CN112322509A - Candida parapsilosis with low temperature resistance and high alcohol yield, and composition and application thereof - Google Patents

Abstract

The invention discloses a new candida parapsilosis, a brewing microbial inoculum containing the candida parapsilosis and application of the candida parapsilosis in liquor brewing. The candida parapsilosis disclosed by the invention has the advantages of strong low temperature and acid resistant growth capability, high alcohol yield, low acetaldehyde and higher alcohol content, strong capability of producing white spirit flavor components such as ethyl phenylacetate and the like, the white spirit produced by fermentation has the advantages of low acetaldehyde and higher alcohol content, aromatic flavor and the like, and the brewing microbial inoculum composition and the brewing reinforced yeast are applied to the solid state fermentation of the white spirit, so that the quality and the yield of the white spirit can be improved.

Description

Candida parapsilosis with low temperature resistance and high alcohol yield, and composition and application thereof

The invention belongs to the field of:

the invention belongs to the field of wine brewing, and particularly relates to candida parapsilosis, a solid microbial inoculum containing the candida parapsilosis, and application of the candida parapsilosis and a composition thereof in liquor brewing.

Background art:

candida parapsilosis (Kazachstania/Candida humulis) and lactic acid bacteria are widely used as natural leavening agents in bread, animal feed, grains and other bakery products (GULLO M, et al. International Journal of Food Microbiology,2003,80), and are considered as a generally recognized safe yeast (DE V L, et al. International Journal of Food Microbiology,2016,239). Liujiali (food science, 2018,39), Wusgugui Nigri (university of inner Mongolia agriculture, 2011), Shaoyojie (modern food technology, 2016, 7) and the like are respectively separated and screened from Chinese traditional starter, acidic dough, traditional glutinous fried cake and the like to obtain Candida parapsilosis. Plum-merron and the like (food and machinery, 2019) are used for compounding and applying 3 excellent fermentation strains of lactobacillus plantarum, saccharomyces cerevisiae and candida parapsilosis in rice steamed sponge cakes. The results show that: the compound volume ratio of the 3 kinds of bacteria is 1: 3: when 6, the obtained rice steamed sponge cake has the best organoleptic quality and texture characteristics. The rice steamed sponge cake obtained by fermenting the compound leaven has better quality.

There have been reports of the presence of Candida parapsilosis in liquor brewing systems (Liujun super et al, brewing science 2015; Pulin Liu, et al, Annals of Microbiology 2017; Yanjianggang et al, food science 2018), but the role of this yeast in liquor brewing is still unclear. Yuya et al (food science, 2018) reported that a low-alcohol-yielding Candida parapsilosis (Candida humulis) and Saccharomyces cerevisiae (Saccharomyces cerevisiae) were subjected to a wine mixing fermentation experiment. The results show that: the mixed fermentation effectively reduces the ethanol content besides producing the glycerol with high yield, and simultaneously improves the content of fragrant substances such as ethyl esters and the like, thereby obviously increasing the flower fragrance and the fruit fragrance. The ethyl phenylacetate has strong and sweet honey fragrance, the threshold value of the substance is 0.1mg/L (Dingyunlian, etc., brewing, 2008), and the substance has an important effect on the flavor characteristics of the white spirit (Wangshang, brewing, 2019). 5 esters of ethyl hexanoate, ethyl phenylacetate, phenethylacetate, ethyl palmitate and ethyl oleate are 'skeleton esters' of Maotai-flavor liquor mechanical brewing turn base liquor (Haitomei and the like, food science 2020). Research shows that the artificially bred special yeast for grape wine/fruit wine and other brewed fruit wine are superior to other yeast brewed products in color, aroma, flavor, taste and wine quality (Kuangyiming, etc., tropical agricultural science, 2016). However, no literature report on the production of ethyl phenylacetate by candida parapsilosis exists at present.

