CN112662575B - Saccharomycetes capulae with high protease activity and high liquor yield, and composition and application thereof - Google Patents

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 saccharomycete with capsula-covering and membrane-covering functions disclosed by the invention has the characteristics of high temperature resistance, strong acid resistance and alcohol resistance, high protease activity and alcohol yield, and strong capacity of producing organic acids such as acetic acid and liquor flavor components such as phenethyl acetate, rice wine produced by fermentation has the advantages of high content of total acid and amino acid nitrogen, aromatic flavor and the like, and the application of the brewing microbial inoculum composition and the brewing reinforced yeast to rice wine and liquor fermentation can improve the quality and yield of rice wine and liquor products.

Description

High protease activity and high alcohol yield of saccharomyces saccharomyces saccharomyces, its composition and application

The field of invention:

The invention belongs to the field of brewing, and in particular, the present invention relates to a yeast with saccharomyces fascicularis, a solid inoculum containing the bacteria, and the application of the yeast and its composition in the brewing of rice wine and liquor.

Background technique:

Saccharomycopsis fibuligera, also known as endosporidium, is one of the main yeasts in a variety of koji and fermented grains (Fan Guangsen, et al. Chinese Journal of Food Science, 2018; Luo Xiaoye, et al. Food and Fermentation Industry , 2016; Hao Wenjun, et al. Brewing Technology, 2019; Li Jingxin, et al. Food and Fermentation Industry, 2018; Tang Jie, et al. Brewing in China, 2020). Some studies have shown that the distribution of S. saccharomyces cerevisiae in 6 brands of rice wine and wine has an absolute advantage, and it plays an important role in the productivity of Shaoxing rice wine and wine (Zang Wei, et al. Journal of Mycology, 2015).

Saccharomyces fascicularis has the ability to secrete amylase, protease, β-glucosidase and other hydrolases, which can provide nutrition for the growth of other liquor-fermenting microorganisms such as Saccharomyces cerevisiae. The synergistic effect of Saccharomyces cerevisiae and other microorganisms has an important contribution to the production of alcohol and the formation of liquor flavor (Wang Juwei, et al. Research progress on yeast Saccharomyces clascens, a common yeast in the fermentation process of liquor, Guangxi Science, 2020 Year; Su Chang, et al. Food Research and Development, 2018). Sun Sijia et al. (Food and Fermentation Industry, 2019) optimized the enzyme production conditions and enzymatic properties of a high-yielding amylase Saccharomyces cerevisiae. After 72h solid-state fermentation, the amylase activity reached 12297u/g, and the amylase produced It can adapt to the acidic and high temperature environment of Daqu fermentation, and has the potential to be applied to high temperature Daqu production. Zhou Yangzi et al. (Food and Fermentation Industry, 2020) isolated an amylase-producing yeast strain from high-temperature Daqu, with a maximum enzyme production of 1452u/mL in liquid culture. The amylase-producing, wine-producing and aroma-producing abilities of the bacteria are relatively strong, and it has good application potential in liquor brewing.

Amino acids are the main source of higher alcohols in liquor, and higher alcohols and their derivatives are important aroma substances in liquor (Zhou Henggang. Brewing Technology, 1998). The quality grade of rice wine is positively correlated with the amino acid nitrogen content in rice wine, and the higher the grade, the higher the amino acid nitrogen content of rice wine (national standard for rice wine GB/T13662-2018). The application of acidic protease for wine in the wine industry has various functions such as dissolving and fermenting raw materials, promoting microbial reproduction, decomposing protein to generate aroma substances, etc., and improving the yield and quality of wine (Tang Shengqiu, et al. Winemaking Technology, 2005). Adding a certain amount of protease can improve the stability of wine, increase the flavor of wine, and improve the sensory properties and nutritional value of wine (Ubeda J, et al. FEMS Yeast Research, 2014; Xu Yanan, et al. Modern Food Technology, 2015).

Organic acids are important aroma components in liquor and rice wine, and are also precursors for the formation of esters. Acetic acid can make liquor feel refreshing (The Complete Book of Liquor Production Technology, p769, edited by Shen Yifang, China Light Industry Press, 2013). (Food Science, 2020) reported that the main acids in koji saccharification samples of Xiaoqu liquor were lactic acid and acetic acid, of which the formation of acetic acid was related to the saccharomyces fascicularis in the samples. Wu Jian et al. (Food and Fermentation Industry, 2020) compared the fermentative acid production capacity of four yeasts including Saccharomyces cerevisiae, Pichia pastoris and Saccharomyces cerevisiae in rice koji juice medium. Yeast has the strongest ability to produce total acid, reaching 3.57g/L.

