CN110923268A

CN110923268A - Culture method of saccharomyces cerevisiae Y3401 and application of saccharomyces cerevisiae Y3401 in white spirit brewing

Info

Publication number: CN110923268A
Application number: CN201911266252.8A
Authority: CN
Inventors: 范光森; 孙宝国; 李秀婷; 滕超; 杨然; 刘朋肖; 富志磊
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2020-03-27

Abstract

The invention discloses a culture method for producing ethanol by saccharomyces cerevisiae Y3401, wherein a culture medium takes glucose as a carbon source, the dosage of the glucose is 250-350g/L, yeast extract powder is a nitrogen source, the dosage of the yeast extract powder is 30-45g/L, and the initial pH of the culture medium is 4.8-5.6; the culture conditions are 28-32 deg.C, and shaking at 80-120 r/min. Under the optimal culture condition of the invention, the final ethanol production concentration of the strain is as high as 117.2g/L, and is improved by 1.24 times compared with the ethanol production of 52.4g/L of the initial strain. Particularly, the culture mode of combining the shaking culture mode and the standing culture mode and firstly shaking culture for 18 hours and then standing culture for 12 hours can reach the optimal state, and the ethanol content is as high as 133.4 g/L. The method disclosed by the invention lays a foundation for the application of the saccharomyces cerevisiae Y3401 in liquor brewing.

Description

Culture method of saccharomyces cerevisiae Y3401 and application of saccharomyces cerevisiae Y3401 in white spirit brewing

Technical Field

The invention belongs to the field of microorganisms and the technical field of brewing, and particularly relates to a culture method of saccharomyces cerevisiae Y3401 and application thereof in a white spirit brewing process.

Background

The white spirit is a traditional Chinese distilled spirit, which is obtained by taking grains such as sorghum, rice and the like as main raw materials and carrying out solid state fermentation by using special leavens such as daqu, bran koji, yeast wine and the like (Zhengfuping, Mayajie, Heimemin and the like, the research overview and the prospect of aroma components of 6 Dadistilled spirits in the world are shown [ J ] in the technical and scientific reports of food, 2017,35(2): 1-12.). Saccharomyces cerevisiae is the dominant strain in the fermentation process of white spirit, and has important influence on the quality, yield and cost reduction of white spirit (Wang Yong. Niuzhan wine factory excellent Saccharomyces cerevisiae screening and identification [ J ] brewing science, 2017(4): 61-64.). Therefore, the deep research on the fermentation conditions of the saccharomyces cerevisiae for producing the ethanol is beneficial to improving the fermentation rate of the white spirit and improving the flavor and the quality of the white spirit.

At present, the Chinese white spirit mostly adopts a solid fermentation process, and compared with liquid and semi-solid-liquid fermentation, the mechanized control is difficult to realize (Wangjiang wave, Wangzhuang, Huangdakang, and the like, Chinese white spirit mechanized brewing technology review and prospect [ J ]. academic newspaper of Hubei university of industry, 2011,26(05): 50-54.). In addition, labor costs are increased, and difficulties are brought to the development of the conventional solid state brewing process. And the liquid state ventilation type fermentation is superior to solid state anaerobic fermentation in the aspects of production safety and convenience of expanded production (Fangusen, Liu Peng Xiao, Wu Qihua, and the like, screening, identification and characteristic research of yeast for producing ethanol with high yield under aerobic condition [ J ] food industry science and technology, 2018 ]. The brewing method of the white spirit is changed in the foreseeable future, and the change of the brewing method of the white spirit can definitely cause the change of the brewing environment, and the prominent change is that the oxygen content in the brewing environment is increased (Van Guansen, Liu Peng Xiao, Wu Qihua, and the like. the fermentation condition of the ethanol produced by the saccharomyces cerevisiae YF1914 is optimized under the aerobic condition and the aroma production characteristic thereof [ J ] food industry science and technology, 2019: 1-13.; Liu selected, Zhangdong Zhao, Yude Yi, and the like. the application of a digital brewing process management system in the mechanized, automatic and intelligent brewing production of the strong aromatic white spirit [ J ] brewing science and technology, 2018(11):70-74 ], and [7] Liyutong, Qishipo, Qili, and the like. Therefore, in order to meet the requirement of changing the brewing mode of semi-solid-liquid fermentation or liquid aerobic fermentation and the like in the liquor brewing industry in the future, the research on the condition that the saccharomyces cerevisiae produces ethanol under the aerobic condition is necessary (Fangson, Liupenxian, Wuqihua, and the like. the saccharomyces cerevisiae YF1914 produces ethanol under the aerobic condition, and the fermentation condition is optimized and the aroma production characteristic [ J ] of the saccharomyces cerevisiae YF1914 is adopted in the food industry science and technology, 2019: 1-13.). In turn, the research can help to promote the change of the white spirit brewing mode, improve the white spirit yield and shorten the production period. The inventor obtains a plurality of strains of Saccharomyces cerevisiae with excellent performance in early stage screening, wherein Saccharomyces cerevisiae Y3401 can produce ethanol with high yield under aerobic conditions by taking sorghum leachate as a culture medium, and provides a precursor substance, namely ethanol, for ethyl acetate produced by the latter when being co-cultured with abnormal hamamelis Y3604, and shows excellent characteristics (FAN G, TENG C, XUD, et al. enhanced process of ethyl acetate using co-culture of Saccharomyces cerevisiae and Saccharomyces cerevisiae [ J ]. Journal of bioscience and bioscience, 2019.; FAN G, TENG C, XU D, et al. 201experimental acetobacter bacterium in basic culture and biological culture [ 1479 ]).

