CN101962620B - Saccharomyces cerevisiae strain and application thereof - Google Patents

Abstract

The invention discloses a strain of Saccharomyces cerevisiae and its application. The present invention provides Saccharomyces cerevisiae ZM1-5 CGMCC No.3761. Saccharomyces cerevisiae ZM1-5 can be used to produce alcohol. Yeast strain ZM1-5 has many advantages such as fast growth, high alcohol yield, high temperature resistance, high sugar resistance, high concentration ethanol resistance and acid resistance, etc. In the alcohol process (synchronous saccharification and fermentation), solve the problems in industrial production that yeast cannot tolerate high osmotic pressure, high concentration of ethanol and low efficiency in the later stage of fermentation, and reduce the cost of industrial alcohol production. Yeast strain ZM1-5 is used in the process of synchronous saccharification and fermentation to produce alcohol, which is expected to solve the problems of high substrate concentration (high osmotic pressure), high temperature in the fermentation tank, and inhibition of yeast by high concentration of alcohol in the late stage of fermentation.

Description

A strain of Saccharomyces cerevisiae and its application

technical field

The invention relates to a strain of Saccharomyces cerevisiae and its application.

Background technique

Yeast is a general term for a class of unicellular fungi, not a unit of phylogenetic classification. Yeast is the earliest microorganism used in the history of human civilization. There are currently more than 1,000 species of yeast known. According to the ability of yeast to produce spores (ascospores and basidiospores), yeast can be divided into three categories: strains that form spores belong to ascomycetes or basidiomycetes, and strains that do not form spores but mainly through Those that reproduce by budding are called incomplete fungi, or "pseudoyeasts" (Kreger-van R, Groningen NY. The yeasts: a taxonomic study. Third revised and enlarged edition[M]. The Netherlands: Elsevier science publishers B.V, 1984.). It is known that most yeasts are classified into the phylum Ascomycota.

Yeasts are widely distributed in nature and mainly grow in acidic and moist sugary environments, for example, they are most common in fruits, vegetables, preserves and on the surface of orchard soil (Yu Jingzhi. Yeast Production and Application Manual[ M]. China Light Industry Press, 2005). Yeast camp obligate or facultative aerobic life, currently unknown obligate anaerobic yeast. In the absence of oxygen, yeast perform anaerobic respiration, and fermentative yeast can convert sugars into pyruvate through the glycolysis pathway (EMP pathway), and pyruvate is further converted into carbon dioxide and ethanol to obtain energy (Wang Jingyan, Zhu Shenggeng , Xu Changfa. Biochemistry (Third Edition, Volume 2). Beijing: Higher Education Press. 2002: 82). In the alcohol industry, the use of fermentative yeast anaerobic respiration can produce ethanol, and the fermentable sugar that yeast can use is converted into alcohol.

As a renewable energy source, ethanol can be directly used as liquid fuel or mixed with gasoline. Fuel ethanol, as a new fuel substitute, can reduce dependence on non-renewable energy-petroleum and ensure national energy security. The current alcohol industry production mostly adopts the fermentation method: using various sugar (disaccharide), starch (polysaccharide) agricultural products or agricultural and forestry by-products as raw materials, after hydrolysis (that is, saccharification) polysaccharides are converted into monosaccharides and then fermented, Or directly ferment disaccharides into ethanol. Under the action of amylases, starch is hydrolyzed into glucose, and then further fermented to produce ethanol (Ma Zanhua. New technology for efficient and clean production of alcohol [M]. Chemical Industry Press, 2003).

Guangxi is located in the subtropical region, and the planting area and output of non-grain cash crops sugarcane and cassava rank first in the country. Sugarcane is mainly used for sugar production, and molasses is a by-product of sugarcane sugar production, which contains a large amount of yeast-fermentable sugar. The starch content of the fresh cassava root reaches 30%, and the cassava starch is converted into yeast fermentable sugar-glucose after being liquefied by α-amylase and saccharified by glucoamylase. Molasses and tapioca starch are excellent raw materials for yeast fermentation to produce alcohol. In 2008, Guangxi COFCO Biomass Energy Co., Ltd. built and put into operation a production facility with an annual output of 200,000 tons of fuel ethanol using molasses and cassava as raw materials in Beihai, Guangxi. In addition, there are two companies in Guangxi that produce ethanol on a large scale using molasses and cassava as raw materials. They are Xintiande Co., Ltd. located in Qinzhou City and Pingguo Kate Biochemical Co., Ltd. located in Pingguo County.