Fermentation of saccharomyces cerevisiae in sorghum and other substrates can produce a variety of esters, lactones, alcohols, organic acids and aldehydes and ketones, which play an important role in the formation of liquor flavor (Hao Wenjun, et al. Brewing Science and Technology, 2019; Huang Hao, et al. Food and Fermentation Industry, 2020). Sun Sijia et al. (Wine Science and Technology, 2018) used microbial enhancement technology to apply the yeast CICC 33077 to the production of high-temperature Daqu in sesame-flavor liquor. Compared with the control Daqu, the surface of the enhanced Daqu was better "dressed" , the koji skin is thinner, the saccharification power and liquefaction power are significantly increased, and the wine obtained from the enhanced Daqu fermentation is mellow, sweet and clean, which is better than the control Daqu fermented wine. (Journal of Guangxi University of Science and Technology, 2016) mixed fermentation with Saccharomyces cerevisiae and Saccharomyces cerevisiae. Compared with the single-strain fermentation of Saccharomyces cerevisiae, the content of total acid and amino acid nitrogen in glutinous rice wine increased by 17.3% and 19.8%, respectively. %; the contents of methanol, isobutanol and isoamyl alcohol decreased significantly. Chang Su et al. (Food Research International, 2020) used Saccharomyces cerevisiae and Saccharomyces cerevisiae mixed yeast as microbial inoculants for Sichuan-style Xiaoqu wine. The activities of enzymes and acid proteases were significantly improved, the ethanol and total ester contents of brewed liquor increased by 42.5% and 11.8%, respectively, while the aldehydes, ketones and heterocyclic compounds decreased by 73.7% and 77.1%, respectively. Wu Jian et al. (China Brewing, 2020.) prepared with Wickerhamomyces anomalus, S. fibuligera, Pichiakudriazevii, Saccharomyces cerevisiae The compound yeast was co-cultured with pure Rhizopus oryzae to prepare flavored rice koji, and with the commercially available Xiaoqu as a control, the quality index detection of flavored rice koji and the verification of the preparation of rice-flavor liquor by semi-solid fermentation were carried out. The results showed that the addition of aroma-producing yeast significantly improved the ester-producing ability and acid-producing ability of flavored rice koji, and greatly improved the aroma of rice-flavor liquor.

et al.Applied Microbiology andBiotechnology,2014)。López-alvarez等(Journal of Bioscience and Bioengineering,2012)将马克斯克鲁维酵母应用于龙舌兰酒发酵中，制备出了具有较高乙醇浓度和芳香化合物的龙舌兰酒。刘梦等(食品与发酵工业，2020)报道了马克斯克鲁维酵母与酿酒酵母液态法混合发酵黄酒的风味与特性，混合发酵黄酒样的乙酸乙酯和乙酸含量分别比单一酿酒酵母提高了2.46倍和1.31倍；高级醇(正丙醇、异丁醇、异戊醇和β-苯乙醇)总量比单一酿酒酵母发酵酒样降低了18.1％。Kluyveromyces marxianus widely exists in yogurt, fruit, kefir and other environments, and is a food-safe yeast (Mo Wenjuan et al. Science in China: Life Science, 2016). Sha Shankar Prasad et al. (Frontiers in microbiology, 2018) reported the co-existence of several yeast species such as Saccharomyces cerevisiae, Saccharomyces clavicularis and Kluyveromyces marxianus in a traditional brewer's inoculant in India. Kluyveromyces marxianus has the ability to produce ethyl acetate and other flavor substances (

et al. Applied Microbiology and Biotechnology, 2014). López-alvarez et al. (Journal of Bioscience and Bioengineering, 2012) applied Kluyveromyces marxianus to tequila fermentation to prepare tequila with higher ethanol concentration and aromatic compounds. Liu Meng et al. (Food and Fermentation Industry, 2020) reported the flavor and characteristics of rice wine fermented by Kluyveromyces marxianus and Saccharomyces cerevisiae in a liquid state. times and 1.31 times; the total amount of higher alcohols (n-propanol, isobutanol, isoamyl alcohol and β-phenylethanol) was reduced by 18.1% compared with that of single Saccharomyces cerevisiae fermented wine samples.

