CN116970500A

CN116970500A - Pichia kudriavzevii and application thereof

Info

Publication number: CN116970500A
Application number: CN202310866329.5A
Authority: CN
Inventors: 夏秀东; 戴意强; 许壮; 王道营; 徐为民; 王喆
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2023-07-14
Filing date: 2023-07-14
Publication date: 2023-10-31

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to pichia kudriavzevii and application thereof. Pichia kudriavzevii (Pichia kudriavzevii) has been deposited in China general microbiological culture collection center (CGMCC) with a accession number of 26178 and a strain number of NL33 in the year 2022, month 12 and 14. The strain can tolerate alcohol concentration of 4-12%vol and SO of 60-180mg/L ₂ 100-300g/L total sugar, pH 2.0-4.0An acid environment. Meanwhile, the strain can generate substances with unique mellow, rose, fruit and other flavors, and can endow the fruit wine and the milk beer with fresh and pleasant mouthfeel when the strain is used for fermenting the fruit wine or the milk beer. The method has wide application prospect in the field of food industry, can be applied to the production of fermented foods such as milk beer, fruit wine, dough and the like, starter and the like, and endows the products with better flavor.

Description

Pichia kudriavzevii and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to pichia kudriavzevii and application thereof.

Background

Pichia kudriavzevii (Pichia kudriavzevii), also known as Issatchenkia orientalis (Issatchenkia orientalis), is milky white in colony, thick in texture, flat in surface, and non-reflective. The thallus is elliptic or rod-shaped, and is germinated with false hypha. The pichia kudriavzevii not only has better acid resistance and high temperature resistance, but also can keep excellent fermentation performance under multiple pressure stresses. The research shows that the pichia kudriavzevii has stronger ester-producing and fragrance-producing capability, is an important microorganism with brewing core function, and the metabolite phenethyl alcohol of the pichia kudriavzevii can obviously inhibit the growth of other fungi in a fermentation system and has a regulating and controlling effect on colony succession in the ecology of the brewing microorganism. The pichia kudriavzevii also has stronger urea degradation capability, and can better reduce the precursor substance of the amino acid methyl ester, namely urea, in a fermentation system, thereby reducing the content of the amino acid methyl ester and ensuring the safety in the fermentation process. In addition, the pichia kudriavzevii can reduce the content of lactic acid in the fermentation system and adjust the acidity of the brewing microorganism environment, thereby promoting the fermentation system.

In the food industry, the Pichia kudriavzevii has been applied to white spirit fermentation, and has also been proved to have a good antibacterial effect in the fermentation process, but the Pichia kudriavzevii has little research on the aspects of fermenting milk beer, fruit wine, fermented dough, pickled Chinese cabbage and the like, and the screening of the Pichia kudriavzevii with good aroma-producing capability and sugar, acid and alcohol resistance has great significance and wide application prospect in the food industry.

Disclosure of Invention

The invention aims to solve the technical problem of providing a Pichia kudriavzevii strain aiming at the defects of the prior art.

The invention also solves the technical problem of providing application of the pichia kudriavzevii in the field of food industry.

In order to solve the technical problems, the invention adopts the following technical scheme:

pichia kudriavzevii Pichia kudriavzevii, classified and named: pichia kudriavzevii Pichia kudriavzevii, strain number NL33, deposited in the China general microbiological culture collection center, accession number: CGMCC No.26178, preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

The NL33 strain was characterized by its milky white colony, thick, flat surface, no reflection, elliptic or rod-like cell, bud, and pseudo hypha.

Molecular biological identification of the NL33 strain described above revealed that the sequence of the 26S rRNA D1/D2 gene region of NL33 strain and Pichia kudriavzevii AMC PK001 was as high as 98% similar in NCBI and that it was closest to the genetic relationship in the same branch of the developmental tree. In conclusion, the NL33 strain can be identified as a Pichia kudriavzevii (Pichia kudriavzevii) strain belonging to non-Saccharomyces cerevisiae.

Wherein, the nucleotide sequence of the 26S rDNA of the Pichia kudriavzevii is shown as SEQ ID No. 1.

The Pichia kudriavzevii can endure alcohol concentration of 4-12%vol and SO of 60-180mg/L ₂ An acidic environment having a total sugar concentration of 100-300g/L and a pH of 2.0-4.0; preferably, an alcohol concentration of 8% -12% vol, SO of 120-180mg/L is tolerated ₂ 200-300g/L of total sugar concentration, and an acidic environment with pH of 2.0-3.0。

Flocculation value (Flocculation value, flo) measurement is an important index of the brewing characteristics of the fruit wine, and the flocculation performance can evaluate the application potential of the saccharomycetes in the fermentation process. The lower the flocculation value, the better the flocculation performance. When the flocculation value (Flo) is less than 30%, high flocculation is obtained; the flocculation property is kept between 30% and 70%; more than 70% is low flocculability.

Flocculation property of the above Pichia kudriavzevii was measured, and it was found that the flocculation value (Flo) was lower than 30%, which was a high flocculation property.

beta-D-Glucosidase (beta-D-Glucosidase) is a hydrolase for catalyzing and hydrolyzing glycosidic bonds between hydrocarbon groups and glycosyl groups to generate aromatic substances and glucose, and the alias is amygdalase, gentiobinase or cellobiase, is an important hydrolase in a cellulase system, and has important significance for generating and releasing aroma substances of fruit wine.

The beta-D-glucosidase activity of the Pichia kudriavzevii is measured, and the beta-D-glucosidase activity of the Pichia kudriavzevii is found to reach 24.19 mu mol/g/min.

The application of the pichia kudriavzevii as described above in any of the following food industry fields is also within the scope of the present invention, in particular:

(1) The application of the pichia kudriavzevii in preparing fermented blackberry wine;

(2) The application of the pichia kudriavzevii in preparing fermented soybean milk beer;

(3) The application of the pichia kudriavzevii in preparing a starter;

in the application (1), the mixed bacteria of the pichia kudriavzevii and the saccharomyces cerevisiae FM-S-115 ferment blackberry juice, and the inoculation amount of the pichia kudriavzevii and the saccharomyces cerevisiae FM-S-115 in the blackberry juice is 3.75X10 ⁵ CFU/mL, fermentation conditions are: fermenting at 25deg.C for 6-8d.

Sensory evaluation of the fermented blackberry wine shows that the total score of the sensory evaluation is the fermented blackberry wine obtained by mixed fermentation of NL33 strain and FM-S-115 strain, and the secondary score of the fermented blackberry wine obtained by mixed fermentation of A1 strain and FM-S-115 strain is 96 and 91 respectively, which are obviously higher than those of other control groups, so that the fermented blackberry wine with better taste and brighter wine body can be obtained by fermentation of NL33 strain and A1 strain, but the comprehensive evaluation of the fermented blackberry wine obtained by mixed fermentation of NL33 strain and FM-S-115 strain is better.

The flavor substance detection of the fermented blackberry wine shows that 53 kinds of fermented blackberry wine obtained by mixed fermentation of NL33 strain and FM-S-115 strain are detected, the content of alcohols and esters is high, the variety of substances is rich, the aroma of the produced fruit wine is overflowed from the aroma of fruit, the sour and sweet are suitable, and the taste is good.

In the application (2), the micro-soymilk prepared from soybeans is fermented by mixing pichia kudriavzevii and lactobacillus paracasei ND1031, the adding ratio of the pichia kudriavzevii and the lactobacillus paracasei ND1031 in the micro-soymilk is 2:1, and the total inoculation amount is 1.75X10 ⁸ log CFU/g micro soy milk, fermentation conditions were: fermenting at 25-30deg.C for 15 hr, preferably at 28deg.C for 15 hr.