Saccharomyces cerevisiae is a microbial strain commonly used in alcohol industrial fermentation production. In the production of alcohol industry, the requirements for bacterial strains are getting higher and higher. Firstly, yeasts are required to have extremely high alcohol production efficiency, high conversion rate of fermentable sugar, and no waste of sugary raw materials; secondly, the alcohol content of fermented liquid after maturation High, reduce distillation energy consumption; Again be that fermentation speed is fast, improves the utilization ratio of equipment, reduces production cost (Jiang Ning. Biological liquid fuel---fuel alcohol [J].Chinese Journal of Nature, 2007,29 (1): 30- 33). The main factors that limit these three requirements are the tolerance of yeast to high concentrations of fermentable sugar and alcohol, the conversion rate of raw materials, and the tolerance to various unfavorable fermentation conditions (such as high temperature, low pH and toxic substances, etc.) . Saccharomyces cerevisiae, which is widely used in the production of domestic alcohol industry, is the high-temperature-resistant active dry yeast (Ange Yeast, referred to as AQ) produced by Hubei Angel Yeast Co., Ltd. Although the fermentation characteristics are good, it is difficult to meet the higher requirements of the industry. .

Contents of the invention

The purpose of the present invention is to provide a strain of Saccharomyces cerevisiae strain and application thereof.

Saccharomyces cerevisiae provided by the present invention, named ZM1-5, referred to as Saccharomyces cerevisiae ZM1-5 or ZM1-5, was isolated from the orchard soil of Qinzhou City, Guangxi Zhuang Autonomous Region, and was preserved in China Microbiology on April 23, 2010. General Microbiology Center of Culture Collection Management Committee (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCC No.3761.

Saccharomyces cerevisiae ZM1-5 can be used to produce alcohol.

When used to produce alcohol, Saccharomyces cerevisiae ZM1-5 can be used alone or mixed with other Saccharomyces cerevisiae.

When using the Saccharomyces cerevisiae ZM1-5 to produce alcohol, the pH value is 3-9 and the temperature is 28-42°C. When using the Saccharomyces cerevisiae ZM1-5 to produce alcohol, the pH value is preferably 4-5. When the Saccharomyces cerevisiae ZM1-5 is used to produce alcohol, the temperature is preferably 28-40°C, and the most preferable temperature is 32°C.

The invention also protects a method for producing alcohol, which is to obtain alcohol by fermenting Saccharomyces cerevisiae ZM1-5.

In the method, the substrate used for fermentation can specifically be glucose and/or sucrose.

In the method, the substrate used for fermentation can be at least one of cassava products (such as cassava flour liquefied mash), molasses and sugarcane juice.

The fermentation temperature can be any temperature from 32°C to 42°C, such as 32°C, 37°C, 40°C or 42°C.

At the beginning of the fermentation, the concentration of Saccharomyces cerevisiae ZM1-5 in the fermentation system may be 1.5×10 ⁷ to 2.5×10 ⁷ cells/mL, preferably 1.5×10 ⁷ -2.0×10 ⁷ cells/mL mL or 2.0×10 ⁷ -2.5×10 ⁷ cells/mL, such as 1.5×10 ⁷ cells/mL, 2.0× ^{10 7} cells/mL or 2.5×10 ⁷ cells/mL.

Saccharomyces cerevisiae ZM1-5 has the following physiological characteristics: (1) When cultured on yeast powder peptone agar plate at 32°C for 2-3 days, the colonies are milky white, raised, smooth in surface and neat in edge; the cells are round or oval, asexual reproduction mode It is budding; (2) The optimum growth and fermentation temperature of the strain: 32°C; pH range: 3-9; the optimum growth pH is 4-5; (3) The growth rate of the strain is fast at 32°C, 37°C and 42°C in Angel yeast; (4) the bacterial strain can grow on 50% glucose medium; the bacterial strain can be cultured in YPD containing inhibitors: 1.6mol/L NaCl, 16% ethanol, 4g/L acetic acid, 200μg/LG418 (5) When it is used to ferment and produce alcohol with cassava flour liquefied mash, molasses and sugarcane juice as raw materials, the alcohol yield and fermentation efficiency are higher than those of Angel high-temperature-resistant active dry yeast used in industrial production at present.