Acetaldehyde is widely present in alcoholic beverages and is recognized as a class I carcinogen by the International agency for research on cancer (IARC). Acetaldehyde has fruity flavor when the concentration is low, and pungent odor is generated when the concentration is high. The average mass concentration (173.24mg/L) of acetaldehyde in the finished white spirit in China is higher than that (less than or equal to 86 mg/L) of acetaldehyde in distilled spirit in other countries. Therefore, considering the current situation of comprehensive intake and low acetaldehyde content in foreign distilled spirits, it is necessary to reduce the acetaldehyde content in white spirits (Zhumengxu, et al, food and fermentation industries, 2016). Higher alcohols are one of the main flavor substances of wines. The higher alcohols mainly include isopropanol, n-propanol, isobutanol, beta-phenylethyl alcohol, etc. (Huangguidong, etc., brewed in China, 2017). If the content of higher alcohols in the spirit is too high, the taste of the spirit is affected and the health of the human body is adversely affected (HofiH, et al. Alcoholism: Clinical and Experimental Research, 2003, 27 (S1): 37S-41S). Higher alcohols are key factors leading to "hangover". Therefore, in the wine making industry, the control of the content of higher alcohols in yellow wine and other wines is more urgent (Huangguidong, et al, Proc. foods and Biotechnology, 2018).

The quality of the fermented grains for brewing the white spirit is better by fermenting the fermented grains in a pool at a lower temperature. For example, the low-temperature cellar entry of the strong aromatic white spirit production is beneficial to the formation of mellow and sweet substances, the control of acidity and ester production, the control of the formation of higher alcohol, the inhibition of the propagation and growth of harmful bacteria, the acceleration of the aging of a new cellar and the stable improvement of the wine quality (Cao Xinli, brewing technology, 2006). The fermentation of white wine, yellow wine and other wines is completed under an acidic condition, so that the yeast strains with low-temperature-resistant and acid-resistant fermentation capacities have better adaptability to the brewing environment, and the fermentation can be better started at a low temperature, so that a better application effect can be obtained.

Chinese patent (CN201910539397.4) reports a yeast with low isoamyl alcohol yield and high beta-phenylethyl alcohol yield, a separation culture method and application thereof, wherein the yeast is candida planifolia (dwarf) and has excellent characteristics of low isoamyl alcohol yield, high beta-phenylethyl alcohol yield and the like. At present, few patents and application reports about candida parapsilosis are provided. Aiming at the application aspects of white spirit and the like, the main defects of the strains reported by the existing research and patents are as follows: the wine yield of the Candida parapsilosis strain is low, the alcoholic strength of fermented mash does not reach 6% (v/v), and the alcohol conversion capacity of the Candida parapsilosis in fermentation liquor with the same sugar degree is generally less than 60% of that of the saccharomyces cerevisiae. The low-temperature resistant growth capacity of the candida parapsilosis is not reported, and the mixed fermentation white spirit of the candida parapsilosis and saccharomyces cerevisiae is not reported. The white spirit is a product of multi-strain mixed fermentation consisting of saccharomyces cerevisiae and non-saccharomyces cerevisiae. In the prior research and patent search, the application report of the candida parapsilosis and the composite microbial inoculum compounded by the candida parapsilosis and the saccharomyces cerevisiae strain, which have the characteristics of high yield of alcohol, low yield of higher alcohol, low temperature resistance and the like, does not exist in the liquor brewing industry at present.

The invention has the technical contents that:

an object of the present invention is to provide a novel Candida parapsilosis strain which is highly capable of producing ethanol by fermentation, has a unique fermentation flavor and is suitable for fermentation in a low temperature environment, and reduces the production of acetaldehyde and fusel oil during fermentation.

It is another object of the present invention to provide liquid fermentation characteristics of Candida parapsilosis.

The invention also aims to provide a solid saccharomyces cerevisiae agent containing candida parapsilosis.

The invention also aims to provide a preparation method of the solid-state saccharomyces cerevisiae agent containing candida parapsilosis.

The invention also aims to provide the application of the solid brewing microbial inoculum of the candida parapsilosis in the fermentation of white spirit.

The invention discloses a novel Candida parapsilosis strain which is characterized by strong low temperature and acid resistant growth capacity, high alcohol yield and low acetaldehyde and higher alcohol content in a fermentation product, wherein the strain is Candida parapsilosis PC12(Candida hunensis PC12) and is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2020656. The preservation date is 10 months and 30 days in 2020.