Disclosure of Invention

The inventor screens 46 strains of ethanol-producing yeasts from Daqu provided by Gujing tribute wine (strong aromatic, Anhui Gujing group) and white spirit (fen-flavor, balanced water, white spirit), wherein one strain of saccharomyces cerevisiae Y3401 is adopted by the invention. The saccharomyces cerevisiae Y3401 is preserved in the China general microbiological culture Collection center (CGMCC) at 10 months and 20 days in 2017, the preservation address is the microbial research institute of China academy of sciences No. 3 of West Lu No.1 Hospital, Kyoho, Beijing City, and the preservation number is CGMCC No.14828 (see the Chinese patent application 201711136361.9).

The saccharomyces cerevisiae Y3401 studied in the early stage has higher ethanol yield when the sorghum leaching solution is used as a culture medium under aerobic conditions, and provides a precursor substance, namely ethanol, for ethyl acetate production of the saccharomyces cerevisiae Y3604 when the saccharomyces cerevisiae is co-cultured with the abnormal saccharomyces verniciae. The invention further takes the yeast as an original strain, and optimizes the fermentation condition of the yeast for producing ethanol with high yield under aerobic condition by adopting a Step-by-Step single factor test and an orthogonal test, so that the yeast has application potential in brewing white spirit in a liquid fermentation mode.

The invention firstly provides a method for producing ethanol by culturing saccharomyces cerevisiae Y3401, which is characterized by comprising the following steps: fermentation conditions are as follows: the culture medium takes glucose as a carbon source, the dosage of the glucose is 250-350g/L, the yeast extract powder is a nitrogen source, the dosage of the yeast extract powder is 30-45g/L, and the initial pH of the culture medium is 4.8-5.6; activating the strain for more than 16 h; and culturing at 28-32 deg.C and 80-120 r/min.

In one embodiment, the culturing is in two stages: after inoculation, shaking culture is carried out and then static culture is carried out, more specifically, after inoculation, shaking culture is carried out for 12-28 hours and then static culture is carried out for 8-16 hours, more preferably, after inoculation, shaking culture is carried out for 16-20 hours and then static culture is carried out for 10-14 hours, and after optimal inoculation, shaking culture is carried out for 18 hours and then static culture is carried out for 12 hours.

Preferably, the amount of glucose in the medium is 280-320g/L, most preferably 300 g/L.

Preferably, the yeast extract powder is used in an amount of 35-40g/L, most preferably 37.8 g/L.