After preparing mixed bacteria fermented micro-soymilk, adding pectin, carrageenan, propylene glycol alginate and acid-resistant carboxymethyl cellulose with mass fractions of 0.1%, 0.2%, 0.4% and 0.6% into hot water at 90 ℃, stirring, mixing uniformly, cooling to obtain a compound stabilizer, adding 2 times of the compound stabilizer into the mixed bacteria fermented micro-soymilk, adding sucralose with mass fractions of 0.03%o into the mixed bacteria fermented micro-soymilk containing the compound stabilizer to obtain soymilk beer beverage, homogenizing at 55 ℃ under the conditions of a first stage homogenizing pressure of 35MPa and a second stage homogenizing pressure of 3.5MPa, and filling CO ₂ And (5) filling and sterilizing to obtain a finished product of the soymilk beer beverage.

The measurement of volatile substances and aroma of the soymilk beer shows that 52 flavor substances, mainly alcohols, esters, acids, aldehydes and the like, are identified in the soymilk beer. The higher alcohol is the product of amino acid or sugar metabolism in the process of yeast alcoholic fermentation, and most alcohols have higher threshold values, but can present bouquet or plant fresh scent, and can generate esterification reaction with organic acids to generate esters, and simultaneously form a very small amount of important flavor substances such as aldehyde, ketone and the like, thereby indirectly affecting the flavor. The alcohol substances contained in the soymilk beer are phenethyl alcohol, benzyl alcohol and 1-octen-3-ol, so the soymilk beer has rose, flower fragrance, honey fragrance and beany flavor; the milk beverage contains butyric acid, caproic acid, caprylic acid and n-capric acid, and has a lower threshold value, so that the milk beverage has stronger milk flavor under low mass concentration; contains aldehydes such as isoamyl butyrate, ethyl decanoate, allyl caproate, allyl heptanoate, diethyl malonate and pineapple ester, so that the beverage has fruit flavor and flower flavor, and has fresh and sweet overall flavor.

The organic acid in the soymilk beer is mainly lactic acid and citric acid. The soybean milk beer prepared by mixed fermentation of NL33 strain and ND1031 strain has highest lactic acid content of 2.152g/L and low lactic acid threshold, and is the main organic acid affecting the flavor of the milk beer. In addition, oxalic acid has astringency, citric acid is fresh and cool, lactic acid has soft sour taste, malic acid has mellow and long-lasting sour taste, and acetic acid has heavy bitter taste, so that the soymilk beer has rich and full flavor characteristics under the combined action of various organic acids.

Aroma measurement was performed on the above-described soybean milk beer, and aroma activity values (aroma activity value, OAV) of the detected flavor compounds were calculated and subjected to radar mapping. OAV refers to aroma compound mass concentration/threshold. As a result, it was found that a soybean milk beer produced by mixed fermentation of the NL33 strain and the ND1031 strain was characterized by a prominent sour and soft feel and a fresh and sour taste, and imparted a good taste and flavor to the soybean milk beer.

Sensory evaluation of the soybean milk beer shows that the soybean milk beer prepared by mixed fermentation of NL33 strain and ND1031 strain has optimal sweet and sour ratio, taste, smell and comprehensive flavor, the highest score is 94, and the soybean milk beer prepared by mixed fermentation of A1 strain and ND1031 strain is 88. It is explained that pichia kudriavzevii NL33 is preferred for fermenting soy milk beer.

In the application (3), the starter is obtained by inoculating pichia kudriavzevii into a PDB liquid culture medium to culture to obtain a fermentation broth, centrifugally collecting thalli of the fermentation broth, washing, adding a protective agent, and freeze-drying.

Specifically, the concentration of the thallus in the fermentation liquid is 10 ⁸ -10 ⁹ CFU/mL。

Specifically, the addition amount of the protective agent is 1/20-1/4 of the volume of the fermentation liquor;

Specifically, the formula of the protective agent is as follows: 6-12g of skim milk powder, 6-12g of sugar substances, 2-4g of glycerin and 100mL of distilled water, wherein the sugar substances are any one of sucrose, trehalose, lactose, maltose and glucose, and preferably trehalose.

The starter containing the pichia kudriavzevii can be applied to fermented flour products, fermented meat and the like in the field of foods.

The beneficial effects are that:

1. the pichia kudriavzevii NL33 can resist high sugar, high alcohol, high acid and high SO ₂ The concentration and other environments can be used for fermenting products with high sugar content and low pH value;

2. the pichia kudriavzevii NL33 disclosed by the invention can be used for high-yielding esters and alcohols, can be used for endowing products with good fragrance and obtaining alcoholic beverages with good taste without adding additional flavoring agents, and meanwhile, the metabolite phenethyl alcohol of the pichia kudriavzevii is used for improving the stability of the products;

3. the pichia kudriavzevii NL33 fermented soybean milk can improve the flavor of the soybean milk beer, can effectively solve the problem of poor taste of the soybean milk beer, generates unique fruit flavor, and improves the nutritional value of the soybean milk beer;

4. the pichia kudriavzevii NL33 can be used as a mixed fermentation agent of fruit wine, can be used for directly fermenting fruit juice with stronger acidity such as blueberries, blackberries and the like, does not need to adjust pH value, and has good taste, and the produced fruit wine has good bouquet and sweet and sour taste.

5. The pichia kudriavzevii NL33 of the present invention can be used as a starter for preparing fermented meat in the food field, thereby imparting a better flavor to meat.

6. The pichia kudriavzevii NL33 can be used as a starter for flour products in the field of foods, can increase the fluffiness of the flour products and improve the texture of the flour products.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 colony plate and microscopic morphology of Pichia kudriavzevii NL 33;

FIG. 2 Pichia kudriavzevii NL33 phylogenetic tree;

FIG. 3 Pichia kudriavzevii NL33 resistance to alcohol;

FIG. 4 Pichia kudriavzevii NL33 vs SO ₂ Tolerance;

FIG. 5 Pichia kudriavzevii NL33 is resistant to sugar;

FIG. 6 Pichia kudriavzevii NL33 is acid tolerant;

FIG. 7 is a aroma profile of aroma components in Pichia kudriavzevii NL33 soymilk beer;

FIG. 8 sensory evaluation of Pichia kudriavzevii NL33 soymilk beer;

FIG. 9 effect of saccharide on survival of Pichia kudriavzevii NL33 during lyophilization;

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

In the following examples, the medium formulation was as follows:

PDB liquid medium: 5g of potato soaked powder, 15g of glucose, 10g of peptone and 5g of sodium chloride, and sterilizing with distilled water to 1000mL and high-pressure steam at 121 ℃ for 20min.

WLN medium: 80 g of powder is weighed, added into 1000mL of distilled water, heated for dissolution, split charging, autoclaved for 20 minutes at 121 ℃, and cooled for standby.

PDA solid medium: 3g of potato soaked powder, 20g of glucose and 14g of agar, and sterilizing with distilled water to 1000mL and high-pressure steam at 121 ℃ for 20min.

Buffer solution with pH of 1.0: 0.2mol/L KCl solution and 0.2mol/L HCl solution were mixed at 25:67 Mixing the materials according to the ratio of (V/V) to obtain the buffer solution.

Buffer solution with pH of 4.5: 1mol/L NaAc, L mol/L HCl and distilled water were mixed at a ratio of 100:60:90 Mixing the materials according to the ratio of (V/V/V) to obtain the buffer solution.

In the following examples, pichia kudriavzevii NL33; the strains G6, G9, G11, P20, P21 and A1 are non-Saccharomyces cerevisiae (NSC).

