CN112226374B

CN112226374B - Pichia kudriavzevii yeast for green production of fruit wine in whole process and application thereof

Info

Publication number: CN112226374B
Application number: CN202010525909.4A
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: CN112226374A

Abstract

The invention relates to a Pichia kudriavzevii (Pichia kudriavzevii) strain, which is deposited in China general microbiological culture Collection center (CGMCC) at 24 days 4.2020, with the collection number of CGMCC No.19721 and is named as Pichia kudriavzevii WTB20042301. The strain can utilize citric acid and tartaric acid, has the degradation capability on citric acid, lactic acid, formic acid, succinic acid and acetic 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 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 deacidification and fermentation of fruit wine, and is an optimal strain for green production of the fruit wine in the whole process.

Description

Pichia kudriavzevii yeast for green production of fruit wine in whole process and application thereof

Technical Field

The invention relates to a functional yeast strain, in particular to pichia kudriavzevii used 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 grape wine is popular in recent years because blueberries contain rich anthocyanin which can resist free radicals and has the function of delaying aging.

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, etc. 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 products 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 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 pichia kudriavzevii 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 bactericides to prevent and control postharvest diseases of the fruits and keep 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, 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 the 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 WL nutrient agar culture 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 acidity reducing capability to alcohol concentration, and the strain is identified to be Pichia kudriavzevii (Pichia kudriavzevii), is named as Pichia kudriavzevii WTB20042301 in classification, and is preserved in China general microbiological culture Collection center in 24 months in 2020 year 4, with the preservation number of CGMCC No.19721.

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

the invention also relates to application of the Pichia kudriavzevii strain P.kudriavzevii WTB20042301 in storage and preservation of picked fruits or deacidification of fruit wine. The method specifically comprises the following steps:

the first scheme is as follows: a fresh fruit preservation method is characterized in that Pichia kudriavzevii P.kudriavzevii WTB20042301 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 Pichia kudriavzevii WTB20042301, fermenting and culturing in YPD liquid culture medium, centrifuging to obtain thallus, washing with sterile water to remove culture medium, and making into 1 × 10 ⁶ CFU/mL～1×10 ⁸ CFU/mL of 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 to inoculate Pichia kudriavzevii WTB20042301 before inoculating Saccharomyces cerevisiae into fruit juice, and then to perform fermentation treatment.

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 pichia dellivialis p. Kudriavzevii WTB20042301 into the blueberry juice, adding saccharomyces cerevisiae after 2-4 days, and performing sugar degree adjustment, primary fermentation, filtration and after-fermentation to obtain the blueberry juice. The delayed addition of Saccharomyces cerevisiae can make the Pichia kudriavzevii WTB20042301 added before survive in large amount and degrade the organic acid, such as citric acid, malic acid, etc. in fruit juice. Sugar is added later to avoid losing the acid reducing effect due to preferential utilization of sugar by pichia kudriavzevii WTB20042301.

Preferably, the bacterial adding amount of the Pichia kudriavzevii WTB20042301 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 pichia kudriavzevii WTB20042301 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 invention has the beneficial effects that:

(1) According to the screened Pichia kudriavzevii P.kudriavzevii WTB20042301, after the Pichia kudriavzevii P.kudriavzevii WTB20042301 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 91.48 percent, so that the Pichia kudriavzevii P.kudriavzevii can utilize 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 44.91 percent, 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 of saccharomyces cerevisiae and pichia kudriavzevii WTB20042301 are added at the same time, the content of lactic acid is reduced by 64.55%, the content of formic acid is reduced by 42.45%, the content of acetic acid is reduced by 45.04%, and the content of succinic acid is reduced by 27.24% at the end point of fermentation. Therefore, the screened Pichia kudriavzevii WTB20042301 has the capacity of degrading citric acid, lactic acid, formic acid, acetic acid and succinic acid in fruit wine, wherein the capacity of degrading citric acid is particularly remarkable.

The Pichia kudriavzevii P.kudriavzevii WTB20042301 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 stabilize the acidity after bottling), and improve the quality of the wine.