The Chinese patent (CN201810274972.8) reported a strain of Saccharomyces fumigatus and its application. The α-amylase activity of the strain was 2.56 times that of the highest known enzyme activity (7425.4U/g), and it also had the ability to produce α-amylase. The ability of 7 kinds of enzymes, such as saccharification enzyme, acid protease, lipase, etc., can be directly and completely decomposed the distiller's grains directly and completely when used in the quick-rot fungicides. The Chinese patent (CN201910264999.3) reported a strain of ester-producing yeast ZB406 and its application, screened and separated co-fermentation of the saccharomyces cerevisiae and Saccharomyces cerevisiae to realize the simultaneous progress of alcohol fermentation and the synthesis of aroma substances, and to realize the aroma and flavor of liquid fermented rice vinegar significantly improved. Chinese patent (CN201710539656.4) reports a kind of Saccharomyces sp. clasus and its application in the production of medium-high temperature Daqu. The amylase activity of the strain can reach 7425.7U/g. The strain was prepared and processed into a bacterial agent, which was applied to the production of sesame-flavored medium and high temperature Daqu. The saccharification power and liquefaction power of the enhanced medium and high temperature Daqu were significantly higher than those of the control Daqu, which improved the utilization rate of raw materials in the preparation of Daqu. The Chinese patent (201510636469.9) reported a kind of Saccharomyces sp. clasus 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 rice wine. The high-temperature saccharification liquor is prepared by mixed culture of Saccharomyces sp. and rice wine yeast screened from Shaoxing rice wine Xiaoqu to improve the quality of rice wine. The purpose of aroma and taste. Chinese patent (201811299636.5) reports a Xiaoqu fortifier and its preparation and application. The Xiaoqu fortifier is prepared by fermenting a yeast strain of Polycystis buckthorne. Chinese patent (201310250102.4) reports a new strain of Kluyveromyces marxianus, a brewer's inoculum composition containing the strain, and the application of Kluyveromyces marxianus and its brewer's inoculum composition in alcohol fermentation and liquor brewing , the strain has the characteristics of good tolerance and high ester production rate.

The shortcoming of the existing patent is that the reported yeast saccharomyces fascicularis generally has a low alcohol production rate, and reports on the amylase-producing ability of the strain are more, and the acid-producing and protease-producing abilities of the strain are less reported. Yang Zilin et al. (China Brewing, 2016) reported that the alcohol content of glutinous rice wine fermented by a single strain 3-1Y of Saccharomyces cerevisiae was 2.6% vol, and the total acid content was 5.04±0.3 (g/L). Wang Xiaodan et al. (Food Science, 2017) isolated a strain numbered FBKL2.0071 saccharomyces cerevisiae from Maotai Daqu. The solid-state fermented product has a strong fruity aroma, among which ethyl acetate, isoamyl acetate, phenylethyl alcohol, The content of phenylethyl acetate and ethyl palmitate was high, but the strain's wine-producing ability was not high, and the fermentation alcohol content in the solid medium containing wheat and sorghum was less than 3.5%. The strains reported by Zhou Yangzi et al. (Food and Fermentation Industry, 2020) can produce 9.3% alcohol by fermentation in liquid medium containing wheat, rice and bran, and can produce 37 kinds of phenethyl alcohol, ethyl acetate, ethyl caprylate and so on. Fragrance and taste substances, but no report on the ability of this strain to produce acid and protease activity. There is no report on the high acid-producing and high protease activity yeast strains, and there is no report on the mixed fermentation of liquor by the yeast and Kluyveromyces marxianus. Rice wine and white wine are the products of mixed fermentation of multiple strains composed of Saccharomyces cerevisiae and non-Saccharomyces cerevisiae. According to the existing research and patent search, there is currently no rice wine and liquor brewing industry that has both high alcohol production, high protease activity, high total acid content, high content of flavor substances such as phenethyl acetate, and high temperature resistance. A report on the application of the saccharomyces fascicularis and its combined inoculum with Kluyveromyces marxianus strains.

Invention technical content:

One object of the present invention is to provide a new strain of yeast saccharomyces fascicularis, which has strong ethanol-producing ability, high protease activity, unique fermentation flavor and is suitable for fermentation in medium and high temperature environment, and can increase total acid and amino acid during fermentation nitrogen content.

Another object of the present invention is to provide the liquid fermentation characteristics of Saccharomyces fasciens.

Another object of the present invention is to provide a solid-state brewer's microbial inoculum containing Saccharomyces cerevisiae.

Another object of the present invention is to provide a method for preparing a solid-state brewer's microbial inoculum containing Saccharomyces cerevisiae.

Another object of the present invention is to provide the application of the solid-state brewing inoculum of the yeast with saccharomyces lata in the fermentation of rice wine and liquor.

The invention discloses a new strain of Saccharomycopsis fibuligera SF31, which is characterized in that Saccharomycopsis fibuligera SF31 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2021104, and the date of preservation is It is January 19, 2021.

The saccharomyces fascicularis SF31 strain disclosed by the invention is obtained from the fermented grains of a winery in Hubei Province through screening. This strain has the following characteristics:

The morphological characteristics of the colony of YPD SF31 cultured on YPD plate for 2 days are: round, white, rough and dry surface, neat outer edge. The microscopic morphological characteristics of culturing in YPD liquid medium shaker for 30 hours were: cells were round or oval, budding and proliferating.

The biochemical characteristics of carbon source utilization of yeast SF31 are: it can utilize saccharides such as sucrose, maltose, raffinose, glycerol and cellobiose, but cannot utilize lactose, xylose and trehalose.

The acid-resistance characteristics of Saccharomyces buckthorne SF31 are: Saccharomyces buckthorne SF31 still grows well in YPD medium with a pH of 3.0, while the standard strain CGMCC 2.1625 grows poorly and is obviously inhibited.