Preferably, the initial pH of the medium is 5.0 to 5.4, most preferably pH 5.2;

further, strain activation is also included before fermentation, and the activation time of the strain is 18-32h, more preferably 20-28 h, and most preferably 24 h. The YPD culture medium is specifically composed of: 20g/L tryptone, 20g/L glucose, 10g/L yeast extract powder, sterilizing at 115 ℃ for 20 min;

further, the amount of the liquid inoculum of the activated strain to be cultured is 2 to 6%, and most preferably 3 to 5%.

Preferably, the culture conditions are 30 ℃ and 100r/min during shaking culture.

Wherein the basic composition of the medium is referred to as ethanol fermentation medium: contains yeast extract powder, glucose, peptone, and KH 1g/L₂PO₄，(NH₄)₂SO₄，MgSO₄. Wherein, the other components except the yeast extract powder and the glucose and the dosage are as follows: 18-22g/L peptone, 0.5-1.5g/L KH₂PO₄，0.5-1.5g/L(NH₄)₂SO₄，0.5-1.5g/L MgSO₄. More preferably, 20g/L peptone, 1g/L KH₂PO₄，1g/L(NH₄)₂SO₄，1g/L MgSO₄。

The invention lays a foundation for the application of the saccharomyces cerevisiae Y3401 in liquor brewing by researching the optimization of the fermentation conditions for producing the ethanol. Under the optimal culture condition of the invention, the final ethanol production concentration of the strain is as high as 117.2g/L, and is improved by 1.24 times compared with the ethanol production of 52.4g/L of the initial strain. In particular, considering the influence of the culture on the ethanol production of the yeast, the inventor optimizes the combination of shaking culture and standing culture, wherein the shaking culture is performed for a period of time, then the standing culture is performed, particularly, the culture mode of shaking culture for 18 hours and then standing culture for 12 hours can reach the optimal state, and the ethanol content is as high as 133.4 g/L.

Drawings

FIG. 1 Effect of initial glucose concentration on the fermentative synthesis of ethanol by yeast Y3401. Note: the inclusion of the same letters indicates that there was no significant difference between groups at p >0.05 using the Tukey test.

FIG. 2 influence of nitrogen source species on the fermentative synthesis of ethanol by yeast Y3401. Note: the inclusion of the same letters indicates that there was no significant difference between groups at p >0.05 using the Tukey test.

FIG. 3 influence of yeast extract concentration on the synthesis of ethanol by fermentation of yeast Y3401. The inclusion of the same letters indicates that there was no significant difference between groups at p >0.05 using the Tukey test.

FIG. 4 Effect of temperature on the fermentative synthesis of ethanol by Yeast Y3401. Note: containing the same letters means that there was no significant difference between groups at p >0.05 using the Tukeytest test.

FIG. 5 influence of pH on the fermentative synthesis of ethanol by yeast Y3401. Note: containing the same letters means that there was no significance between groups at p >0.05 using the Tukeytest test.

FIG. 6 influence of rotational speed on the synthesis of ethanol by fermentation of yeast Y3401. Note: the inclusion of the same letters indicates that there was no significant difference between groups when p >0.05 using the Tukeytest test.

FIG. 7 shows the effect of the liquid loading on the synthesis of ethanol by fermentation with yeast Y3401. Note: containing the same letters means that there was no significant difference between groups at p >0.05 using the Tukeytest test.

FIG. 8 Effect of age-old on the fermentative synthesis of ethanol by yeast Y3401. Note: containing the same letters means that there was no significant difference between groups at p >0.05 using the Tukeytest test.

FIG. 9 Effect of inoculum size on ethanol synthesis by fermentation of yeast Y3401. Note: containing the same letters means that there was no significant difference between groups at p >0.05 using the Tukeytest test.

FIG. 10 shows the course of ethanol synthesis by fermentation with yeast Y3401.

FIG. 11 glucose concentration and pH changes during shake fermentation of Yeast Y3401.

FIG. 12 is a graph of the trend of yeast Y3401 fermentation for ethanol production versus factor levels.

Detailed Description

The invention is further illustrated by the following specific embodiments or examples in order to provide a better understanding of the invention.

The operations or methods described in the following examples are conventional in the art unless otherwise specified. Reagents and instruments are conventionally available or commercially available unless otherwise specified.