In the following examples, the G6, G9, G11, P20, P21 and A1 strains are all derived from the academy of agricultural sciences of Jiangsu province; the saccharomyces cerevisiae FM-S-115 strain is derived from the academy of agricultural sciences of Jiangsu province, and the detailed information of the strain is disclosed in patent CN 103205369B; the lactobacillus paracasei ND1031 is derived from the academy of agricultural sciences of Jiangsu province, and the detailed information of the strain is disclosed in patent CN 115838658A.

In the following examples, the expression of pichia kudriavzevii NL33 and NL33 strains are the same, and refer to NL33 yeasts other than saccharomyces cerevisiae.

Example 1 identification of species of Pichia kudriavzevii NL33

Pichia kudriavzevii NL33 was isolated from cheese at 11.12 of 2021 at the academy of agricultural sciences of Jiangsu province, and its growth characteristics were characterized as follows.

(1) Colony morphology characterization: activating and culturing NL33 strain preserved at-80deg.C in PDB liquid culture medium for 12 hr, streaking and inoculating on WLN solid culture medium, culturing at 28deg.C in inverted mode, and classifying according to the morphological characteristics of NL33 strain when the colony morphology of yeast is clear. The colony of NL33 strain shown in FIG. 1 on the culture medium plate is white and opaque, is round with uneven size, slightly convex, matt and not easy to pick up, and has light fruity flavor. Microscopic examination of image 1 revealed that the bacterial cells were oval.

(2) Molecular biology identification: the NL33 strain obtained by pure strain activation culture was subjected to extraction of total genomic DNA using a microbial DNA extraction kit (purchased from the dix biochemical technology (beijing)) and PCR amplification was performed using the same as a template. Sample DNA was subjected to Qubit fluorometer and agarose gel electrophoresis and stored in a-80℃refrigerator if it was acceptable (concentration 10 ng/. Mu.L, and band clear).

PCR amplification primer pair: NL1 primer (sequence 5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 primer (sequence 5'-GGTCCGTGTTTCAAGACGG-3').

PCR amplification reaction system: 2 XPCR Mix 25. Mu.L, primer F (10. Mu.M) 2.5. Mu.L, primer R (10. Mu.M) 2.5. Mu.L, genomic DNA 200ng, ddH ₂ O was added to 50. Mu.L.

PCR amplification procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 1min, annealing at 52℃for 1min, extension at 72℃for 3min,36 cycles; after the completion, the mixture was extended at 72℃for 8min.

The PCR amplified products were subjected to 2% agarose gel electrophoresis, and the DNA fragments were recovered and purified using an agarose gel recovery kit, and quantitatively detected using Nanodrop 2000. A1682 bp DNA fragment was obtained, the sequences obtained from the test were compared with sequences in the NCBI database, 5 similar species were selected which have a sequence similarity of more than 99% with the NL33 strain, and a phylogenetic tree was constructed by the adjacent (Neighbor-Joining) method.

The nucleotide sequence of 26S rDNA of NL33 strain is shown in SEQ ID No. 1. Homology alignment and treelet results, as can be seen in FIG. 2: the sequence similarity of the NL33 strain to the 26S rRNA D1/D2 gene region of Pichia kudriavzevii AMC PK001 is up to 98% at NCBI and on the same branch of the developmental tree, indicating closest relatedness. In conclusion, the NL33 strain can be identified as a strain of pichia kudriavzevii (Pichia kudriavzevii).

The identified pichia kudriavzevii is sent to a preservation agency for preservation, and is classified and named: pichia kudriavzevii Pichia kudriavzevii, strain number NL33, deposited in the China general microbiological culture collection center, accession number: CGMCC No.26178, preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

EXAMPLE 2 tolerance analysis of Pichia kudriavzevii NL33

In this embodiment, the following combinations are used as basal fermentation media: the yeast extract 10g, peptone 20g and glucose 20g are dissolved in 1000mL distilled water, heated, boiled and prepared into YPD liquid culture medium, and each 100mL YPD liquid culture medium is packaged into conical flasks and sterilized by high pressure steam at 121deg.C for 20min.

1. The tolerability analysis used the following test methods:

(1) Alcohol tolerance: and under the aseptic condition, respectively adding different volumes of absolute ethyl alcohol into the cooled YPD liquid culture medium to ensure that the ethanol content in the YPD liquid culture medium is respectively 4%, 8% and 12% (v/v). The fully activated Pichia kudriavzevii NL33 strain was inoculated into conical flasks, respectively, with an initial cell density of 1.75X10 ⁵ CFU/mL. Stationary culture is carried out for 72 hours at the constant temperature of 25 ℃, and the growth condition is recorded every 24 hours. The experiment was performed three times in parallel with the control group without adding exogenous alcohol. Different non-Saccharomyces cerevisiae (NSC) G6, G9, G11, P20, P21, A1 were used as references for Pichia kudriavzevii NL 33.

(2)SO ₂ Tolerance: adding filtered and sterilized potassium metabisulfite solution into the cooled YPD liquid culture medium respectively, and regulating SO ₂ The content of (C) is 60mg/L, 120mg/L, 180mg/L. Inoculating activated Pichia kudriavzevii NL33 strain with initial cell density of 1.75X10 ⁵ CFU/mL. Stationary culture is carried out for 72 hours at the constant temperature of 25 ℃, and the growth condition is recorded every 24 hours. To be free of exogenous SO ₂ The medium of (2) was used as a control group and the experiment was performed three times in parallel. Different non-Saccharomyces cerevisiae (NSC) G6, G9, G11, P20, P21, A1 were used as references for Pichia kudriavzevii NL 33.

(3) Sucrose tolerance: YPD liquid culture containing sucrose concentration of 100g/L, 200g/L, 300g/L is sterilized by high pressure steam at 121deg.C for 20min, cooled, inoculated with activated Pichia kudriavzevii NL33, and initial cell density of 1.75X10 ⁵ CFU/mL, stationary culture at 25 ℃ for 72h, and recording growth every 24 h. Experiments were performed in triplicate using medium without exogenous sucrose as control. Not to take The reference to Saccharomyces cerevisiae (NSC) G6, G9, G11, P20, P21, A1 was Pichia kudriavzevii NL 33.

(4) High acid tolerance: the cooled YPD liquid culture medium is sterilized by high pressure steam at 121 ℃ for 20min after the pH values are respectively adjusted to 2.0, 3.0 and 4.0 by adopting 1mol/L of dilute hydrochloric acid solution and 1mol/L of sodium hydroxide solution, and after the YPD liquid culture medium is cooled, the fully activated Pichia kudriavzevii NL33 is inoculated, and the initial thallus density is 1.75X10 ⁵ CFU/mL, stationary culture at 25 ℃ for 72h, and recording growth every 24 h. Experiments were performed in triplicate using medium with neutral pH as control. Different non-Saccharomyces cerevisiae (NSC) G6, G9, G11, P20, P21, A1 were used as references for Pichia kudriavzevii NL 33.

2. The test results are as follows:

(a) Alcohol tolerance: as can be seen from FIG. 3, growth of non-Saccharomyces cerevisiae (NSC) was inhibited at various test periods when the test culture was conducted for 72 hours with an alcoholicity of greater than 8% vol.

In an alcohol content environment of 12% vol, the remaining yeast strains were not substantially able to grow except for the G6 and NL33 strains, and the lg value was reduced to around 5.20, indicating that the maximum alcohol tolerance concentration was less than 12% vol.

The strain activity of the G6, G9 and NL33 strains can be maintained under the condition of higher alcoholicity within 48 hours before the test, which proves that the three strains have better alcohol tolerance.

In the 24h-72h period, NL33 strain has a thallus concentration lg value rising from 6.87 to 7.17 and G6 rising from 6.75 to 7.10 under the test condition of alcohol content of 8%vol, so that both strains can still keep steadily proliferating under the alcohol content, the growth rate of NL33 strain is faster than that of G6, and the final thallus concentration is higher than that of G6 strain.

