CN112662575A - Saccharomycetes fibuligera with high protease activity and high wine yield, and composition and application thereof - Google Patents
Saccharomycetes fibuligera with high protease activity and high wine yield, and composition and application thereof Download PDFInfo
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Abstract
The invention discloses a novel Saccharum-covering composite saccharomycete, a brewing microbial inoculum containing the saccharomycete and application of the saccharomycete in brewing rice wine and white spirit. The saccule-covering saccharomycete has the features of high temperature resistance, high acid and alcohol resistance, high proteinase activity, high alcohol yield, high capacity of producing organic acid, such as acetic acid, and other spirit flavor components, and the fermented rice wine has high total acid and amino acid nitrogen content, rich fragrance, etc. and the application of the composite brewing microbial inoculum and the reinforced brewing yeast in rice wine and spirit fermentation can raise the quality and yield of rice wine and spirit product.
Description
The invention belongs to the field of:
the invention belongs to the field of brewing, and particularly relates to a saccharomycete with a capsule covering membrane, a solid microbial inoculum containing the saccharomycete, and application of the saccharomycete and a composition of the saccharomycete in brewing rice wine and white spirit.
Background art:
saccharomycosis fibuligera (also known as Neurospora pseudochinensis) is one of the main yeasts in various distiller's yeast and fermented grains (Fangansen, et al, Chinese food science, 2018, Luo lobule, et al, food and fermentation industry, 2016, Huowen et al, brewing science, 2019, Lijing, et al, food and fermentation industry, 2018, Tangjie, et al, Chinese brewing, 2020). Research shows that the distribution of the saccharomyces bayanus in 6 brands of yellow wine medicines has absolute advantages and plays an important role in the productivity of the Shaoxing yellow wine medicines (zangwei, et al. fungi science, 2015).
The enveloped yeast has the capability of secreting hydrolytic enzymes such as amylase, protease, beta-glucosidase and the like, and can provide nutrition for the growth of other white spirit fermentation microorganisms such as saccharomyces cerevisiae and the like. The synergistic effect of the saccharomycete tectorial membrane yeast and other microbes has important contribution to the generation of alcohol and the formation of white spirit flavor (Wangchuang, et al. research progress of common saccharomycete tectorial membrane yeast in the white spirit fermentation process, Guangxi science, 2020; Suchang, et al. food research and development, 2018). The enzyme production condition of a strain of high-yield amylase, namely ascochyta sporotrichum is optimized and the enzymology property is researched by Sunjiao and the like (food and fermentation industry, 2019), through 72-hour solid state fermentation, the amylase activity reaches 12297u/g, and the produced amylase can adapt to the acidic high-temperature environment of Daqu fermentation and has the potential of being applied to high-temperature Daqu production. Zhouyangzi (food and fermentation industry, 2020) isolated 1 amylase-producing strain of Saccharum covering yeast from high temperature Daqu, with the maximum enzyme production of 1452u/mL in liquid culture. The strain has stronger amylase production capacity, wine production capacity and aroma production capacity, and has good application potential in liquor brewing.
Amino acids are the main source of higher alcohols in white spirit, and higher alcohols and their derivatives are important flavor-producing substances in white spirit (Zhongchanggang. brewing science, 1998). The quality grade of the yellow wine is positively correlated with the content of amino acid nitrogen in the yellow wine, and the higher the grade of the yellow wine, the higher the content of the amino acid nitrogen in the yellow wine is (the national standard GB/T13662-2018 of the yellow wine). The acid protease for wine has the functions of dissolving fermentation raw materials, promoting microbial propagation, decomposing protein to generate fragrant substances and the like, and improves the yield and quality of wine (Tangshengball, et al. brewing science and technology, 2005). The stability of wine, flavor substances of wine and sensory properties and nutritional value of wine can be improved by adding a certain amount of protease (Ubeda J, et al. FEMS Yeast Research, 2014; Xuyaman, et al. modern food science 2015).
Organic acid is an important flavor component of white spirit and rice wine, and is also a precursor for generating esters. Acetic acid can make the liquor refreshing (liquor production technical book, p769, Shen Yi Fang Shu, China light industry Press 2013). Wangchun et al (food science, 2020) reported that lactic acid and acetic acid are the main acids in a saccharification sample of finished koji of Xiaoqu liquor, wherein the formation of acetic acid is related to the enveloped yeast in the sample. Wujian and the like (food and fermentation industry, 2020) compare the acid production capability of 4 yeasts such as sacculus tectorial membrane yeast, pichia pastoris, saccharomyces cerevisiae and the like in a rice koji juice culture medium, and the result shows that the total acid production capability of the sacculus tectorial membrane yeast is strongest and reaches 3.57 g/L.