The characteristics of high temperature resistant growth ability of Saccharomyces fasciatus SF31 are: the strain still grows well at 45℃, and the OD600nm value of SF31 strain cultured at 45℃ is 4.17 times that of its standard strain 2.1625. Compared with the growth at 25°C, the relative growth was 75.6%.

The PCR amplification and sequencing experiments of 26S rDNA of Saccharomycopsis fibuligera strain SF31 were carried out. The analysis showed that the homology between strain SF31 and the 26S rDNA of Saccharomycopsis fibuligeraCGMCC2.1625 reached 100%. Membrane yeast, as shown in Figure 1.

The invention also discloses the application of the yeast SF31 and its microbial inoculum in fermenting rice wine and liquor.

The invention also discloses the application of the yeast SF31 bacterial agent composition in the fermentation of rice wine and liquor.

The reference strain CGMCC 2.1625 used in the present invention is listed in the catalogue of the China General Microorganism Culture Collection and Management Center, and is in an open state, and scientific workers can obtain it from the culture collection center. Kluyveromyces marxianus CCTCC M2013258 is a patented strain, which has been disclosed in Chinese Patent No. 201310250102.4. Any qualified researchers can obtain it from the China Type Culture Collection.

The bacterial agent disclosed in the present invention is solid or liquid. Preferably, the SF31 yeast preparation disclosed in the present invention is solid.

The invention also discloses a method for preparing a solid inoculum, comprising the following steps:

(1) Strain activation: Inoculate yeast SF31 strain and Kluyveromyces marxianus CCTCCM2013258 in sterilized wort conical flask medium with a sugar content of 12 Bahrain, respectively, and cultivate at 28°C for 24 hours on a shaker.

(2) solid culture: the ingredients of solid culture are: bran 68%, corn flour 30%, sucrose 2%, moistened with water, after cooking and cooling, the two kinds of yeast liquids of (1) activated and cultured were respectively 4- 8% were respectively inoculated and packed into boxes, maintained at 25-35°C fermentation temperature, and cultured for 2 days to complete the fermentation.

(3) Vacuum freeze-drying and pulverization: the fermented solid samples are subjected to vacuum freeze-drying treatment at -45°C. After the moisture content of the sample is lower than 10%, the dried culture is pulverized with a universal pulverizer to prepare solid powder bacteria. put it in a sealed plastic bag and store in the freezer. The number of viable cells of Saccharomyces buckthorne SF31 strain and Kluyveromyces marxianus CCTCCM2013258 in each sample was detected by dilution plate counting method, both reaching 3 billion cfu/g.

(4) mixing and packaging: the solid inoculum containing the SF31 strain is packed into a plastic bag and sealed to make a brewing inoculum containing a single bacterial species; by Kluyveromyces marxianus CCTCC M2013258 and SF31 by the viable count ratio=1: After mixing in 2 proportions, a plastic bag is sealed to prepare a brewing fungus composition.

Advantages of the present invention:

The Saccharomyces cerevisiae SF31 strain provided by the present invention is a new strain, and the advantages are as follows:

1. The strain has strong resistance to high temperature, alcohol and acid growth. The strain can still grow well at 45℃, which is 4.17 times the relative biomass of the standard strain CGMCC 2.1625, and its relative growth reaches 75.6% compared with the culturing temperature of 25℃. The SF31 strain can tolerate the acidic growth environment of pH 3.0, which is stronger than the standard strain.

2. The protease activity of yeast SF31 strain is high, and its protease activity is as high as 565U/mL, which is 138.8% of the enzyme activity of the standard strain CGMCC 2.1625; the amino acid nitrogen content of the fermented rice wine of the SF31 strain is high, which is 52.1% higher than that of the standard strain.

3. The yeast SF31 strain has a high alcohol production rate. The yield of fermented glutinous rice saccharified liquid by strain SF31 was 9.68% (v/v), which was 68.1% higher than that of standard strain CGMCC2.1625. Under the solid state fermentation condition, the wine yield of the SF31 composition was 19.3% higher than that of the standard strain CGMCC 2.1625 composition.

4. Strain SF31 has strong ability to produce acid and phenethyl acetate flavor substances. Compared with the standard strain 2.1625, the acetic acid and total acid of the liquid fermented rice wine of the SF31 strain were increased by 44.2% and 46.3%, respectively. The content of phenethyl acetate produced by SF31 was 35.23ug/L, which was 75.3% higher than that of the standard strain 2.1625.

5. The advantages of the composition fermentation: in the combined fermentation with Kluyveromyces marxianus, the acetaldehyde and total higher alcohol contents of the rice wine fermented by the liquid method of the composition are lower than those of the control, respectively. The wine sample added with SF31 compound brewing inoculum has high wine yield and ethyl acetate content, moderate total higher alcohol content, good flavor and sense of fermented rice wine and liquor, and strong aroma.

