sap had dried (about 4 hours), the experimental group was added with 20. mu.L of yeast suspension of the desired concentration, and after 4 hours (essentially complete absorption of the yeast suspension) 20. mu.L of pathogenic bacteria 5X 10 was added⁴CFU/mL spore suspension, control, also added 20. mu.L of sterile water, and in the case of framing, two groups of water-wetted toilet paper rolls were placed in the frame.

And 4, step 4: statistics of disease (rot)

The statistical method of the rotting rate adopts an observation method, and the calculation formula is as follows: rotten rate (%) — rotten fruit count/total fruit count × 100%. The diameter of the lesion is measured by the cross method in mm.

(3) Results of the experiment

The incidence rate of grape is counted at 96h, 144h and 192h of grape inoculated with botrytis cinerea (B.cinerea), and the concentration of biocontrol bacteria is1 × 10⁶CFU/mL, sterile water as blank control, different treatment between different cases of the difference of the disease. The result shows that the incidence rate of the strain I.orientalis WTB20042304 added in the invention is 42.22%; under the condition of the same concentration of the biocontrol bacteria, the morbidity of the existing biocontrol bacteria (the existing strain with the preservation number of CGMCC No.14909) is 71.11 percent. The incidence rate of the I.orientalis WTB2 strain is 70%, the incidence rate of the I.orientalis WTB 3 strain is 93%, the incidence rate of the I.orientalis WTB 4 strain is 93%, and the incidence rate of the I.orientalis WTB 5 strain is 90%. The concentration of biocontrol bacteria is1 multiplied by 10⁶The soaking morbidity of fruits soaked in CFU/mL by using the strain I.orientalis WTB20042304 is 43.00 percent, the morbidity of the strain I.orientalis WTB2 is 63.00 percent, the morbidity of the strain I.orientalis WTB 3 is 88.00 percent, the morbidity of the strain I.orientalis WTB 4 is 88.00 percent, the morbidity of the strain I.orientalis WTB2 is 93.00 percent, and the morbidity of the prior known biocontrol strain P.cactophia is 60 percent.

Therefore, compared with the existing biocontrol bacteria, the yeast strain disclosed by the invention has a better inhibiting effect on botrytis cinerea, and the biocontrol effects of different strains of the same strain are obviously different.

As can be seen from the comparison, Issatchenkia orientalis I.orientalis WTB2 of the present invention0042304 has certain inhibitory effect on Botrytis cinerea (B.cinerea). When the concentration of the biocontrol bacteria is increased to 1 x 10⁸The biocontrol effect of the Issatchenkia orientalis I.orientalis WTB20042304 bacterial suspension is not obviously increased (the morbidity is 31.11%) at the time of CFU/mL. Therefore, from the economical point of view, when fresh fruits are preserved using the suspension of Issatchenkia orientalis I.orientalis WTB20042304 of the present invention, it is preferable to set the concentration at 1X 10⁶CFU/mL。

Based on the above description, issatchenkia orientalis WTB20042304(CGMCC No.19724) has uniqueness in degrading citric acid, lactic acid, formic acid, acetic acid and the like in fruit wine, compared with other acid-reducing yeasts, and has excellent acid-reducing performance and wide adaptability. By reducing the content of organic acid in the fruit wine, the quality of the fruit wine such as taste, color and the like can be improved.

In addition, experiments also prove that in the process of preparing fruit wine by fermentation, the sugar content at the fermentation end point shows that the fermentation process of the saccharomyces cerevisiae is not influenced after the saccharomyces cerevisiae strain is added, and the ethanol concentration has no obvious influence on the capability of the saccharomyces cerevisiae strain to utilize organic acid.

When the fruit wine, particularly the blueberry fruit wine is prepared, the Issatchenkia orientalis I.orientalis WTB20042304 is added before saccharomyces cerevisiae is added, the alcoholic strength of all treatments at the fermentation end point is 11.73%, SSC is 6.87 degrees Bx, the pH value at the fermentation end point is higher and is 3.92, the contents of total phenols, total flavones and anthocyanin in the fruit wine are increased, and the oxidation resistance and the total reduction capability are improved.

In addition, the Issatchenkia orientalis I.orientalis WTB20042304 also proves to have certain inhibition effect on botrytis cinerea.

Therefore, the Issatchenkia orientalis I.orientalis WTB20042304 can prevent the fresh fruits from being rotted and deteriorated after the fresh fruits are picked to ensure the freshness of the fresh fruits, can play a role in reducing acid when the fruit juice is used for fermentation and brewing in the later period, is not influenced by the alcohol concentration, does not influence the normal fermentation process of the saccharomyces cerevisiae, can play a role in reducing acid of most kinds of organic acids in the fruit wine, improves the taste and the appearance of the fruit wine (the fruit wine is turbid in high acidity wine juice), is a preferable strain for the whole-process green production of the fruit wine, and can still play the role in reducing acid of residual strains in the fermentation process even if the biological control bacterium suspension sprayed during the fresh-keeping and storage of the fresh fruits is not cleaned.

Claims

1. Issatchenkia orientalis (Issatchenkia orientalis) is deposited in China general microbiological culture Collection center at 24.04.2020 under the preservation number of CGMCC No.19724 and named as Issatchenkia orientalis WTB 20042304.

2. Use of the issatchenkia orientalis WTB20042304 in fruit postharvest storage and preservation or wine deacidification according to claim 1.

3. Use according to claim 2, characterized in that: the fruit is blueberry, and the fruit wine is blueberry fruit wine.

4. A fresh fruit preservation method is characterized by comprising the following steps: issatchenkia orientalis I.orientalis WTB20042304 is prepared into bacterial suspension, and the bacterial suspension is used for spraying or dip-coating fresh fruits.

5. The fresh-keeping method of claim 4, wherein: activating Issatchenkia orientalis I.orientalis WTB20042304, fermenting and culturing with YPD liquid culture medium, centrifuging to obtain thallus, and making into 1 × 10⁶CFU/mL～1×10⁸CFU/mL of bacterial suspension; putting fruits into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a fresh-keeping box, sealing, and storing at room temperature.

6. The fresh-keeping method of fresh fruit according to claim 4 or 5, characterized in that: the fresh fruit is grape or blueberry.

7. A fruit wine deacidification fermentation method is characterized in that before saccharomyces cerevisiae is inoculated in fruit juice, issatchenkia orientalis I.orientalis WTB20042304 is inoculated, and then fermentation treatment is carried out.

8. The acid-reducing fermentation method of fruit wine according to claim 7, characterized in that: the fruit wine is blueberry fruit wine, and the fruit juice is blueberry fruit juice.

9. The acid-reducing fermentation method of fruit wine according to claim 7, characterized in that: crushing fresh blueberries, adjusting the components to obtain blueberry juice, adding Issatchenkia orientalis WTB20042304 into the blueberry juice, adding saccharomyces cerevisiae after 2-4 days, and performing the process flows of sugar degree adjustment, primary fermentation, filtration and post-fermentation.

10. The acid-reducing fermentation method of fruit wine according to claim 7, characterized in that: the addition amount of the Issatchenkia orientalis I.orientalis WTB20042304 is1 × 10⁶CFU/mL。