The fermentation of the yeast covered with the capsule in the matrix such as sorghum can generate various esters, lactones, alcohols, organic acids and aldehyde ketones compounds, and has an important effect on the formation of the flavor of the white spirit (Haowen et al, brewing science 2019, Huanghao et al, food and fermentation industry 2020). Sunxiaojia and the like (brewing science and technology, 2018) adopt a microorganism strengthening technology, and the ascochyta fibuligera CICC 33077 is applied to the production of the sesame flavor type white spirit high-temperature Daqu. Yanzilin, etc. (the university of Guangxi science and technology, 2016) performs mixed fermentation on the envelope-covering yeast and the saccharomyces cerevisiae, and compared with single-strain fermentation of the saccharomyces cerevisiae, the contents of total acid nitrogen and amino acid nitrogen in the glutinous rice wine are respectively improved by 17.3 percent and 19.8 percent; the contents of methanol, isobutanol and isoamylol are obviously reduced. Chang Su et al (Food Research International,2020) adopt a mixed yeast of enveloped yeast and saccharomyces cerevisiae as a microorganism strengthening inoculant for Sichuan process Xiaoqu liquor, and the results show that compared with the traditional Xiaoqu liquor, the activities of saccharifying enzyme and acid protease of the strengthening leaven prepared by inoculating the strengthening inoculant are obviously improved, the contents of ethanol and total esters of brewed liquor are respectively improved by 42.5% and 11.8%, and the contents of aldehyde ketone and heterocyclic compound are respectively reduced by 73.7% and 77.1%. Wujian et al (Chinese brewing, 2020.) use Hansenula anomala (Wickerhamomyces anomallus), Saccharomyces cerevisiae (S.fibuligera), Pichia kluyveri (Pichia kurarazevii) and Saccharomyces cerevisiae (Saccharomyces cerevisiae) to prepare compound yeast, co-culture with pure rhizopus oryzae to prepare flavor rice-koji, and perform quality index detection and semi-solid fermentation on the flavor rice-koji to prepare the rice-flavor liquor by using the commercial rice-koji as a contrast. The result shows that the addition of the aroma-producing yeast obviously improves the ester-producing capability and the acid-producing capability of the flavor rice koji, and greatly improves the aroma of the rice-flavor liquor.
Kluyveromyces marxianus (Kluyveromyces marxianus) is widely present in yogurt, fruit, kefir and other environments, and is food safety grade yeast (Muwenjuan and other Chinese science: Life sciences, 2016). Shankar Prasad et al (Frontiers in microbiology, 2018) reported the simultaneous presence of multiple yeast species such as Saccharomyces cerevisiae, Saccharomycopsis fibuligera and Kluyveromyces marxianus in a traditional inoculant of Saccharomyces cerevisiae in India. Kluyveromyces marxianus has the capability of producing various flavor substances such as ethyl acetate (
Applied Microbiology and Biotechnology, 2014). Lopez-alvarez et al (Journal of Bioscience and Bioengineering,2012) applied Kluyveromyces marxianus to the fermentation of agave wine to prepare agave wine with higher ethanol concentration and aromatic compounds. Liu Meng et al (food and fermentation industry, 2020) reported that the flavor and characteristics of Kluyveromyces marxianus and Saccharomyces cerevisiae mixed fermentation yellow wine by liquid state method, the ethyl acetate and acetic acid contents of the mixed fermentation yellow wine sample are respectively improved by 2.46 times and 1.31 times than those of single Saccharomyces cerevisiae; the total amount of higher alcohols (n-propanol, isobutanol, isoamyl alcohol and beta-phenylethyl alcohol) is reduced by 18.1 percent compared with that of a single saccharomyces cerevisiae fermented wine sample.