Compared with the angel high-temperature resistant yeast commonly used in the existing industrial production, the yeast strain ZM1-5 has many advantages such as fast growth, high alcohol production, high temperature resistance, high sugar resistance, high concentration ethanol resistance and acid resistance. Saccharomyces cerevisiae ZM1-5 can be applied to the alcohol production process (synchronous saccharification and fermentation) using cassava flour, molasses and sugarcane juice as raw materials to solve the problem that yeast cannot tolerate high osmotic pressure, high concentration of ethanol and Problems such as low efficiency in the later stage of fermentation can reduce the cost of alcohol industrial production. Compared with angelic high temperature resistant yeast, Saccharomyces cerevisiae ZM1-5 has higher alcohol yield when fermented under high temperature conditions. Yeast strain ZM1-5 is used in the process of synchronous saccharification and fermentation to produce alcohol, which is expected to solve the problems of high substrate concentration (high osmotic pressure), high temperature in the fermentation tank, and inhibition of yeast by high concentration of alcohol in the late stage of fermentation.

Description of drawings

Figure 1 is the photos of ZM1-5 and AQ on YPD flat plate under various high temperature conditions.

Figure 2 is a photograph of ZM1-5 and AQ on YPD plates with various concentrations of NaCl at 30°C.

Figure 3 is a photo of ZM1-5 and AQ on YPD plates with various concentrations of ethanol at 30°C.

Figure 4 is a photograph of ZM1-5 and AQ on YPD plates with various concentrations of acetic acid at 30°C.

Figure 5 is a photo of ZM1-5 and AQ on YPD plates with various concentrations of G418 at 30°C.

Figure 6 is a photograph of ZM1-5 and AQ on a YPD plate with 50% glucose at 30°C.

Figure 7 shows the morphology of ZM1-5 and AQ on the YPD plate at different culture temperatures; A: the colony morphology of AQ at 32°C; B: the colony morphology of ZM1-5 at 32°C; C: the colony of AQ at 37°C Morphology; D: the colony morphology of ZM1-5 at 37°C; E: the colony morphology of AQ at 42°C; F: the colony morphology of ZM1-5 at 42°C.

Figure 8 shows the morphology of ZM1-5 and AQ under the scanning electron microscope; A: the microscopic morphology of AQ cells when cultured at 32 °C; B: the microscopic morphology of cells when ZM1-5 was cultured at 32 °C; C: the microscopic morphology of AQ cells when cultured at 32 °C; Microscopic morphology of cells when cultured at 42°C; D: Microscopic morphology of cells when ZM1-5 was cultured at 42°C.

Figure 9 is a comparison of the bacterial mass of ZM1-5 and AQ grown under different pH conditions for 24 hours.

Figure 10 is a comparison of the bacterial mass of ZM1-5 and AQ grown under different temperature conditions for 20 hours.

Figure 11 is a comparison of the growth curves of ZM1-5 and AQ under different temperature conditions; A: AQ; B: ZM1-5.

Fig. 12 is a comparison of wines after ZM1-5 and AQ fermented glucose for 72 hours at different temperatures.

Fig. 13 is a comparison of the wine parts of ZM1-5 and AQ after fermenting sucrose at different temperatures for 72 hours.

Figure 14 shows the results analysis of ZM1-5 and AQ fermented cassava flour liquefaction mash; A: wine portion; B: live bacteria concentration; C: residual total sugar; D: residual reducing sugar.

Fig. 15 is the result analysis of ZM1-5 and AQ fermentation sugarcane juice product; A: wine part (sterilized sugarcane juice is substrate); B: wine part (fresh sugarcane juice is substrate); C: residual sugar content (sterilized sugarcane juice as substrate); D: residual sugar content (fresh sugarcane juice as substrate); E: _CO2 weight loss (sterilized sugarcane juice as substrate); F: _CO2 weight loss (fresh sugarcane juice as substrate).

Figure 16 is the analysis of the results of ZM1-5 and AQ fermenting sugarcane molasses at different temperatures; A: alcohol; B: CO ₂ weight loss.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Angel yeast (AQ, high temperature resistant active dry yeast): purchased from Hubei Angel Yeast Co., Ltd. % in the following examples, unless otherwise specified, are mass percentages.

YPD medium (pH4.5): yeast extract 10g/L, peptone 20g/L, glucose 20g/L, solid medium containing agar 15g/L.

YPD+50% Glu medium (pH4.5): yeast extract 10g/L, peptone 20g/L, glucose 500g/L, solid medium containing agar 15g/L.

YPD+20% Glu medium (pH4.5): yeast extract 10g/L, peptone 20g/L, glucose 200g/L, solid medium containing agar 15g/L.

YPD+20% Suc medium (pH4.5): yeast extract 10g/L, peptone 20g/L, sucrose 200g/L, solid medium containing agar 15g/L.

Embodiment 1, the acquisition of yeast strain ZM1-5

1. The acquisition of yeast strain ZM1-5

1. Sample collection

Sample: Orchard soil. Collection location: rural area of Qinzhou City, Guangxi Zhuang Autonomous Region.

