Saccharomyces cerevisiae as well as application and fermentation method thereof

Abstract

The invention relates to a Saccharomyces cerevisiae strain as well as application and a fermentation method thereof, and provides Saccharomyces cerevisiae HKB-145051 which is preserved in China General Microbiological Culture Collection Center (CGMCC) on June 9, 2022, and the preservation number is CGMCC No.25020. The Saccharomyces cerevisiae HKB-145051 has the advantages that the Saccharomyces cerevisiae HKB-145051 can be applied to fermentation of the Saccharomyces cerevisiae HKB-145051; the saccharomyces cerevisiae can be used for simultaneously fermenting and producing three cellulolytic enzymes, namely cellulase, xylanase and ligninase. The invention also provides a corresponding method for fermenting and producing three cellulolytic enzymes, namely cellulase, xylanase and ligninase, and the yeast strain can be induced to efficiently ferment and produce the three cellulolytic enzymes through anaerobic fermentation by utilizing corncob powder or wheat bran and matching with inorganic salt.

Description

Technical Field

[0001] The invention relates to the technical field of fungi, and in particular to a brewer's yeast, an application thereof and a fermentation method thereof. Background Art

[0002] my country has abundant fiber resources. With the development of industrial technology, the function of fiber has gradually been valued and is known as the seventh nutrient. Fiber has different nutritional value requirements in human and animal production. Adding a certain level of dietary fiber to animal feed plays an important role in animal growth, reproductive performance and intestinal health. It can reduce the pollution of nitrogen residues in livestock and poultry manure to the environment, realize the recycling of waste feed resources, save feed costs, and further improve the efficiency of breeding. In the process of reducing and replacing antibiotics in animal production in developed countries in Europe and the United States, dietary fiber has been widely used as an important product. At present, most of my country's feed fiber products are bran, rice husks, etc. The direct use of such crude fiber raw materials has biosafety problems such as low dietary fiber utilization and excessive mycotoxins.

[0003] In actual production, feed processing will choose to use products such as mildew inhibitors and fiber-degrading enzymes to reduce the mildew of natural fiber feed raw materials, reduce the content of long-chain cellulose, lignin and high-polymer xylan in feed raw materials, and increase the content of soluble fiber and short-chain insoluble fiber, thereby improving the efficacy of natural fiber products. However, chemically synthesized fiber-degrading enzymes are expensive; a more promising method is the microbial fermentation method, which uses fruit, vegetable and grain by-products as raw materials, and uses microorganisms to secrete fiber-degrading enzymes to ferment the resistant starch in the raw materials. In addition, during the fermentation process, microorganisms utilize and decompose nutrients such as proteins and fats and metabolize them to produce active substances such as organic acids and small peptides, thereby obtaining natural dietary fiber products with multiple activities. In addition, the dietary fiber purified by fermentation has the advantages of high quality, high color and fragrant smell, and has high palatability.

[0004] The selection of fermentation strains is the basis and focus of microbial fermentation. At present, mixed bacteria fermentation or fungi such as Aspergillus oryzae and Aspergillus niger are mostly used as fermentation bacteria for the fermentation of high-fiber feed raw materials. However, the smell and palatability of Aspergillus fermentation products are not high, and the fermentation parameters of mixed bacteria fermentation are greatly affected by the materials, which has high requirements for pretreatment technology.

[0005] Yeast has been used by humans for a very long time. As early as prehistoric times, human ancestors learned to make wine from the natural fermentation of ripe fallen fruits, and they have been eating fermented bread for 6,000 years. At present, yeast has been widely used in food, medicine, feed, cosmetics, bioengineering, industry and other fields. In actual production, the production process of yeast fermentation can be determined according to different purposes based on the demand for the number of yeast cells, cell components, or yeast metabolites. At the same time, yeast is also the most widely used chassis strain used by humans for biomanufacturing. At present, in my country, there are only three types of yeast that are legally used in feed raw materials and additives: brewer's yeast ( Saccharomyces cerevisiae ), Candida utilis ( Candida utilis ) and Phaffia rhodozyma ( Phaffia rhodozyma ).