(2) The screened Pichia kudriavzevii P. Kudriavzevii WTB20042301 is added into blueberry juice fermentation liquor, if the blueberry juice fermentation liquor only added with saccharomyces cerevisiae is taken as a reference, the total phenols in the blueberry fruit wine are increased by 8.75 percent, the total flavonoids are increased by 8.74 percent, and the content of anthocyanin is increased by 7.54 percent when the blueberry juice fermentation liquor added with saccharomyces cerevisiae and Pichia kudriavzevii WTB20042301 is at the fermentation end point. Phenols and anthocyanins are important active substances in blueberry fruit wine, and have activities of resisting oxidation, scavenging free radicals, resisting variation and preventing canceration. The DPPH free radical scavenging capacity is improved by 8.54 percent, the total oxidation resistance of FRAP is improved by 10.13 percent, the ABTS free radical scavenging capacity is improved by 8.69 percent, the total reducing power is improved by 8.99 percent, and the improvement of the oxidation resistance of the fruit wine effectively prevents aging and pathological changes of organisms caused by aging. Therefore, if the pichia kudriavzevii wtb20042301 screened by the method is applied to brewing of blueberry wine, the quality of the blueberry wine can be greatly improved.

(3) 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 pichia kudriavzevii WTB20042301 is added. Therefore, the blueberry juice can be normally fermented after the Pichia kudriavzevii WTB20042301 is added, the alcoholic strength at the end point of fermentation is 12.13 percent, and SSC is 7.03-degree Bx. If the blueberry juice fermentation broth only added with saccharomyces cerevisiae is taken as a reference (the pH value at the end of fermentation is = 3.61), and the pH value at the end of fermentation is 3.89 after the pichia kudriavzevii WTB20042301 and the saccharomyces cerevisiae are added, the pichia kudriavzevii WTB20042301 of the pichia kudriavzevii provided by the invention is further proved to have obvious acid reducing effect.

(4) The incidence of grapes inoculated with botrytis cinerea in advance is counted by taking the pichia kluyveriavzevii wtb20042301 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, so that the yeast strain has better inhibiting effect on the botrytis cinerea compared with the existing biocontrol bacteria.

Therefore, the Pichia kudriavzevii P.kudriavzevii WTB20042301 can not only prevent the fresh fruits from rotting and deteriorating 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 wine in the later period, the role in reducing acid is not influenced by the alcohol concentration, the normal fermentation process of the saccharomyces cerevisiae is not influenced, the acid reducing effect can be achieved on most kinds of organic acids in the fruit wine, the taste and the quality of the fruit wine are improved (the fruit wine with high acidity is turbid), the Pichia kudriavzevii WTB is an optimal strain which can be applied to the whole process of the fruit wine preparation process, and the acid reducing effect can still be achieved in the fermentation process even if part of biocontrol bacterium suspension sprayed during the fresh-keeping storage of the fresh fruits remains. In addition, the content of active substances such as phenols, anthocyanin and the like 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 pichia kudriavzevii WTB20042301 screened in the present invention.

FIG. 2 is a PCR electrophoresis detection map of total DNA of Pichia kudriavzevii WTB20042301 screened in the present invention.

Fig. 3 is the result of the evolutionary tree analysis of pichia kudriavzevii WTB20042301 screened by the present invention.

Detailed Description

For a better understanding of the present invention, reference will now be made in detail to the present embodiments of the invention, which are illustrated in the accompanying drawings.

1. 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. Selecting a single colony on a WL nutrient agar plate for separation and purification, culturing for 48h at the constant temperature of 26 ℃, selecting a suspected colony according to morphological characteristics of the saccharomycetes, and obtaining a purified strain through scribing and separation on a PDA plate.

Experiments prove that the Pichia kudriavzevii P.kudriavzevii WTB20042301 claimed by the invention has better comprehensive capacities in acid reduction, biocontrol and fruit wine quality improvement than other 6 P.kudriavzevii strains which are subjected to an acid reduction experiment, a biocontrol experiment and a fermentation experiment simultaneously.