The Candida parapsilosis PC12 strain disclosed by the invention is obtained by separating and screening fermented grains in a certain brewery in Hubei province. The strain has the following characteristics:

the colony morphology characteristics of 2d of Candida parapsilosis PC12 cultured on YPD plates are as follows: round, dark brown, smooth and moist in appearance, and neat in outer edge. The microscopic morphological characteristics of shaking culture in YPD liquid medium for 30h are: the cells are round or oval, have a size of (2.0-5.0) μm x (2.2-6.0) μm, proliferate by budding, and generally clump into 4-6 cells.

The biochemical characteristics of C source utilization by Candida parapsilosis PC12 are as follows: the ability to utilize sucrose, trehalose, D-raffinose, inulin, and D-galactose was strong, and glycerol could be used for growth, but L-arabinose and D-xylose could only be weakly utilized.

The acid resistance of Candida parapsilosis PC12 is characterized in that: candida parapsilosis PC12 still grows well in YPD medium with pH of 3.5, and standard strain CGMCC2.2346 grows poorly and is inhibited obviously.

The low temperature resistant growth capacity of the candida parapsilosis PC12 is characterized in that: the strain is a low-temperature yeast strain, can still grow well at 15 ℃, has a relative growth amount which is 17.3 percent higher than that of a standard Candida parapsilosis CGMCC2.2346, and has a relative growth amount which reaches 96 percent compared with the culture temperature of 25 ℃.

26S rDNA PCR amplification and sequencing experiments are carried out on the Candida parapsilosis PC12 strain, and analysis shows that: the homology of the strain PC12 and 26S rDNA of Candida hupemis CGMCC2.2346 reaches 100 percent, and the strain PC12 is determined to be Candida parapsilosis.

The invention also discloses Candida parapsilosis PC12 and application of the microbial agent thereof in white spirit fermentation.

The invention also discloses application of the candida parapsilosis PC12 microbial inoculum composition in white spirit fermentation.

The reference strains adopted in the invention are common microbial strains, wherein the Candida parapsilosis CGMCC2.2346 is uploaded in a China common microbial strain preservation management center catalog and is in an open state, and scientists can ask for the strain preservation center. Saccharomyces cerevisiae CCTCC AY92017 is common microorganism strains, is loaded in a catalog of China center for type culture collection and is in an open state, and scientists can ask for the China center for type culture collection.

The microbial inoculum disclosed by the invention is in a solid state or a liquid state. Preferably, the PC12 yeast agent disclosed by the invention is in a solid state.

The invention also discloses a method for preparing the solid microbial inoculum, which comprises the following steps:

(1) activating strains: inoculating Candida parapsilosis PC12 strain and Saccharomyces cerevisiae CCTCC AY92017 respectively in sterilized wort triangular flask culture medium with sugar degree of 12 barlin, and performing shake culture at 28 deg.C for 24 hr;

(2) solid culture: the ingredients for solid culture were: moistening wheat bran 80% and wheat flour 20% with water, steaming, cooling, inoculating 5-10% of the activated and cultured 2 yeast liquid respectively, packaging, maintaining fermentation temperature at 20-25 deg.C, and culturing for 2 days;

(3) vacuum freeze drying and crushing: and (3) carrying out vacuum freeze drying treatment on the fermented solid sample at the temperature of minus 45 ℃, crushing the dried culture by using a universal crusher after the water content of the sample is lower than 10%, preparing into a solid powdery microbial inoculum, filling into a sealed plastic bag, and placing in a refrigerator for preservation. Sampling, and detecting the number of viable bacteria of the Candida parapsilosis PC12 strain and the Saccharomyces cerevisiae CCTCC AY92017 yeast in each sample by adopting a dilution flat plate number measurement method to reach 30 hundred million cfu/g.

(4) Mixing and packaging: packing the solid microbial inoculum containing the PC12 strain in a plastic bag, and sealing to prepare the brewing microbial inoculum containing a single strain; mixing Saccharomyces cerevisiae CCTCC AY92003 and PC12 at a ratio of viable bacteria of 1:5, sealing in plastic bag to obtain the Saccharomyces cerevisiae composition.