[0006] Therefore, breeding a strain of brewer's yeast with high cellulase fermentation production and applying it to feed raw material processing and product development will help my country's feed raw material resources to "increase revenue and reduce expenditure." Summary of the invention The object of the present invention is to provide a strain of brewer's yeast and its application and fermentation method. The brewer's yeast can simultaneously ferment and produce three fiber-degrading enzymes, namely, cellulase, xylanase and ligninase, through a specific anaerobic fermentation technology.

[0007] To this end, in a first aspect, the present invention provides a strain of Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) HKB-145051, the brewer's yeast was deposited on June 9, 2022 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 25020.

[0008] The brewer's yeast HKB-145051 was isolated and screened from nature, obtained after natural domestication, and has not been modified by genetic engineering technology.

[0009] The second aspect of the present invention provides a fermentation method of the brewer's yeast, which comprises inoculating the brewer's yeast into a liquid culture medium for aerobic fermentation to prepare an aerobic fermentation liquid.

[0010] The brewer's yeast can be prepared in large quantities through the fermentation method.

[0011] Furthermore, the liquid culture medium comprises: 10%-15% sugarcane molasses, 20-30 g / L peptone, 0.1%-0.15% KH2PO4, 0.1%-0.15% MgSO4·7H2O and 0.05%-0.15% CaCl2.

[0012] In some embodiments, the liquid culture medium comprises: 10% cane molasses, 20 g / L peptone, 0.1% KH2PO4, 0.1% MgSO4·7H2O and 0.05% CaCl2.

[0013] Furthermore, the conditions of the liquid culture medium include: initial fermentation temperature 25-28°C, initial fermentation pH 6.5-7.0, rotation speed 180-200 rpm, and fermentation time 32-36h.

[0014] Furthermore, the aerobic fermentation further includes a seed solution preparation step; the seed solution preparation step includes: inoculating the brewing yeast into a seed solution culture medium, performing seed culture, and obtaining the brewing yeast seed solution.

[0015] In some embodiments, the seed liquid culture medium is the same as the liquid culture medium.

[0016] Furthermore, when the step of preparing a seed solution is included, the seed solution is inoculated into the liquid culture medium in an inoculation amount of 10%-12%.

[0017] Furthermore, before the seed solution preparation step, the method further comprises the following steps: bacterial strain activation; the bacterial strain activation comprises: inoculating the brewer's yeast into a slant culture medium to perform bacterial strain activation culture.

[0018] Furthermore, the slant culture medium is a solid malt extract culture medium, including malt extract powder, chloramphenicol, and agar; the content of each component is preferably 130 g / L malt extract powder, 0.1 g / L chloramphenicol, and 20 g / L agar.

[0019] Furthermore, the temperature for the activation culture of the strain is 28°C-32°C, and the culture time is 24-48 h.

[0020] The third aspect of the present invention provides the use of the Saccharomyces cerevisiae in the fermentation production of fiber degrading enzymes.

[0021] The fourth aspect of the present invention provides a fermentation method for simultaneously producing three fiber-degrading enzymes using the brewer's yeast, comprising inoculating the brewer's yeast into a liquid fermentation medium for anaerobic fermentation; the fiber-degrading enzymes include cellulase, xylanase and ligninase.

[0022] Furthermore, the anaerobic fermentation medium includes: 10%-15% sugarcane molasses, 20-30 g / L peptone, 20-25 g / L corn cob, 0.1%-0.15% MgSO4·7H2O, 0.05%-0.15% KH2PO4, 0.05%-0.15% NaCl; and may also contain 0.08-0.1 g / L chloramphenicol.

[0023] In some embodiments, the anaerobic fermentation medium includes: 10% sugarcane molasses, 20 g / L peptone, 20 g / L corn cob, 0.1% MgSO4·7H2O, 0.1% KH2PO4, 0.05% NaCl; and may further include 0.08 g / L chloramphenicol.

[0024] Furthermore, the conditions of the anaerobic fermentation include: an initial temperature of 25-30°C, such as 25°C, 28°C, 30°C, etc.; an initial pH value of 6.0-7.0, such as pH 6.0, 6.5, 7.0, etc.; a fermentation time of 60-72h, such as 60h, 64h, 68h, 72h, etc.