Biological Properties of Pichia kudriavzevii WTB20042301

(1) Morphological characteristics

Referring to FIG. 1A and FIG. 1B, A represents the colony morphology of cultured 48h at 26 deg.C on WL nutrient agar (fungus culture medium, 75gWL nutrient agar culture medium dissolved in 1000mL water, subpackaged, sterilized at 121 deg.C for 20 min), with scale of 1cm; b represents the cell morphology observed under a 40-fold microscope (10 μm scale). According to observation, the colony has irregular shape, milk white, flat and rough surface; the cells were observed under a microscope to be sausage-shaped and about 10 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 using NL-1 and NL-4 as primers, and performing agarose gel electrophoresis analysis (electrophoresis conditions: 180V, 30min), the result is shown in FIG. 2; and (4) detecting that the size of the target fragment is correct, and carrying out 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 pichia kudriavzevii (KX 118628.1) on the same branch with 100% confidence and very close affinity (< 0.05) to identify the strain as pichia kudriavzevii.

2. Acid reducing properties of Pichia kudriavzevii WTB20042301

(one) using citric acid and tartaric acid as the only carbon source of the culture medium, test the acid reducing ability of Pichia kudriavzevii WTB20042301

(1) Experimental materials

(1) Strain: pichia kudriavzevii WTBB 20042301 (from blueberry wine), P.kudriavzevii WTBa2 (from hawthorn wine), P.kudriavzevii WTBa3 (from grape wine), P.kudriavzevii WTBa4 (from grape wine), P.kudriavzevii WTBa5 (from grape wine), P.kudriavzevii WTBa6 (from hawthorn wine), P.kudriavzevii WTBa7 (from hawthorn wine), P.cactophila (preservation number CGMCC No. 14909)

(2) Drugs and reagents

TABLE 1 drugs and reagents

(3) Laboratory apparatus

TABLE 2 Main Instrument of the experiment

(4) Culture medium

Citric acid culture 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 method

(1) Respectively picking up single bacteria to a test tube filled with 5ml of YPD culture medium, carrying out shaking for 16-24h on a shaking table to ensure the activity and the purity, taking out, removing supernatant, adding water, cleaning, centrifuging for 3 times, and finally adding 1ml of water to fix the volume to prepare a 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-added 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 made into three replicates. 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. 3 plants were inoculatedRespectively adding the yeast into corresponding triangular flasks 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: 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 3 strains of acid-reducing yeast added organic acid culture medium fermented 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 times with ultrapure water, loaded for testing, and then diluted and tested for 48h and 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

(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 is clear from table 3, when the p.kudriavzevii WTB20042301 strain was added to a citric acid medium (citric acid was used as a sole carbon source), the absorbance increased by 1.6 or more at 48 hours and 72 hours of culture, and only the p.kudriavzevii WTBA6 and the p.kudriavzevii WTBA7 increased the absorbance by 1.6 or more at 72 hours, and the other strains were 1.5 or less at each time point. Cactophila BY35 increased BY 0.9 and 1.2 at 48h and 72h, respectively.

As is clear from table 4, when the p.kudriavzevii WTB20042301 strain was added to a tartaric acid medium (tartaric acid was used as the sole carbon source), the absorbance increased by 0.8 or more at 48 hours and 72 hours of culture. While other p.kudriavzevii strains were below 0.6 and appeared in 72h increments of less than 48h. Cactophila BY35 increased BY 0.3 and 0.65 at 48h and 72h, respectively.

From table 5, it can be seen that p.kudriavzevii wtb20042301 has reached 91.48% of citric acid degradation in 72h, and other strains are below 90%; the degradation of tartaric acid already reaches 44.91%, and other strains are below 40%.

Thus, the ability of the p.kudriavzevii WTB20042301 screened by the invention to grow, reproduce and utilize citric acid in citric acid is significantly higher than that of p.kudriavzevii WTBA2 (derived from hawthorn fruit wine), p.kudriavzevii WTBA3 (derived from grape fruit wine), p.kudriavzevii WTBA4 (derived from grape fruit wine), p.kudriavzevii WTBA5 (derived from grape fruit wine), p.kudriavzevii WTBA6 (derived from hawthorn fruit wine), p.kudriavzevii WTBA7 (derived from hawthorn fruit wine) and p.cactophila (accession number of CGMCC No. 14909); and the utilization rate of citric acid is higher than that of tartaric acid.