The strains G6, G9, NL33 and A1 can still keep relatively high thallus concentration at 72h under the condition of alcohol concentration of 8%vol, which is beneficial to prolonging the co-fermentation time and accumulating more flavor substances in the fermentation process.

From 24-72 h variation in cell density and comparison with the pure medium control group, NL33 strain can tolerate alcohol concentrations ranging between 8-12% vol; while the G6, G9, G11, P21 and A1 strains can only tolerate 4% vol-8% vol alcohol concentrations; the alcohol tolerance of the P20 strain is below 4%vol. It was demonstrated that the alcohol tolerance of the G6, G9, G11, P20, P21, A1 strains was generally less than 10% vol, while the NL33 strain still maintained a certain bacterial activity at 12% vol of alcohol.

In conclusion, the NL33 strain, i.e., pichia kudriavzevii NL33, has better alcohol tolerance and higher cell concentration.

(b)SO ₂ Tolerance: SO (SO) ₂ Has the functions of resisting oxidation, inhibiting the growth of mixed bacteria, protecting color and preventing food from deteriorating, and is one of the most common food additives in the food industry. Potassium metabisulfite can be decomposed to generate about 57% mass fraction of SO after being dissolved in water ₂ There is a certain loss in the process, and the actual operation is usually calculated as 50% (weight ratio). SO in fruit wine according to the requirements of GB2760-2014 national food safety Standard food additive use Standard ₂ The addition amount is less than 250mg/L, and the test dosage is within the specification.

As can be seen from FIG. 4, at 24h of the test, the concentration of each strain of non-Saccharomyces cerevisiae was dependent on SO ₂ The addition amount is increased and the concentration of the thalli is obviously higher than that of the control group (P<0.05). But at 48h and 72h of non-Saccharomyces cerevisiae proliferation, dissolved SO ₂ Increased content of SO ₂ The growth inhibitory effect on non-Saccharomyces cerevisiae began to be enhanced, with the cell concentrations of the G6 and G9 strains starting to be significantly lower than the control group. This is probably due to the addition of potassium metabisulfite to adjust the pH of the medium, the early growth environment being more suitable for the growth of test non-Saccharomyces cerevisiae, and the later SO ₂ The inhibition of the non-Saccharomyces cerevisiae is enhanced, so that the proliferation of non-Saccharomyces cerevisiae is slowed down.

7 strains of non-Saccharomyces cerevisiae against SO ₂ Has stronger tolerance. Each strain is free of exogenous SO ₂ Growth in the medium is mainly affected by the activity of the yeast itself. At 72h of the test, the concentration of the cells of NL33 and A1 strain was significantly higher than that at 24 and 48h samples, indicating that it was possible to use it in SO ₂ In a medium with a concentration of 120mg/L over timeCan still grow normally but when SO ₂ When the concentration is increased to 180mg/L, the growth is obviously inhibited, and the maximum lg value of the strain concentration of the NL33 strain is 6.87, and the lg value of the strain A1 is 6.75, which indicates that the two strains can tolerate SO ₂ In the range of 120-180mg/L, but strain NL33 is somewhat more tolerant than strain A1.

(c) Sucrose tolerance: the sugar content of the fermentation substrate of the fruit wine is between 22% and 30%, and some of the fermentation substrate is even approximately 44%, so that the excellent saccharomycetes have high sugar tolerance.

As can be seen from FIG. 5, in the culture medium of 100g/L of exogenous sucrose within 24 hours before the test, the concentration of each thallus is obviously higher than that of the control group, which shows that a small amount of sucrose added has obvious growth promoting effect on each strain. In the 48 th to 72 th of the test, the stress effect of the high sucrose concentration solution on non-saccharomyces cerevisiae is obvious, the growth of the NL33 strain is obviously inhibited when the sucrose concentration is 200 g/L to 300g/L, but the thallus concentration lg value can still be kept at 6.98 when the thallus concentration lg value is 72h, which is higher than the strain concentration of most of the selected bacteria in the test. Thus, NL33 strain has a better high sugar tolerance than other non-saccharomyces cerevisiae.

(d) High acid tolerance: the pH value fluctuation range of the freshly squeezed blackberry juice and the blueberry juice is 2.9-3.1, and under the environment, the growth of part of non-saccharomyces cerevisiae can be inhibited, so that the non-saccharomyces cerevisiae for mixed fermentation has certain acid resistance.

As can be seen from FIG. 6, the initial inoculum size of each non-Saccharomyces cerevisiae was 1.75X10 ⁵ CFU/mL. Between 24h and 48h, the NL33 strain maintained at 6.88 lg at pH 2.0, indicating that the strain proliferated at a slower rate under this acidic condition. In the whole, when the pH is more than or equal to 3.0, the growth activity can be kept high except for the P20 strain. When the pH was lowered to 2.0, the concentration of cells of each strain was reduced as the test was conducted except for the NL33 strain, and the NL33 strain was able to maintain a higher concentration of cells and proliferated slowly over 24-72 hours. Therefore, according to the test results, the NL33 strain is selected for the blackberry wine mixed fermentation process, and the adjustment of the pH value of the blackberry juice for fermentation is not needed.

Example 3 analysis of fermented blackberry fruit wine of Pichia kudriavzevii NL33

(1) Flocculation measurement: flocculation value (Flocculation value, flo) determination method Pichia kudriavzevii NL33 was cultured in YPD medium to stationary phase (about 24 h), the culture solution was removed by centrifugation, washed twice with deflocculating buffer and sterile water, resuspended in flocculation buffer, sampled, and OD was measured ₆₀₀ Values, the rest of the samples were incubated for 2h on a constant temperature shaker (30 ℃,100 rpm). 5mL of the cell suspension is taken into a 15mL test tube, the cell suspension is vertically stood for 30min at room temperature, 350 mu L of the sample below the concave liquid surface is sucked, the OD value is measured at 600nm, and the experiment is repeated for 3 times. Different non-Saccharomyces cerevisiae (NSC) G6, G9, G11, P20, P21, A1 were used as references for Pichia kudriavzevii NL 33.

Wherein, flocculation value calculation formula: flo=b/a×100%

In the above formula: a represents the OD of the bacterial suspension after resuspension of the flocculation buffer ₆₀₀ A value; b value represents OD of each strain after 30min of standing precipitation ₆₀₀ A value; flo represents flocculation value.

(2) Determination of beta-D-glucosidase Activity: the enzyme activity of the beta-D-glucosidase is determined by taking p-nitrobenzene-beta-glucoside (pNPG) as a substrate and adopting a spectrophotometry. Drawing a standard curve: weighing 139.0mg of p-nitrophenol, and metering distilled water to 1000mL, respectively sucking 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0mL into 100mL volumetric flask, and adding 1mol/L Na ₂ CO ₃ The solution is mixed evenly after the volume is fixed. Distilled water was used as a blank, and absorbance was measured at 400 nm. P-nitrophenol and Na ₂ CO ₃ The mixture turns yellow, and the concentration of the mixture is proportional to the absorbance in a certain range. And drawing a standard curve by taking the mass concentration of the p-nitrophenol as an abscissa and the absorbance as an ordinate.