That is, compared with the standard strain CGMCC2.1625, the SF31 strain has higher high temperature resistance and acid resistance growth ability, high protease activity, high wine yield, strong ability of phenethyl acetate, the flavor component of wine production, and aroma. It has the characteristics of low content of acetaldehyde and higher alcohol, and good taste of fermented rice wine and liquor.

Description of drawings

Figure 1. Phylogenetic tree of 26S rDNA D1/D2 region sequences of Saccharomyces fascicularis SF31 strain

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention, but not to limit the protection scope of the present invention.

Example

Example 1 Isolation and identification of yeast SF31 strain

1.1 Isolation of Saccharomyces cerevisiae SF31 strain: a fermentation substrate sample was collected from a winery in Hubei Province, 10 g of the sample was weighed, and added to 90 mL of sterilized liquid acidic YPD medium (pH 3.0), 40 °C, 180 rpm Shaker enrichment culture for 90h. Take out 1 mL of bacterial suspension for serial dilution, and apply three dilution gradients of 10 ^-2 , 10 ^-3 , and 10 ^-4 to Martin-Bengal red medium. Each dilution gradient is repeated three times, and incubated at 40°C. Incubate in the box for 40h, observe the morphology of the colonies growing on the plate, select the single colony with rough surface of the colony, which is the yeast cell morphology after microscopic examination, and further streak separation, purification and preservation for the determination of acid protease activity. Twenty-one yeast strains and one reference yeast strain isolated from the fermentation matrix samples were activated by YPD plates, then inoculated into 100 mL of glutinous rice saccharification liquid conical flask medium with a sugar content of 20%, and placed on a shaker at 180 rpm/min. After culturing at 30°C for 24 hours, 10 mL of each fermentation broth was centrifuged to collect the fermentation supernatant of each strain, and the protease activity of each fermentation broth was determined by the Folin reagent method, and 5 yeast strains with protease activity ≥ 500 U/mL were screened out (Table 1) . Then, from the 5 strains of yeast with high protease activity, through the comparison of alcohol fermentation ability and flavor production in YPD liquid medium, a yeast strain with high wine yield and unique fruity flavor was finally obtained. No. SF31 strain, its wine yield in YPD liquid medium was 7.16%, and the fruity aroma was strong (Table 2).

Table 1 The detection results of protease activity of different strains of Saccharomyces fumigatus

Table 2 Comparison results of wine yield and aroma production ability of glutinous rice saccharified liquid fermented by different strains of Saccharomyces cerevisiae

The inventor has deposited a new strain of Saccharomycopsis fibuligera SF31 (Saccharomycopsis fibuligera SF31) isolated in the China Center for Type Culture Collection, a preservation institution designated by the State Intellectual Property Office, with the deposit number CCTCC NO: M2021104, and the deposit date is 2021 January 19, 2009. The Chinese Collection of Type Cultures is abbreviated as CCTCC. It is located in Wuhan University, Wuhan City, Hubei Province, 430072, Tel: 027-68752319, Email: cctcc@whu.edu.cn.

1.2 Identification of Saccharomyces cerevisiae SF31 strain

SF31 was identified by the observation of morphological characteristics, physiological and biochemical assays and the results of gene sequence analysis of 26S rDNA D1/D2 region. In the identification, the standard strain 2.1625 of Saccharomyces cerevisiae was selected as the control strain. The standard strain 2.1625 of Saccharomyces fascicularis is a commonly used common microbial strain, from the Beijing Culture Collection Center of the Chinese Academy of Sciences, and can be obtained by scientific workers.

1.2.1 Observation of morphological characteristics of yeast SF31 strain

The morphological characteristics of the colony of YPD SF31 cultured on YPD plate for 2 days are: round, white, rough and dry surface, neat outer edge. The microscopic morphological characteristics of culturing in YPD liquid medium shaker for 30h were: cells were round or oval.

1.2.2 Phylogenetic status identification by 26S rDNA D1/D2 region sequence analysis

The genomic DNA of the yeast strain was used as a template for PCR amplification reaction to amplify the 26S rDNA D1/D2 region of the yeast strain, and the forward primer NL1 (5'-GCATATCAATAA GCGGAGGAAAAG-3') of the yeast 26S rDNA was amplified; the reverse primer NL4 ( 5'-GGTCCGTGTTTCAAGAC GG-3').

A 50 μL reaction system (25 μL Taq DNA polymerase, 22 μL triple distilled water, 1 μL forward primer, 1 μL reverse primer, 1 μL extracted DNA template) was used for PCR amplification. The PCR cycle program was: pre-denaturation at 94°C for 5 min; 33 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 60s; extension at 72°C for 10 min; incubation at 16°C for 10 min. The PCR product was detected by 0.8% agarose gel electrophoresis and sent to Wuhan Jimei Biotechnology Co., Ltd. for sequencing.