2. Isolation of yeast strains

Separation by dilution plate method: Weigh 1g of soil into a 100mL Erlenmeyer flask, add 10mL of sterilized distilled water, place on a constant temperature shaker at 28°C with a rotation speed of 180rpm for 2 hours, take 1mL of the suspension and dilute to 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 dilutions, take 100 μL of each dilution gradient to coat the YPD plate, place in a constant temperature incubator at 32°C for 2 days, and pick out a single colony.

3. Screening of yeast strains

The specific screening steps of yeast are as follows:

①Observe the shape of the colony grown on the plate, and examine its cell shape under the microscope, purify the colony determined to be yeast, and use it as an alternative strain to carry out the high temperature resistance growth test of the strain;

②Purified strains were cultured at 32°C, 37°C, and 42°C for 3-5 days, observed the growth of the bacteria, and selected high-temperature-resistant yeast strains.

③The high-temperature-resistant yeast strains were spot-inoculated on the YPD medium, cultured at 32°C for 48 hours, and poured into the upper TTC medium after the colonies grew out (red tetrazolium TTC 0.05g, glucose 0.5g, agar 1.5g, water 100mL ), keep it away from light for 2-3 hours, and select the yeast strain with darker color.

④The yeast strains screened by TTC were put into YPD liquid medium respectively, cultured on a shaker at 28°C and 180rpm overnight, and the bacterial liquid was put into the YPD liquid medium with Duchenne tubules at the final concentration of OD ₆₀₀ =0.01, and placed in Cultivate at 32°C, 37°C, 40°C, and 42°C, observe and record the gas production in the Duchenne tubules at 12h, 24h, and 48h, respectively, and select the yeast strains that produce more gas.

⑤ Into 50mL of YPD liquid culture medium, cultured on a shaker at 28°C and 180rpm for 24 hours, and inserted into 100mL YPD + 20% Glu The 250mL Erlenmeyer flask of liquid medium was distilled after 72 hours of fermentation to detect its alcohol production.

⑥ Stress-resistant growth detection of yeast on various media

Using the resting cell gradient growth experiment, the yeast was inoculated in 20 mL of YPD liquid medium, cultured at 32° C. for 12 hours (OD ₆₀₀ =0.7), and the cells were collected by centrifugation to prepare resting cells. Adjust the concentration of the bacterial solution to OD ₆₀₀ = 1.0, and dilute to the concentration of 10 ⁰ , 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , take 4 μl and spot in various high osmotic pressure medium [YPD+ NaCl (1.2mol/L, 1.4mol/L, 1.6mol/L), YPD+50%Glu] plate, and YPD+ethanol (12%, 14%, 16%), YPD+acetic acid (3g/L, 4g/L ), YPD+G418 (100 μg/L, 200 μg/L) plates, cultured at 32°C for 2-3 days, and observed the growth of the colonies. The % in this paragraph is volume percentage.

Through the above isolation and screening, 33 yeast strains were obtained, one of which was good in all aspects, and was named as strain ZM1-5.

The photos of ZM1-5 and AQ on the YPD plate under various high temperature conditions are shown in Figure 1. The photos of ZM1-5 and AQ on the YPD plate under various concentrations of NaCl at 30°C are shown in Figure 2. ZM1 at 30°C The photos of -5 and AQ on the YPD plate under various concentrations of ethanol are shown in Figure 3. The photos of ZM1-5 and AQ on the YPD plate under various concentrations of acetic acid at 30°C are shown in Figure 4. At 30°C, ZM1 The photos of ZM1-5 and AQ on the YPD plate under the condition of various concentrations of G418 are shown in Figure 5. The photos of ZM1-5 and AQ on the YPD plate under the condition of 50% glucose at 30°C are shown in Figure 6.

2. Identification of Saccharomyces cerevisiae ZM1-5

ZM1-5 was cultured on YPD plates at 32°C, 37°C, and 42°C for 2 days, respectively, and grew vigorously at 32°C and 37°C, and could also grow at 42°C. The colonies are milky white, raised, with smooth surfaces and neat edges (see Figure 7). Under the microscope, the cells are round or oval, and the asexual reproduction method is multilateral budding (see Figure 8).

ZM1-5 can assimilate fructose, raffinose, galactose, maltose, cellobiose, melibiose, trehalose, melezitose, α-methyl D-glucoside, N-acetylglucosamine; ZM1-5 can assimilate Ammonium sulfate, potassium nitrate; ZM1-5 can ferment glucose, sucrose, maltose, galactose, but not lactose.

