CN112094761B

CN112094761B - Abnormal hamamelis virginiana for green production of fruit wine in whole process and application of abnormal hamamelis virginiana

Info

Publication number: CN112094761B
Application number: CN202010525908.XA
Authority: CN
Inventors: 王友升
Original assignee: Shandong Kaipu Fite Biotechnology Co ltd
Current assignee: Shandong Kaipu Fite Biotechnology Co ltd
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2022-11-04
Anticipated expiration: 2040-06-10
Also published as: CN112094761A

Abstract

The invention relates to an abnormal Wickerhamomyces anomalus strain, which is deposited in the China general microbiological culture Collection center at 24.04.2020, has the preservation number of CGMCC No.19723 and is named as Wickerhamomyces anomalus WTB20042303. The strain can effectively utilize citric acid and tartaric acid, has the degradation capability on citric acid, lactic acid, formic acid, acetic 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 flavones and anthocyanin, and improve the antioxidation level of the fruit wine. In addition, the strain has a biocontrol effect superior to that of the existing known biocontrol strains, and has a remarkable 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 applied to the whole process of a fruit wine preparation process.

Description

Abnormal hamamelis virginiana for green production of fruit wine in whole process and application of abnormal hamamelis virginiana

Technical Field

The invention relates to a functional yeast strain, in particular to an abnormal yeast Weikehan for the whole process green production of fruit wine 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 Ficus eight can be used for brewing fruit wine, and fructus Actinidiae chinensis, fructus Myricae Rubrae, fructus Citri Junoris, fructus Vitis Viniferae, fructus Myrtilli, fructus Jujubae, fructus Pruni Pseudocerasi, fructus litchi, fructus Persicae, fructus kaki, and strawberry are more ideal. 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 fruit wine products, 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, etc. L-malic and tartaric acids are among the most prominent organic acids in wine and play a vital role in the wine brewing process, including the organoleptic quality and physical, biochemical and microbial 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 nutritional ingredients in fresh fruits. In the prior art, acid reducing agents (edible alkali), anion exchange resin columns, low-temperature freezing acid reduction and the like are usually added into fruit wine, the flavor and taste of the fruit wine are influenced by the acid reducing mode of the former strain due to the introduction of alkali, the organic acid can be controlled only in the production process by the acid reducing mode of the latter strain, the cost is high, the efficiency is low, and the organic acid in the wine can not be controlled 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 an abnormal yeast Weikehan for green production of fruit wine in the whole process and application thereof. The strain has excellent biocontrol effect, has obvious inhibition effect on postharvest pathogenic fungi of fruits, can replace chemical bactericide to prevent postharvest diseases of the fruits, keeps the fruits fresh and prevents the fruits from rotting and deteriorating; meanwhile, the strain has an excellent function of reducing organic acids such as citric acid, tartaric acid, malic acid, lactic acid, formic acid, succinic acid and acetic acid in fruit wine, and can be used for reducing acid in the fermentation of 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 adopts a Montgomera medium to screen and purify microorganisms in naturally fermented blueberry fruit wine to obtain a strain which can effectively utilize citric acid and tartaric acid and has insensitive acid-reducing capability to alcohol concentration, and the strain is identified to be abnormal yeast Wickham (W.anomalus), is classified and named as W.anomalus WTB20042303, is preserved in China general microbiological culture Collection center (CGMCC) 24.04.2020, and has a preservation number of CGMCC No.19723.

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

the invention also relates to application of the abnormal hank yeast strain W.anomalus WTB20042303 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 abnormal Wickham yeast W.anomallus WTB20042303 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 abnormal yeast W.anomallus WTB20042303, fermenting and culturing in YPD liquid culture medium, centrifuging to obtain thallus, cleaning thallus with sterile water to remove culture medium, and making into 1 × 10 thallus ⁶ CFU/mL～1×10 ⁸ CFU/mL bacterial suspension; putting fruits into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a preservation box, sealing, and storing at normal 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 and 200r/min for 24h, centrifuging at 4000rpm for 5min, collecting thallus, and washing with sterile water for 3 times.

Scheme two is as follows: a fruit wine deacidification fermentation method is characterized in that before saccharomyces cerevisiae is inoculated into fruit juice, abnormal hamamelis w.