1. Basal Medium used in the examples

YPD medium: 20g/L tryptone, 20g/L glucose, 10g/L yeast extract powder, sterilizing at 115 ℃ for 20 min;

ethanol fermentation medium (basal amount): 10g/L yeast extract powder, 200g/L glucose, 20g/L peptone and 1g/LKH₂PO₄，1g/L(NH₄)₂SO₄，1g/L MgSO₄Sterilizing at 115 deg.C for 20 min;

2. main reagents and instruments used in the examples

The reagents such as glucose, yeast extract powder, peptone, soybean peptone, agar powder, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate and the like are domestic biological or analytical pure reagents; octanol, ethanol were purchased from Sigma; sorghum is purchased from Shandong, Yinan.

An autoclave (YQX-SG46-280S), Shanghai Boxun industries, Inc. medical facilities; biological clean bench (BCN-1360), Beijing Toyobo Harr Instrument manufacturing, Inc.; a constant temperature and humidity incubator (LHS-100CL), Shanghai-constant instruments and Equipment Co., Ltd; ultraviolet-visible spectrophotometer (TU-19), beijing general analysis instruments ltd; centrifuge (Microfuge 2R), hengtai technologies ltd, beijing forest; high performance liquid chromatography (1260series), Agilent technologies ltd; triple quadrupole mass spectrometer (TSQTM8000 evo), ThermoFisher scientific, USA.

3. Basic method adopted in the examples

(1) Fermentation broth pH and yeast biomass measurements: the pH of the fermentation broth was measured using a pH meter, the absorbance was measured at 560nm using a spectrophotometer, and the yeast biomass was measured by the turbidity method.

(2) Glucose and ethanol concentration determination: centrifuging the fermentation liquid at room temperature at 10000r/min for 10min, sucking supernatant, filtering with 0.22 μm filter membrane, and measuring glucose and ethanol concentration by HPLC. The specific measurement method parameters are as follows: BioRadaminex HPX-87H (300 mm. times.7.8 mm); the mobile phase is 0.01mol/L H₂SO₄(ii) a Flow rate: 0.6 mL/min; the column temperature was 25 ℃ and the amount of sample was 10. mu.L, and the amount was determined by external standard method.

(3) Drawing a standard curve: preparing ethanol and glucose standard solutions with different concentrations according to a certain concentration gradient, and drawing a standard curve by adopting the method.

The first embodiment is as follows: single factor assay of Saccharomyces cerevisiae Y3401 culture conditions

To investigate that yeast Y3401 fermentations produced more ethanol, this example performed optimization experiments on the culture conditions of this strain.

1. Initial concentration of glucose

Saccharomyces cerevisiae Y3401 was inoculated into YPD for activation culture for 24 hours. Inoculating 100 μ L of seed liquid into ethanol fermentation culture medium with different initial glucose concentrations (100, 150, 200, 250, 300, 350 and 400g/L), culturing at 30 deg.C and 120r/min for 24h, and determining ethanol production concentration.

As can be seen from FIG. 1, the concentration of ethanol produced by yeast Y3401 increased first and then decreased as the initial concentration of glucose increased, and when the initial concentration of glucose was 300g/L, the ethanol concentration reached a maximum of 85.7g/L, which was 63.5% higher than that of the initial fermentation medium (52.4 g/L). Glucose is the feedstock for ethanol production by yeast conversion, and thus at a suitable initial concentration, facilitates ethanol production by the yeast. When the concentration of the glucose is lower, the requirement of yeast for growth, reproduction, metabolism and conversion into ethanol is not met, so that the ethanol production capacity is limited, and the ethanol accumulation is not enough; however, when the glucose is high, the enzyme activity in the normal growth and metabolism process of the yeast is inhibited due to the high osmotic pressure formed by the high-concentration glucose, and finally the ethanol production capability of the yeast is reduced. The initial concentration of ethanol produced by glucose fermentation varies from strain to strain due to differences in glucose tolerance.

2. Kind of nitrogen source

Inoculating activated saccharomyces cerevisiae Y3401 into ethanol fermentation culture media prepared by different nitrogen sources for culture, wherein the culture comprises the following steps: the nitrogen source, yeast extract powder, tryptone, soybean peptone, beef peptone and ammonium sulfate are initially compounded, and the other conditions are selected from the optimal conditions.