30mL of a bacterial solution of Pichia kudriavzevii NL33 was centrifuged at 10000g at 4℃for 10min, and the bacterial pellet was washed with deionized water for 2 times, and then the bacterial pellet was resuspended in distilled water to obtain a test sample. Accurately transferring 500 mu L of sample, and adding 1mL of 5mM p-nitrobenzene-beta-glucoside solution (pH 5.0 phosphoric acid-citric acid buffer solution is used as a solvent) for mixing to obtain a reaction solution. The reaction solution was found to be 37After 30min of reaction in a water bath at the temperature of 10000g, centrifuging (4 ℃ C., 10 min) to obtain 1mL of supernatant, adding 2mL of 1mol/L Na ₂ CO ₃ The reaction was stopped and developed, and the color was developed under 400nm conditions. The control group replaced the sample with distilled water. The method for measuring the dry weight of the thalli comprises the following steps: 30mL of the bacterial liquid of Pichia kudriavzevii NL33 is centrifuged (10000 g,4 ℃ C., 10 min) to obtain bacterial cells, and the bacterial cells are dried to constant weight and weighed. The enzyme activity is defined as: under the above reaction conditions, the amount of p-nitrophenol produced per minute is under the action of each gram of bacteria (dry weight). The unit is: p-nitrophenol (. Mu.M)/dry weight of bacteria (g)/time (min). Different non-Saccharomyces cerevisiae (NSC) G6, G9, G11, P20, P21, A1 were used as references for Pichia kudriavzevii NL 33.

(3) Sensory evaluation fermented blackberry wine:

the preparation method of the blackberry juice comprises the following steps: the method comprises the steps of taking a 'hel' blackberry as a raw material, naturally thawing the 'hel' blackberry, pulping, taking a proper amount of blackberry pulp, putting the blackberry pulp into a sterilized stainless steel container, adding 0.15% pectase, 150mg/L potassium metabisulfite and 150g/L white granulated sugar, stirring and uniformly mixing, and carrying out enzymolysis for 1-2h at room temperature to obtain the blackberry juice.

The strains G6, G9, G11, P20, P21 and A1 and the Pichia kudriavzevii NL33 are non-saccharomyces cerevisiae (NSC), so that more volatile substances can be generated when the fermented blackberry wine is prepared by fermentation, the taste level of the fruit wine is enriched, and more extracellular enzymes such as polysaccharase, glucanase, proteolytic enzyme and the like can be generated. These hydrolases facilitate the hydrolytic release of the bound aroma. In addition, the interaction between NSC and Saccharomyces cerevisiae may result in more free aroma. The FM-S-115 strain is saccharomyces cerevisiae, has the physiological advantages of strong stress resistance, rapid proliferation, anti-bacteria, convenient pure culture and the like, has excellent fermentation performance, has strong sugar consumption capacity, ensures stable and rapid alcohol fermentation process, can perform the alcohol fermentation process better under the stress environments of high alcohol degree, high osmotic pressure and the like, and is an essential strain for the blackberry fruit wine fermentation process. Therefore, the invention adopts a mixed fermentation mode in the fermentation preparation of the fermented blackberry wine. The fermented blackberry wine is prepared by pure-bred fermentation of FM-S-115, and the fermented blackberry wine is prepared by mixed fermentation of G6, G9, G11, P20, P21 and A1 strains and FM-S-115 strains respectively, and is used as a test reference group for preparing the fermented blackberry wine by mixed fermentation of Pichia kudriavzevii NL33 and FM-S-115 strains.

Taking 30mL of bacterial liquid of each bacterial strain, respectively placing the bacterial liquid into 50mL of sterilized centrifuge tubes, centrifuging for 10min at 4 ℃ and 10000g parameters by using a refrigerated centrifuge, washing the precipitate by using sterile water, repeating the steps for two times, adding the sterile water, stirring uniformly by vortex, and adjusting to the same OD value. Cell density measurement was performed on the seed solution using a hemocytometer at 3.75X10 respectively ⁵ Seed solutions of all strains are respectively inoculated into the prepared blackberry juice by CFU/mL inoculum size, and fermented blackberry wine prepared by different strains is obtained after fermentation for 7d under the constant temperature condition of 25 ℃.

Analysis referring to GB/T15038-2006 general analysis methods for wine and fruit wine and GB/T10221-2021 sensory analysis terminology standards, in a standard taste house, a panel of 10 wine tasters (5 men and 5 women) was composed, and fermented blackberry wine samples prepared from different strains were scored in multiple ways using the scoring criteria in Table 1, including evaluation and description of color, clarity, aroma, taste and typical properties of the wine samples, taking 100 minutes of preparation, with a greater score indicating a better quality of fermented blackberry wine.

Table 1 blackberry wine sensory evaluation scoring criteria

(4) And (3) detecting flavor substances:

volatile matter extraction: accurately transferring 2.0mL of fermented blackberry wine sample to be detected, respectively injecting the sample into 20mL of headspace bottles, inserting an aged solid-phase microextraction sampling needle into the sealed headspace bottles under a sealed condition, pushing out an extraction head, extracting the sample in a water bath at 60 ℃ for 1h, immediately inserting the extraction head into a GC-MS sampling port, and performing thermal analysis for 2min. Before each sample extraction, the extraction head needs to be aged for 5min at 250 ℃ to reduce the memory effect.

Wherein, GC-MS gas chromatography conditions: the chromatographic column is a TG-5MS (30 m×0.25mm×0.25 μm) elastic quartz capillary column; the carrier gas is high-purity helium (purity 99.999%); the flow rate of the carrier gas is 1.2mL/min; adopting non-split sampling; the temperature of the sample inlet is 250 ℃; programming temperature: the initial temperature is maintained at 40deg.C for 2min, then at 3deg.C/min to 100deg.C for 1min, then at 5deg.C/min to 160deg.C for 1min, and then at 10deg.C/min to 280 deg.C for 1min.

Mass spectrometry conditions: the ion source is an EI source, and the transmission line temperature is as follows: 280 ℃; ion source temperature: 300 ℃; electron energy: 70eV; scan range (m/z): 33-800amu, and adopts a full scanning acquisition mode.

Qualitative analysis: the relative content of volatile components in the fermented blackberry wine was analyzed by peak area normalization by computer retrieval of the components and comparison with a standard mass spectrum provided by the NIST17 mass spectrum library.

2. The experimental results are as follows:

(a) Flocculation measurement: the non-saccharomyces cerevisiae with poor flocculation property can exist in a large amount in a wine body in the later fermentation period of the fruit wine, so that the probability of autolysis of the saccharomyces cerevisiae is increased, the fruit wine is difficult to clarify, and the yeast taste is too heavy. Good flocculation property is beneficial to clarification and later management of fruit wine. Therefore, the flocculation property of the yeast is an important index of the brewing property of the fruit wine. The lower the flocculation value, the better the flocculation performance of the non-Saccharomyces cerevisiae. When the flocculation value (Flo) is less than 30%, high flocculation is obtained; the flocculation property is kept between 30% and 70%; more than 70% is low flocculability.

The saccharomycete has strong flocculation property, which is beneficial to the clarification process of the blackberry wine, so that the flocculation property can evaluate the application potential of the saccharomycete in the fermentation process. As can be seen from table 2: the NL33 strain has the lowest flocculation value, i.e. the best flocculation.

TABLE 2 flocculation property differences of different yeasts

(b) Determination of beta-D-glucosidase Activity: beta-D-Glucosidase (beta-D-Glucosidase) is a hydrolase for catalyzing and hydrolyzing glycosidic bonds between hydrocarbon groups and glycosyl groups to generate aromatic substances and glucose, and the alias is amygdalase, gentiobinase or cellobiase, is an important hydrolase in a cellulase system, and has important significance for generating and releasing aroma substances of fruit wine.

As can be seen from table 3: the NL33 strain, A1 strain, G6 strain and G11 strain have stronger glycosidase activity, wherein the NL33 strain is the highest, and can reach 24.19 mu mol/G/min.