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 abnormal Wilm's yeast W.anomallus WTB20042303 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 can make the early added abnormal yeast W.anomalus WTB20042303 survive in a large amount and can degrade the organic acid, such as citric acid, malic acid, etc. in the fruit juice. Sugar is added later to avoid losing the acid reducing effect because the abnormal yeast Wikazakholderia W.anomallus WTB20042303 preferentially utilizes sugar.

Preferably, the addition amount of the abnormal yeast Wickerhamia willebrand WTB20042303 is 1 × 10 ⁶ CFU/mL。

Preferably, the bacterial adding amount of the saccharomyces cerevisiae is 1 × 10 ⁶ CFU/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.

Before use, the abnormal yeast Wirkinje W.anomallus WTB20042303 comprises an activation treatment: taking single colony on solid culture medium, culturing in YPD liquid culture medium at 26 deg.C and 200r/min for 24h, centrifuging at 4000rpm for 5min, collecting thallus, and washing with sterile water for 3 times.

(III) advantageous effects

The beneficial effects of the invention are:

(1) According to the abnormal yeast Wickham yeast W.anomallus WTB20042303, after the abnormal yeast W.anomallus WTB20042303 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 44.91%, and the tartaric acid can be utilized. After the culture medium is cultured for 96 hours on the citric acid (citric acid is used as the sole carbon source) screening culture medium, the content of the citric acid in the culture medium is reduced by 88.92 percent, which indicates that the citric 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 of saccharomyces cerevisiae and abnormal yeast wikam w.anomalus WTB20042303 are added at the same time, the content of lactic acid is reduced by 31.39%, the content of formic acid is reduced by 7.32%, the content of acetic acid is reduced by 6.29% and the content of succinic acid is reduced by 11.94% at the end point of fermentation. Therefore, the abnormal Hanm yeast W.anomalus WTB20042303 screened by the invention has the capability of degrading citric acid, formic acid, acetic acid and lactic acid in fruit wine, wherein the capability of degrading citric acid is particularly remarkable.

The abnormal yeast Wtromlus WTB20042303 is used as an 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 abnormal Wilm's yeast W.anomallus WTB20042303 screened by the invention is added into blueberry juice fermentation liquor, if the blueberry juice fermentation liquor only added with saccharomyces cerevisiae is used as a reference, and the saccharomyces cerevisiae and the blueberry juice fermentation liquor added with the abnormal Wilm's yeast W.anomalus WTB20042303 are added at the fermentation end point, the total phenol content in the blueberry fruit wine is increased by 7.08%, the total flavone content is increased by 3.80%, the anthocyanin content is increased by 6.83%, the anthocyanin is an important active substance in the blueberry fruit wine, and the blueberry fruit wine has the activities of resisting oxidation, removing free radicals, resisting variation and preventing canceration. The DPPH free radical scavenging capacity is improved by 7.61%, the total oxidation resistance of FRAP is improved by 9.76%, the ABTS free radical scavenging capacity is improved by 10.42%, and the total reducing power is 5.85%. The improvement of the oxidation resistance of the fruit wine can effectively prevent aging and pathological changes of organisms caused by aging. The anthocyanin is an important active substance in the blueberry fruit wine, and has the activities of resisting oxidation, removing free radicals, resisting variation and preventing canceration. Therefore, if the abnormal Weikehan yeast W.anomalus WTB20042303 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 on the blueberry wine is not influenced after the abnormal hamamelis w. Therefore, the blueberry juice can be normally fermented after the abnormal yeast Wickham W.anomallus WTB20042303 is added, the alcoholic strength at the end point of the fermentation is 12.92, and the SSC is 6.87 degrees Bx. If the blueberry juice fermentation liquor only added with saccharomyces cerevisiae is taken as a reference (the pH value at the end point of fermentation is = 3.61), and after the abnormal hamamelis w.anomalus WTB20042303 and saccharomyces cerevisiae are added, the pH value at the end point of fermentation is 3.90, the obvious acid reducing effect of the abnormal hamamelis w.anomalus WTB20042303 is further proved.

(5) The abnormal yeast Wikazakholderia WTB20042303 is used as a biocontrol bacterium, and the concentration of a bacterial suspension of grapes inoculated with botrytis cinerea in advance is 1 multiplied by 10 ⁶ The morbidity at CFU/mL is 36.50%; under the condition of the same concentration of biocontrol bacteria, the disease rate is lower than that of the existing biocontrol bacteria (the existing strain with the preservation number of CGMCC No. 14909), and the disease rate is 71.11 percent. Therefore, compared with the existing biocontrol bacteria, the yeast strain disclosed by the invention has a better inhibiting effect on botrytis cinerea.