Chinese patent (CN201810274972.8) reports a strain of Saccharopolyspora fibuligera and application thereof, the activity of the strain for producing alpha-amylase is 2.56 times of the highest enzyme activity (7425.4U/g) known at present, and the strain also has the capability of producing 7 enzymes such as saccharifying enzyme, acid protease, lipase and the like, and can directly and thoroughly decompose vinasse rapidly when being applied to fast-decomposing microbial inoculum. Chinese patent (CN201910264999.3) reports an ester-producing yeast ZB406 and application thereof, co-fermentation of screened and separated saccharomyces cerevisiae realizes synchronous alcohol fermentation and synthesis of aroma substances, and realizes obvious improvement of aroma and flavor of liquid fermented rice vinegar. Chinese patent (CN201710539656.4) reports a Saccharopolyspora fibuligera and application thereof in medium-high temperature yeast production, and the amylase activity of the strain can reach 7425.7U/g. The strain is prepared and processed into a microbial inoculum, the microbial inoculum is applied to the production of the sesame flavor type medium-high temperature yeast, the saccharification capacity and the liquefaction capacity of the enhanced medium-high temperature yeast are obviously higher than those of a control yeast, and the utilization rate of raw materials in the yeast preparation process is improved. Chinese patent (201510636469.9) reports a Saccharopolyspora fibuligera and its use, the strain can produce isoamyl alcohol in Maotai-flavor liquor. Chinese patent (application No. 201910622949.8) reports a brewing method for improving the quality of liquefied yellow wine, and the aim of improving the aroma and taste of the yellow wine is achieved by preparing high-temperature saccharified yeast through mixed culture of Saccharopolyspora fibuligera and yellow wine yeast screened from Shaoxing yellow wine Xiaoqu. Chinese patent (201811299636.5) reports a Xiaoqu enhancer and its preparation and application. The Xiaoqu reinforcer is prepared by fermenting a Fujian fungus strain. Chinese patent (201310250102.4) reports a new Kluyveromyces marxianus strain, a wine brewing microbial inoculum composition containing the strain, and application of the Kluyveromyces marxianus strain and the wine brewing microbial inoculum composition thereof in alcohol fermentation and white spirit brewing, wherein the strain has the characteristics of good tolerance and high ester yield.
The defects of the prior patent are that the reported sacculus-covering membrane yeast generally has low wine yield, more reports on the amylase producing capability of the strain and less reports on the acid producing and protease producing capabilities of the strain. Yanzilin et al (Chinese brewing, 2016) report that a 3-1Y strain of Saccharomycopsis fibuligera coated yeast fermented glutinous rice wine has an alcoholic strength of 2.6% vol and a total acid content of 5.04 +/-0.3 (g/L). Wangxiangdan and the like (food science, 2017) separate a strain of Saccharomycopsis fibuligera with the number of FBKL2.0071 from Maotai Daqu, and a solid fermentation product has strong fruit flavor, wherein the content of ethyl acetate, isoamyl acetate, phenethyl alcohol, phenethyl acetate and ethyl palmitate is high, but the wine production capacity of the strain is not high, and the fermentation alcoholic strength in a solid culture medium containing wheat and sorghum is less than 3.5%. The bacterial strain reported by Zhouyangzi (food and fermentation industry, 2020) produces 9.3% of alcohol by fermentation in a liquid culture medium containing wheat, rice and bran, and can produce 37 aroma-producing substances such as phenethyl alcohol, ethyl acetate, ethyl caprylate and the like, but the bacterial strain does not report the capability of producing acid and producing protease activity. The sacculus-covering yeast strains with high acid production and high protease activity are not reported, and the mixed fermentation of the sacculus-covering yeast and Kluyveromyces marxianus for white spirit is not reported. Rice wine and Chinese liquor are the product of mixed fermentation of several kinds of yeast including Saccharomyces cerevisiae and non-Saccharomyces cerevisiae. According to the existing research and patent search, no application report of the composite bacteria agent which has the characteristics of high yield of alcohol, high protease activity, high total acid content, high content of flavor substances such as phenethyl acetate and the like, high temperature resistance and the like, and is compounded with the Kluyveromyces marxianus strain exists in the rice wine and liquor brewing industry at present.
The invention has the technical contents that:
the invention aims to provide a novel sacculus-covering yeast strain which has strong ethanol production capacity by fermentation, high protease activity and unique fermentation flavor, is suitable for fermentation in medium-high temperature environment and can improve the content of total acid and amino acid nitrogen in the fermentation.
It is another object of the present invention to provide liquid fermentation characteristics of the Saccharomycopsis fibuligera.
The invention also aims to provide a solid saccharomyces cerevisiae agent containing the saccharomyces cerevisiae.
The invention also aims to provide a preparation method of the solid saccharomyces cerevisiae agent containing the saccharomyces cerevisiae.
