Coix straw hydrolysate and method for culturing oleaginous yeast under non-sterile condition thereof
Through the preparation of coix straw hydrolysate, the problem of sterile conditions required for traditional fermentation is solved, and efficient cultivation of oil-producing yeast under non-sterile conditions is achieved, reducing costs, and improving resource utilization and oil production.
Patent Information
- Application Number
- CN202411869598.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-03-14
AI Technical Summary
The traditional fermentation process needs to be carried out under sterile conditions, resulting in increased production costs and time costs. High-temperature autoclave may damage enzyme activity. It is difficult for the existing technology to effectively utilize agricultural by-products such as coix straw, resulting in waste of resources and environmental pollution.
Through the preparation of coix straw hydrolysate, including cleaning, drying, heating treatment, filtration, enzymatic lysis, etc., a culture medium suitable for culturing oil-producing yeast under non-sterile conditions was prepared.
It realizes the cultivation of oil-producing yeast under non-sterile conditions, which reduces production costs and time costs, improves resource utilization and environmental protection benefits, and improves oil and fat yield and yeast growth performance.
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Abstract
Description
Technical Field
[0001] The invention discloses coix straw hydrolyzate and a method for culturing oil-producing yeast under non-sterile conditions, belonging to the field of biotechnology. Background Art
[0002] In the production and processing of traditional Chinese medicine, the comprehensive development and utilization of non-medicinal parts of traditional Chinese medicine is relatively low, which not only leads to different types of waste in the entire industrial chain of traditional Chinese medicine resources, but also causes serious waste of resources and environmental pollution. Therefore, it is urgent to strengthen the comprehensive utilization of non-medicinal parts.
[0003] Job's tears is one of the biomass resources containing cellulose, hemicellulose and lignin. Job's tears seeds are the dried and mature kernels of Job's tears. They have rich nutritional value and unique medicinal value and have been widely used in the fields of medicine, food and cosmetics. In particular, anticancer drugs using kernels as raw materials have been widely used in clinical practice. However, the plant of Job's tears is very tall, and its fruit accounts for a small proportion of the whole plant. A large number of non-kernel parts have not been effectively utilized, and more than 80% of Job's tears straw is burned or discarded.
[0004] At present, the production and utilization of bioenergy by using straw biomass has good advantages in reducing carbon emissions and environmental protection. However, traditional fermentation must be carried out under sterile conditions, and bacteria from the surrounding environment or polluted input waste streams may cause the quality of fermentation products to deteriorate, the yield to decrease, and even lead to the failure of the fermentation process. Therefore, during the high-temperature sterilization process, the fermentation equipment and fermentation broth need to be strictly sterilized and carefully sealed, and the fermentation operation is carried out under strict aseptic conditions to ensure the strict sterility required by the yeast during the fermentation process. However, this method increases the production cost and time cost to a certain extent, thereby limiting the large-scale production of high-value compounds. In addition, high-temperature and high-pressure sterilization converts monosaccharides after enzymatic hydrolysis into harmful substances that inhibit cell growth. Although there are reports that bacterial growth can be inhibited by adding antibiotics, this may cause serious drug resistance problems. Therefore, bypassing the sterilization process without affecting productivity can save a lot of time and cost.
[0005] Most high-value natural compounds have a wide range of pharmacological effects and are in great demand. Traditionally, these compounds are mainly derived from plants, but their content in plants is low, extraction and separation are difficult, and the yield is low. In addition, some medicinal plants have problems such as long growth cycles and continuous cropping obstacles, which hinder the development and utilization of natural products. In recent years, many researchers have used synthetic biology technology and metabolic engineering to transform yeast to synthesize natural products to obtain high-value-added products. This method has excellent properties such as high yield, short production cycle, and saving land resources. It is an important source to solve the shortage of plant resources. Yeast is one of the most widely studied microbial chassis cells. It can build reasonable recombinant biosynthetic pathways and construct high-yield microbial strains of corresponding products to achieve the production of high-value compounds from renewable raw materials. Summary of the invention
[0006] The purpose of the present invention is to provide coix straw hydrolyzate for culturing oil-producing yeast; the non-sterile cyclic application of coix straw provided by the present invention not only provides a suitable substrate for yeast biosynthesis, but also realizes its high-value utilization.