Therefore, the abnormal Weikehan yeast W.anomallus WTB20042303 disclosed by the invention can prevent 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 a 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, and can improve the taste and the appearance of the fruit wine (the fruit juice with high acidity is turbid), is an optimal strain which can be applied to the whole process of the fruit wine preparation process, and can still play the acid reducing effect in the fermentation process even if part of biocontrol bacterium suspension sprayed during fresh-keeping storage of the fresh fruits remains. In addition, the content of active substances such as anthocyanin in the fruit wine can be increased, and the quality of the fruit wine is further improved.

Drawings

Fig. 1 is a photograph showing morphological characteristics of an abnormal hamamelis w.

FIG. 2 is a PCR electrophoresis detection map of total DNA of abnormal yeast Wickerhamia weckerensis WTwomalus WTB20042303 screened by the present invention.

FIG. 3 shows the result of the evolutionary tree analysis of the abnormal yeast Wickham, W.anomalus WTB20042303 screened by 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.

1. Screening and identification of strains

(I) 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 Bengal red culture medium by 1000 times, 10000 times and 100000 times, paving the sample, and culturing at the constant temperature of 26 ℃ for 48 hours to obtain single bacterial colonies, wherein two experiments are performed in parallel. And (3) selecting a single colony to separate and purify on a Bengal red culture medium plate, culturing at a constant temperature of 26 ℃ for 48h, selecting a suspected colony according to morphological characteristics of the yeast, performing microscopic examination, and performing streak separation on the Bengal red culture medium plate to obtain a purified strain.

Experiments prove that the abnormal Wilm's yeast W.anomallus WTB20042303 disclosed by the invention has better comprehensive capabilities of reducing acidity, preventing and treating bio-control and the like, and particularly has better prevention and treatment effect on botrytis cinerea than the known bio-control patent strains.

Biological characteristics of abnormal Hansenula Velcro W.anomallus WTB20042303

(1) Morphological characteristics

Referring to FIGS. 1A and 1B, A represents a strain obtained by culturing Bengal in fungal culture medium, peptone 5g, glucose 10g, potassium dihydrogen phosphate 1g, and magnesium sulfate (MgSO 2) ₄ ·7H ₂ O) 0.5g, agar 20g, 1/3000 Bengal red solution 100mL, distilled water 1000mL, chloramphenicol 0.1g; the manufacturing method comprises the following steps: dissolving the above components in distilled water, adding Bengal red solution, subpackaging, sterilizing at 121 deg.C for 20min, and culturing at 26 deg.C for 48 hr to obtain colony morphology with scale of 1cm; b represents the cell morphology observed under a 40-fold microscope (scale 10 μm). According to observation, the colony has irregular shape, milk white, flat and rough surface; the cells were observed under a microscope to be circular and about 8 μm in diameter.

(1) Molecular biological identification

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

(2) Homology alignment and evolutionary tree results

And comparing the sequencing Gene sequence with the corresponding sequence of the known strain in the Gene Bank, and constructing a phylogenetic evolutionary tree through a homology comparison result. As shown in fig. 3. The results show that: the strain was identified as abnormal hankholderia wekawaken (w. Anomalus) on the same branch with 100% confidence and close affinity (< 0.02) with w.anomalus (KT 895977.1).

2. Acid reduction characteristics of abnormal Hansenula vechicus W.anomallus WTB20042303

(one) the acid reducing capability of abnormal yeast Wickham's yeast W.anomallus WTB20042303 is tested by using a culture medium with citric acid and tartaric acid as unique carbon sources

(1) Experimental Material

(1) The strain is as follows: abnormal hamamelis virginiana w.

(2) Drugs and reagents

TABLE 1 drugs and reagents

Name of reagent	Specification of	Manufacturer of the product
			Phenol and its preparation	Analytical purity	BEIJING CHEMICAL PLANT
Citric acid	Analytical purity	BEIJING CHEMICAL PLANT
			Tartaric acid	Analytical purity	BEIJING CHEMICAL PLANT
Ethanol	Analytical purity	BEIJING CHEMICAL PLANT
			Dipotassium hydrogen phosphate	Analytical purity	Tianjin City Fuchen chemical reagent plant
Methanol	HPLC grade	Fisher

(3) Laboratory apparatus

TABLE 2 Main Instrument of the experiment

(4) 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 future use.