The invention also aims to provide the application of the solid brewing microbial inoculum of the sacculus-covering yeast in the fermentation of rice wine and white spirit.
The invention discloses a novel bursa-buckled membrane-coated yeast strain, which is characterized in that bursa-buckled membrane-coated yeast SF31 (Saccharomyces fibuligeraSF31) is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2021104, and the preservation date is 2021 year, 1 month and 19 days.
The invention discloses a capsule-covering yeast SF31 which is obtained by separating and screening fermented grains of a certain brewery in Hubei province. The strain has the following characteristics:
the colony morphology characteristics of the enveloped yeast SF31 cultured on YPD plates for 2d are as follows: is round and white, has rough and dry surface and tidy outer edge. The microscopic morphological characteristics of shaking culture in YPD liquid medium for 30h are: the cells are round or oval and bud and proliferate.
The biochemical characteristics of the buckling capsule envelope yeast SF31 for utilizing the carbon source are as follows: sugars such as sucrose, maltose, raffinose, glycerol, and cellobiose can be utilized, but lactose, xylose, and trehalose cannot be utilized.
The acid resistance of the saccule-covering yeast SF31 is characterized in that: the enveloped yeast SF31 still grows well in YPD medium with pH of 3.0, and the standard strain CGMCC2.1625 grows poorly and is inhibited obviously.
The high-temperature resistant growth capability of the saccule-covering yeast SF31 is characterized in that: the strain still grows well at 45 ℃, and the OD600nm value of the SF31 strain cultured at 45 ℃ is 4.17 times that of the standard strain 2.1625. The relative growth amount was 75.6% as compared with the growth amount cultured at 25 ℃.
26S rDNA PCR amplification and sequencing experiments are carried out on the strain of the Saccharomycopsis fibuligera SF31, and analysis shows that: the homology of the strain SF31 and 26S rDNA of Saccharomyces fibuligera CGMCC2.1625 reaches 100%, and the strain SF31 is determined to be a Saccharomycopsis fibuligera yeast, as shown in figure 1.
The invention also discloses application of the Saccharum-covering yeast SF31 and a microbial agent thereof in fermenting rice wine and white spirit.
The invention also discloses application of the capsule-coated yeast SF31 microbial inoculum composition in fermenting rice wine and white spirit.
The reference strain capsule coated yeast CGMCC2.1625 adopted in the invention is loaded in the catalog of China general microbiological culture collection center and is in an open state, and scientists can ask for the culture collection center. Kluyveromyces marxianus CCTCC M2013258 is a patent preservation strain and is disclosed in Chinese patent 201310250102.4, and any researcher meeting the conditions can ask for the strain from the Chinese typical culture collection center.
The microbial inoculum disclosed by the invention is in a solid state or a liquid state. Preferably, the SF31 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 Saccharomycopsis fibuligera SF31 strain and Kluyveromyces marxianus CCTCC M2013258 respectively in sterilized triangular flask culture medium of wort with sugar degree of 12 Balin, and shake culturing at 28 deg.C for 24 hr.
(2) Solid culture: the ingredients for solid culture were: 68% of bran, 30% of corn flour and 2% of cane sugar, moistening the materials with water, steaming, cooling and dispersing, respectively inoculating 4-8% of the activated and cultured 2 yeast liquid in the step (1), packing the yeast liquid, keeping the fermentation temperature of 25-35 ℃, and culturing for 2 days to finish fermentation.
(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. The sampling adopts a dilution flat plate number measurement method to detect that the viable count of the saccule-covering yeast SF31 strain and the Kluyveromyces marxianus CCTCCM2013258 yeast in each sample reaches 30 hundred million cfu/g.
(4) Mixing and packaging: packing the solid microbial inoculum containing SF31 strain in plastic bag, sealing to obtain brewing microbial inoculum containing single strain; mixing Kluyveromyces marxianus CCTCC M2013258 and SF31 according to the ratio of viable bacteria to 1:2, sealing a plastic bag, and preparing the brewing microbial inoculum composition.
The invention has the advantages that:
the sacculus-covering yeast SF31 strain provided by the invention is a new strain and has the following advantages:
1. the strain has strong high-temperature resistance, alcohol resistance and acid-resistant growth capacity. The strain can still grow well at 45 ℃, is 4.17 times of the relative biomass of the standard strain CGMCC2.1625 of the saccule-covering yeast, and has a relative growth amount of 75.6 percent compared with the culture temperature of 25 ℃. The SF31 strain can endure acid growth environment with pH3.0, and has stronger acid resistance than standard strains.