[0025] Furthermore, the anaerobic fermentation also includes aerobic fermentation before the anaerobic fermentation, and the aerobic fermentation is carried out according to the fermentation method described in the second aspect of the present invention.

[0026] Furthermore, when the anaerobic fermentation includes the aerobic fermentation before the anaerobic fermentation, the inoculation amount of the aerobic fermentation liquid into the anaerobic culture medium is 10%-15%, for example, about 10%, 12%, 15%, etc.

[0027] Compared with the prior art, the advantages of the present invention are: 1) The present invention provides a Saccharomyces cerevisiae strain HKB-145051 having high cellulase expression activity; 2) The Saccharomyces cerevisiae strain HKB-145051 provided by the present invention can simultaneously produce three fiber-degrading enzymes, namely, cellulase, xylanase and ligninase; 3) The brewer's yeast strain HKB-145051 provided by the present invention can induce the yeast strain to efficiently ferment and simultaneously produce the above three fiber-degrading enzymes by using a specific anaerobic fermentation technology, corn cob powder or wheat bran, and inorganic salts. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings: Figure 1 :Effects of different molasses concentrations in culture medium on the growth of Saccharomyces cerevisiae HKB-145051; Figure 2 :Effects of different nitrogen sources in culture medium on the growth of Saccharomyces cerevisiae HKB-145051; Figure 3 :Effects of different nitrogen source concentrations in culture medium on the growth of Saccharomyces cerevisiae HKB-145051; Figure 4:Effects of different inorganic salts and concentrations in the culture medium on the growth of Saccharomyces cerevisiae HKB-145051; Figure 5 :The effect of different inoculation amounts on the fermentation performance of Saccharomyces cerevisiae HKB-145051; Figure 6 :The effect of different fermentation temperatures on the fermentation performance of Saccharomyces cerevisiae HKB-145051; Figure 7 :The effect of different fermentation times on the fermentation performance of Saccharomyces cerevisiae HKB-145051; Figure 8 :The effect of different pH values ​​on the fermentation performance of Saccharomyces cerevisiae HKB-145051; Fig. 9 :The effect of different rotation speeds on the fermentation effect of brewer's yeast HKB-145051; Fig.10 :The effects of aerobic and anaerobic fermentation on cellulose production by Saccharomyces cerevisiae HKB-145051; Fig.11 :The effect of different raw materials on cellulose production by Saccharomyces cerevisiae HKB-145051 in anaerobic fermentation; Fig.12 :Effects of different concentrations of corncobs and wheat bran on cellulose production by Saccharomyces cerevisiae HKB-145051 in anaerobic fermentation; Fig.13 :Effects of different inorganic salts and addition concentrations on cellulose production by Saccharomyces cerevisiae HKB-145051 during anaerobic fermentation; Fig.14 :The effect of different inoculation amounts on cellulose production by Saccharomyces cerevisiae HKB-145051 in anaerobic fermentation; Fig.15 :The effect of different initial temperatures on cellulose production by Saccharomyces cerevisiae HKB-145051 during anaerobic fermentation; Fig.16 :The effect of different initial pH values ​​on cellulose production by Saccharomyces cerevisiae HKB-145051 in anaerobic fermentation; Fig.17 :The effect of different fermentation time on cellulose production by Saccharomyces cerevisiae HKB-145051 in anaerobic fermentation; Fig.18 : Schematic diagram of the technical route of the present invention. DETAILED DESCRIPTION

[0029] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0030] The technical route of the technical solution of the present invention is as follows Fig.18 Reference Fig.18 The present invention first screens a Saccharomyces cerevisiae strain that can simultaneously produce cellulase, xylanase and ligninase from nature, and obtains the Saccharomyces cerevisiae HKB-145051 after strain selection; then, through fermentation process research (including liquid aerobic fermentation and liquid anaerobic fermentation), mainly through research on substrates that stimulate the production of cellulose degrading enzymes, the optimal fermentation conditions are explored, and an efficient fermentation process for Saccharomyces cerevisiae and cellulose degrading enzymes is formed.