(2) Experimental method

(1) Respectively picking up single strains of two cultured commercial acid-reducing yeasts (No. 15#, no. 16 #) and abnormal Wickham yeast W.anomalus WTB20042303, carrying out shake flask on a shaking table for 16-24h to ensure the purity and the activity of the test tubes, taking out the test tubes, removing supernatant, adding water for cleaning, centrifuging for 3 times, and finally adding 1ml of water for constant volume to prepare bacterial 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 × 10 ⁶ CFU/mL。

(2) mu.L of the above-mentioned acid-addition medium (citric acid medium and tartaric acid medium) and 100. Mu.L of the bacterial suspension were added to a 96-well plate, and each strain was divided into three groups. And scanning the absorbance under 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 picking out single strains of the cultured 3 strains of bacteria, dropping the single strains into a test tube filled with 5mLYPD culture medium, shaking the test tube on a shaking table for 16-24h to ensure the purity and the activity of the single strains of bacteria, taking out the test tube, removing supernatant, adding water, centrifuging for 3 times, finally adding 1ml of water to fix the volume, preparing bacterial suspension, and counting blood cells. Adding 3 strains of yeast into corresponding triangular flasks respectively to make the final concentration of yeast be 10 ⁶ And (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: agi nut 5TC-C18 (250 x 4.6 mm), using diammonium phosphate with pH of 2.4 as mobile phase, column temperature of 45 ℃, flow rate of 0.8ml/min, sample injection amount of 20 muL, respectively centrifuging and diluting samples of 48h and 96h fermentation of culture medium containing citric acid and tartaric acid and organic acid culture medium added with 3 strains of acid-reducing yeast, 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 the organic acid standard substance with ultrapure water to prepare a mother solution with the concentration of 10mg/ml, diluting the organic acid standard substance with a mobile phase to obtain a 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

(1) The acid reducing 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 experiment ₅₉₀ Increase in value

TABLE 4 relative OD in tartaric acid at the beginning of the experiment ₅₉₀ Increase value

Table 5 contents of citric acid and tartaric acid

As can be seen from table 3, the addition of the p.kluyveri WTB20042303 strain to citric acid medium (citric acid as the sole carbon source) increased the absorbance by 1.34 at 72h of culture. When the strain P.lactophila BY35 was added to the citric acid medium, the absorbance increased only BY about 1.2 at 72 hours of culture. As can be seen from table 4, the absorbance increased BY about 1.0 after 48 hours of adding the p.kluyveri WTB20042303 strain to the tartaric acid medium (tartaric acid is the only carbon source), and the absorbance increased BY only 0.3 for p.cactophila BY 35; after 72h of addition of the P.kluyveri WTB20042303 strain, the absorbance of the P.ctophila BY35 strain is increased by 1.24, and the absorbance of the P.ctophila BY35 strain is only increased by 0.65. As can be seen from Table 5, the degradation of citric acid was substantially completed in 72 hours for P.kluyveri WTB20042303 and P.cactophila BY35 (the preservation strain number is CGMCC No. 14909), and the acid reduction rates were 88.92% and 3.83%, respectively; the deacidification rate of the tartaric acid is 44.91 percent and 39.93 percent respectively.

Therefore, the ability of the P.kluyveri WTB20042303 screened BY the invention to grow, reproduce and utilize citric acid in citric acid is obviously higher than that of the known patent strain P.cactophila BY35, and the utilization rate of citric acid is higher than that of tartaric acid.

Effect of abnormal Wilm's yeast W.anomallus WTB20042303 on organic acids, active substances and antioxidant levels in blueberry mash

(1) Experimental Material

(1) The strain is as follows: abnormal Wilm yeast W.anomallus WTB20042303 and Pichia Capabilis BY35 (the existing strain and the preserved strain number are CGMCC No. 14909)

Blueberry: provided by a sunshine good gift winery 2019 year 1 month 6 days, and the variety is Beiluo

(2) Drugs and reagents

TABLE 6 drugs and reagents

(3) Laboratory apparatus

TABLE 7 main apparatus for experiment

Name of the instrument	Type number
		Ultraviolet spectrophotometer	T6-New world
PH meter	PHS-3D
		Analytical balance	BP3100S
Saccharimeter	PR-201
		Enzyme-linked immunosorbent assay (ELISA) instrument	Molecular Devices SpectraMax 190