Experimental results show that the organic nitrogen source is obviously superior to the inorganic nitrogen source in terms of promoting the fermentation of Y3401 to produce ethanol (figure 2), and under the same effective nitrogen concentration (3.4g/L), the ethanol yield is the highest when yeast extract powder is taken as a single nitrogen source and reaches 88.5g/L, and is slightly higher than the ethanol yield under the condition of an initial compound nitrogen source (yeast extract powder and peptone) (figure 2). The yeast extract powder is a nutrient-rich organic nitrogen source, is rich in protein, sufficient amino acids, peptides, nucleic acids, vitamins, trace elements and the like, and can effectively promote the growth and propagation of yeast and activate the metabolism of the yeast, particularly genes and enzymes related to ethanol conversion and synthesis.

3. Concentration of Nitrogen Source

The method of the thallus activation stage is as above, culturing in ethanol fermentation medium with different nitrogen concentrations (initial concentration, 20, 30, 40, 50, 60, 70 and 80g/L), and selecting the above optimal conditions for other conditions.

The results show that the concentration of the yeast extract powder has certain influence on the ethanol production of the yeast fermentation, and the concentration of the ethanol produced by the yeast Y3401 is increased and then reduced along with the increase of the concentration of the yeast extract powder (figure 3). When the concentration of the yeast extract powder is the corresponding concentration of the effective nitrogen of the original ethanol fermentation culture medium, namely 41.6g/L, the highest ethanol yield is 90.7 g/L. The high or low concentration of nitrogen source can cause the yeast to grow too vigorously or insufficiently, thereby leading to premature death or insufficient growth of the yeast and finally affecting the ethanol production capability of the yeast.

4. Temperature of fermentation

The method of the thallus activation stage is as above, fermentation is carried out under different temperature conditions (22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 ℃), and the optimal conditions are selected as above for other conditions.

Considering the growth temperature range of the yeast, 22-40 ℃ is selected to study the influence of the temperature on the ethanol production by the fermentation of the yeast Y3401. The results showed that yeast Y3401 produced up to 88.7g/L of ethanol when fermented at 30 ℃ (FIG. 4). Temperature not only affects the growth of the microorganism, but also its metabolic activity, altering the amount of metabolite synthesis. The growth temperature of the saccharomyces cerevisiae and the test result of ethanol production by fermentation are comprehensively considered, and the subsequent test still selects fermentation culture at the temperature of 30 ℃.

5. Initial pH of the Medium

The method of the thallus activation stage is as above, the ethanol fermentation culture medium is prepared according to the above optimal culture medium formula, the initial pH is respectively adjusted to (initial pH, 2, 3, 4, 5, 6, 7, 8, 9 and 10), and the other conditions are selected to be the above optimal conditions.

Optimization of initial pH of the ethanol producing medium by yeast Y3401 fermentation found that as initial pH increased, the ethanol producing capacity of the yeast fermentation increased first and then decreased slowly (fig. 5). Notably, the ethanol production by yeast fermentation was relatively high when the initial pH was over a wide range of pH (4-9) (FIG. 5). The pH value can be greatly changed due to the metabolic activity of various microorganisms in the liquor brewing process, and the yeast Y3401 is in the liquor brewing environment for a long time and is adapted to the pH change in the liquor brewing process, so that the yeast Y340can grow and ferment to produce ethanol in a wider pH range. As can be seen from FIG. 5, the concentration of ethanol produced by fermentation reached the highest value of 89.4g/L for yeast Y3401 at pH5.2 (natural pH). In order to ensure the accuracy and consistency of the subsequent tests, the pH of the medium used for the fermentation was subsequently adjusted to 5.2 in a uniform manner for the study.

6. Rotational speed and liquid loading

For the rotational speed screening experiment, the method of the thallus activation stage is as above, the rotational speed is selected to be respectively cultured under the conditions of (0, 40, 60, 80, 100, 120, 140, 160, 180 and 200r/min) in the fermentation stage, and the other conditions are selected to be the optimal conditions as above.