According to the morphological, physiological and biochemical characteristics of ZM1-5, referring to "The Yeast" and "Common and Commonly Used Fungi", ZM1-5 was identified as Saccharomyces cerevisiae. Saccharomyces cerevisiae ZM1-5 was deposited in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation number was CGMCC No.3761.

Embodiment 2, the comparison of the optimal growth pH value of Saccharomyces cerevisiae ZM1-5 and Angelica AQ

Saccharomyces cerevisiae ZM1-5 and Angelica AQ were treated under exactly the same conditions to determine the optimum pH value for growth.

1. Cultivate the yeast (Saccharomyces cerevisiae ZM1-5 or Angelica AQ) overnight, and detect the OD ₆₀₀ value of the bacterial liquid.

2. According to the final concentration of OD ₆₀₀ =0.01, put the bacterial solution into the YPD liquid medium with different pH values (3, 4, 5, 6, 7, 8 or 9), cultivate it at 28°C and 180rpm for 24 hours, and measure it by spectrophotometry Measure the optical density value (OD ₆₀₀ value) at a wavelength of 600 nm.

Three replicates were set up, and the results were averaged. See Figure 9 for the comparison of the bacterial mass of ZM1-5 and AQ grown for 24 hours. The optimum growth pH of Saccharomyces cerevisiae ZM1-5 and Angelica AQ was 4-5.

Embodiment 3, the comparison of the optimal growth temperature of Saccharomyces cerevisiae ZM1-5 and Angelia yeast AQ

Saccharomyces cerevisiae ZM1-5 and Angelica AQ were treated under exactly the same conditions to determine the optimum growth temperature.

1. Cultivate the yeast (Saccharomyces cerevisiae ZM1-5 or Angelica AQ) overnight, and detect the OD ₆₀₀ value of the bacterial liquid.

2. Put the bacterial solution into the YPD liquid medium (pH4.5) according to the final concentration of OD ₆₀₀ =0.01, and culture at different temperatures (28°C, 32°C, 35°C, 37°C, 40°C or 42°C) and 180rpm for 20 Hours, the OD ₆₀₀ value of the bacterial solution was detected.

Three replicates were set up, and the results were averaged. See Figure 10 for the comparison of the bacterial mass of ZM1-5 and AQ grown for 20 hours under different temperature conditions. The optimal growth temperature of Saccharomyces cerevisiae ZM1-5 and Angelica AQ was 32°C, and the growth of Saccharomyces cerevisiae ZM1-5 at 28-42°C was higher than that of Angelica AQ.

Embodiment 4, comparison of growth curves of Saccharomyces cerevisiae ZM1-5 and Angelica AQ at different temperatures

Saccharomyces cerevisiae ZM1-5 and Angelica AQ were treated under exactly the same conditions, and the growth curves at different temperatures were compared.

1. Cultivate the yeast (Saccharomyces cerevisiae ZM1-5 or Angelica AQ) overnight, and detect the OD ₆₀₀ value of the bacterial liquid.

2. Put the bacterial solution into YPD liquid medium (pH4.5) according to the final concentration of OD ₆₀₀ = 0.01, and cultivate them at different temperatures (28°C, 32°C, 35°C, 37°C, 40°C or 42°C) and 180rpm After 72 hours, samples were taken at 4h, 16h, 22h, 28h, 48h, and 68h to detect the OD ₆₀₀ value of the bacterial solution.

Three replicates were set up, and the results were averaged. The comparison of the growth curves of ZM1-5 and AQ under different temperature conditions is shown in Fig. 11 . The two basically reached the stable phase at 22h, and the growth curve of Angelica AQ at 32°C tended to be stable after 22h, while the growth curve of Saccharomyces cerevisiae ZM1-5 still tended to the slow growth stage after 22h. Both strains grew fastest at 32°C and had the largest amount of bacteria. At 42°C, the survival ability of Saccharomyces cerevisiae ZM1-5 was significantly higher than that of Angelica AQ, indicating that Saccharomyces cerevisiae ZM1-5 had a stronger ability to tolerate high temperature than Angelica AQ.

Embodiment 5, the comparison of Saccharomyces cerevisiae ZM1-5 and Angelia yeast AQ fermentation glucose

Insert the yeast liquid (Saccharomyces cerevisiae ZM1-5 or Angelica AQ) into 100mL YPD+20% Glu liquid medium, the inoculum size is 1.5× ¹⁰⁷ /mL (final concentration), and place them at different temperatures (32°C, 37°C, 40°C or 42°C) for fermentation, after 72 hours of fermentation, take the fermentation broth to measure the alcohol content (wine content).