(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 abnormal yeast W.anomallus WTB20042303 and P.cactophila BY35 (the existing strain and the preserved strain are CGMCC No. 14909) into 2 fermentation tanks, wherein the bacterial adding amount is 1 × 10 ⁶ CFU/mL, control group without any acid reducing yeast. Adding Saccharomyces cerevisiae at day 3, with concentration of 1 × 10 ⁶ CFU/mL, sugar addition 120g/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 determination: 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 L5% phenol solution +2.5mL concentrated sulfuric acid, mixing for 5min, taking out and cooling to room temperature, and measuring light absorption value 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. colorimetric of total phenols with phosphophenolic acids reagents [ J ]. American Journal of Enlogy and Viticulture,1965, 16. The content of total flavonoids is determined by methods of Wang you Yan, wang Yongli, zhao Rubia, etc. the extraction conditions of antioxidant and free radical scavenging active substances in Sophora flower are studied [ J ] food industry science, 2009 (12): 130-132 ], rutin is used as a standard curve, and the content of total phenols in mash is converted into the content of rutin in each gram of sample. The anthocyanin content is determined by referring to 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 with a syringe and tested in a liquid phase vial under optimal conditions.

Sample pretreatment: the samples were first diluted 20 times with ultrapure water, tested in the load, and then diluted and tested in 48h, 96h media, respectively.

Liquid phase conditions: a chromatographic column: agilent 5TC-C18 (250X 4.6 mm), 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 organic acid culture medium after adding 2 strains of acid-reducing yeast 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

(1) Variation of citric acid content

The citric acid content in the blueberry juice is higher and is 6.81g/L at the beginning of fermentation, and no citric acid is detected in the blueberry wine added with the P.kluyveri WTB20042303 and the P.cactophila BY35 (the existing strain and the preserved strain have the serial number of CGMCC No. 14909) strains at the end of fermentation.

Therefore, the yeast W.anomalus WTB20042303 has strong degradation capability on citric acid in the blueberry wine.

(2) Change in lactic acid content

The lactic acid content at the beginning of the fermentation was very low, only 0.08g/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 0.94g/L. Specific results are shown in table 8:

TABLE 8

In addition, when the yeast strain screened by the invention is added, the content of lactic acid at the fermentation end point is reduced by 31.91 percent relative to the control group. And at the end of fermentation, the content of lactic acid in the blueberry fruit wine is lower than that of the blueberry fruit wine added with P.

(3) 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 Phila BY35 is added into the blueberry mash, the content of formic acid is increased, and it is supposed that other organic acids are decomposed to generate partial formic acid. The W.anomallus WTB20042303 screened by the invention has the capability of degrading formic acid, the formic acid content is 0.38g/L at the fermentation end point, and is reduced by 7.32 percent compared with a control group.

In addition, the addition of the acid reducing yeast screened from the hawthorn wine mash and the persimmon wine mash also resulted in an increase in the formic acid content at the end of the fermentation. Therefore, compared with other strains, the W.anomalus WTB20042303 strain has excellent formic acid reduction capability.

(4) 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, the saccharomyces cerevisiae can generate a certain amount of acetic acid in the fermentation process, and the acetic acid content of a control group reaches 14.79g/L at the end of fermentation. Thus, the saccharomyces cerevisiae can easily generate acetic acid in the fermentation process. The results are shown in Table 11.

TABLE 10

After the W.anomallus WTB20042303 is added, the content of acetic acid is reduced BY 6.29 percent compared with a control group, but the content of the acetic acid in mash is slightly increased BY the patent strain P.cactophila BY35.

(5) Change in 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 0.67g/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 specific results are shown in Table 11.

TABLE 11

In addition, when the screened yeast strains of the invention are added, the succinic acid content of the fermentation end point is reduced by 11.94 percent relative to the control. And the succinic acid content of the blueberry wine added with the P.cactophila BY35 strain is increased BY about 10 percent relative to that of a control group at the end of fermentation.

(6) Influence of kluyveri WTB20042303 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 120.29mg/L.