For the liquid loading experiment, the method of the cell activation stage was as above, the ethanol fermentation medium was prepared under the optimized conditions, and the cells were dispensed at different liquid loading amounts (15, 30, 60, 90, 120, 150, 180 and 210mL/250mL), and the optimum conditions were selected as above.

As a result, it was revealed from FIG. 6 that yeast Y3401 produces ethanol not only by fermentation in the standing culture mode but also by high yield under the shaking culture condition, and that even the shaking fermentation at a high rotation speed produces ethanol at 24 hours of culture in a higher content than that in the standing, which is likely to be an advantage in the density of high concentration bacteria. In general, the concentration of the ethanol produced by fermentation is increased and then slowly decreased along with the increase of the fermentation culture rotating speed of the yeast Y3401, and when the rotating speed reaches 100r/min, the yield of the ethanol is 93.8g/L which is slightly higher than other culture conditions (figure 6), so that 100r/min is selected as the rotating speed for producing the ethanol by subsequent fermentation. The same conclusion can be drawn through the liquid loading amount experiment result, and the oxygen content in the fermentation environment can be regulated and controlled by adjusting the liquid loading amount, so that the ethanol fermentation capacity of the yeast is influenced (figure 7). When the amount of the liquid is 150mL/250mL or less, the effect of ethanol production by fermentation is good, and when the amount of the liquid is 30mL/250mL, the concentration of ethanol is 95.5g/L (FIG. 7), and therefore, the amount of the liquid is considered to be the other conditions. In conclusion, the proper dissolved oxygen is beneficial to the growth and the propagation of the yeast in the early stage of fermentation, the cell number basis of the fermentation for producing the ethanol is ensured, and more oxygen is consumed in the later stage due to the large cell number, so that an anaerobic environment is created for the self fermentation, and the ethanol is produced in the later stage through anaerobic respiration fermentation. According to the research result, ethanol can be produced by fermenting in stages in the later stage, namely, cell accumulation is carried out in the early stage in a ventilation mode, and ethanol accumulation is carried out by anaerobic fermentation in the later stage.

7. Age of the seed

The effect of the age of the cells on the ethanol production was investigated by inoculating with cells at different activation times (0, 6, 12, 18, 24, 30 and 36h), and the yeast biomass and ethanol concentration were measured separately using the above optimal conditions for the other conditions.

The ethanol production by yeast culture and fermentation of different ages is shown in FIG. 8, and the results show that when yeast of different ages are inoculated, the ethanol production concentration can be different: the ethanol production by seed liquid fermentation is at a low level (FIG. 8) at the age of 0-12h, probably because yeast growth is in lag phase and pre-log phase, when the cell number is small and in an environment-adapted state; the ethanol content produced by seed liquid fermentation at the age of 18-36h is higher, not only because of the high cell number, but also because the cells are adapted to the environment (figure 8). In view of economic efficiency, yeast Y3401 is preferably activated for 18 hours, and the ethanol yield reaches 105.1g/L (FIG. 8).

8. Amount of inoculation

The method of the thallus activation stage is as above, the ethanol fermentation culture medium is prepared according to the optimized conditions, and is inoculated according to different inoculation amounts (0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10 and 15 percent) respectively, and other conditions are as above.

The result shows that the influence of the inoculation amount on the ethanol production of the yeast Y3401 shows that when the inoculation amount is in the range of 0.05-10%, the ethanol production concentration of the yeast increases along with the increase of the inoculation amount; when the inoculation amount is higher than 10%, the ethanol production amount of the fermentation is reduced (figure 9). When the inoculation amount is low, the delay period is relatively long, and the yield of the ethanol is low because the cell number is lower than that of the proper inoculation amount (3-10% for the yeast) during 24h of culture; when the inoculation amount is high, the ethanol yield is reduced because the nutrient substances are mostly used for the growth and the propagation of the yeast. In the range of suitable inoculation amount, higher inoculation amount can not only quickly reach high-density bacterium amount and timely create an anoxic environment, thereby improving the yield of products, but also can inhibit the growth and metabolism of other bacteria and reduce the pollution of other mixed bacteria. The results showed that the ethanol yield was 105.6g/L at an inoculum size of 5%, and experiments were conducted at this optimum inoculum size in subsequent studies.