[0031] Example 1 Isolation of Saccharomyces cerevisiae HKB-145051 (1) Culture medium: malt extract culture medium. Take 500 g of malt, crush it and put it into a beaker, add 2000 mL of distilled water, stir and place in a 45℃ water bath for 30 min, then raise the temperature to 70℃ and keep it for 1 h; filter through 8 layers of gauze, and wash the beaker and filter tank with distilled water to make the filtrate reach 2000 mL, boil and filter, cool, and store at 10℃ for later use.

[0032] (2) Isolation of brewer's yeast: Using dairy cow manure, beef cattle manure, goat manure, and soil from cattle and sheep pastures as raw materials, collect 400 mL (liquid sample) or 400 g (crushed solid sample) of raw material samples, place them in sterile conical bottles, cover with 8 layers of gauze and ferment naturally at 28°C for 3 days. Under sterile conditions, shake the fermentation culture sample evenly, use a sterile pipette to draw an appropriate amount of fermentation liquid into the sterilized malt extract culture medium, and culture at 28°C. When there is a wine flavor, use the gradient dilution method to dilute to a concentration of 10 -5 , 0.1 mL of the diluted culture solution was evenly spread on the malt extract medium (malt extract culture solution + 2% agar), and inverted at 28°C for 48 h. After the colonies grew, single colonies with typical yeast colony characteristics were selected for further separation and purification until a pure strain was obtained. After identification and confirmation, the strain was transferred to the malt extract solid slant medium and stored at 4°C for later use.

[0033] (3) Isolation of yeast with fiber-degrading properties Cellulose culture medium: NaNO3 2.0 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.5 g / L, KClKCl 0.5 g / L, peptone 2.0 g / L, carboxymethyl cellulose 0.5 g / L; Xylan medium: NaNO3 2.0 g / L, KH2PO4 1.0 g / L, MgSO4·7H2O 0.5 g / L, KClKCl 0.5 g / L, peptone 2.0 g / L, xylan 0.5 g / L; Lignin medium: KH2PO4 1.0 g / L, yeast extract 0.01 g / L, C4H 12 N2O6 ammonium tartrate 1.0 g / L, CuSO4 0.001 g / L, MgSO4·7H2O 0.5 g / L, Fe2(SO4)3 0.001 g / L, MnSO4 0.001 g / L, CaCl2 0.01 g / L, agar 1.6% w / v, 0.25% w / v lignin.

[0034] After the above three culture media were sterilized by high pressure, the yeast isolated above was inoculated; then 10 mL of 20% (w / v) glucose solution and 0.1 g / L chloramphenicol were added respectively, the pH value was adjusted to 6.0, the fermentation temperature was 25°C, and the culture was carried out for 2.5 days.

[0035] (4) Qualitative analysis of fiber-degrading enzymes produced by Saccharomyces cerevisiae The bacterial colonies with obvious colony growth on the above three culture media were separated and processed as follows; (1) On cellulose and xylan culture media: Pour about 15 mL of 0.1% Congo red staining solution into the yeast colony, soak for 15 min, then pour off the staining solution, and then stain with 1 mol / L NaCl solution. The yellow halo from the inoculation point to the outside indicates that the yeast metabolites degrade the substrate (CMC or xylan), and red indicates no degradation; (2) For yeast colonies on lignin culture medium, the yeast colonies were immersed in a newly prepared 1% (w / v) FeCl3 and K3 (Fe(CN)6) aqueous solution. The blue-green color around the colonies indicated that lignin was degraded into phenols by bacterial metabolites. The screening results are shown in Table 1.

[0036] Table 1 Screening of yeasts with fiber degradation properties

[0037] Note: + indicates degradable, - indicates non-degradable.

[0038] (5) Identify and preserve yeast 145051.

[0039] The yeast 145051 was identified as Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), named HKB-145051, and deposited on June 9, 2022 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, with the deposit number CGMCC No. 25020.

[0040] Example 2 Selection of fermentation medium for Saccharomyces cerevisiae HKB-145051 Saccharomyces cerevisiae HKB-145051 was activated and cultured in seed solution, and then aerobic fermentation was carried out in the following culture media, and the temperature condition was 28°C.