Influence of Pichia kudriavzevii WTB20042301 on organic acids and active substances in blueberry mash

(1) Experimental Material

(1) Strain: pichia kudriavzevii WTB20042301 and Pichia Capphila (accession number CGMCC No. 14909)

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

(3) Laboratory apparatus

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. Activated Pichia kudriavzevii WTBb 20042301P. Kudriavzevii WTBa2 (from wine of hawthorn), P. Kudriavzevii WTBa3 (from wine of grape), P. Kudriavzevii WTBa4 (from wine of grape), P. Kudriavzevii WTBa5 (from wine of grape), P. Kudriavzevii WTBa6 (from wine of hawthorn), P. Kudriavzevii WTBa7 (from wine of hawthorn), and P. Cactophila (deposited strain number CGMCC No. 14909) were added to each fermentation tank, and the bacterial addition amount was 1X 10 ⁶ CFU/mL, control 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 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 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 described in Singleton [ Singleton V.L., rossi J.A.colorimetry of total phenolics with phospho-molar phosphoric acid reagents [ J ]. American Journal of Enlogology and Viticulture,1965, 16. The total flavone content is researched according to the extraction conditions of antioxidant and free radical scavenging active substances in sophora flower [ J ] research in food industry science and technology, 2009 (12): 130-132 ], according to the method of Wang you Yan, wang you Sheng, zhao Qian, and the like, rutin is taken as a standard curve, and the total phenol content in mash is converted into the rutin content 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 by loading, and then diluted and tested for 48h, 96h of culture medium, 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 was high at 6.81g/L at the beginning of fermentation, and at the end of fermentation, no citric acid was detected in any of the other treatment groups except the control group (without the addition of the acid-reducing yeast).

Thus, the yeast P.kudriavzevii WTB20042301 has strong capacity of degrading citric acid in 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 selected yeast strain of the present invention was added, the content of lactic acid was reduced by 64.55% relative to the control fermentation end point. While p.kudriavzevii wtba2 and p.kudriavzevii wtba3 are about 20% higher than the control, the other strains are reduced, but the effects are not as good as those of the strain p.kudriavzevii wtb20042301, and the lactic acid reducing ability of the yeast strain of the present invention is most remarkable compared with other strains.

(3) Variation in 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 P.kudriavzevii WTB20042301 screened by the invention has the capability of degrading formic acid, the formic acid content is 0.24g/L at the fermentation end point, and the formic acid content is reduced by 42.45 percent compared with the control group. The end points of the fermentation were elevated in formic acid, and p.cappillava BY35, p.kudriavzevii wtba2, p.kudriavzevii wtba3, p.kudriavzevii wtba4 and p.kudriavzevii wtba5 were almost no different from the control. Compared with other strains, the yeast strain has the most remarkable formic acid reducing 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, 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

At the end of the fermentation, the acetic acid content of p.cactophila BY35, p.kudriavzevii wtba2, p.kudriavzevii wtba3 was elevated relative to the control; after the P.kudriavzeviii WTB20042301 is added, the content of acetic acid is reduced by 45.04 percent compared with a control group, and the acetic acid is the most excellent strain of all acid-reducing yeasts.

(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 results are shown in Table 10.

TABLE 11

In addition to the strain p. Kudriavzevii wtb20042301, which is 27.24% lower than the control, succinic acid was reduced by the addition of the acid-reducing yeast, other strains did not only degrade succinic acid but also produce a part of succinic acid.

And the other substances can be metabolized BY P.ctophila BY35 to generate a small amount of succinic acid, and only BY adding the blueberry fermentation liquid of the invention P.kudriavzevii WTB20042301, the succinic acid is reduced BY 27.24 percent compared with a control group at the end point of the fermentation.

Therefore, different pichia kudriavzevii have different acid reducing abilities, and different strains of the same yeast have obvious difference in acid reducing abilities.