9. Experiment of fermentation process for producing ethanol by yeast Y3401

The ethanol is produced by yeast fermentation mainly through anaerobic respiration, but the inventor discovers through preliminary optimization that when the yeast is cultured for 24 hours, a certain amount of oxygen exists to be better than the case of oxygen deficiency, which is probably because the cell volume of the yeast is higher than that of the oxygen deficiency under the aerobic condition. For validation, the fermentation history of ethanol production under these two conditions was studied: other conditions with the above optimal results, the influence of fermentation time on ethanol production was investigated in both shaking (0, 8, 16, 24, 32, 40, 48 and 56h) and standing (0, 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 128, 136 and 144h) culture modes, respectively; the pH of the fermentation broth, the yeast biomass, the ethanol concentration and the residual amount of glucose were determined.

FIG. 10 shows the ethanol production of yeast Y3401 in the static culture mode, and the results show that the ethanol production of yeast Y3401 fermentation in the culture mode is increased gradually with the time, but the ethanol production is increased slowly, and the ethanol production is highest at 83.9g/L after 104h of culture. In the shaking culture mode, the early stage of fermentation is in a growth delay period, the cell amount of yeast is low, the ethanol yield is not high, the cell amount is greatly increased along with the growth of yeast in a logarithmic phase, the ethanol yield is also greatly increased, and the ethanol concentration reaches 107.8g/L after the yeast is cultured for 32 hours (figure 10). In addition, the ethanol production capacity of the yeast Y3401 is positively correlated with the cell amount regardless of the static culture or the shaking culture, and the ethanol production capacity is highest when the cell amount is highest in both the static culture and the shaking culture, which belongs to a product formation and cell growth correlation type. Under aerobic conditions, the tolerance of the yeast to high-concentration glucose can be improved, and the yeast cell number can be greatly increased, so that the ethanol yield of the shaking culture mode is higher than that of the standing culture mode, the shaking culture mode is faster than that of the standing culture mode, and the fermentation period is shortened.

The analysis of the glucose content and the pH change in the shaking culture mode shows that the glucose concentration decreases rapidly with the increase of the culture time and is almost exhausted after 32h (figure 11), which is consistent with the growth trend of the strain; the pH is then rapidly reduced and then slightly increased with increasing time, which correlates with the glucose content of the medium. The yeast Y3401 at the early stage of culture utilizes sufficient glucose to grow and metabolize to generate a large amount of acid substances, so that the pH of the culture medium is rapidly reduced; in the later stage, the nutrient substances such as glucose are depleted, and the yeast lacks the nutrient substances required for growth and metabolism, so that the yeast begins to die and break cells, and the pH is slightly increased.

Example two: orthogonal experiments of culture conditions for Saccharomyces cerevisiae Y3401

According to the result of the single-factor test, after the data are subjected to significance analysis, 4 factors of fermentation temperature, initial pH, rotating speed and inoculation amount are selected for L₉(3⁴) Orthogonal experiments are carried out to determine the optimal process conditions for producing the ethanol by fermenting the yeast Y3401. The levels of the orthogonality test factors are shown in table 1.

TABLE 1 orthogonal test factor horizon

The experimental results show that the influence of the inoculation amount on the ethanol production effect of the yeast Y3401 is the largest, and the influence of the rotation speed and the initial pH and the temperature on the fermentation effect is the smallest as shown in the table 2 by the extremely poor R analysis that D is larger than C, and B is larger than A. The analysis of K value results shows that the optimal process combination for producing ethanol by fermenting the yeast Y3401 is A₂B₂C₂D₁I.e. at a temperature of 30 ℃, an initial pH of 5.2, a rotation speed of 100r/min and an inoculum size of 3% (Table 2). The effect of four factors on ethanol production by yeast Y3401 is shown in FIG. 12. As can be seen from FIG. 12, the ethanol yield showed a tendency of increasing and then decreasing with the increase of fermentation temperature, initial pH and rotation speed, so the experimental level should be selected to be 30 ℃ for fermentation temperature, 5.2 for initial pH and 100r/min for rotation speed, i.e. A₂B₂C₂(ii) a The ethanol yield of the strain shows a descending trend along with the increase of the inoculation amount, so that the inoculation amount is 3 percent at the test level, namely D₁. According to the optimal combination, the yeast Y3401 is fermented to produce ethanol with the concentration of 117.2g/L under the conditions of initial pH of 5.2, temperature of 30 ℃, rotating speed of 100r/min and inoculation amount of 3 percent.