The invention has the advantages that:

the Candida parapsilosis PC12 strain provided by the invention is a new strain and has the following advantages:

1. the strain is a low-temperature fermentation strain and has strong low-temperature resistance and acid-resistant growth capacity. The strain can still grow well at 15 ℃, the relative growth amount of the strain is 17.3 percent higher than that of a Candida parapsilosis standard strain CGMCC2.2346, compared with the culture temperature of 25 ℃, the relative growth amount of the strain reaches 96.1 percent, and the standard strain CGMCC2.2346 grows poorly at 15 ℃. The PC12 strain can tolerate an acid growth environment with pH of 3.5, and has stronger acid resistance than a standard strain.

2. The yield of alcohol of the Candida parapsilosis PC12 strain is high. The liquor yield of the sorghum saccharification liquid fermented by the strain PC12 reaches 8.63% (v/v), is 28.2% higher than that of the standard strain CGMCC2.2346, and reaches 91.9% of that of the Saccharomyces cerevisiae AY 92017. Under the condition of solid fermentation, the wine yield of the PC12 composition is 20.9 percent higher than that of the standard strain CGMCC2.2346 composition.

3. Candida parapsilosis PC12 strain has strong capability of producing ethyl phenylacetate flavor substances. The content of ethyl phenylacetate produced by PC12 reaches 1.09mg/L, which is 87.9% higher than that of standard strain 2.2346, and the honey flavor of the fermented wine is prominent.

4. The Candida parapsilosis PC12 strain has low acetaldehyde and higher alcohol content. The acetaldehyde and total higher alcohol content of the PC12 sorghum saccharified liquid fermented wine was only 62.3% and 69.5% of that of the standard strain 2.2346.

5. The advantages of the fermentation of the composition: the content of acetaldehyde and the content of total high-grade alcohol in the white spirit fermented by the composition liquid method are respectively lower than those of a contrast. The composite brewing microbial inoculum added with the Candida parapsilosis PC12 has high wine yield, low acetaldehyde content, moderate total high-grade alcohol content, good flavor and sense of the fermented white wine and strong fragrance.

Compared with the standard strain CGMCC2.2346, the Candida parapsilosis PC12 strain has the characteristics of higher low temperature resistance and acid growth resistance, high liquor yield, strong capacity of producing ethyl phenylacetate serving as a flavor component of the white liquor, prominent fragrance, low content of acetaldehyde and higher alcohol and good taste of the fermented white liquor.

Drawings

FIG. 1 is a phylogenetic tree of 26S rDNA D1/D2 region sequence of Candida parapsilosis PC12 strain

Detailed Description

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Examples

Example 1 isolation and identification of Candida parapsilosis PC12 Strain

1.1 isolation of Candida parapsilosis PC12 Strain: a fermentation substrate sample was collected from a winery in Hubei province, 10g of the sample was weighed and added to 90mL of sterilized liquid acidic YPD medium (pH3.5) and subjected to shake cultivation at 15 ℃ and 150rpm for 80 hours. Taking out 1mL of bacterial suspension for serial dilution, and taking out 10^-3、10^-4、10^-5And (3) coating the three dilution gradients on a Martin-Bengal red culture medium, repeating each dilution gradient for 3 times, culturing in a constant-temperature incubator at 20 ℃ for 36 hours to obtain a single colony, performing microscopic examination to obtain a yeast cell morphology, and further performing streaking, separation, purification and storage for measuring the growth temperature. Activating 20 strains of yeast and 2 strains of reference yeast separated from fermentation substrate sample by YPD plate, inoculating into 100mL YPD liquid triangular flask culture medium with pH of 3.5, respectively, culturing at 15 deg.C for 24 hr at 180rpm/min in shaking table, and determining OD of bacterial liquid_600nmThe growth ability of each strain was measured by comparison, and 6 OD strains were selected from them_600nmA yeast strain of ≧ 1.1 (Table 1). Then, from 6 yeasts with acid resistance and low temperature resistance, through comparison of alcoholic fermentation capacity and flavor production in a YPD liquid culture medium, 1 yeast with high liquor yield and unique honey flavor is finally screened, wherein the strain is the PC12 strain, the liquor yield in the YPD liquid culture medium reaches 7.45%, and the honey flavor is thick (Table 2).