[0041] (1) Effects of different molasses concentrations on the growth of Saccharomyces cerevisiae HKB-145051 5%, 8%, 10%, 13%, and 15% sugarcane molasses were selected as culture medium, and the inoculation amount of bacterial solution was 2%. After culturing for 24 h, 8 mL of bacterial solution was washed twice with sterile water, and the bacteria were dried to constant weight and weighed. Three replicates were used in each group and the average value was calculated. The statistical results are shown in the figure. Figure 1 As shown. Figure 1 The results showed that when the concentration of sugarcane molasses was above 10%, the change of molasses concentration in the culture medium had no significant effect on the growth of Saccharomyces cerevisiae HKB-145051, so increasing the amount of molasses above 10% was meaningless for the fermentation of the strain.

[0042] (2) Effects of different nitrogen sources on the growth of Saccharomyces cerevisiae HKB-145051 Nitrogen sources were added to the 10% sugarcane molasses. Specifically, beef extract, yeast extract, peptone, ammonium chloride, ammonia water, and urea were selected as nitrogen sources, and the final nitrogen content was 2.2 g / L. The inoculation amount of the bacterial solution was 2%. After 24 h of culture, 8 mL of the bacterial solution was washed twice with sterile water, and the cells were dried to constant weight and weighed. Three parallels were added to each group and the average value was calculated. The statistical results are shown in Figure 2 As shown. Figure 2 The results showed that the growth of Saccharomyces cerevisiae HKB-145051 was best when yeast extract and peptone were used.

[0043] (3) Selection of nitrogen source concentration Nitrogen source was added on the basis of 10% sugarcane molasses. Specifically, peptone was selected as the nitrogen source based on production convenience and versatility, and its concentration was selected as 10, 15, 20, 25, and 30 g / L respectively. The inoculation amount of bacterial liquid was 2%. After 24 h of culture, 8 mL of bacterial liquid was washed twice with sterile water, and the bacterial cells were dried to constant weight and weighed. Three parallels were made in each group and the average value was calculated. The statistical results are shown as follows: Figure 3 As shown. Figure 3 The results showed that when the peptone concentration in the culture medium was 20 g / L, the growth effect of Saccharomyces cerevisiae HKB-145051 was the best.

[0044] (4) Effects of different inorganic salts and concentrations on the growth of Saccharomyces cerevisiae HKB-145051 Inorganic salts were added to 10% cane molasses and 20 g / L peptone. Specifically, KH2PO4, K2HPO4·3H2O, MgSO4·7H2O, KCl, and CaCl2 were selected, and different addition concentrations were set at 0.00%, 0.05%, 0.10%, and 0.15%, respectively. The inoculation amount of the bacterial solution was 2%. After 24 h of culture, 8 mL of the bacterial solution was washed twice with sterile water, and the bacterial cells were dried to constant weight and the dry weight was weighed.

[0045] Each group has three parallels and the average value is calculated. The statistical results are as follows Figure 4 As shown. Figure 4 The results show that KCl and K2HPO4·3H2O have little effect on yeast growth, so they are no longer added. 0.1% and 0.15% KH2PO4 and MgSO4·7H2O have basically the same effect on growth, so the concentration is 0.1%. 0.05% CaCl2 has basically the same effect on growth as 0.1% and 0.15%, so 0.05% is selected.

[0046] In summary, the optimized culture medium composition was: 10% cane molasses, 20 g / L peptone, 0.1% KH2PO4, 0.1% MgSO4·7H2O and 0.05% CaCl2.

[0047] Example 3 Selection of fermentation parameters for Saccharomyces cerevisiae HKB-145051 Saccharomyces cerevisiae HKB-145051 was activated and cultured with seed solution, and then aerobic fermentation was carried out using the culture medium optimized in Example 2. The specific culture conditions were as follows: (1) Selection of inoculation volume Different inoculation amounts of bacterial solution were used: 2%, 5%, 8%, 10%, 12%, 15%, and 18%. The final dry weight of yeast was weighed after 24 hours of fermentation. Three parallels were run in each group and the average value was calculated. The statistical results are shown in the figure. Figure 5 As shown. Figure 5 It can be seen that when the inoculation amount is 10~12%, a higher fermentation effect can be achieved.