Alcohol testing method: Take 100ml of fermentation broth and add 100mL of distilled water, mix well and then heat and distill. Collect 100mL of distillate, add 100mL of distilled water, mix well, measure the alcohol content with an alcohol meter, and measure the temperature of the mixture with a thermometer; check the obtained value from the alcohol content temperature correction table and convert it into alcohol % (volume percentage) at 20°C , and then multiplied by 2 to get the alcohol content of the fermented liquid.

Three replicates were set up, and the results were averaged. See Figure 12 for a comparison of the alcohol content of ZM1-5 and AQ after glucose was fermented at different temperatures for 72 hours. Saccharomyces cerevisiae ZM1-5 had the highest alcohol content (11.3%) at 32°C, slightly lower (10.8%) at 37°C, 10.5% at 40°C, and 7.4% at 42°C. Angel Yeast AQ has 9.9% alcohol at 40°C and 6.7% alcohol at 42°C. The wine content of Saccharomyces cerevisiae ZM1-5 was significantly higher than that of Angelica AQ at 40°C and 42°C, indicating that Saccharomyces cerevisiae ZM1-5 had a stronger ability to use glucose to produce wine at high temperature than Angelica AQ.

Embodiment 6, Saccharomyces cerevisiae ZM1-5 compares with Angelica yeast AQ fermentation sucrose

Insert the yeast liquid (Saccharomyces cerevisiae ZM1-5 or Angelica AQ) into 100mL YPD+20% Suc liquid medium, the inoculum size is 1.5× ¹⁰⁷ /mL (final concentration), and place them at different temperatures Ferment at (32°C, 37°C, 40°C or 42°C), ferment for 72 hours and take the fermented liquid to measure the alcohol content (wine content). Alcohol detection method is the same as in Example 5.

Three replicates were set up, and the results were averaged. See Figure 13 for a comparison of the alcohol content of ZM1-5 and AQ after sucrose fermentation at different temperatures for 72 hours. Both strains had the highest alcohol content when fermented at 32°C, and slightly lower alcohol yields at 37°C and 40°C. At 42°C, the wine content of Saccharomyces cerevisiae ZM1-5 was significantly higher than that of Angelia yeast AQ, which indicated that Saccharomyces cerevisiae ZM1-5 had a stronger ability to produce wine from sucrose at high temperature than Angelia yeast AQ.

Example 7. Comparison of production of ethanol by Saccharomyces cerevisiae ZM1-5 and Angelica AQ using cassava flour liquefied mash fermentation

Cassava powder liquefied mash: Guangxi COFCO Biomass Energy Co., Ltd., containing 27g total sugar per 100mL mash.

1. Cultivate the yeast (Saccharomyces cerevisiae ZM1-5 or Angelica AQ) in YPD liquid medium at 28°C and 180rpm on a shaker for about 24 hours, so that the concentration of viable bacteria reaches OD ₆₀₀ = 2.0 or more, and put it on a hemocytometer under a microscope Calculate the bacterial concentration.

2. According to the inoculum amount (final concentration) of 1.5×10 ⁷ cells/mL, 2.0× ^{10 7} cells/mL, and 2.5× ^{10 7} cells/mL, respectively, put the bacterial solution into 9 liquefied tubes containing 500 mL of cassava flour Fermented mash was cultured at 32° C. for 70 hours in 1 L Erlenmeyer flasks (3 flasks for each inoculum) to obtain a fermented liquid.

3. Determination of the contents of various components (alcohol, number of viable bacteria, residual total sugar, residual reducing sugar) in the fermentation broth.

Alcohol detection method is the same as in Example 5.

Viable count detection method (plate counting method): take 10 times as the gradient to dilute the fermentation broth, take 100 μl of each gradient and coat three YPD plates, culture at a constant temperature of 32°C for 2-3 days, observe the plates coated with each dilution in turn, Calculate with the flat plate of the diluent of coating maximum dilution and have single bacterium colony growth, calculate the single bacterium colony number of growth (on the flat plate of the maximum dilution that can grow bacterium colony); Calculation formula: number of viable bacteria/ml=(three The number of single colonies grown on the block plate/3)×10×dilution factor.

Determination of residual total sugar: ① Take 1mL fermentation broth and add 5mL ddH ₂ O, 1mL concentrated hydrochloric acid; ② React in boiling water for 20 minutes and cool in cold water; ③ Add a drop of phenolphthalein, and use 4M NaOH aqueous solution to drop to reddish, and finally dissolve the solution Set the volume to 100mL to obtain the reaction solution; ④ Take 400μL of the reaction solution and add 800μL of DNS solution (200g sodium potassium tartrate tetrahydrate, 10g sodium hydroxide, 0.5g anhydrous sodium sulfite, 2g crystalline phenol, 10g DNS, 1L distilled water, keep in room temperature It can be used after standing still for 7 days), take a boiling water bath for 5 minutes, then cool in cold water, take 200 μL on the microplate, and measure the value at OD ₅₄₀ ; ⑤ Substitute the obtained value into the glucose standard curve to obtain the amount of reducing sugar (the amount of total residual sugar).