(6) Influence of kudriavzevii WTB20042301 on active substances in blueberry 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 the control, the contents of total phenols, total flavones and anthocyanin of the blueberry wine only added with the strain P.kudriavzevii WTB20042301 are improved, and the total phenols are improved by 8.75 percent; the total flavone is increased by 8.74 percent; the anthocyanin is increased by 7.54 percent.

(7) Effect of kudriavzevii wtb20042302 on antioxidant levels of blueberry wine table 13

Compared with the blueberry wine only added with the strain P.kudriavzevii WTB20042301, all the oxidation resistance indexes are improved, the DPPH is improved by 8.54 percent, the FRAP is improved by 10.13 percent, the ABTS is improved by 8.69 percent, and the total reducing power is improved by 8.99 percent.

(8) Influence of kudriavzevii WTB20042301 on pH in blueberry 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 about 12%, and SSC is about 7-degree B. The pH of the control at the end of fermentation was 3.61. After addition of pichia kudriavzevii WTB20042301, the fermentation end point pH was 3.89. The P.kudriavzevii WTB20042301 has obvious acid reduction effect.

Biocontrol effect of (tetra) Pichia kudriavzevii WTB20042301 on fresh fruits

(1) Experimental Material

(1) The strain is as follows: yeast: pichia kudriavzevii WTBB 20042301 (from blueberry wine), P.kudriavzevii WTBa2 (from hawthorn wine), P.kudriavzevii WTBa3 (from grape wine), P.kudriavzevii WTBa4 (from grape wine), P.kudriavzevii WTBa5 (from grape wine), P.kudriavzevii WTBa6 (from hawthorn wine), P.kudriavzevii WTBa7 (from hawthorn wine), P.cactophila (preservation number CGMCC No. 14909)

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

Fruit: grape, purchased from Beijing New-onset

(2) Main instruments of the experiment, see 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 thallus is left, 1mL of sterile water is added and mixed evenly, and the mixture is diluted to a proper time.

(3) And (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. 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 × 10 ⁴ Spore suspension at CFU/mL concentration.

And step 3: fruit preparation

Pricking wound methods: selecting bought grapes, placing the bought grapes with bruise and damage independently, cleaning the selected good fruits with consistent maturity and size with clear water, treating 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 of each fruit with a pricking needle, and burning the inoculating needle one box at each 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 also added 20. Mu.L of pathogen, and in the case of framing, two groups of toilet paper balls wetted with water were placed in the frame.

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 preservation box, sealing, and storing at normal temperature.

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

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 a crisscross 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) is used as positive control, and the difference of the disease incidence between different treatments is obvious. The incidence of grapes inoculated with the p.kudriavzevii wtb20042301 strain was 42.22%; whereas the incidence of inoculation p.kudriavzevii wtba2 (from crataegus pinnatifida) was 48.89%, p.kudriavzevii wtba3 (from grapeseed wine) was 60.00%, p.kudriavzevii wtba4 (from grapeseed wine) was 63.00%, p.kudriavzevii wtba5 (from grapeseed wine) was 75.56%, p.kudriavzevii wtba6 (from crataegus pinnatifida) was 57.78%, and p.kudriavzevii wtba7 (from crataegus pinnatifida) was 82.22%. 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.

1×10 ⁶ The CFU/mL bacterial suspension was used for soaking, the incidence of grapes soaked with the P.kudriavzevii WTB20042302 strain was 30.00%, the incidence of grapes soaked with the P.sectorhiavzevii WTBB 20042302 strain was 60.00%, and the incidence of inoculated P.kudriavzevii WTBa2 (from Hawthorn fruit wine) was 63.00%, the incidence of P.kudriavzevii WTBa3 (from Hawthorn fruit wine) was 51.00%, the incidence of P.kudriavzevii WTBa4 (from Hawthorn fruit wine) was 40.00%, the incidence of P.kudriavzevii WTBa5 (from Hawthorn fruit wine) was 51.00%, the incidence of P.kudriavzevii WTBa6 (from Hawthorn fruit wine) was 51.00%, and the incidence of P.kudriavzevii WTBaB 7 (from Hawthorn fruit wine) was 72.00%.