TABLE 3 differential beta-D-glucosidase Activity of Yeast

(c) Sensory evaluation fermented blackberry wine: given that the beta-D-glucosidase activity of the G9, P20 and P21 strains was significantly lower than that of the other strains, there was a distinct "short plate" and thus no sensory evaluation of the fermented blackberry wine was performed. The color and clarity reflect the appearance quality of the fermented blackberry wine, while the aroma, taste and typical property better represent the flavor quality of the fermented blackberry wine, and as can be seen from the table 4, the color and clarity score is the fermented blackberry wine obtained by mixing and fermenting the NL33 strain and the FM-S-115 strain, the wine body is bright and transparent, the centrifugal effect is better, and no obvious suspended matters exist. In addition, the fermented blackberry wine obtained by mixed fermentation of the A1 strain and the FM-S-115 strain is also clearer, and has good flocculation relationship with the A1 strain and the NL33 strain. The wine sample with the highest aroma score is fermented blackberry wine obtained by mixed fermentation of NL33 strain and FM-S-115 strain, the aroma of the wine body overflows with the aroma of the wine, and pleasant feeling is given to people. The wine sample with higher taste score is obtained from fermentation tests of mixed fermentation of NL33 strain and FM-S-115 strain, and A1 strain and FM-S-115 strain, and has plump wine body, proper sweet and sour taste and fragrance retention of lips and teeth. The higher representativeness shows that the fruit wine has obvious self characteristics, unique style and highest scoring, and the wine sample is the fermented blackberry wine obtained by mixed fermentation of NL33 strain and FM-S-115 strain. After comprehensive comparison, the total score of the sensory score is the fermented blackberry wine obtained by mixed fermentation of the NL33 strain and the FM-S-115 strain, and the secondary score of the fermented blackberry wine obtained by mixed fermentation of the A1 strain and the FM-S-115 strain is 96 and 91 respectively, which are obviously higher than those of other control groups, so that the fermented blackberry wine with better taste and brighter wine body can be obtained by utilizing the fermentation of the NL33 strain and the A1 strain, but the comprehensive evaluation of the fermented blackberry wine obtained by mixed fermentation of the NL33 strain and the FM-S-115 strain is better.

Table 4 sensory evaluation score table for fermented blackberry wine

(d) And (3) detecting flavor substances: the alcohol, ester, acid and aldehyde ketone substances form the volatile flavor substance component of the fermented blackberry wine. The higher alcohols and esters form the main body of the aroma substances of the fermented blackberry wine, and the trace substances can increase the aroma level and complexity of the fermented blackberry wine, endow the fermented blackberry wine with unique flavor characteristics and meet the requirements of more consumers.

Since the beta-D-glucosidase activity of the G9, P20 and P21 strains was significantly lower than that of the other strains in the beta-D-glucosidase activity assay, there was a clear "short plate", and thus no volatile matter detection test of fermented blackberry wine was performed. In sensory evaluation of fermented blackberry fruit wine, the sensory evaluation total score of the G6 and G11 strains is not high, and the sensory evaluation total score of the fermented blackberry wine prepared by using NL33 and A1 strains is higher than 90, so that the quality improvement effect of the fermented blackberry wine is good, and the GC-MS volatile matter relative content detection is carried out on the fermented blackberry wine obtained by mixing and fermenting NL33 strain and A1 strain with FM-S-115 strain respectively, and the results are shown in Table 5.

TABLE 5 different non-Saccharomyces cerevisiae and FM-S-115 Mixed fermentation to obtain blackberry wine with different volatile content

/>

As can be seen from Table 5, all the test samples are combined, 54 volatile components are detected in total, 53 fermented blackberry wines obtained by mixed fermentation of NL33 strain and FM-S-115 strain are detected in total, the content of alcohols and esters is high, the variety of substances is rich, and the variety of substances is only 49 fermented blackberry wines obtained by mixed fermentation of A1 strain and FM-S-115 strain, and the abundance of volatile substances is small. The NL33 strain and the A1 strain increase the relative content of alcohol substances in the fermented blackberry wine under the mixed fermentation condition, and reduce the relative content of the ester substances.

The alcohol and ester volatile substances are the main part of the detection result of the flavor substances of the fermented blackberry wine, wherein the total content of the two substances of the fermented blackberry wine obtained by pure fermentation of the FM-S-115 strain is 89.43%, and the total content of the two substances of the fermented blackberry wine obtained by mixed fermentation of the NL33 strain and the FM-S-115 strain and the A1 strain and the FM-S-115 strain is 74.68% and 76.25% respectively. Thus, the mixed fermentation can reduce the ratio of alcohols and esters in the total volatile substance content, and can relatively increase the relative content of aldehyde ketone and acid substances, such as the relative content of nonanal and octanoic acid. Among the alcohol substances, phenethyl alcohol which is representative of typical volatile substances of the fermented blackberry wine has the highest relative content, and under the mixed fermentation condition, the test group is obviously higher than the control group. The phenethyl alcohol has rose fragrance in a proper concentration range, and can endow the fruit wine with characteristic flavor. The relative content of phenethyl alcohol in the fermented blackberry wine obtained by mixed fermentation is increased to be respectively 12.61 percent and 20.06 percent, which is obviously higher than that in the fermented blackberry wine obtained by pure fermentation of FM-S-115 strain. It has been found that the aroma of fruit wine can be significantly enhanced when the higher alcohol concentration is maintained below a certain threshold, and that fruit wine can produce a pungent and pungent odor when the content is above the threshold.

The variety and content of the esters have a great influence on the flavor quality of the blackberry wine. In the process of fermenting the NL33 strain and the FM-S-115 strain in a mixed mode to produce the fermented blackberry wine, the relative content of acetic acid-2-phenethyl ester (floral fragrance and rubber odor), ethyl octanoate (pear fragrance), ethyl decanoate (pineapple fragrance), ethyl dodecanoate (fruit fragrance and flower fragrance), ethyl tetradecanoate (coconut fragrance), ethyl hexadecanoate (cream fragrance), 9, 12-octadecadienoic acid n-propyl ester and ethyl oleate (floral fragrance and fruit fragrance) is higher than 1%, so that the fresh and pleasant taste of the fermented blackberry fruit wine is endowed, and the obtained wine-like acid substances are higher than that of an FM-S-115 strain pure-seed control group and an A1 strain and FM-S-115 strain mixed fermentation test group by 15.08%, especially the relative content of acetic acid (3.53%), octanoic acid (3.63%) and n-decanoic acid (3.75%) is higher, so that the organic acid content of the fermented blackberry wine can be increased while the flavor is increased.

In conclusion, the sensory quality of the fermented blackberry wine obtained by mixed fermentation of the NL33 strain and the FM-S-115 strain is higher than that of the fermented blackberry wine obtained by mixed fermentation of the A1 strain and the FM-S-115 strain, and the volatile substances are more abundant than those of the fermented blackberry wine obtained by mixed fermentation of the A1 strain and the FM-S-115 strain, so that the Pichia kudriavzevii NL33 is more suitable for fermenting fruit wine.