2. The activity of protease of the buckling capsule membrane-covering yeast SF31 strain is high, the protease activity is up to 565U/mL, and the protease activity is 138.8 percent of that of a standard strain CGMCC 2.1625; the content of amino acid nitrogen in the SF31 fermented rice wine is 52.1 percent higher than that of the standard strain.
3. The ethanol production rate of the strain of the Saccharum covering yeast SF31 is high. The liquor yield of the strain SF31 fermented glutinous rice saccharified liquid reaches 9.68% (v/v), which is 68.1% higher than that of the standard strain CGMCC 2.1625. Under the condition of solid fermentation, the liquor yield of the SF31 composition is 19.3 percent higher than that of the standard strain CGMCC2.1625 composition.
4. The saccule-covering yeast SF31 has strong capacity of producing acid and phenethyl acetate flavor substances. The acetic acid and total acid of the SF31 liquid fermented rice wine sample are respectively improved by 44.2 percent and 46.3 percent compared with the standard strain 2.1625. The content of the ethyl phenylacetate produced by SF31 reaches 35.23ug/L, which is 75.3 percent higher than that of the standard strain 2.1625.
5. The advantages of the fermentation of the composition: the acetaldehyde and the total high-grade alcohol content of the rice wine fermented by the liquid method of the composition are respectively lower than those of a contrast by combining the fermentation with Kluyveromyces marxianus. The wine sample added with the composite brewing microbial inoculum of the sacculus-covering yeast SF31 has high wine yield and ethyl acetate content, moderate total high alcohol content, good flavor sense of fermented rice wine and white spirit and strong fragrance.
Compared with the standard strain CGMCC2.1625, the strain of the saccharomyces cerevisiae SF31 has the characteristics of higher high temperature resistance and acid growth resistance, high protease activity, high wine yield, strong capacity of producing flavor component ethyl acetate of wine, prominent fragrance, low content of acetaldehyde and higher alcohol, and good mouthfeel of fermented rice wine and white wine.
Drawings
FIG. 1 is a phylogenetic tree of 26S rDNA D1/D2 region sequence of Saccharomycopsis fibuligera SF31 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 Saccharomycopsis fibuligera strain SF31
1.1 isolation of Saccharomycopsis Fungiensis strain SF 31: 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 (pH3.0), and subjected to shake cultivation at 40 ℃ and 180rpm for 90 hours. Taking out 1mL of bacterial suspension for serial dilution, and taking out 10-2、10-3、10-4And (3) coating the three dilution gradients on a Martin-Bengal red culture medium, repeating each dilution gradient for 3 times, culturing for 40 hours in a constant-temperature incubator at 40 ℃, observing the morphology of a bacterial colony growing on a flat plate, selecting a single bacterial colony with a rough surface of the bacterial colony, performing microscopic examination to obtain the cell morphology of the yeast, and further performing streak separation, purification and storage for determining the activity of the acid protease. After 21 yeasts separated from a fermentation substrate sample and 1 reference yeast strain are activated by a YPD plate, respectively inoculated into 100mL of a triangular flask culture medium of glutinous rice saccharification liquid with the sugar degree of 20%, placed in a shaking table at 180rpm/min, cultured for 24 hours at 30 ℃, 10mL of fermentation liquor is respectively taken for centrifugation and collection of fermentation supernatant of each strain, the protease activity of each fermentation liquor is determined by adopting a Fulin reagent method, and 5 yeast strains with the protease activity more than or equal to 500U/mL are screened out (Table 1). Then, from 5 yeasts having high protease-producing activity, 1 yeast having high alcohol-yielding rate and unique fruity flavor was finally screened out by comparing alcohol fermentation ability and flavor-producing ability in a YPD liquid medium, and the strain number is SF31, which shows an alcohol-yielding rate of 7.16% in a YPD liquid medium and is rich in fruity flavor (Table 2).