[0007] The method for preparing coix straw hydrolyzate provided by the present invention comprises the following steps: S1, the coix straw powder is washed and descaled and then dried to a constant weight; S2, adding NaOH aqueous solution or sulfuric acid solution to the coix straw powder and heating; S3, after cooling to room temperature, filtering and separating to obtain coix residue; S4. Add deionized water to the coix residue dried to constant weight for redissolution, adjust the pH value to 4.5-5.5, add cellulase and bovine serum albumin, or cellulase and PEG10000 for enzymatic hydrolysis, and obtain coix straw hydrolyzate.
[0008] In the above preparation method, in step S1, the coix straw is crushed and then sieved through an 80-mesh sieve to obtain the coix straw powder; Dry at 50 °C-105 °C to constant weight.
[0009] In the above preparation method, in step S2, the mass concentration of the NaOH aqueous solution is 0.5%-2%, and the concentration of the sulfuric acid solution is 0.1-0.2 M; The dosage ratio of the coix straw powder to the NaOH aqueous solution or the sulfuric acid solution is 0.02-0.1 g / mL, preferably 0.02-0.08 g / mL, 0.02 g / mL, 0.05 g / mL and 0.08 g / mL; The heating temperature is 80°C-121°C, and the heating time is 60 min-90 min.
[0010] In the above preparation method, in step S4, the coix seed residue is washed with deionized water and then dried.
[0011] In the above preparation method, in step S4, during the re-dissolution, the dosage ratio of the coix residue to the deionized water is 0.02-0.1 g / mL.
[0012] In the above preparation method, in step S4, the conditions for the enzymatic hydrolysis are: temperature of 40 °C-55 °C, and time of 36-72 h.
[0013] In the above preparation method, in step S4, the amount of the cellulase is as follows: Aspergillus glaucus Cellulase and Aspergillus riparianus The total dosage of the cellulase is 5-15 FPU / g of the coix residue, the dosage of the β-glucosidase is 10-30 CBU / g of the coix residue; and the dosage of the cellulose mixed enzyme (Cellic CTec3) is 10-20 FPU / g of the coix residue.
[0014] The amount of the bovine serum albumin and the PEG10000 is 0.05-0.10 g / g of the coix seed residue, preferably 0.075 g / g.
[0015] The coix straw hydrolyzate prepared by the above method of the present invention also belongs to the protection scope of the present invention.
[0016] Based on the coix straw hydrolyzate, the present invention further provides a method for culturing oleaginous yeast under non-sterile conditions, comprising the following steps: Inoculating the oil-producing yeast into the coix straw hydrolyzate, adding trace elements, and fermenting and culturing at 150-250 rpm and 25° C.-30° C.; The trace elements consist of CaCl 2 ·2H 2 O 4.0 g, FeSO 4 7H 2 O 0.55 g, ZnSO 4 7H 2 O 0.10 g, MnSO 4 ·H 2 O 0.076 g, 100 µL 18 MH 2 SO 4 Composition, dilute to 1 L with deionized water; The oleaginous yeast comprises Lipomyces starkeyi ATCC 58680, Rhodosporidium round-shapedAS 2.1389, Yarrowia lipolytica AS 2.1398 and Cryptococcus curvatus ATCC20509.
[0017] The present invention has the following beneficial technical effects: 1. Resource utilization and environmental protection: Using agricultural by-product Job's tears straw as raw material can reduce agricultural waste and improve the recycling of resources, which is of environmental significance.
[0018] 2. Cost-effectiveness: Using coix straw hydrolyzate as culture medium is expected to reduce the cost of cultivating oil-producing yeast, thereby improving economic benefits.