Example 4 fermented soymilk beer analysis of Pichia kudriavzevii NL33

1. The test method comprises the following steps:

cleaning soybeans, and then, according to the mass ratio of the soybean water of 1:8, soaking the soybeans in water for 12 hours, fully grinding the soaked soybeans by using a pulping machine, filtering the soybean residues by using 100-mesh gauze to obtain coarse soybean milk, further grinding the coarse soybean milk by colloid grinding for 2 times to obtain micro soybean milk, and sterilizing the micro soybean milk at 75 ℃ for 30 minutes. The lactobacillus paracasei ND1031 (Lactobacillus paracasei, strain preservation number: CGMCC No.22115, the detailed information of the strain is disclosed in patent CN 115838658A) is selected as lactobacillus. NL33, A1, G6, G11 strain and Lactobacillus paracasei ND1031 strain were added simultaneously to sterilized and cooled micro soy milk at a ratio of 2:1, respectively. The total microbial inoculation amount is 1.75X10 ⁸ log CFU/g micro-soymilk is fermented for 15 hours at 28 ℃ to obtain the mixed bacteria fermented micro-soymilk. Adding pectin 0.1%, carrageenan 0.2%, propylene glycol alginate 0.4% and acid-resistant carboxymethyl cellulose 0.6% into 90deg.C hot water, stirringUniformly mixing and cooling to obtain a compound stabilizer, adding 2 times of the compound stabilizer into the mixed fermentation micro-soymilk, adding 0.03 per mill of sucralose into the mixed fermentation micro-soymilk containing the compound stabilizer to obtain a soymilk beer beverage, homogenizing at 55 ℃ under the first stage of homogenizing pressure of 35MPa and the second stage of homogenizing pressure of 3.5MPa, and filling CO ₂ And (5) filling and sterilizing to obtain a finished product of the soymilk beer beverage.

And (3) carrying out volatile matter measurement, organic acid measurement, aroma measurement and sensory evaluation on the soybean milk beer beverage finished product obtained in the above way.

(1) Volatile matter determination: adopting headspace adsorption, respectively sucking 2mL soybean milk beer samples, placing into a 15mL headspace sample injection bottle, adding 1g sodium chloride, sealing, balancing at 60deg.C on a magnetic heating stirrer for 15min, pushing out an aged extraction head (50/30 μm DVB/CAR/PDMS), performing headspace adsorption for 40min, taking out the extraction head, injecting sample, desorbing for 5min, and extracting. Wherein, chromatographic conditions: chromatographic column: DB-WAX (60 m. Times.0.25 mm. Times.0.25 μm); programming temperature: the temperature of the sample inlet is 230 ℃, the initial temperature is 50 ℃ and kept for 3min, the temperature is raised to 70 ℃ at 1 ℃/min, the temperature is raised to 140 ℃ at 4 ℃, the temperature is raised to 180 ℃ at 7 ℃, and the temperature is raised to 230 ℃ at 4 ℃ and kept for 8min; sample injection mode: split-flow sample injection; split ratio: 10:1, a step of; carrier gas: high purity helium (He); flow rate: 1.76mL/min. Mass spectrometry conditions: the ionization mode is an electron ionization (electron ionization, EI) source; the ion source temperature is 230 ℃; the collection mode Scan; electron energy 70eV; the mass scanning range is 35-500m/z.

(2) Organic acid determination: sample pretreatment: accurately sucking 2mL of soymilk beer sample respectively, fixing volume to 10mL by using a mobile phase, sucking a proper amount of solution, centrifuging at 10000r/min for 10min at high speed, sucking supernatant, and passing through a 0.22 mu m microporous filter membrane for organic acid analysis. Preparing a standard substance: respectively and accurately weighing a certain amount of oxalic acid, lactic acid, fumaric acid, malic acid, citric acid and propionic acid standard substances, preparing a standard solution of 1mg/mL, 5mg/mL, 1mg/mL, 20mg/mL and 99mg/mL by flow dependence, and sequentially diluting the standard solution into a mixed standard solution of 0.083mg/mL, 0.520mg/mL, 0.083mg/mL, 1.670mg/mL and 8.250mg/mL for sample injection analysis. Measurement conditions: ther mo Acclaim Origanic Acid column (4 mm. Times.250 mm,5 μm); mobile phase: preparation of 100mmol/L Na ₂ SO ₄ The solution was adjusted to pH 2.65 with methanesulfonic acid; flow rate: 0.5mL/min; sample injection amount: 5. Mu.L; elution type: isocratic elution; a detector: a UV detector; detection wavelength: 210nm; column temperature: 30 ℃.

(3) Aroma measurement: aroma activity values (aroma activity value, OAV) were calculated and radar mapped for the detected flavor compounds. OAV refers to aroma compound mass concentration/threshold.

(4) Sensory evaluation: in a standard tasting room, a panel of 10 tasters (5 men, 5 women) was composed, and soymilk beer samples were scored for multiple aspects using the scoring criteria in table 6, including the evaluation and description of the sweet and sour ratio, smell, taste, mouthfeel, and overall flavor of the milk beer, taking a 100 point score, with a greater score indicating a better quality soymilk beer.

TABLE 6 Soy milk beer sensory evaluation criteria

2. The experimental results are as follows:

(a) Volatile matter determination: the aroma components are the main indexes for measuring the flavor quality of the soymilk beer, and the aroma components and the contents of the four saccharomycete soymilk beer are shown in Table 7. The results show that 52 flavors, mainly alcohols, esters, acids and aldehydes, are identified in the soymilk beer. The higher alcohol is the product of amino acid or sugar metabolism in the process of yeast alcoholic fermentation, and most alcohols have higher threshold values, but can present bouquet or plant fresh scent, and can generate esterification reaction with organic acids to generate esters, and simultaneously form a very small amount of important flavor substances such as aldehyde, ketone and the like, thereby indirectly affecting the flavor.

The four kinds of soymilk beer contain alcohol substances such as phenethyl alcohol, benzyl alcohol and 1-octene-3-alcohol, and have rose, flower fragrance, honey fragrance and beany flavor. Wherein, the content of alcohols in the soybean milk beer prepared by NL33 strain is highest, namely 33.12%, and the content of isoamyl alcohol, benzyl alcohol and phenethyl alcohol is respectively 10.21%, 3.98% and 8.34% higher than that of other soybean milk beer, but the content of 1-octen-3-ol capable of generating the fishy smell of the soybean is lower than that of 3.36%. The alcohol content in the other 3 soymilk beer types is 25.41%, 29.03% and 28.92% respectively.

Among the four soymilk beer, the soymilk beer prepared by mixed fermentation of NL33 strain and ND1031 strain contains butyric acid, caproic acid, caprylic acid and n-capric acid, has lower threshold value and has stronger milk flavor under low mass concentration. The ester flavor substances are mainly generated by esterification reaction of alcohols and organic acids, and most of the ester flavor substances have low flavor threshold value, but have great flavor effect, and have fruit flavor and flower flavor, so that the whole flavor is fresh and sweet. The total content of esters in the four soymilk beer is 22.13%, 22.12%, 23.46% and 21.01%, respectively. The kind of esters in the soybean milk beer prepared by mixing and fermenting NL33 strain and ND1031 strain is obviously different from other 3 kinds, and the soybean milk beer contains isoamyl butyrate, ethyl decanoate, allyl caproate, allyl heptanoate, diethyl malonate and pineapple ester in addition to 6 kinds of esters such as ethyl acetate and the like. Aldehydes and ketones are carbonyl compounds, the aldehyde flavor threshold is low, and due to the unsaturation of aldehyde groups, the aldehydes and ketones are unstable intermediate compounds, and the aldehydes and ketones are fast in oxidation, and are easy to generate acid substances in a long-time fermentation process or reduced to corresponding alcohols, so that the formation of beer aroma of the soymilk is facilitated. The higher aldehyde substance in the soybean milk beer prepared by the mixed fermentation of the NL33 strain and the ND1031 strain is benzaldehyde, the soybean milk beer has sweet almond flavor and 1.64 percent of content, and compared with the soybean milk beer prepared by the mixed fermentation of the NL33 strain and the ND1031 strain, the soybean milk beer prepared by the mixed fermentation of the A1 strain, the G6 strain, the G11 strain and the ND1031 strain has higher hexanal content and nonanal content, so that the beany flavor in the soybean milk beer is heavier. Therefore, the soybean milk beer prepared by the mixed fermentation of the NL33 strain and the ND1031 strain has rich ester substance content and less beany flavor substance content, and can give the soybean milk beer a better flavor.