The sequencing length was about 500 bp, and the sequencing results were manually proofread with DNA Star software. The corrected sequences were searched for homologous sequences in the nucleic acid sequence database of NCBI, and the phylogenetic relationship and phylogenetic status of the tested strains and known yeast strains were compared. According to the homologous sequence search results, the D1/D2 region sequences of the type strains with the closest relationship to the experimental strains were taken, and the sequences were aligned with Clustal X, MEGA5.2 software neighbor joining method, and 1000 Bootstrap tests were used to construct a phylogenetic tree.

After molecular sequencing and comparison of homologous sequences, it was found that the 26S rDNA homology between strain SF31 and Saccharomycopsis fibuligera reached 100%, and the strain SF31 was determined to be a buckthorn-fusculum yeast. Figure 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 Saccharomyces fascicularis SF31 strain

The utilization results of different carbon sources by SF31 strain are shown in Table 3.

Table 3 Utilization results of different carbon sources for yeast SF31

Note: "-" means unavailable, "+" means available.

Example 2 Tolerance comparison of saccharomyces cerevisiae SF31

2.1 Comparison of acid resistance of Saccharomyces cerevisiae SF31 with standard strains and Saccharomyces cerevisiae

Seed solution was prepared by inoculating activated yeast slant strains in YPD liquid medium at 30° C., 180 rpm, and culturing in a shaker for 24 hours. Take the cultured seed solution and inoculate it into YPD liquid medium with different pH values (3.0-6.5, pH adjusted with lactic acid) at 1×10 ⁶ /mL, cultivate at 30°C, 180rpm, shaker for 24h, and dilute the bacterial solution After a certain multiple, UV spectrophotometer was used to measure the absorbance at OD _{600 nm} of the bacterial solution. The results showed (Table 4) that the SF31 strain could tolerate the acidic growth environment of pH 3.0, while the standard strain CGMCC 2.1625 had poor acid resistance, and its growth was significantly inhibited at pH 3.0.

Table 4 Results and analysis of acid resistance of two strains of Saccharomyces cerevisiae and Saccharomyces cerevisiae (OD _600nm value)

2.2 Comparison of high-temperature growth ability of saccharomyces cerevisiae SF31

The activated three kinds of yeast seed solutions were inoculated into YPD liquid medium in the same proportion, and cultured in a shaker at 180 rpm for 24 hours at different temperatures of 25-45 °C, and the OD ₆₀₀ value was determined. The test results are shown in Table 5. The results show that the SF31 strain still grows well at 45°C and has strong high temperature resistance. Compared with the growth at 25°C, the relative growth at 45°C reaches 75.6%. The OD _600nm value of the SF31 strain cultured at 45°C was 4.17 times that of the standard strain 2.1625.

Table 5 Determination results of the OD _600nm value of two strains of Saccharomyces cerevisiae at different growth temperatures

2.3 Comparison of alcohol tolerance between SF31 strain and standard strain CGMCC2.1625

The seed liquids of strain SF31 and standard strain CGMCC2.1625 were inserted into YPD liquid medium containing ethanol concentration (v/v) of 2%-10%, respectively, placed in 30 ° C, 170 rpm shake flask for 24 hours, and the OD was measured. ₆₀₀ value. As shown in Table 6, in the medium supplemented with 6% (v/v) ethanol, both SF31 and CGMCC2.1625 could grow, but SF31 was more tolerant than CGMCC2.1625, and the OD ₆₀₀ value of SF31 was CGMCC2 159.2% of .1625; in the medium supplemented with 8% ethanol, the tolerance of SF31 was still stronger than that of CGMCC2.1625, and the OD ₆₀₀ of SF31 was 239.3% of that of CGMCC2.1625.

Table 6. Comparison of alcohol-tolerant growth ability between SF31 strain and standard strain CGMCC2.1625 (OD ₆₀₀ )

Example 3 Comparison of the production of alcohol, acid and phenethyl acetate in fermented rice wine by Saccharomyces cerevisiae SF31 strain

Saccharomyces fasciatus strain SF31 has higher ethanol production ability. The fermentation results of glutinous rice saccharification liquid rice wine are shown in Table 7, and the fermented ethanol yield of Saccharomyces cerevisiae SF31 is high. Fermented at 28℃ for 7 days, the yield of fermented glutinous rice saccharified liquid of SF31 strain reached 9.68% (v/v), which was 1.68 times that of standard strain 2.1625. The results of gas chromatography detection of flavor components of fermented rice wine showed that the content of phenethyl acetate produced by SF31 strain reached 35.23ug/L, which was 1.75 times that of standard strain 2.1625, and the fermented rice wine had outstanding aroma. The contents of acetic acid, total acid and amino acid nitrogen in the liquid fermented rice wine of SF31 strain were 569.57 mg/L, 3.98 g/L and 0.73 g/L, which were 144.2%, 146.3% and 152.1% of the standard strain 2.1625, respectively. It indicated that the yeast SF31 had more advantages in the application of rice wine fermentation.