OD of Table 120 strains of Low temperature resistant Yeast grown at 15 ℃_600nmResults of value measurement

TABLE 2 comparison of the alcohol yield and aroma-producing ability of YPD culture solutions fermented by different yeasts

The inventor deposits a new separated Candida parapsilosis PC12 strain (Candida humilis PC12) in China type culture collection (CCTCC NO: M2020656) of a preservation organization designated by the national intellectual property office, wherein the preservation date is 10, 10 and 30 days in 2020. China Center for Type Culture Collection (CCTCC) for short, located in the university of Wuhan, Hubei province, zip 430072, telephone: 027-68752319, Email: cctcc @ whu.

1.2 identification of Candida parapsilosis PC12 Strain

The PC12 is identified by morphological feature observation, physiological and biochemical determination and 26S rDNA D1/D2 region gene sequence analysis results. In the identification, Candida parapsilosis standard strain 2.2346 was selected as a control strain. The Candida parapsilosis standard strain 2.2346 is a common microorganism strain, comes from Beijing strain preservation center of Chinese academy of sciences, and can be requested by scientists.

1.2.1 Observation of morphological characteristics of Candida parapsilosis PC12 strains

The colony morphology characteristics of 2d of Candida parapsilosis PC12 cultured on YPD plates are as follows: round, dark brown, smooth and moist appearance, and regular outer edges, and the microsoform characteristics after shaking culture in YPD liquid medium for 30h are as follows: the cells are round or oval, have a size of (2.0-5.0) μm x (2.2-6.0) μm, proliferate by budding, and generally clump into 4-6 cells.

1.2.2 phylogenetically status identification by sequence analysis of the 26S rDNA D1/D2 region

Performing PCR amplification reaction by using genome DNA of the yeast strain as a template, amplifying a 26S rDNA D1/D2 region of the yeast strain, and amplifying a 26S rDNA forward primer NL1 of the yeast strain (5'-GCATATCAATAA GCGGAGGAAAAG-3'); reverse primer NL4 (5'-GGTCCGTGTTTCAAGAC GG-3').

PCR amplification was performed using 50. mu.L of the reaction system (25. mu.L of Taq DNA polymerase, 22. mu.L of triple distilled water, 1. mu.L of forward primer, 1. mu.L of reverse primer, 1. mu.L of extracted DNA template). The PCR cycling program was: pre-denaturation at 94 ℃ for 5 min; circulating for 33 times at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 60 s; extending for 10min at 72 ℃; keeping the temperature at 16 ℃ for 10 min. The PCR product was detected by 0.8% agarose gel electrophoresis and then sequenced by Wuhan Tianyihui Biotech Ltd.

The sequencing length is about 500bp, and the sequencing result is manually corrected by adopting DNA Star software. The corrected sequences were subjected to homologous sequence search in the nucleic acid sequence database of NCBI (national center for biotechnology information), and the genetic relationship and the systematic position thereof between the test strain and the known yeast strain were compared. According to the homologous sequence search result, taking the D1/D2 region sequence of the model strain with the closest genetic relationship with the experimental strain, using Clustal X to calibrate the alignment sequence, using MEGA5.2 software adjacent method, and using 1000 times Bootstrap test to construct phylogenetic tree

After molecular sequencing and homologous sequence comparison, the homology of the strain PC12 with 26S rDNA of Candida humilis is found to reach 100%, and the strain PC12 is determined to be Candida parapsilosis. FIG. 1 is a phylogenetic tree based on the 26S rDNA D1/D2 region sequence.

1.2.3 determination of carbon Source utilization ability of Candida parapsilosis PC12 Strain

The results of the utilization of different carbon sources by the PC12 strain are shown in Table 3.

TABLE 3 fermentation test results of different carbon sources of Candida parapsilosis PC12

Note: "-" is not available, "+" is available, the more available the effect is.

Example 2 comparison of acid resistance and Low temperature growth resistance of Candida parapsilosis PC12

2.1 acid resistance comparison of Candida parapsilosis PC12 with Standard Strain and Saccharomyces cerevisiae

Inoculating activated slant strain of yeast into YPD liquid culture medium, culturing at 28 deg.C and 170rpm for 24 hr in shaking bed to obtain seed liquid. Taking cultured seed liquid according to the proportion of 1 × 10⁶each/mL was inoculated into YPD liquid medium of different pH values (3.0-5.0, pH adjusted 1:1 with acetic acid and lactic acid) at 28 ℃ and 17%0rpm, shake culturing for 24h, diluting the bacterial liquid by a certain multiple, and measuring the OD of the bacterial liquid by using an ultraviolet spectrophotometer_600nmAn absorbance value. The results show (Table 4) that the PC12 strain can tolerate an acidic growth environment with a pH of 3.5, while the standard strain CGMCC2.2346 has poor acid resistance and obviously inhibits the growth at a pH of 3.5.

