CN112322509B - 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 humilis) and lactic acid bacteria are widely used as natural ferments in bakery products such as bread, animal feed, grains, etc. (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). Liujianli (food science, 2018, 39), wusigulianglang (university of inner Mongolia agriculture, 2011), shaoyangjie (modern food technology, 2016, 7) and the like are respectively separated and screened from Chinese traditional noodle starter, acidic dough, traditional glutinous fried cake and the like to obtain the 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 not only can produce glycerol at high yield, but also can effectively reduce the content of ethanol, and simultaneously can improve 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 fruit wine brewed by the artificially bred special yeast for wine/fruit wine and the like is superior to products brewed by other yeasts in color, aroma, flavor, taste and vinosity (Kuangyiming et al, 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 its concentration is low, and pungent odor is generated when its concentration is high. The average mass concentration (173.24 mg/L) of acetaldehyde in the finished product of the Chinese liquor is higher than that (less than or equal to 86 mg/L) of acetaldehyde in distilled liquor of 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 in causing "hangover". Therefore, in the wine 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 fermented grains for brewing the white spirit are fermented in the tank at a lower temperature, so that the quality of the white spirit is better. 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 (CN 201910539397.4) 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 is generally less than 60% of that of the saccharomyces cerevisiae in fermentation liquor with the same sugar degree. 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, no 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, exists in the liquor brewing industry at present.

The technical content of the invention is as follows:

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 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 PC 12) 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 winery 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 Candida parapsilosis PC12 is characterized in that: the strain is low-temperature yeast, can still grow well at 15 ℃, has 17.3 percent higher relative growth than that of a standard Candida parapsilosis CGMCC2.2346, and has 96 percent relative growth 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 humilis 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 Candida parapsilosis CGMCC2.2346 is loaded in China catalog of common microbial strain preservation management center and is in an open state, and scientists can ask for the strain preservation center. Saccharomyces cerevisiae CCTCC AY92017 is common microbe strain, is loaded in China center for type culture collection and is in open state, and may be requested by scientist from 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 into 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 the activated 2 yeast liquid obtained in step (1) at a ratio of 5-10%, packaging, maintaining fermentation temperature at 20-25 deg.C, and culturing for 2 days;

(3) Vacuum freeze drying and crushing: and (3) performing 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 solid powdery microbial inoculum, filling into a sealed plastic bag, and placing in a fresh-keeping layer of a refrigerator for preservation. Sampling, and detecting the viable count 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: packaging the solid microbial inoculum containing the PC12 strain in a plastic bag, and sealing to prepare a brewing microbial inoculum containing a single strain; mixing the saccharomyces cerevisiae CCTCC AY92003 and the PC12 according to the viable bacteria ratio =1, then sealing a plastic bag, and preparing the saccharomyces cerevisiae microbial inoculum 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 the pH value of 3.5, and has stronger acid resistance compared with 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 AY92017. Under the condition of solid state fermentation, the wine yield of the PC12 composition is 20.9 percent higher than that of the standard strain CGMCC2.2346 composition.

3. The 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 percent higher than that of the 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 sample is 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 total high-grade alcohol in the white spirit fermented by the composition liquid method is respectively lower than that of a contrast. The composite saccharomyces cerevisiae microbial inoculum added with the candida parapsilosis PC12 has high wine yield, low acetaldehyde content, moderate total high-grade alcohol content, good flavor sense of 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 high-grade 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 characterization 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 (pH 3.5), and subjected to enrichment culture at 15 ℃ for 80 hours by shaking at 150 rpm. 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, and obtaining a single colony which is subjected to microscopic examination to obtain the cell morphology of the yeast, and further streaking, separating, purifying and storing 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, the strain number is PC12, the liquor yield in the YPD liquid culture medium reaches 7.45%, and the honey flavor is thick (Table 2).

TABLE 1 OD of low temperature resistant Yeast strains grown at 15 ℃_600nmResults of value measurement

TABLE 2 comparison of the liquor 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 PC 12) in a preservation organization designated by the national intellectual property office, namely China center for type culture preservation, with the preservation number of CCTCC NO: M2020656, and the preservation date is 2020, 10, 30 days. China Center for Type Culture Collection (CCTCC) for short, in Wuhan university school of Wuhan city, hubei province, post code 430072, telephone: 027-68752319, email.

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. Candida parapsilosis standard strain 2.2346 is a common microbial strain, comes from Beijing strain preservation center of Chinese academy of sciences, and can be obtained by scientists.

1.2.1 Observation of morphological characteristics of Candida parapsilosis PC12 plants

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 phylogenetic position identification by sequence analysis of the 26S rDNA D1/D2 region

Taking the genome DNA of the yeast strain as a template to carry out PCR amplification reaction, amplifying the 26S rDNA D1/D2 region of the yeast strain, and amplifying the NL1 (5; reverse primer NL4 (5-.