Table 7 Gas chromatography of liquid fermented rice wine with different yeast strains and analysis results of physicochemical indexes of rice wine samples

Embodiment 4 The preparation method of saccharomyces cerevisiae SF31 brewing inoculum and its composition

Follow these steps:

4.1 Activation of strains: Inoculate yeast SF31 strain and Kluyveromyces marxianus CCTCCM2013258 in sterilized wort flask medium with a sugar content of 12 Bahrain, respectively, and culture in a shaker at 28°C for 24 hours.

4.2 Solid culture: The ingredients of solid culture are: bran 68%, corn flour 30%, sucrose 2%, moistened with water, boiled and cooled and dispersed, respectively 4-8% of the two yeast liquids of (1) activated and cultured. The proportions were respectively inoculated and boxed, and the fermentation temperature was maintained at 25-35 °C, and the fermentation was completed in 2 days.

4.3 Vacuum freeze-drying and pulverization: The fermented solid samples are subjected to vacuum freeze-drying treatment at -45°C. After the moisture content of the sample is lower than 10%, the dried culture is pulverized with a universal pulverizer to prepare a solid powdery inoculum and then prepared. Store in an airtight plastic bag and store in the freezer. Sampling, the number of viable cells of Saccharomyces buckthorne SF31 strain and Kluyveromyces marxianus CCTCC M2013258 yeast in each sample was detected by dilution plate counting method, both reaching 3 billion cfu/g.

4.4 Mixing and packaging: put the solid inoculum containing SF31 strain in a plastic bag and seal it to make a brewing inoculum containing a single strain; mix Kluyveromyces marxianus CCTCC M2013258 and SF31 according to the ratio of viable bacteria = 1:2 After mixing, the plastic bag is sealed to prepare a brewing fungus composition.

Example 5 The application of saccharomyces cerevisiae SF31 brewing agent in liquid rice wine

Kluyveromyces marxianus CCTCC M2013258 and Saccharomyces saccharomyces saccharomyces SF31 brewer's inoculum were mixed and inoculated into the glutinous rice saccharification solution at the bacterial count ratio of 1:0, 2:1, 1:1, 1:2 and 0:1 respectively. Fermentation of Famí. After the fermentation, the fermented wine samples were analyzed by gas chromatography, and the results are shown in Table 8. As can be seen from the table, when Kluyveromyces marxianus CCTCCM2013258 and Saccharomyces saccharomyces saccharomyces SF31 were mixed and fermented at a ratio of 1:2, the contents of ethanol, ethyl acetate, phenethyl acetate, and acetic acid in the fermented wine samples were relatively high. High, the proportion of flavor substances is coordinated, and the sensory score is the highest. It shows that after adding a certain proportion of Kluyveromyces marxianus SF31 brewing agent, the protease produced by it can hydrolyze protein into amino acids, which can promote yeast fermentation, and has a great effect on improving the yield of rice wine and the content of flavor substances. It has obvious promoting effect, the total higher alcohol content of the wine sample is moderate, and the taste is good.

Table 8 Analysis of flavor substances, physicochemical indexes and sensory evaluation results of M2013258 and SF31 mixed fermented rice wine in different proportions

Example 6 Application of SF31 brewing combination bacterial agent in improving liquor yield and liquor quality 6.1 Test method

The fermented grains after the distillation in the brewing workshop of a certain liquor enterprise were cooled and piled up according to 250kg/group, the proportion of inoculated traditional koji in the control group was 6% of the feeding amount, and the ratio of inoculating traditional koji in the two experimental groups was 3% of the feeding amount ( Reduce the amount of koji by 50%), and simultaneously add the SF31 brewing inoculum composition (SF31 group) prepared in Example 6 and the CGMCC2.1625 brewing inoculum composition (CGMCC2.1625 group) that replaces SF31 with the standard strain of Saccharomyces cerevisiae ), and the inoculation ratio was 0.3% (volume percentage) of the feeding amount, replacing 50% of traditional koji. After inoculation, it was fermented in the pond for 30 days, and sampled for distillation. Three parallel samples were processed each. The contents of alcohols, acids, esters and other components in the distilled samples were analyzed by gas chromatography. Main instrument: gas chromatography analyzer.

6.2 Chromatographic analysis results and sensory evaluation of wine quality

It can be seen from Table 9 that the alcohol content of the fermented grains in the experimental group containing the SF31 brewing fungus composition can reach 6.8%, which is 19.3% higher than that of the CGMCC2.1625 group. The contents of n-propanol, isobutanol and isoamyl alcohol in SF31 group were decreased by 10.9%, 8.7% and 18.5% respectively compared with CGMCC2.1625 group. The contents of ethyl acetate and phenethyl acetate were increased by 7.6% and 47.2% respectively, and the total acid and total ester were increased by 17.5% and 8.4%, respectively, which had obvious effects on reducing the content of higher alcohol in the original wine, improving the flavor and improving the taste of the original wine. It shows that adding the SF31-containing brewing fungus composition to the fermented grains has a significant promoting effect on improving the original wine yield and wine quality, and can reduce the amount of koji by 50%.