TABLE 4 acid resistance results and analysis (OD) of two Candida parapsilosis and Saccharomyces cerevisiae strains_600nmValue)

2.2 comparison of the Low temperature growth resistance of Candida parapsilosis PC12

Inoculating the activated three yeast seed solutions into YPD liquid culture medium at the same ratio, shaking-culturing at 15-42 deg.C and 180rpm for 24 hr, and measuring OD₆₀₀The value is obtained. The detection results are shown in Table 5, and the results show that the PC12 strain still grows well at 15 ℃, has strong low temperature resistance, and has a relative growth amount of 96.1% at 15 ℃ compared with the growth amount cultured at 25 ℃. OD of PC12 strain cultured at 15 ℃_600nmThe values are 17.3% and 60.5% higher than those of the standard strain 2.2346 and the Saccharomyces cerevisiae AY92017, respectively.

TABLE 5 Low temperature resistant growth Capacity OD of three yeasts_600nmResults of value measurement

Example 3 comparison of ability to produce alcohol, ethyl phenylacetate and higher alcohol by Candida parapsilosis PC12 Strain fermented sorghum saccharification liquid

The Candida parapsilosis PC12 strain has higher ethanol production capacity. The fermentation results of sorghum liqueur are shown in Table 6, and the yield of the ethanol fermented by the Candida parapsilosis PC12 strain is high. Under the condition of liquid state fermentation of liquor at 28 ℃, the liquor yield of the sorghum saccharified liquid fermented by the PC12 strain reaches 8.63% (v/v), is 28.2% higher than that of the standard strain 2.2346, and reaches 91.9% of that of the Saccharomyces cerevisiae AY 92017. The gas chromatography detection result of the flavor components of the distilled liquor sample of the fermented sorghum saccharified liquid shows that the content of ethyl phenylacetate produced by the PC12 strain reaches 1.09mg/L, is 87.9 percent higher than that of the standard strain 2.2346, and the honey flavor of the fermented liquor sample is outstanding. The acetaldehyde and total high alcohol (n-propanol, isobutanol, isoamyl alcohol and beta-phenylethyl alcohol) contents of the liquid fermented wine of the PC12 strain were 75.80mg/L and 159.59mg/L, respectively, which were 62.3% and 69.5% of that of the standard strain 2.2346, respectively. The Candida parapsilosis PC12 is proved to have more advantages in the application of white spirit fermentation.

TABLE 6 gas chromatographic analysis results (mg/L) of distilled liquor samples of different yeast strains for liquid fermentation of Baijiu

Note: "-" indicates no detection.

Example 4 Candida parapsilosis PC12 Saccharomyces cerevisiae agent and preparation method of composition thereof

The method comprises the following steps:

4.1 strain activation: inoculating Candida parapsilosis PC12 strain and Saccharomyces cerevisiae CCTCC AY92017 respectively in sterilized wort triangular flask culture medium with sugar degree of 12 barlin, and performing shake culture at 28 deg.C for 24 hr;

4.2 solid culture: the ingredients for solid culture were: moistening wheat bran 80% and wheat flour 20% with water, steaming, cooling, inoculating 5-10% of the activated and cultured 2 yeast liquid respectively, packaging, maintaining fermentation temperature at 20-25 deg.C, and culturing for 2 days;

4.3 vacuum freeze drying and crushing: and (3) carrying out vacuum freeze drying treatment on the fermented solid sample at the temperature of minus 45 ℃, crushing the dried culture by using a universal crusher after the water content of the sample is lower than 10%, preparing into a solid powdery microbial inoculum, filling into a sealed plastic bag, and placing in a refrigerator for preservation. Sampling, and detecting the number of viable bacteria of the Candida parapsilosis PC12 strain and the Saccharomyces cerevisiae CCTCC AY92017 yeast in each sample by adopting a dilution flat plate number measurement method to reach 30 hundred million cfu/g.