[0048] (2) Selection of fermentation temperature The inoculation volume was 10%, and the fermentation was carried out at the following temperatures: 25°C, 28°C, 30°C, 32°C, and 35°C. The culture was carried out for 24 hours, and the dry weight of the yeast was weighed. Three parallels were prepared for each group, and the average value was calculated. The statistical results are shown in the figure. Figure 6 As shown. Figure 6 It can be seen that an initial fermentation temperature of 25-28°C will achieve better fermentation results.

[0049] (3) Selection of fermentation time The inoculation volume was 10%, and the fermentation temperature was 28°C. The fermentation culture was carried out, and samples were taken every 4 hours from 12h to 48h, and the dry weight of the yeast was weighed. Three parallels were made in each group and the average value was calculated. The statistical results are shown in the figure. Figure 7 As shown. Figure 7 It can be seen that when the fermentation time is 32~36h, excellent fermentation effect can be achieved.

[0050] (4) Selection of fermentation pH The inoculation volume was 10%, the fermentation temperature was 28°C, and the following initial pH values ​​were used for fermentation culture: 5.0, 5.5, 6.0, 6.5, and 7.0. The final dry weight of yeast was weighed after 32 hours of fermentation. Three parallels were performed in each group and the average value was calculated. The statistical results are shown in the figure. Figure 8 As shown. Figure 8 It can be seen that the fermentation effect is better under the condition of pH 6.5-7.0.

[0051] (5) Selection of speed The inoculation volume was 10%, the fermentation temperature was 28°C, the initial pH was 7.0, and the following shaking speeds were used for fermentation culture: 150, 180, 200, 220, 240, 260 rpm. The final yeast dry weight was weighed after 32 hours of fermentation. Three parallels were used in each group and the average value was calculated. The statistical results are shown in the figure. Fig. 9 As shown. Fig. 9 It can be seen that the fermentation effect obtained under the condition of 180-200 rpm is better.

[0052] In summary, through a series of experimental studies, the following conditions were used to ferment the brewer's yeast HKB-145051 to achieve better fermentation results: inoculation amount 10-12%, initial fermentation temperature 25-28℃, initial fermentation pH 6.5-7.0, rotation speed 180-200rpm, and fermentation time 32-36h.

[0053] Example 4 Liquid aerobic fermentation of Saccharomyces cerevisiae HKB-145051 (1) Activation of slant strains: The frozen Saccharomyces cerevisiae HKB-145051 was inoculated onto a slant culture medium containing 130 g / L malt extract powder, 0.1 g / L chloramphenicol, and 20 g / L agar, and cultured at a constant temperature of 30°C for 48 h to obtain the activated yeast strain.

[0054] (2) Preparation of seed solution: Inoculate one loop of activated yeast strain into a liquid culture medium containing 10% cane molasses, 20 g / L peptone, 0.1% KH2PO4, 0.1% MgSO4·7H2O, 0.05% CaCl2, 0.08 g / L chloramphenicol, and pH 6.5. Incubate the culture in a constant temperature shaker at 28°C and 200 r / min for 24 h to prepare the seed solution.

[0055] (3) Aerobic fermentation: The seed liquid was inoculated into a liquid culture medium containing 10% sugarcane molasses, 20 g / L peptone, 0.1% KH2PO4, 0.1% MgSO4·7H2O and 0.05% CaCl2, 0.08 g / L chloramphenicol, and a pH value of 7.0 at a 10% inoculation rate, and cultured in a constant temperature shaker at 28°C, a speed of 200 r / min, and cultured for 32 h to prepare an aerobic fermentation liquid. The aerobic fermentation liquid was inoculated into the culture medium in Example 5 for subsequent fermentation.

[0056] Example 5 Study on the fermentation method for efficiently producing fiber-degrading enzymes by Saccharomyces cerevisiae HKB-145051 (1) Selection of fermentation methods to induce Saccharomyces cerevisiae HKB-145051 to efficiently produce fiber-degrading enzymes Under the same liquid medium conditions (10% cane molasses, 20 g / L peptone, 0.1% KH2PO4, 0.1% MgSO4·7H2O and 0.05% CaCl2, 0.08 g / L chloramphenicol), the inoculation was 10%, and aerobic and anaerobic fermentations were carried out, respectively, with an initial temperature of 25°C, an initial pH of 6.5, and fermentation for 24 h.