[0019] 3. Cultivation under non-sterile conditions: Cultivating oil-producing yeast under non-sterile conditions simplifies the production process and reduces the dependence on sterile facilities, which may reduce production costs and equipment requirements.
[0020] 4. Improve oil production: The culture conditions and the formula of the hydrolyzate are optimized to increase the oil production of yeast and increase the supply of raw materials for biodiesel or food industry.
[0021] 5. Promote yeast growth: Job's tears straw hydrolyzate may provide the nutrients needed for yeast growth and promote the growth and reproduction of yeast cells.
[0022] 6. Simplified process flow: The method of the present invention simplifies the culture process from raw materials to oil-producing yeast, and improves the convenience and efficiency of operation.
[0023] 7. Wide application: The method of the present invention may be applicable not only to laboratory-scale research but also to industrial-scale production, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Figure 4 shows the changes in sugar during the pretreatment and fermentation of coix straw; Figure a shows the changes in sugar during the enzymatic hydrolysis of coix straw; Figure b shows the glucose consumed by four oil-producing yeasts in sterilized coix straw hydrolysate; Figure c shows the glucose consumed by four oil-producing yeasts in non-sterilized coix straw hydrolysate; Figure d shows the glucose consumed by four oil-producing yeasts in non-sterilized coix straw hydrolysate (with Escherichia coli added); in the figure, M represents culture under sterile conditions; F represents culture under non-sterile conditions; E represents the addition of Escherichia coli under non-sterile conditions.
[0025] Figure 2Figure 2 shows the effects of different treatment conditions on the hydrolysis of coix straw; Figure a shows the effects of different substrate loads on hydrolysis under 121°C sulfuric acid (S) and sodium hydroxide (N) treatment conditions; Figure b shows the effects of different substrate loads on hydrolysis under 80°C sulfuric acid (S) and sodium hydroxide (N) treatment conditions; Figure c shows the effects of cellulase on enzymatic hydrolysis; Figure d shows the effects of enzymatic hydrolysis aids on enzymatic hydrolysis; in the figure, N121 / 80 represents alkaline treatment at 121°C and 80°C, respectively, and S121 / 80 represents acid treatment at 121°C and 80°C, respectively; N / S121 / 80-1 / 2 / 3 represent 8%, 5% and 2% substrate loads, respectively; -Y / -S / -N represent substrates from Aspergillus glaucus Cellulase, Aspergillus riparianus Cellulase and Cellic CTec3; BSA / PEG 10000-1 / 2 / 3 / 4 / 5 represent the added amounts of 0.01, 0.025, 0.05, 0.075 and 0.1 g / g, respectively. DETAILED DESCRIPTION
[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0028] Example 1: Preparation of coix straw hydrolyzate 1. Crush the coix straw and sieve it through an 80-mesh sieve.
[0029] 2. Wash the screened coix straw powder to remove dirt and dry it at 105 °C to constant weight.
[0030] 3. Add 1% NaOH solution to the coix straw powder at a solid-liquid ratio of 8:100 (w / v, g / ml) and heat at 80 °C for 60 min.
[0031] 4. After the pretreatment is completed, it is cooled to room temperature and the solution and solid residue are separated by filtration.
[0032] 5. Wash the pretreated coix residue repeatedly with deionized water several times.
[0033] 6. The pretreated residue was placed in an oven at 65°C and dried to constant weight.
[0034] 7. Add deionized water at a solid-liquid ratio of 8:100 (w / v, g / ml) to redissolve the dried residue, adjust the pH of the solution to 4.8, add 15 FPU / g Cellic CTec 3 and 0.075 g / g BSA, and enzymatically hydrolyze at 50°C for 36 h.
[0035] After testing, the hydrolyzate prepared in this example has a glucose yield of 44.93 g / L (56.15%) and a xylose yield of 26.75 g / L (33.44%).