Determination of residual reducing sugar: ① Take 1mL of fermentation broth and add 19mL ddH ₂ O, mix well; ② Take 400μL of the mixed solution and add 800μL of DNS solution, cook in a boiling water bath for 5min, then cool in cold water, take 200μL on a microtiter plate, OD ₅₄₀ Measured value; ③ The obtained value is substituted into the glucose standard curve to obtain the amount of reducing sugar (the amount of residual reducing sugar).

The experiments were repeated three times, and the results were averaged. The results are shown in Figure 14.

When the inoculum amount was 2.0×10 ⁷ cells/mL, the wine fermented by Saccharomyces cerevisiae ZM1-5 was the highest, reaching 15.2%, which was 0.8% higher than that of Angelica AQ (t-test, p<0.05).

At each inoculum size, the number of viable cells of Saccharomyces cerevisiae ZM1-5 was significantly higher than that of Angelica AQ; after 70 hours of fermentation at an inoculum size of 1.5×10 ⁷ cells/mL, Saccharomyces cerevisiae ZM1-5 multiplied to 3.20×10 ⁸ live bacteria concentration/mL; it shows that Saccharomyces cerevisiae ZM1-5 can survive well in the environment of thick mash and high alcohol.

The residual total sugar content of Saccharomyces cerevisiae ZM1-5 and Angelica AQ at the inoculum concentration ^of 2.0×10 ⁷ cells/mL was lower than that of the other two inoculum amounts, indicating that fermentation The effect was the best; when the inoculum amount was 2.0×10 ⁷ cells/mL, the residual total sugar content and residual reducing sugar content of Saccharomyces cerevisiae ZM1-5 were 0.2g/100mL and 0.09g/100mL lower than that of Angelica AQ, respectively.

Example 8. Comparison of Saccharomyces cerevisiae ZM1-5 and Angelica AQ producing alcohol by fermentation of sugarcane juice

1. Cultivate the yeast (Saccharomyces cerevisiae ZM1-5 or Angelica AQ) in YPD liquid medium at 28°C and 180rpm on a shaker for about 24 hours, so that the concentration of viable bacteria reaches OD ₆₀₀ =2.0 or above.

2. Take 10mL of the bacterial liquid from Step 1 and put it into a 250mL Erlenmeyer flask filled with 100mL of sugarcane juice (obtained by pressing fresh sugar cane, with a total sugar content of 18%), and place them at different temperatures (32°C, 37°C, 40°C) , or 42°C) for 70 hours to obtain a fermentation broth.

3. Take 10mL of the bacterial solution from Step 1 and insert it into a 250mL Erlenmeyer flask filled with 100mL of sterilized sugarcane juice (obtained by pressing fresh sugar cane, with a total sugar content of 18%, and sterilized at 121°C for 20 minutes after packing into Erlenmeyer flasks). They were cultured at different temperatures (32°C, 37°C, 40°C, or 42°C) for 70 hours to obtain fermentation broth.

4. Measure the carbon dioxide weight loss of the fermented liquid of step 2 and step 3 and the content of various components (alcohol, residual total sugar) in the fermented liquid.

Carbon dioxide weight loss: The weight difference of the fermentation broth before and after fermentation is the carbon dioxide weight loss (g).

Alcohol detection method is the same as in Example 5.

Determination method of residual sugar: ① Take 1mL fermentation broth and add 3mL ddH ₂ O and 1mL concentrated hydrochloric acid; ②React at 66°C-68°C for 10min and cool in cold water; ③Add a drop of phenolphthalein, and drop it with 4M NaOH aqueous solution until it turns reddish, Finally, dilute the solution to 10 mL to obtain the reaction solution; ④ take 400 μL of the reaction solution and add ₈₀₀ μL of DNS solution; Glucose standard curve obtains the amount of reducing sugar (the amount of residual total sugar).