TABLE 12

Compared with a control group, after different acid-reducing yeasts are added into the blueberry fruit wine, the total phenol content in the fruit wine can be increased, and after the P.kluyveri WTB20042303 is added, the total phenol content at the end of fermentation is 1.21mg/g, the total flavone content is 1.09mg/g, the anthocyanin content is 128.50mg/L, the total phenol content is increased by 7.08 percent, the total flavone content is increased by 3.80 percent, and the anthocyanin content is increased by 6.83 percent. The content of total phenols of the blueberry fruit wine added with P.cactophila BY35 is also increased but is increased BY less than P.kluyveri WTB20042303, the total flavonoids are hardly changed relative to a control group, and the anthocyanin is increased but is increased BY less than P.kluyveri WTB20042303 relative to a control group.

(7) Influence of Kluyveri WTB20042303 on antioxidant level of blueberry fruit wine

Watch 13

Compared with a control group, after pichia kluyveri p.kluyveri WTB20042303 and p.cactophila BY35 (the existing strain and the preserved strain number is CGMCC No. 14909) are added into the blueberry fruit wine, FRAP of the sterilization strain p.cactophila BY35 is slightly reduced, and each antioxidant index of all strains is increased. After the P.kluyveri WTB20042303 is added, the DPPH free radical scavenging capacity is improved BY 7.61 percent, the total antioxidant capacity of FRAP is improved BY 9.76 percent, the ABTS free radical scavenging capacity is improved BY 10.42 percent, and the total reducing power is improved BY 5.85 percent and is superior to P.cactophila BY35 (the number of the existing strain and the number of the preserved strain are CGMCC No. 14909). The Kluyveri WTB20042303 has an obvious effect of improving the antioxidant level of the blueberry fruit wine.

(8) Effect of anomalus WTB20042303 on pH in 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 12.92, and SSC is 6.87 degrees Bx. The pH of the control at the end of the fermentation was 3.61. After the addition of Hanm's Exo Weikemei yeast W.anomallus WTB20042303, the fermentation end point pH was 3.90. The W.anomalus WTB20042303 disclosed by the invention has an obvious acid reduction effect.

Biocontrol effect of abnormal yeast Wikazakholdi W.anomallus WTB20042303 on fresh fruits

(1) Experimental Material

(1) The strain is as follows: yeast: anomalus WTB20042303 and P.cactophila BY35 (preservation number CGMCC No. 14906)

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

Fruit: grape, purchased from New places in Beijing

(2) Main instruments of the experiment are shown in Table 2

(2) Experimental methods

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

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

(2) And (3) collecting thalli: centrifuging, removing culture medium, washing with 1mL sterile water (blowing with pipette or shaking, mixing well), centrifuging at 12000r/min,4 deg.C, and 2 min. This operation was repeated three times. After the water is removed and the thalli are left, 1mL of sterile water is added and mixed evenly, and the mixture is diluted to a proper multiple.

(3) Counting blood corpuscle plates: cover the glass slide first, add 10 uL bacterial suspension in the upper chamber and lower chamber separately, find the square with 10 times of mirror first, then change 40 times of mirror to count. After counting, diluting the suspension into a suspension with a target concentration.

And 2, step: preparing 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 × 10 ⁴ Spore suspension at CFU/mL concentration.

And 3, step 3: fruit preparation

The puncture method comprises the following steps: selecting bought grapes, independently placing the grapes with bruise and damage, cleaning the selected good grapes with consistent maturity and size with clear water, treating the fruits with sodium hypochlorite aqueous solution (0.5%) for 5min, placing the fruits into a cleaned box, pricking 15 grapes in each box, after the surface moisture of the grapes is dried, pricking 1 hole in each fruit with a pricking needle, and burning the inoculating needle one box at a time. 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 added ⁴ CFU/mL spore suspension, control group with 20. Mu.L of pathogen, and in the case of casing, two water-wetted toilet paper rolls were placed.

The soaking method comprises the following steps: putting fruit into 1 × 10 ⁶ Soaking the strain in CFU/mL bacterial suspension for 30 seconds, taking out the strain, and air-drying the strain; putting into a fresh-keeping box, sealing, and storing at room temperature.

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: rotting rate (%) = rotted fruit number/total fruit number × 100%. The diameter of the lesion is measured by the cross method in mm.