[0036] Example 2: Cultivation of oleaginous yeast using coix straw hydrolyzate under non-sterile conditions A portion of the hydrolyzate after enzymatic hydrolysis in Example 1 was sterilized at 121° C. for 15 min, and the other portion was not sterilized.
[0037] Yeast cells from YPD solid medium were inoculated into 5 mL YPD liquid medium and cultured at 30 °C and 200 rpm for 24 h. The activated yeast was centrifuged, the YPD medium was discarded, and it was resuspended in an equal volume of deionized water. Then, 10% (v / v) was inoculated into 50 mL sterilized YPD medium, 50 mL non-sterile YPD medium, 50 mL sterilized coix straw hydrolyzate, 50 mL non-sterile coix straw hydrolyzate, and 50 mL non-sterile coix straw hydrolyzate with Escherichia coli added.
[0038] At the same time, trace elements (v:v, 1:100) were added. Fermentation was carried out at 200 rpm and 30 °C. Trace elements consisted of CaCl 2 ·2H 2 O 4.0 g, FeSO 4 7H 2 O 0.55 g, ZnSO 4 7H 2 O 0.10 g, MnSO 4 ·H 2 O 0.076 g, 100 µL 18 MH 2 SO 4 Make up to 1 L with deionized water.
[0039] Example 3, culture results under different conditions 1. If Figure 1 As shown in Figure b, under sterile conditions, R. toruloides , C. curved , L. starkeyi and Y. lipolytica All of them can grow normally in the sterilized coix straw hydrolyzate and can completely consume the glucose in the hydrolyzate. Y. lipolytica Compared with the strain, the other strains could completely consume the glucose in the culture medium within 72 h. R. toruloidesThe oil content in the strains was the highest, reaching 63.96% (Table 1). This indicates that these strains have good adaptability to the sterilized coix straw hydrolyzate and R. toruloides The sterilized coix straw hydrolyzate not only has the ability to quickly consume glucose, but also has a high oil accumulation capacity.
[0040] Table 1 Study on lipid fermentation performance of coix straw hydrolyzate
[0041] Note: M represents culture under sterile conditions; F represents culture under non-sterile conditions; E represents the addition of E. coli under non-sterile conditions; three parallel experiments were randomly selected, and the data are mean ± standard deviation. CC represents C. curvatus, RT stands for R. toruloides 、YR stands for Y. lipolytica and LS represents L. starkeyi
[0042] 2. Under non-sterile conditions, R. toruloides , C. curved , L. starkeyi and Y. lipolytica All strains were able to grow normally in non-sterile coix straw hydrolysate and completely consume glucose in the hydrolysate. The glucose consumption of all strains within 48 h was significantly higher than that under sterile conditions. R. toruloides and C. curved The glucose in the culture medium was consumed within 72 h. L. starkeyi and Y. lipolytica At 96 h, the glucose in the culture medium was consumed ( Figure 1 Compared with the sterile conditions, the sugar consumption rate and growth rate of these yeast strains were not significantly different, indicating that these strains can also grow normally in non-sterile coix straw hydrolyzate.
[0043] 3. Under non-sterile conditions where exogenous E. coli was introduced, R. toruloides , C. curved , L. starkeyi and Y. lipolytica All strains were able to grow normally in non-sterile coix straw hydrolysate and completely consume glucose in the hydrolysate. Compared with non-sterile conditions without the introduction of other contamination sources, all strains completely consumed glucose in the culture medium within 96 h. L. starkeyi The growth rate was faster, and the glucose in the culture medium was almost consumed at 48 h ( Figure 1 The results show that the growth and metabolic characteristics of these strains under non-sterile conditions are somewhat different from those under sterile conditions, but they still maintain a higher growth rate and glucose consumption capacity overall.
[0044] 4. Using YPD medium as a control, the sugar concentration in the medium was consistent with the glucose and xylose concentrations in coix straw hydrolyzate, yeast powder 10 g / L, and peptone 20 g / L. The above four strains were cultured under sterile and non-sterile conditions. The results showed that glucose was consumed in 72 hours under sterile conditions, while glucose consumption was slower under non-sterile conditions, and contamination occurred. This indicates that other microorganisms exist in the non-sterile environment, and the presence of these competitive microorganisms may limit the growth and metabolic activity of yeast strains.