4.4 mixing and packaging: packing the solid microbial inoculum containing the PC12 strain in a plastic bag, and sealing to prepare the brewing microbial inoculum containing a single strain; mixing Saccharomyces cerevisiae CCTCC AY92003 and PC12 at a ratio of viable bacteria of 1:5, sealing in plastic bag to obtain the Saccharomyces cerevisiae composition.

Example 5 application of Candida parapsilosis PC12 brewing microbial inoculum in white spirit by liquid method

Mixing Saccharomyces cerevisiae AY92003 and Candida parapsilosis PC12 Saccharomyces cerevisiae strains according to the strain number ratio of 1:0, 5:1, 1:5 and 0:1 respectively, inoculating sorghum saccharified liquid, and fermenting with liquid Chinese liquor. After the fermentation was completed, the fermentation broth was subjected to gas chromatography, and the results are shown in Table 7. As can be seen from the table, when the strains of the Saccharomyces cerevisiae AY92003 and the Candida parapsilosis PC12 are mixed and fermented according to the ratio of 1:5, the contents of ethanol, ethyl acetate, ethyl phenylacetate, acetic acid, caproic acid and the like in the fermentation liquor are the highest, the contents of acetaldehyde and higher alcohol are all in lower levels, the contents of flavor substances are moderate, and the mouthfeel is coordinated. The method shows that after the candida parapsilosis PC12 saccharomyces cerevisiae is added into the saccharomyces cerevisiae in a certain proportion, the method has obvious promotion effect on improving the liquor yield and the liquor flavor substance content, the acetaldehyde content of the liquor sample is low, the total high-grade alcohol content is moderate, and the taste is good.

TABLE 7 gas chromatographic analysis results (mg/L) of the wine samples fermented by mixing Saccharomyces cerevisiae AY92017 and PC12 at different ratios

Note: "-" indicates no detection.

Example 6 application of PC12 brewing combined bacterial agent in improving liquor yield and liquor quality

6.1 test methods

Spreading and cooling fermented grains distilled in a brewing workshop of a liquor enterprise, stacking according to 250 kg/group, wherein the proportion of the medium-temperature Daqu inoculated in a control group is 10% of the feeding amount, the proportion of the medium-temperature Daqu inoculated in two experimental groups is 6% of the feeding amount (the using amount of the Daqu is reduced by 40%), simultaneously respectively adding the PC12 brewing microbial inoculum composition (PC12 group) prepared in the example 6 and the CGMCC2.2346 brewing microbial inoculum composition (CGMCC2.2346 group) for replacing PC12 by the Candida parapsilosis standard strain, wherein the inoculation proportion is 0.5% (volume percentage) of the feeding amount and 40% of the medium-temperature Daqu is replaced. Inoculating, fermenting in a tank for 35d, sampling, and distilling. Three replicates were processed each. And (4) carrying out gas chromatography analysis on the contents of components such as alcohols, acids, esters and the like in the distilled sample. The main apparatus is as follows: a gas chromatograph.

6.2 wine quality chromatography results and sensory evaluation

As can be seen from table 8: the alcohol content in the fermented grains of the experimental group containing the PC12 brewing microbial inoculum composition can reach 5.2 percent, and is improved by 20.9 percent compared with the fermented grains of CGMCC2.2346 group. The content of acetaldehyde and total high-grade alcohol in the PC12 group is respectively reduced by 42.2 percent and 28.0 percent compared with the content of total high-grade alcohol in the CGMCC2.2346 group, the content of acetic acid, caproic acid and ethyl phenylacetate is respectively improved by 10.7 percent, 38.9 percent and 28.6 percent, and the effects of reducing the content of acetaldehyde and high-grade alcohol in the wine base, improving the flavor of the wine base and improving the taste are obvious. The PC 12-containing brewing microbial inoculum composition added in the fermented grains has obvious promotion effect on improving the yield of the raw wine and the quality of wine products, and can reduce the consumption of high-temperature Daqu by 40 percent.