TABLE 1 protease activity assay results of different strains of Saccharomycopsis fibuligera
TABLE 2 comparison of the liquor yield and aroma-producing ability of different strains of Saccharum-covering yeast for fermenting glutinous rice saccharified liquid
The inventor deposits a new separated strain of the sacculus-covering membrane yeast SF31 (Saccharomyces fibuligeraS F31) in a preservation organization designated by the national intellectual property office, namely China center for type culture Collection, with the preservation number of CCTCC NO: M2021104 and the preservation date of 2021 year, 1 month and 19 days. 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 Saccharomycopsis Functoria Yeast strain SF31
SF31 is identified through morphological feature observation, physiological and biochemical determination and 26S rDNA D1/D2 region gene sequence analysis results. In the identification, the Saccharomycopsis fibuligera reference strain 2.1625 was selected as a control strain. The Saccharomycopsis fibuligera standard strain 2.1625 is common microorganism strain, and is available for scientific workers from Beijing culture Collection of Chinese academy of sciences.
1.2.1 Observation of morphological characteristics of Saccharomycopsis Fungiensis strain SF31
The colony morphology characteristics of the enveloped yeast SF31 cultured on YPD plates for 2d are as follows: is round and white, has rough and dry surface and tidy outer edge. The microscopic morphological characteristics of shaking culture in YPD liquid medium for 30h are: the cells are circular or oval.
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 sent to Wuhan Gimerae Biotechnology Ltd for sequencing.
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 NCBI's nucleic acid sequence database, and the genetic relationship and its systematic position 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, adopting MEGA5.2 software adjacency method, and performing Bootstrap test 1000 times to construct a phylogenetic tree.
After molecular sequencing and homologous sequence comparison, the homology of the strain SF31 and 26S rDNA of Saccharomyces fibuligera is found to reach 100%, and the strain SF31 is determined to be a Saccharomycopsis fibuligera. FIG. 1 is a phylogenetic tree based on the 26S rDNA D1/D2 region sequence.
1.2.3 measurement of carbon Source utilization ability of Saccharomycopsis Fungiensis strain SF31
The results of using different carbon sources by the SF31 strain are shown in Table 3.
TABLE 3 results of utilization of different carbon sources by Saccharomycopsis fibuligera SF31
Note: "-" is not available and "+" is available.
Example 2 comparison of tolerance of Saccharomycopsis fibuligera SF31
2.1 comparison of acid resistance of Saccharomycopsis Fungiensis SF31 with Standard Strain and Saccharomyces cerevisiae
Inoculating activated slant strain of yeast into YPD liquid culture medium, culturing at 30 deg.C and 180rpm for 24 hr in shaking bed to obtain seed liquid. Taking cultured seed liquid according to the proportion of 1 × 106Inoculating each/mL of the mixture to different pH values (3.0-6.5, pH adjusted with lactic acid)Culturing in YPD liquid culture medium at 30 deg.C and 180rpm for 24 hr, diluting the bacterial liquid by a certain times, and measuring bacterial liquid OD with ultraviolet spectrophotometer600 nmAn absorbance value. The results show (table 4) that SF31 strain can tolerate an acidic growth environment with pH of 3.0, whereas the standard strain CGMCC2.1625 has poor acid resistance and growth is significantly inhibited at pH 3.0.
TABLE 4 acid resistance results and analysis (OD) of two strains of Saccharomycopsis and Saccharomyces cerevisiae600nmValue)
2.2 comparison of the high temperature resistant growth of Saccharomycopsis Fungiensis SF31
Inoculating the activated three yeast seed solutions into YPD liquid culture medium at the same ratio, performing shake culture at 25-45 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 SF31 strain still grows well at 45 ℃, has strong high temperature resistance, and has a relative growth amount of 75.6% at 45 ℃ compared with the growth amount cultured at 25 ℃. OD of SF31 strain cultured at 45 ℃600nmThe value was 4.17 times that of the standard strain 2.1625.
TABLE 5 OD of two strains of Saccharomycopsis fibuligera at different growth temperatures600nmResults of value measurement
2.3SF31 compared with the standard strain CGMCC2.1625 for alcohol resistance
Inoculating seed liquid of strain SF31 and standard strain CGMCC2.1625 into YPD liquid culture medium with ethanol concentration (v/v) of 2-10%, shake-flask culturing at 30 deg.C and 170rpm for 24 hr, and measuring OD600The value is obtained. As shown in Table 6, both SF31 and CGMCC2.1625 grew in the medium supplemented with 6% (v/v) ethanol, but SF31 was more tolerant than the OD of CGMCC2.1625 and SF31600The value is 159.2% of CGMCC 2.1625; adding 8% ethanolIn nutrient medium, the tolerance of SF31 is still stronger than OD of CGMCC2.1625 and SF31600Is 239.3% of CGMCC 2.1625.