[0045] 5. As shown in Table 1, R. toruloides and C. curved The biomass, lipid titer and lipid content under sterile conditions were relatively close, showing good applicability in coix straw hydrolysate. L. starkeyi Compared with the other three oil-producing yeasts, Y. lipolytica The cell biomass of was the lowest (2.78 g / L). This indicates that different strains have different adaptability to specific substrates, and R. toruloides and C. curved It may be more suitable to utilize the nutrients in coix straw hydrolyzate for growth and lipid accumulation.
[0046] In order to effectively explore whether the hydrolyzate of coix straw can be used to culture yeast without strict sterilization conditions, Escherichia coli was inoculated as a control under non-sterile conditions. R. toruloides , L. starkeyi , Y. lipolytica and C. curved The results of cell biomass and lipid titer of the strain were almost the same as those obtained under sterile conditions. At the same time, compared with YPD medium, the biomass, lipid titer and lipid content of the strain cultured with coix straw hydrolyzate were all increased.
[0047] In summary, under non-sterile conditions, coix straw hydrolyzate can be used as an effective culture medium for the cultivation of R. round-shaped , L. starkeyi , Y. lipolytica and C. curved The growth and lipid production capacity of yeast strains were not affected. Compared with YPD medium, coix straw hydrolysate can significantly increase the biomass, lipid titer and lipid content of bacteria, providing a new and potential choice for the cultivation of yeast strains and lipid production.
[0048] Example 4: Preparation of coix straw hydrolyzate under different conditions 1. Effect of matrix loading on hydrolysis During the hydrolysis process, a high solid content will cause stirring difficulties, making it impossible for the pretreatment liquid to fully contact the straw powder, which will affect the subsequent glucose concentration. When the solid-liquid ratio exceeds 8%, the solution cannot completely wet the powder. Therefore, coix seed treatment liquid with solid loading of 2% (-3), 5% (-2) and 8% (-1) was used as the research object. After being treated with sulfuric acid (S) and sodium hydroxide (N) at 80 ℃ / 121 ℃, cellulase and β-glucosidase were used for enzymatic hydrolysis for 24 hours to produce glucose. Figure 2 As shown in Figures a and b, when the coix matrix loading is 8%, the glucose yield of all coix samples is the highest. This shows that within a certain range, a higher matrix loading can produce more products. Beyond a certain range, higher solid content will be affected by mixing difficulties and low heat transfer efficiency, resulting in low hydrolysis efficiency. The above solid loading of 8% refers to the addition of 8g of coix straw powder to 100 mL of 1% sodium hydroxide solution.
[0049] 2. Effect of pretreatment methods on hydrolysis Different amounts of Job's tears straw were treated with traditional dilute acid and dilute alkali, such as Figure 2 As shown in Figures a and b, when the coix seed matrix loading was 8%, the glucose yield was the highest under the dilute alkali treatment at 121 ℃ and 80 ℃, reaching 22 g / L. Under the dilute acid treatment at 121 ℃, the glucose yield was similar to that under the alkali treatment. The difference was that under the acid treatment at 80 ℃, the glucose yield was significantly lower than all other treatments in the same period. This shows that the coix seed straw sample can be hydrolyzed under the dilute alkali treatment at 80 ℃, and 121 ℃ is better for acid treatment of coix seed straw.