PCR amplification was performed using a 50. Mu.L reaction system (25. Mu.L Taq DNA polymerase, 22. Mu.L triple distilled water, 1. Mu.L forward primer, 1. Mu.L reverse primer, 1. Mu.L extracted DNA template). The PCR cycling program was: pre-denaturation at 94 ℃ for 5min; circulation is carried out for 33 times at 94 ℃ 30s,55 ℃ 30s and 72 ℃ 60 s; extending for 10min at 72 ℃; keeping the temperature at 16 ℃ for 10min. 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 at NCBI (national center for biotechnology information), and the genetic relationship between the test strain and the known yeast strain and the systematic position thereof 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 adjacency method, and performing Bootstrap test 1000 times to construct phylogenetic tree

After molecular sequencing and homologous sequence comparison, the homology of the strain PC12 and 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 sequence of the 26S rDNA D1/D2 region.

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

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

TABLE 3 fermentation test results of C12 Candida parapsilosis on different carbon sources

Note: "-" is not available, and "+" 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 comparison of acid resistance of Candida parapsilosis PC12 with the 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 multiplied by 10⁶Inoculating the strain/mL into YPD liquid culture medium with different pH values (3.0-5.0, adjusting pH by adopting acetic acid and lactic acid 1)_600nmAnd (4) an 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-resistant growth ability of Candida parapsilosis PC12

Inoculating the activated three yeast seed solutions into YPD liquid culture medium at the same ratio, shake culturing at 180rpm at 15-42 deg.C for 24 hr, and determining 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 percent and 60.5 percent 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 result of sorghum liquid state white spirit is shown in table 6, and the yield of ethanol fermented by the Candida parapsilosis PC12 strain is high. Under the condition of liquid state fermentation of the white spirit at the temperature of 28 ℃, the liquor yield of the PC12 fermented sorghum saccharified liquid 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 AY92017. The result of the gas chromatography detection 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, which 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 PC12 liquid fermented wine are 75.80mg/L and 159.59mg/L respectively, and are 62.3% and 69.5% 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 state fermentation of white liquor

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 into 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 solid culture comprises the following ingredients: 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 viable count 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: packaging the solid microbial inoculum containing the PC12 strain in a plastic bag, and sealing to prepare a brewing microbial inoculum containing a single strain; mixing the saccharomyces cerevisiae CCTCC AY92003 and the PC12 according to the viable bacteria ratio =1, then sealing a plastic bag, and preparing the saccharomyces cerevisiae microbial inoculum composition.

Example 5 application of Candida parapsilosis PC12 Saccharomyces cerevisiae in liquor by liquid method

And (2) mixing the saccharomyces cerevisiae AY92003 with the candida parapsilosis PC12 saccharomyces cerevisiae agent according to the bacterial count ratio of 1. 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 bacteria number of the Saccharomyces cerevisiae AY92003 and the Candida parapsilosis PC12 is 1. The method shows that after the candida parapsilosis PC12 saccharomyces cerevisiae is added into saccharomyces cerevisiae in a certain proportion, the method has obvious promotion effects on improving the liquor yield and the liquor flavor substance content, the acetaldehyde content of a liquor sample is low, the total high-grade alcohol content is moderate, and the taste is good.

TABLE 7 gas chromatography analysis results (mg/L) of the Saccharomyces cerevisiae AY92017 and PC12 mixed fermented distilled liquor samples 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 adding the PC12 brewing microbial inoculum composition (PC 12 group) prepared in the embodiment 6 and the CGMCC2.2346 brewing microbial inoculum composition (CGMCC 2.2346 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. Fermenting in a pool for 35d after inoculation, sampling and distilling. Three replicates were processed each. And (3) 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 vinosity chromatography results and organoleptic 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, which is 20.9 percent higher than that of the fermented grains of the 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 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, 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 score of the control group is 85-90, and the comment is that the wine sample is sweet and mellow, has light fragrance and heavy stimulation taste when being eaten; the wine sample of CGMCC2.234 group has a score of 90-95, and the score is soft and sweet, thick fragrance and thick wine body; the wine sample score of the PC12 group is 95-100, and the comment is soft and sweet, mellow, rich and harmonious in fragrance and full in wine body. The medium temperature Daqu is matched with the Candida parapsilosis PC12 microbial inoculum to obviously improve the wine quality, because the content of the irritating acetaldehyde and the high alcohol such as isoamyl alcohol which easily causes the 'head-up' drinking in the original wine is obviously reduced, the content of the main fragrance components of the white spirit such as acetic acid, caproic acid, ethyl phenylacetate and the like is obviously increased, the fragrance 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 PC 12), is preserved in China center for type culture collection, and has the preservation number of CCTCC NO: M2020656.

2. A Saccharomyces cerevisiae microbial inoculum containing Candida parapsilosis PC12 strain as claimed in claim 1, characterized in that the active ingredients are Candida parapsilosis PC12 strain and its metabolites.

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

4. The brewer's inoculant composition according to claim 3, wherein it 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: AY92017.

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

5 percent of wine-brewing microbial inoculum composition

Wherein the traditional yeast is Daqu or Xiaoqu prepared by a traditional method and used for brewing white spirit, and the brewing microbial inoculum composition is the component of 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 into 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: 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 viable count 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: packaging the solid microbial inoculum containing the PC12 strain in a plastic bag, and sealing to prepare a brewing microbial inoculum containing a single strain; mixing the saccharomyces cerevisiae CCTCC AY92003 and the PC12 according to the viable bacteria ratio =1, then sealing a plastic bag, and preparing the saccharomyces cerevisiae microbial inoculum 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 composition described in claim 3 in the brewing of white spirit.

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