TABLE 2 results and analysis of orthogonal experiments

Example three: saccharomyces cerevisiae Y3401 shaking and standing culture experiment

Based on the experiment of the ethanol production fermentation process of the yeast Y3401 in the first embodiment, the inventors of the present invention further performed the mixed culture mode of the early shaking culture (8, 16, 24, 32) and the later standing culture in combination with the optimal conditions (except the rotation speed) obtained in the orthogonal experiment of the second embodiment to search for better culture conditions and obtain the effect of higher ethanol production.

Since the analysis in the first embodiment shows that the initial stage of the culture is mainly an ethanol production preparation stage, namely a yeast biomass accumulation process, and the middle and later stages are ethanol production stages in relative anaerobic environment, we consider that the yeast biomass is rapidly accumulated by shaking in the early stage to provide sufficient oxygen, and the mixed culture mode of producing ethanol by anaerobic fermentation is adopted in the later stage to provide anaerobic conditions for the yeast to fully perform anaerobic fermentation, and the concrete steps are as follows: in the case of shaking culture, the glucose concentration rapidly decreased with the increase of the culture time, and almost exhausted after 32 hours, 8, 16, 24, and 32 hours were set for shaking culture, and experiments were carried out while selecting 4, 8, 12, 16, 20, and 24 hours for later-stage static culture. Experimental results show that the best state can be achieved by a culture mode of firstly shaking culture for 18 hours and then standing culture for 12 hours, and the ethanol content can reach 133.4 g/L.

Claims

1. A method for producing ethanol by culturing Saccharomyces cerevisiae Y3401, wherein the preservation number of the Saccharomyces cerevisiae Y3401 is CGMCC No.14828, and is characterized in that:

the culture medium during culture takes glucose as a carbon source, the dosage of the glucose is 250-350g/L, the yeast extract powder is a nitrogen source, the dosage of the yeast extract powder is 30-45g/L, and the initial pH of the culture medium is 4.8-5.6;

the culture conditions are 28-32 deg.C, and shaking at 80-120 r/min.

2. The method of claim 1, wherein: also comprises a standing culture stage after shaking culture.

3. The method of claim 2, wherein: the culture is performed by shaking culture for 12-28 hr and then static culture for 8-16 hr, more preferably by inoculating and then shaking culture for 16-20 hr and then static culture for 10-14 hr, and most preferably by shaking culture for 18 hr and then static culture for 12 hr.

4. The method of claim 1 or 2, wherein: the amount of glucose in the medium is 280-320g/L, most preferably 300 g/L.

5. The method of claim 1 or 2, wherein: the usage amount of the yeast extract powder is 35-40g/L, and the most preferable is 37.8 g/L.

6. The method of claim 1 or 2, wherein: the initial pH of the medium is 5.0 to 5.4, most preferably 5.2.

7. The method of claim 1 or 2, wherein: the method also comprises the steps of activating the strains for 16-30h before fermentation, and then inoculating and culturing, more preferably activating the strains for 20-28 h, and most preferably activating the strains for 18-24 h; preferably, the culture used for strain activation is YPD medium.

8. The method of claim 1 or 2, wherein: the culture conditions were 30 ℃ and 100r/min during shaking culture.

9. The method of claim 1 or 2, wherein: wherein the basic composition of the culture medium is as follows: the yeast extract powder and glucose in the above amount, in addition, comprise peptone 18-22g/L and KH 0.5-1.5g/L₂PO₄，0.5-1.5g/L(NH₄)₂SO₄，0.5-1.5g/L MgSO₄More preferably, 20g/L peptone, 1g/L KH₂PO₄，1g/L(NH₄)₂SO₄，1g/LMgSO₄。

10. The method of claim 1 or 2, wherein: the amount of inoculum during cultivation is 2-6%, most preferably 3-5%, based on the activated strain liquid.