(3) Results of the experiment

The incidence rate of grape inoculated with Botrytis cinerea (B. Cinerea) is counted, and the concentration of biocontrol bacteria is 1 × 10 ⁶ CFU/mL, P.cactophila BY35 (existing strain, preservation number CGMCC No. 14909) as positive control, and difference of disease occurrence between different treatmentsThe comparison is remarkable. The incidence of grapes inoculated with the P.kluyveri WTB20042303 strain is 36.50%; under the condition of the same concentration of biocontrol bacteria, the morbidity of the existing biocontrol bacteria P.cactophila BY35 (the existing strain with the preservation number of CGMCC No. 14909) is 71.11 percent. 1X 10 ⁶ The morbidity of grapes soaked BY the strain P.kluyveri WTB20042303 after being soaked in the CFU/mL bacterial suspension is 40.00 percent, and the morbidity of grapes soaked BY the strain P.ctophila BY35 is 60.00 percent.

The comparison shows that the inhibition effect of the pichia kluyveri p.kluyveri WTB20042303 on botrytis cinerea (b.cinerea) is stronger than that of a biocontrol bacterium p.cactophila BY35 (the existing strain, the preservation number is CGMCC No. 14909). When the concentration of the biocontrol bacteria is increased to 1 x 10 ⁸ The biocontrol effect of the pichia pastoris P.kluyveri WTB20042303 bacterial suspension is not obviously increased (the morbidity is 26.67%) at the time of CFU/mL. Therefore, from the economical point of view, when fresh fruits are preserved using the suspension of the Pichia pastoris P.kluyveri WTB20042303 of the present invention, it is preferable to set the concentration at 1X 10 ⁶ CFU/mL。

Based on the above description, it can be known that the abnormal wilk hank yeast w.anomallus WTB20042303 (CGMCC No. 19723) has uniqueness in degrading citric acid, tartaric acid, lactic acid, formic acid, succinic acid, acetic acid and the like in fruit wine, compared with other acid-reducing yeasts, and has very 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 abnormal yeast Wickham W.anomallus WTB20042303 is added before saccharomyces cerevisiae is added, the alcoholic strength of all treatments at the fermentation end point is 12.92, SSC is 6.87 degrees Bx, the pH value at the fermentation end point is higher and is 3.90, the anthocyanin content in the fruit wine is increased, and the total phenol content and the total ketone content are not obviously changed.

In addition, the abnormal hank yeast Wickingw.anomalus WTB20042303 is proved to have good inhibition effect on botrytis cinerea.

Therefore, the abnormal Weikeham yeast W.anomallus WTB20042303 can not only prevent the fresh fruits from being rotted and deteriorated after the fresh fruits are picked and ensure the freshness of the fresh fruits, but also 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 and the normal fermentation process of the saccharomyces cerevisiae, can play a role in reducing acid of vast varieties of organic acids in the fruit wine and improve the taste and the quality of the fruit wine (the fruit wine with high acidity is turbid), is a preferable strain which can be applied to the whole process of the fruit wine preparation process, and can still play the role in reducing acid of the residual strains in the fermentation process even if the biocontrol bacterium suspension sprayed during the fresh-keeping storage of the fresh fruits is not cleaned.

Claims

1. A strain of abnormal Wickerhamomyces anomalus is deposited in China general microbiological culture Collection center in 24.04.2020, with the preservation number of CGMCC No.19723.

2. Use of the abnormal Hanover yeast according to claim 1 for post-harvest storage and preservation of fruit or deacidification of wine.

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: the method is characterized in that the abnormal Hanm yeast Velcro as the claim 1 is prepared into a bacterial suspension, and the bacterial suspension is used for spraying or dipping the fresh fruits.

5. The fresh-keeping method of fresh fruit according to claim 4, characterized in that: activating the abnormal Hanm's yeast of claim 1, fermenting in YPD liquid culture medium, centrifuging to obtain thallus, washing with sterile water to remove culturePost-basal formulation to a concentration of 1X 10 ⁶ CFU/mL~1×10 ⁸ CFU/mL bacterial suspension; putting fruits into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a preservation box, sealing, and storing at normal 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 acid-reducing fermentation method, which is to inoculate the abnormal yeast Wickham according to claim 1 before inoculating the saccharomyces cerevisiae in fruit juice, and then to perform fermentation treatment.

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 fruit wine acid-reducing fermentation method according to claim 7, characterized in that: crushing fresh blueberries, adjusting the components to obtain blueberry juice, adding the abnormal Wickham yeast into the blueberry juice, adding saccharomyces cerevisiae after 2-4 days, and performing processes such as sugar degree adjustment, primary fermentation, filtration, after-fermentation and the like.

10. The fruit wine acid-reducing fermentation method according to claim 7, characterized in that: the addition amount of the abnormal yeast is 1 × 10 ⁶ CFU/mL。