TABLE 7 composition and content of different Yeast soymilk beer volatile matters

/>

(b) Organic acid determination: the content, threshold and flavor profile of various organic acids in each soymilk beer are shown in Table 8. As can be seen from Table 8, the organic acids in the beer were mainly lactic acid and citric acid. The soybean milk beer prepared by mixed fermentation of NL33 strain and ND1031 strain has highest lactic acid content of 2.152g/L and low lactic acid threshold, and is the main organic acid affecting the flavor of the milk beer. In addition, oxalic acid has astringency, citric acid is fresh and cool, lactic acid has soft sour taste, malic acid has mellow and long-lasting sour taste, and acetic acid has heavy bitter taste, so that the soymilk beer has rich and full flavor characteristics under the combined action of various organic acids.

TABLE 8 determination of organic acid content in soymilk beer

(c) Aroma measurement: to evaluate the effect of organic acids on flavor, aroma compounds for all OAV > 1 (note: the material of OAV > 1 contributed to the overall aroma exhibited by the sample) were listed and analyzed for aroma profile, with the results shown in table 9 and fig. 7. As shown in fig. 7, the four soymilk beer has very similar taste characteristics, but the soymilk beer prepared by fermenting the NL33 strain and the ND1031 strain in a mixed mode has the main characteristics of prominent sour soft feel and fresh sour taste, and has good taste and flavor.

TABLE 9 OAV of organic acids in soymilk beer

(d) Sensory evaluation: as shown in FIG. 8, the four kinds of beer have optimal sweet-sour ratio, taste, smell and comprehensive flavor, and the highest score is 94, and the beer produced by fermenting NL33 strain and ND1031 strain is 88. It is explained that pichia kudriavzevii NL33 is preferred for fermenting soy milk beer.

Example 5 preparation of a starter Using Pichia kudriavzevii NL33

Inoculating Pichia kudriavzevii NL33 into PDA culture medium, activating for 2 times, inoculating activated strain into PDA culture solution at 2% (v/v), and culturing at 30deg.C for 18 hr until thallus concentration in fermentation solution is 10 ⁸ -10 ⁹ CFU/mL. The fermentation broth containing pichia kudriavzevii NL33 was centrifuged at 5000rpm for 5min to obtain a bacterial pellet, and washed 3 times with 0.85% physiological saline. Mixing the cleaned thallus precipitate with different protective agents (the protective agents contain 8g of skim milk powder, 10g of sugar substances (sucrose, trehalose, lactose, maltose or glucose), 3g of glycerol and dissolved in 100mL of distilled water), pre-freezing for 5 hours at-80 ℃, freeze-drying (cold trap temperature-45 ℃ and vacuum degree 10-20 Pa) for 20-24 hours, stopping drying when the water content of the thallus powder is reduced to 3%, obtaining the dry powder type fermenting agent, and measuring the survival rate of the thallus in the freeze-dried fermenting agent. Comparing the freeze-drying protection effect on the thalli when the protective agent contains different saccharides, the result of the survival rate of the thalli is shown in figure 9.

As a result, it was found that the bacterial species survival rate was improved to 89.15% when trehalose was added to the saccharide in the protectant. The second protection effect is better sucrose, and the strain survival rate is 84.35%. When trehalose is added into saccharide in the protecting agent, the number of viable bacteria in the fermenting agent is higher than 1×10 ¹¹ CFU/g. The obtained starter can be used in food field for preparing fermented flour product, fermented meat, etc.

Example 6 application of a leavening agent comprising Pichia kudriavzevii NL33 in the preparation of fermented meat

(1) Preparing a fermentation solvent A: the dry powder form of the fermentation agent obtained in example 5 was added to a sucrose mass fraction of 0.5% (g/100 mL) solution in an amount of 0.5-1% (g/100 mL), and thoroughly mixed to obtain fermentation solvent A. Sterile water was used instead of pichia kudriavzevii NL33 to prepare fermentation solvent B as a control.

(2) Preparing brine: glucose with the mass fraction of 1.5%, sodium nitrite with the mass fraction of 0.02% and sodium erythorbate with the mass fraction of 0.1% (g/100 mL) are added into the starter of the step (1) to prepare brine.

Cutting fresh pork leg meat into pieces with weight of 2+ -0.1 kg, uniformly injecting prepared saline water into the cut pork leg meat by using a syringe, independently wrapping each piece of injected pork leg meat with a bag, vacuum rolling and kneading for 24h at 4 ℃, removing the bag after rolling and kneading, burying the pork leg meat in crude salt at 4 ℃ for pickling for 2 days, brushing salt particles on the surface of the pork piece, hanging and drying at 12 ℃ for 4 days, and hanging and fermenting at 18 ℃ and 70% humidity under a fermentation condition until the pork leg meat is ripe (3 months).

The color, texture, mouthfeel and flavor of the different groups of fermented meat were evaluated. As can be seen from the results in Table 10, the NL33 fermented meat was superior to the control group in texture, mouthfeel and flavor.

TABLE 10 evaluation of quality characteristics of fermented meat with different yeasts

The invention provides pichia kudriavzevii, the thought and the method for applying the same, and the method for realizing the technical scheme is a plurality of methods and paths, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. Pichia kudriavzevii Pichia kudriavzevii with the strain number NL33 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number 26178 in the year of 2022 and 12 and 14.

2. The pichia kudriavzevii according to claim 1, wherein the nucleotide sequence of the 26S rDNA of the pichia kudriavzevii is shown in SEQ ID No. 1.

3. The pichia kudriavzevii according to claim 1, wherein the pichia kudriavzevii tolerates alcohol concentrations of 4% to 12% vol and SO of 60-180mg/L ₂ An acidic environment having a total sugar concentration of 100-300g/L and a pH of 2.0-4.0; preferably, an alcohol concentration of 8% -12% vol, SO of 120-180mg/L is tolerated ₂ 200-300g/L of total sugar concentration, and an acidic environment with pH of 2.0-3.0.

4. The pichia kudriavzevii according to claim 1, wherein the pichia kudriavzevii has a flocculation value Flo of less than 30% and a β -D-glucosidase activity of 24.19 μmol/g/min.

5. Use of pichia kudriavzevii according to any one of claims 1 to 4 in any one of the following food industry fields:

(3) The application of the pichia kudriavzevii in preparing a starter.

6. The use according to claim 5, wherein in the use (1), pichia kudriavzevii and saccharomyces cerevisiae FM-S-115 are mixed and fermented to blackberry juice Both of which were inoculated in blackberry juice at 3.75X10 ⁵ CFU/mL, fermentation conditions are: fermenting at 25deg.C for 6-8d.

7. The use according to claim 5, wherein in the application (2), the micro-soymilk made of soybean is fermented by mixing pichia kudriavzevii with lactobacillus paracasei ND1031, the ratio of the two to the micro-soymilk is 2:1, and the total inoculation amount is 1.75x10 ⁸ log CFU/g micro soy milk, fermentation conditions were: fermenting at 25-30deg.C for 15 hr.

8. The use according to claim 5, wherein in the application (3), the starter is obtained by inoculating pichia kudriavzevii into PDB liquid medium, culturing to obtain fermentation broth, centrifuging the fermentation broth to collect thalli, washing, adding a protective agent, and freeze-drying.

9. The use according to claim 8, wherein the concentration of the bacterial cells in the fermentation broth is 10 ⁸ -10 ⁹ CFU/mL。

10. The use according to claim 8, wherein the protective agent is added in an amount of 1/20 to 1/4 of the volume of the fermentation broth; the formula is as follows: 6-12g of skim milk powder, 6-12g of sugar substances, 2-4g of glycerin and 100mL of distilled water, wherein the sugar substances are any one of sucrose, trehalose, lactose, maltose and glucose, and preferably trehalose.