TABLE 8 gas chromatographic analysis results of each component in distilled liquor sample of fermented grains

The sensory evaluation comparison is as follows: the wine sample of the control group has the score of 85-90, and the comment is that the wine is sweet and mellow, has light fragrance and has heavy stimulation taste in the mouth; the wine sample of CGMCC2.234 group has a score of 90-95, and the score is that the wine is sweet and mellow, has strong fragrance and mellow wine body; the PC12 group wine sample score is 95-100, and the comment is that the wine is sweet and mellow, the fragrance is rich and harmonious, and the wine body is full. The medium temperature Daqu is matched with Candida parapsilosis PC12 microbial inoculum to obviously improve the quality of wine, because the content of the acetaldehyde with irritation in the original wine and the higher alcohols such as isoamyl alcohol which easily causes the 'top' of the wine is obviously reduced, the content of the main flavor components of the white spirit such as acetic acid, caproic acid, ethyl phenylacetate and the like is obviously increased, the flavor in the wine is more coordinated, and the taste is better.

Claims (10)

1. The Candida parapsilosis can be used for brewing white spirit, and is characterized in that the strain has strong low temperature and acid resistant growth capacity, high alcohol yield and low acetaldehyde and higher alcohol content in a fermentation product, is Candida parapsilosis PC12(Candida hunensis PC12), is preserved in China center for type culture collection (CCTCC NO: M2020656).

2. A Saccharomyces cerevisiae agent containing the Candida parapsilosis PC12 strain as claimed in claim 1, characterized in that the active ingredient is the Candida parapsilosis PC12 strain and its metabolite.

3. A saccharomyces cerevisiae bacterial agent composition comprising the strain as claimed in claim 1.

4. The composition of the brewer's inoculant according to claim 3, wherein the composition comprises the following components (inoculum size):

candida parapsilosis 10x10⁸cfu/g

Saccharomyces cerevisiae 2x10⁸cfu/g

Wherein the Candida parapsilosis is CCTCC NO: M2020656, and the Saccharomyces cerevisiae is CCTCC NO: AY 92017.

5. A enhanced koji for brewing wine comprising the strain of claim 1.

6. The enhanced brewing koji as claimed in claim 5, characterized in that it comprises the following components:

95 percent of traditional yeast

Brewing microbial inoculum composition 5%

Wherein the traditional yeast is Daqu or Xiaoqu for brewing white spirit prepared by a traditional method, and the brewing microbial inoculum composition is the component in claim 4.

7. The saccharomyces cerevisiae bacterial agent composition according to claim 3, wherein said saccharomyces cerevisiae bacterial agent composition is a solid bacterial agent.

8. A process for preparing the solid saccharomyces cerevisiae composition described in claim 3 comprising the steps of:

(1) activating strains: inoculating Candida parapsilosis PC12 strain and Saccharomyces cerevisiae CCTCC AY92017 respectively in sterilized wort triangular flask culture medium with sugar degree of 12 barlin, and performing shake culture at 28 deg.C for 24 hr;

(2) solid culture: the ingredients for solid culture were: 70% of bran and 30% of wheat flour, moistening the materials with water, steaming and cooling the materials, respectively inoculating 5-10% of the 2 yeast liquid obtained by the activation culture in the step (1), boxing the inoculated yeast liquid, maintaining the fermentation temperature at 20-25 ℃, and culturing for 2 days;

(3) vacuum freeze drying and crushing: and (3) carrying out vacuum freeze drying treatment on the fermented solid sample at the temperature of minus 45 ℃, crushing the dried culture by using a universal crusher after the water content of the sample is lower than 10%, preparing into a solid powdery microbial inoculum, filling into a sealed plastic bag, and placing in a refrigerator for preservation. Sampling, and detecting the number of viable bacteria of the Candida parapsilosis PC12 strain and the Saccharomyces cerevisiae CCTCC AY92017 yeast in each sample by adopting a dilution flat plate number measurement method to reach 30 hundred million cfu/g;

(4) mixing and packaging: packing the solid microbial inoculum containing the PC12 strain in a plastic bag, and sealing to prepare the brewing microbial inoculum containing a single strain; mixing Saccharomyces cerevisiae CCTCC AY92003 and PC12 at a ratio of viable bacteria of 1:5, sealing in plastic bag to obtain the Saccharomyces cerevisiae composition.

9. Use of the Candida parapsilosis PC12 strain according to claim 1 for brewing white spirit.

10. The use of the saccharomyces cerevisiae bacterial agent composition as claimed in claim 3 in brewing of white spirit.

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