TABLE 6 comparison of ethanol-resistant growth of Saccharomyces cerevisiae SF31 with that of standard strain CGMCC2.1625 (OD)600)
Example 3 comparison of ability of Saccharum-covering yeast SF31 to produce alcohol, acid and phenethyl acetate in fermented Rice wine
The strain of Saccharomycopsis fibuligera SF31 has high ethanol production capacity. The results of rice wine fermentation using a saccharified glutinous rice solution are shown in Table 7, and the ethanol yield in fermentation using the Saccharum covering yeast strain SF31 was high. The fermented glutinous rice is fermented for 7 days at the temperature of 28 ℃, and the liquor yield of the SF31 strain fermented glutinous rice saccharified liquid reaches 9.68% (v/v), which is 1.68 times that of the standard strain 2.1625. The gas chromatography detection result of the flavor components of the fermented rice wine shows that the content of the ethyl phenylacetate produced by the SF31 strain reaches 35.23ug/L, which is 1.75 times of that of the standard strain 2.1625, and the fermented rice wine has outstanding fragrance. The contents of acetic acid, total acid and amino acid nitrogen in the SF31 liquid fermented rice wine are 569.57mg/L, 3.98g/L and 0.73g/L respectively, and are 144.2%, 146.3% and 152.1% of the standard strain 2.1625 respectively. It shows that the Saccharum subcrenatum covered yeast SF31 has more advantages in rice wine fermentation application.
TABLE 7 gas chromatography of liquid fermented rice wine with different yeast strains and analysis results of physical and chemical indexes of rice wine samples
Example 4 preparation method of Saccharomycopsis fibuligera SF31 Saccharomyces cerevisiae agent and composition thereof
The method comprises the following steps:
4.1 strain activation: inoculating Saccharomycopsis fibuligera SF31 strain and Kluyveromyces marxianus CCTCC M2013258 respectively in sterilized triangular flask culture medium of wort with sugar degree of 12 Balin, and shake culturing at 28 deg.C for 24 hr.
4.2 solid culture: the ingredients for solid culture were: 68% of bran, 30% of corn flour and 2% of cane sugar, moistening the materials with water, steaming, cooling and dispersing, respectively inoculating 4-8% of the activated and cultured 2 yeast liquid in the step (1), packing the yeast liquid, keeping the fermentation temperature of 25-35 ℃, and culturing for 2 days to finish fermentation.
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. The sampling adopts a dilution flat plate number measurement method to detect that the viable count of the saccule-covering yeast SF31 strain and the Kluyveromyces marxianus CCTCC M2013258 yeast in each sample reaches 30 hundred million cfu/g.
4.4 mixing and packaging: packing the solid microbial inoculum containing SF31 strain in plastic bag, sealing to obtain brewing microbial inoculum containing single strain; mixing Kluyveromyces marxianus CCTCC M2013258 and SF31 according to the ratio of viable bacteria to 1:2, sealing a plastic bag, and preparing the brewing microbial inoculum composition.
Example 5 application of Saccharomycopsis fibuligera SF31 Saccharomyces cerevisiae in liquid Rice wine
Mixing Kluyveromyces marxianus CCTCC M2013258 and Saccharomycotina fibuligera SF31 brewing microbial agents according to the ratio of bacteria number of 1:0, 2:1, 1:2 and 0:1 respectively, inoculating glutinous rice saccharification liquid, and carrying out liquid rice wine fermentation. After the fermentation, the fermented wine sample was subjected to gas chromatography, and the results are shown in Table 8. As can be seen from the table, when the Kluyveromyces marxianus CCTCC M2013258 and the Saccharomycopsis fibuligera SF31 are mixed and fermented according to the ratio of 1:2, the contents of ethanol, ethyl acetate, phenethyl acetate, acetic acid and the like in the fermented wine sample are high, the proportion of the contents of flavor substances is coordinated, and the sensory score is highest. The results show that after a certain proportion of the saccharomyces cerevisiae preparation of the saccharomyces cerevisiae SF31 is added into kluyveromyces marxianus, the protease generated by the kluyveromyces marxianus preparation can hydrolyze protein into amino acid, can promote yeast fermentation, has obvious promotion effect on improving the wine yield of rice wine and the content of flavor substances, and has moderate total high-grade alcohol content and good taste of wine samples.