The experiments were repeated three times, and the results were averaged. Figure 15 shows the results of fermentation of sugarcane juice products by S. cerevisiae ZM1-5 and Angeliae AQ. After sterilized sugarcane juice alcoholic fermentation, the alcohol yields of Saccharomyces cerevisiae ZM1-5 and Angelica AQ were equivalent (10.3%) at 32°C, and the alcohol yields of Saccharomyces cerevisiae ZM1-5 were 10.3% at 37°C (10.3%) and 42°C (7.1%). ) was higher than that of AQ (10%, 6.5%); the residual total sugar content of Saccharomyces cerevisiae ZM1-5 was lower than that of AQ at 40℃ and 42℃. In the alcoholic fermentation of fresh sugarcane juice, the alcohol content of Saccharomyces cerevisiae ZM1-5 was 10.1% at 32°C and 7.6% at 42°C, and the alcohol content of Angelica AQ was 9.6% at 32°C and The alcohol content is 7%, and the alcohol content of Saccharomyces cerevisiae ZM1-5 is higher than that of Angelica AQ; at 32℃ and 37℃, the residual sugar content of Saccharomyces cerevisiae ZM1-5 and Angelica AQ are quite low; 40 At ℃ and 42℃, the total sugar content of Saccharomyces cerevisiae ZM1-5 was lower than that of Angelica AQ. In the fermentation of fresh cane juice, the _CO2 weight loss of S. cerevisiae ZM1-5 was significantly higher than that of Angelica AQ at 42°C. The results showed that Saccharomyces cerevisiae ZM1-5 fermented better in fresh sugarcane juice than in sterilized sugarcane juice, and the residual sugar content was low. If it is applied to actual production, it will save a lot of energy.

Example 9, Saccharomyces cerevisiae ZM1-5 and Angelia yeast AQ use molasses fermentation to produce alcohol comparison

1. Dilute molasses and tap water at a volume ratio of 1:2, mix well, and adjust the pH to 4.0, which is the molasses medium.

2. Cultivate the yeast (Saccharomyces cerevisiae ZM1-5 or Angelica AQ) in YPD liquid medium at 28°C and 180rpm on a shaker for about 24 hours, so that the concentration of viable bacteria reaches about OD ₆₀₀ =2.0.

3. Take 10 mL of the bacterial liquid and put it into a 250 mL Erlenmeyer flask containing 100 mL of molasses medium, and culture it at different temperatures (32°C, 37°C, 40°C or 42°C) for 70 hours to obtain a fermentation broth.

4. Measure the carbon dioxide weight loss of the fermented liquid in step 3 and the alcohol content of the fermented liquid.

Carbon dioxide weight loss: The weight difference of the fermentation broth before and after fermentation is the carbon dioxide weight loss (g).

Alcohol detection method is the same as in Example 5.

The experiments were repeated three times, and the results were averaged. The results of ZM1-5 and AQ fermenting sugarcane molasses at different temperatures are shown in Figure 16. The alcohol content and carbon dioxide weight loss of Saccharomyces cerevisiae ZM1-5 at different temperatures were significantly higher than those of Angelia yeast AQ, indicating that it could use molasses as a substrate for fermentation, and the fermentation results were better than Angelia yeast AQ.

Claims (11)

1. yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZM1-5, its deposit number is CGMCC No.3761.

2. the application of the described yeast saccharomyces cerevisiae ZM1-5 of claim 1 in producing alcohol.

3. application as claimed in claim 2 is characterized in that: when using described yeast saccharomyces cerevisiae ZM1-5, the pH value is 3-9, and temperature is 28-42 ℃.

4. application as claimed in claim 3 is characterized in that: when using described yeast saccharomyces cerevisiae ZM1-5, the pH value is 4-5.

5. as claim 3 or 4 described application, it is characterized in that: when using described yeast saccharomyces cerevisiae ZM1-5, temperature is 28-40 ℃.

6. application as claimed in claim 5 is characterized in that: when using described yeast saccharomyces cerevisiae ZM1-5, temperature is 32 ℃.

7. method of producing alcohol, the described yeast saccharomyces cerevisiae ZM1-5 of the claim 1 that comprises the steps: to ferment obtains alcohol.

8. method as claimed in claim 7 is characterized in that: in the described method, the substrate that is used to ferment is glucose and/or sucrose.

9. method as claimed in claim 7 is characterized in that: in the described method, the substrate that is used for fermenting is at least a of cassava, molasses and sugar cane juice.

10. as claim 7 or 8 or 9 described methods, it is characterized in that: the temperature of described fermentation is 32 ℃ to 42 ℃.

11. as arbitrary described method in the claim 7 to 10, it is characterized in that: when described fermentation was initial, the concentration of the described yeast saccharomyces cerevisiae ZM1-5 of claim 1 in fermentation system was 1.5 * 10 ⁷To 2.5 * 10 ⁷Individual cell/mL.

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