From the comparison, it can be seen that pichia kudriavzevii WTB20042301 has a good inhibitory effect on botrytis cinerea (b.cinerea) in the early stage of disease onset. In addition, other Pichia kudriavzevii (P. Kudriavzevii) from hawthorn wine and wine were used in the experiment) The incidence was higher for each time period than for the control group. Thus, the different strains of the same yeast strain have great differences in biocontrol ability. Over time, the inhibition effect of the control group on the botrytis cinerea (B.cinerea) is gradually weakened, while the inhibition effect of the screened Pichia kudriavzevii P.kudriavzevii WTB20042301 bacterial suspension on the botrytis cinerea is stable, and the incidence rate is not increased after 144 hours. When the concentration of the biocontrol bacteria is increased to 1 x 10 ⁸ The biocontrol effect of the Pichia kudriavzevii WTB20042301 bacterial suspension is not obviously increased (the morbidity is 36.67%) when CFU/mL is adopted. Therefore, from the economical point of view, when fresh fruits are preserved using the Pichia kudriavzevii WTB20042301 suspension of the present invention, it is preferable to set the concentration at 1X 10 ⁶ CFU/mL。

Based on the foregoing description, pichia kudriavzevii p. Kudriavzevii WTB20042301 (CGMCC No. 19721) has uniqueness in degrading citric acid, lactic acid, formic acid, succinic 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 yeast strain is added, and the ethanol concentration has no obvious influence on the capability of utilizing organic acid by the yeast strain.

When the fruit wine, particularly the blueberry fruit wine is prepared, the Pichia kudriavzevii WTB20042301 is added before saccharomyces cerevisiae is added, the alcoholic strength of all treatments at the fermentation end point is about 12 percent, SSC is about 7 degrees Bx, the pH value at the fermentation end point is higher and is more than 3.8, the contents of total phenols and anthocyanin in the fruit wine are increased, and the content of total ketone is almost unchanged. Is the only P.kudriavzevii strain with all oxidation resistance indexes being increased at the fermentation end point of the fruit wine.

In addition, the Pichia kudriavzevii WTB20042301 has a good inhibition effect on botrytis cinerea.

Therefore, the Pichia kudriavzevii P.kudriavzevii WTB20042301 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 wine in the later period, the effect of reducing acid is not influenced by the alcohol concentration, the normal fermentation process of the saccharomyces cerevisiae is not influenced, the effect of reducing acid can be achieved on the vast variety of organic acids in the fruit wine, the taste and the quality of the fruit wine are improved (the fruit wine with high acidity is turbid), the Pichia kudriavzevii WTB is an optimal strain which can be applied to the whole process of the fruit wine preparation process, and the biocontrol bacterium suspension sprayed during the fresh-keeping storage of the fresh fruits can still play the effect of reducing acid even if the bacteria are not cleaned.

Claims

1. A Pichia kudriavzevii (Pichia kudriavzevii) is deposited in China general microbiological culture Collection center (CGMCC) at 24 months 4 in 2020 with the preservation number of CGMCC No.19721.

2. The use of pichia kudriavzevii, according to claim 1, in the storage and preservation of fruit after harvest or in the deacidification of fruit 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 in that: the pichia kudriavzevii of claim 1 is prepared into a 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 the Pichia kudriavzevii yeast of claim 1, fermenting and culturing in YPD liquid culture medium, centrifuging to obtain thallus, and making into 1 × 10 thallus ⁶ CFU/mL～1×10 ⁸ CFU/mL bacterial suspensionLiquid; 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, before inoculating Saccharomyces cerevisiae into fruit juice, inoculating the Pichia kudriavzevii yeast of claim 1, and then fermenting.

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 ingredients to obtain blueberry juice, adding the pichia kudriavzevii in claim 1 into the blueberry juice, adding saccharomyces cerevisiae after 2-4 days, and performing processes 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 Pichia kudriavzevii strain of claim 1 having a inoculum size of 1X 10 ⁶ CFU/mL。