Table 8 results of analysis of flavor substances, physical and chemical indexes and sensory evaluation of mixed fermented rice wine prepared by mixing M2013258 and SF31 in different proportions
Example 6 application of SF31 combined bacterium agent for brewing wine in improving liquor yield and liquor quality 6.1 test method
Fermented grains distilled in a brewing workshop of a liquor enterprise are spread and cooled, and then stacked according to 250 kg/group, a control group is inoculated with traditional distiller's yeast in a proportion of 6% of the fed amount, two experimental groups are inoculated with traditional distiller's yeast in a proportion of 3% of the fed amount (the amount of the distiller's yeast is reduced by 50%), and simultaneously SF31 brewing microbial inoculum composition (SF31 group) prepared in example 6 and CGMCC2.1625 brewing microbial inoculum composition (CGMCC2.1625 group) using capsule membrane-covered yeast standard strains to replace SF31 are respectively added, wherein the inoculation proportion is 0.3% (volume percentage) of the fed amount to replace 50% of the traditional distiller's yeast. Inoculating, fermenting in a tank for 30d, 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 9: the alcohol content in the fermented grains of the experimental group containing the SF31 brewing microbial inoculum composition can reach 6.8 percent, and is improved by 19.3 percent compared with the fermented grains of CGMCC2.1625 group. The contents of n-propanol, isobutanol and isoamylol in the SF31 group are respectively reduced by 10.9 percent, 8.7 percent and 18.5 percent compared with the content in the CGMCC2.1625 group. The contents of ethyl acetate and phenethyl acetate are respectively increased by 7.6 percent and 47.2 percent, the total acid and total ester are respectively increased by 17.5 percent and 8.4 percent, and the functions of reducing the content of higher alcohol in the raw wine, improving the flavor of the raw wine and improving the taste are obvious. The addition of the SF 31-containing brewing microbial inoculum composition in 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 distiller's yeast by 50 percent.
TABLE 9 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 SF31 group wine sample score is 95-100, and the comment is sweet and mellow, fragrant and harmonious, and full wine body. The results show that the wine quality can be obviously improved by using the capsule-covering membrane yeast SF31 microbial inoculum in the traditional distiller's yeast, the contents of main flavor components of white spirit such as total acid, total ester, phenethylacetate and the like are obviously increased, the flavor in the white spirit is more coordinated, and the taste is better.
Claims (11)
1. The strain is the sacculus-covering yeast SF31, Saccharomyces fibuligera SF31 which is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2021104.
2. A Saccharomyces cerevisiae preparation containing the strain of Saccharomycopsis fibuligera SF31 as claimed in claim 1, wherein the active ingredient is strain of Saccharomycopsis fibuligera SF31 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):
saccharomycopsis fibuligera 20x108cfu/g
Kluyveromyces marxianus 10x108cfu/g
Wherein the Saccharomycopsis fibuligera is CCTCC NO: M2021104, and the Kluyveromyces marxianus is CCTCC NO: M2013258.
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:
90 percent of traditional distiller's yeast
The wine brewing microbial inoculum composition is 10 percent
Wherein the traditional distiller's yeast is Daqu or Xiaoqu for brewing white spirit prepared by traditional method, 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: respectively inoculating buckling bag film-coating yeast SF31 strain and Kluyveromyces marxianus CCTCC M2013258 into sterilized wort triangular flask culture medium with sugar degree of 12 Balin, and shake-culturing at 28 deg.C for 24 hr;
(2) solid culture: the ingredients for solid culture were: 68% of bran, 30% of corn flour and 2% of cane sugar, moistening the materials with water, steaming, cooling and dispersing, respectively inoculating 4-8% of the activated and cultured 2 yeast liquid respectively, then boxing, keeping the fermentation temperature at 25-35 ℃, 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 viable count of the saccule-covering yeast SF31 strain and the Kluyveromyces marxianus CCTCC M2013258 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 SF31 strain in plastic bag, sealing to obtain brewing microbial inoculum containing single strain; mixing Kluyveromyces marxianus CCTCC M2013258 and SF31 according to the ratio of viable bacteria to 1:2, sealing a plastic bag, and preparing the brewing microbial inoculum composition.
9. The use of the strain of the Saccharomycopsis fibuligera SF31 in the brewing of rice wine and white spirit as claimed in claim 1.
10. The use of the composition of brewers' inoculant according to claim 3 for the brewing of rice wine and white spirit.
11. Use of the enhanced koji as claimed in claim 5 for brewing rice wine and white spirit.
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