[0057] 50 mM citric acid buffer (pH 4.8) was added to 5 mL / g fermentation mud to extract crude enzymes, and stirred at 200 rpm for 30 min. The filtrate was collected after filtration and separation, and the supernatant was selected after centrifugation at 1500 rpm for 20 min. The cellulase and xylanase activities were determined by 3,5-dinitrosalicylic acid (DNS) method, and the ligninase activity was determined by Lip peroxidation method.

[0058] The activities of cellulase, xylanase and ligninase were compared (three replicates in each group and the average value was calculated. For specific results, see Fig.10 As shown in the figure), anaerobic fermentation was selected as the main fermentation method.

[0059] (2) Selection of anaerobic fermentation substrates for efficient production of fiber-degrading enzymes by Saccharomyces cerevisiae HKB-145051 Anaerobic fermentation is ideal when sugar is exhausted, and some cellulose raw materials can improve the expression of cellulase. Soybean meal, corn, corn cob, wheat bran, and beet pulp were selected as raw materials, crushed and then anaerobic fermented. After 24 hours, the fiber degradation enzyme activity of the fermentation products was compared. Three parallels were run in each group and the average value was calculated. The results are shown in the figure. Fig.11 As shown, it was found that corn cobs and wheat bran can well induce the production of fiber-degrading enzymes in Saccharomyces cerevisiae HKB-145051 under anaerobic conditions.

[0060] (3) Composition of anaerobic fermentation medium for efficient production of fiber-degrading enzymes by Saccharomyces cerevisiae HKB-145051 In the basic medium containing 10% sugarcane molasses, 20 g / L peptone and 0.08 g / L chloramphenicol, 10, 15, 20, and 25 g / L corn cobs and wheat bran cooled to 40°C after high temperature sterilization were added, the bacterial liquid inoculation amount was 10%, the initial fermentation temperature was 25°C, and the cellulase activity was detected after 24 hours of fermentation under natural pH conditions. Three parallels were added to each group and the average value was calculated. The results are shown in Fig.12 As shown, it was found that the production of fiber-degrading enzymes in Saccharomyces cerevisiae HKB-145051 could be induced by using 20 g / L corncobs.

[0061] Based on the application of 20 g / L corn cob, KH2PO4, K2HPO4·3H2O, MgSO4·7H2O, NaCl, and CaCl2 were selected as inorganic salts, and different inorganic salt addition concentrations were set at 0.00%, 0.05%, 0.10%, and 0.15%, respectively. The inoculation amount of the bacterial liquid was 10%, the initial fermentation temperature was 25℃, and the activity of the three fiber-degrading enzymes was detected after 24 hours of fermentation under natural pH conditions. Three parallels were used in each group and the average value was calculated. The results are shown in Figure 2. Fig.13 As shown, it can be seen that when 20 g / L corn cobs are used in combination with an organic salt solution containing 0.1% MgSO4·7H2O, 0.1% KH2PO4, and 0.05% CaCl2 as an aerobic fermentation medium for Saccharomyces cerevisiae HKB-145051, a higher fiber-degrading enzyme can be obtained.

[0062] (4) Selection of anaerobic fermentation conditions for efficient production of fiber-degrading enzymes by Saccharomyces cerevisiae HKB-145051 The aforementioned liquid fermentation medium (10% sugarcane molasses, 20 g / L peptone, 0.08 g / L chloramphenicol, 20 g / L corn cob, 0.1% MgSO4·7H2O, 0.1% KH2PO4, 0.05% NaCl) was used, and the inoculation amount was selected to be 5%, 8%, 10%, 12%, and 15%, respectively. The initial fermentation temperature was 25°C, and the natural pH value was used. After 24 hours of fermentation, the activities of the three fiber-degrading enzymes were detected. Three parallels were added to each group and the average value was calculated. The results are shown in Figure 2. Fig.14 shown.