Table 9 Gas chromatographic analysis results of each component in the fermented fermented grain distilled wine sample

The sensory evaluation comparison is as follows: the scores of the wine samples in the control group are 85-90, and the comments are that the taste is more mellow, the aroma is lighter, and the entrance is more irritating; the scores of the wine samples in the CGMCC2.234 group are 90-95, and the comments are that the wine is sweet and mellow. , strong aroma, and mellow wine body; SF31 group wine sample score is 95-100, and the comments are sweet and mellow, rich and harmonious aroma, and full-bodied wine. It shows that the use of SF31 yeast in traditional distiller's yeast can significantly improve the quality of the wine, and the content of the main aroma components of liquor such as total acid, total ester and phenethyl acetate can be significantly increased, which makes the aroma in the wine more harmonious and the taste is better. better.

Claims (11)

1. a strain of saccharomyces cerevisiae, can be used for rice wine and liquor brewing, it is characterized in that, this bacterial strain is resistant to high temperature and acid-resistant growth ability, protease activity and alcohol yield are high, total acid and amino acid nitrogen content in the fermented product Gao, the strain is Saccharomycopsis fibuligera SF31 and Saccharomycopsis fibuligera SF31. 2. A Saccharomyces cerevisiae agent containing the Saccharomyces buckthorne SF31 strain described in claim 1, characterized in that its active component is the Saccharomyces buckthorne SF31 and its metabolites whose deposit number is CCTCC NO: M2021104. 3. A yeast composition containing the storage number CCTCC NO: M2021104 described in claim 1. 4. according to the deposit number of CCTCC NO:M2021104 described in claim 3, it is characterized in that it contains the following components, by inoculum size: Saccharomyces cerevisiae 20x10 ⁸ cfu/g, Kluyveromyces marxianus 10x10 ⁸ cfu/g, Among them, Saccharomyces buckthorne is CCTCC NO: M2021104, and Kluyveromyces marxianus is CCTCC NO: M2013258. 5. A brewing-enhanced koji containing the yeast with the deposit number CCTCC NO: M2021104 described in claim 1. 6. according to the Saccharomyces cerevisiae fortified koji of CCTCC NO:M2021104 with deposit number in claim 5, it is characterized in that it contains the following components: Traditional koji 90%, Brewing fungus composition 10%, Wherein the traditional koji is Daqu or Xiaoqu prepared by a traditional method for liquor brewing, and the brewing inoculum composition is the composition of claim 4 , the capsule-encapsulating and laminating yeast composition. 7. according to the inoculum composition of the brewing-enhanced koji of the saccharomyces buckthorne with the deposit number of CCTCC NO:M2021104 described in claim 6, it is characterized in that the described brewing inoculum composition is a solid microbial inoculum. 8. prepare the method for the fortified koji of the saccharomyces cerevisiae described in claim 5, comprising the steps: (1) Activation of strains: respectively inoculate Saccharomyces buckthorne with the deposit number of CCTCC NO: M2021104 and Kluyveromyces marxianus CCTCC M2013258 in sterilized wort conical flask medium with a sugar content of 12 Bahrain, shake at 28°C Bed culture for 24 hours; (2) Solid culture: The ingredients for solid culture are: bran 68%, corn flour 30%, sucrose 2%, moistened with water, boiled and cooled, and then the two yeast strains activated and cultured in the above step (1) were separated. After inoculation according to the proportion of 4-8%, it is boxed, and the fermentation temperature is maintained at 25-35 °C, and the fermentation is completed in 2 days; (3) Vacuum freeze-drying and pulverization: The fermented solid samples are subjected to vacuum freeze-drying treatment at -45 °C. After the moisture content of the sample is less than 10%, the dried culture is pulverized with a universal pulverizer to prepare solid powder bacteria. and put them into sealed plastic bags, put them in the fresh-keeping layer of the refrigerator for preservation, and use the dilution plate counting method to detect the viable counts of yeast SF31 strain and Kluyveromyces marxianus CCTCC M2013258 in each sample. 3 billion cfu/g; (4) Mixing and packaging: put the solid inoculum containing SF31 strain in a plastic bag and seal it to make a brewing inoculum containing a single strain; the ratio of Kluyveromyces marxianus CCTCC M2013258 to SF31 according to the number of viable bacteria = 1: After mixing in 2 proportions, a plastic bag is sealed to prepare a brewing fungus composition. 9. the application of the saccharomyces cerevisiae with the deposit number of CCTCC NO:M2021104 described in claim 1 in rice wine and liquor brewing. 10. The application of the buckwheat multi-film yeast composition described in claim 3 with the deposit number of CCTCC NO: M2021104 in the brewing of rice wine and liquor. 11. The application of the wine-enhancing koji described in claim 5 in the brewing of rice wine and liquor.

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