[0063] On this basis, the initial temperature was further optimized: using a 10% inoculation amount and natural pH conditions, the following initial temperatures were used for fermentation: 25°C, 28°C, 30°C, 32°C, and 35°C. After 24 hours of fermentation, the activities of the three fiber-degrading enzymes were detected. Three parallels were added to each group and the average value was calculated. The results are shown in Figure 2. Fig.15 shown.

[0064] On this basis, the pH value was further optimized: 10% inoculation, initial temperature 28°C, and the following initial pH values ​​were used for fermentation: 5.5, 6.0, 6.5, 7.0. After 24 hours of fermentation, the activities of three fiber-degrading enzymes were detected. Three parallels were added to each group and the average value was calculated. The results are shown in Figure 2. Fig.16 shown.

[0065] On this basis, the fermentation time was further optimized: 10% inoculation, initial temperature 28°C, initial pH 6.0, and after 24 hours of fermentation, samples were taken every 4 hours to detect the activities of the three fiber-degrading enzymes. Three parallels were added to each group and the average value was calculated. The results are shown in Figure 2. Fig.17 shown.

[0066] According to the above research, the efficiency of brewer's yeast HKB-145051 in producing three fiber-degrading enzymes was relatively high under the anaerobic fermentation conditions of 10% inoculation amount, 28°C initial temperature, 6.0 initial pH value and 60-72h fermentation.

[0067] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A strain of Saccharomyces cerevisiae, characterized in that: For Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) HKB-145051, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 9, 2022, with the deposit number CGMCC No. 25020.

2. The fermentation method of Saccharomyces cerevisiae according to claim 1, characterized in that: The method comprises inoculating the brewer's yeast into a liquid culture medium for aerobic fermentation to prepare an aerobic fermentation liquid.

3. The fermentation method of Saccharomyces cerevisiae according to claim 2, characterized in that: The liquid culture medium comprises: 10%-15% sugarcane molasses, 20-30 g / L peptone, 0.1%-0.15% KH2PO4, 0.1%-0.15% MgSO4·7H2O and 0.05%-0.15% CaCl2.

4. The fermentation method of Saccharomyces cerevisiae according to claim 2 or 3, characterized in that: The conditions of the liquid culture medium include: initial fermentation temperature of 25-28° C., initial fermentation pH of 6.5-7.0, rotation speed of 180-200 rpm, and fermentation time of 32-36 hours.

5. The fermentation method of brewer's yeast as claimed in claim 2, characterized in that: The aerobic fermentation also includes a seed liquid preparation step; The seed solution preparation step comprises: inoculating the brewing yeast into a seed solution culture medium, performing seed culture, and obtaining the brewing yeast seed solution.

6. Use of the saccharomyces cerevisiae according to claim 1 in the fermentation production of fiber degrading enzymes.

7. A fermentation method for simultaneously producing three fiber-degrading enzymes using Saccharomyces cerevisiae, characterized in that: The method comprises inoculating the brewer's yeast of claim 1 into an anaerobic fermentation medium to perform anaerobic fermentation; the fiber degrading enzyme comprises cellulase, xylanase and ligninase.

8. The fermentation method for simultaneously producing three fiber degrading enzymes using Saccharomyces cerevisiae as claimed in claim 7, characterized in that: The anaerobic fermentation medium comprises: 10%-15% sugarcane molasses, 20-30 g / L peptone, 20-25 g / L corn cob, 0.1%-0.15% MgSO4·7H2O, 0.05%-0.15% KH2PO4, and 0.05%-0.15% NaCl.

9. The fermentation method for simultaneously producing three fiber degrading enzymes using Saccharomyces cerevisiae according to claim 7 or 8, characterized in that: The conditions of the anaerobic fermentation include: initial temperature 25-30° C., initial pH value 6.0-7.0, and fermentation time 60-72 hours.

10. The fermentation method for simultaneously producing three fiber degrading enzymes using Saccharomyces cerevisiae according to claim 7, characterized in that: The anaerobic fermentation also includes aerobic fermentation, and the aerobic fermentation is carried out according to the fermentation method of brewer's yeast according to any one of claims 2 to 5; Furthermore, when the anaerobic fermentation includes the aerobic fermentation before the anaerobic fermentation, the aerobic fermentation liquid is inoculated into the anaerobic culture medium in an inoculation amount of 10%-15%.

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