[0050] 3. Effect of cellulase on enzymatic hydrolysis In order to more effectively treat high-concentration coix straw and save resources, a batch enzymatic hydrolysis strategy was performed using cellulase (Y) from Trichoderma viride, cellulase (S) from Trichoderma reesei, and Cellic CTec3 (N) at a solid loading of 8% (w / v), respectively, using 121 °C acid treatment and 80 °C alkali treatment. Figure 2As shown in Figure c, 2 g / L of glucose can be obtained from coix straw under 121 ℃ acid treatment, and no glucose was detected in the alkaline treatment solution. After 12 h of reaction between the enzyme and substrate, the glucose content in the alkaline treatment hydrolyzate gradually increased to that in the acid treatment, until 72 h, the glucose content obtained by the three different types of cellulases in the alkaline treatment was higher than that in the acid treatment, among which Cellic CTec3 had the highest glucose yield, which could reach 29 g / L. However, the different types of enzymes in the acid treatment had reached a plateau at 60 h. This shows that the enzymatic hydrolysis effect of the cellulase Cellic CTec3 after 80 ℃ alkaline treatment is significantly better than that of the other two cellulases, and is more conducive to the biomass conversion of coix straw.
[0051] 4. Effect of enzymatic hydrolysis aids on enzymatic hydrolysis In order to improve the enzymatic hydrolysis ability of coix straw by cellulase, different amounts of BSA and PEG10000 enzymatic hydrolysis aids were added during the enzymatic hydrolysis process (BSA / PEG-1 / 2 / 3 / 4 / 5 represent the addition amounts of 0.01, 0.025, 0.05, 0.075 and 0.1 g / g, respectively), and the changes in glucose concentration were measured every 12 h. Figure 2 As shown in Figure d, compared with the hydrolyzate without enzymatic hydrolysis aid, the hydrolyzate with BSA and PEG10000 added had significantly higher glucose yields at the same enzymatic hydrolysis time, and its glucose yield reached the highest at 60 h. For BSA and PEG10000, the glucose yields of 0.075 g / g and 0.1 g / g were close at 60 h of enzymatic hydrolysis, among which BSA could reach a maximum of 32 g / L, slightly higher than PEG10000. Therefore, 0.075 g / g BSA and PEG10000 were able to meet the enzymatic hydrolysis of coix straw. It is worth noting that BSA not only helps to stabilize the enzyme during the enzymatic hydrolysis process, but also prevents enzyme decomposition and nonspecific adsorption, reducing enzyme loss. Therefore, 0.075 g / g BSA is the most preferred enzymatic hydrolysis aid for coix straw.
Claims
1. A method for preparing coix straw hydrolyzate, comprising the following steps: S1, the coix straw powder is washed and descaled and then dried to a constant weight; S2, adding NaOH aqueous solution or sulfuric acid solution to the coix straw powder and heating; S3, after cooling to room temperature, filtering and separating to obtain coix seed residue; S4. Add deionized water to the coix residue dried to constant weight for redissolution, adjust the pH value to 4.5-5.5, add cellulase and bovine serum albumin, or cellulase and PEG10000 for enzymatic hydrolysis, and obtain coix straw hydrolyzate.
2. The preparation method according to claim 1, characterized in that: In step S1, the coix straw is crushed and then sieved through an 80-mesh sieve to obtain the coix straw powder; Dry at 50 °C-105 °C to constant weight.
3. The preparation method according to claim 1 or 2, characterized in that: In step S2, the mass concentration of the NaOH aqueous solution is 0.5%-2%; The concentration of the sulfuric acid solution is 0.1-0.2 M; The dosage ratio of the coix straw powder to the NaOH aqueous solution or the sulfuric acid solution is: 0.05-0.1 g / mL; The heating temperature is 80°C-121°C, and the heating time is 60-90 min.
4. The preparation method according to any one of claims 1 to 3, characterized in that: In step S4, the coix seed residue is washed with deionized water and then dried.
5. The preparation method according to any one of claims 1 to 4, characterized in that: In step S4, during the re-dissolution, the dosage ratio of the coix residue to the deionized water is 0.02-0.1 g / mL.
6. The preparation method according to any one of claims 1 to 5, characterized in that: In step S4, the conditions for the enzymatic hydrolysis are: temperature of 40 °C-55 °C, and time of 36 h-72 h.
7. The preparation method according to any one of claims 1 to 6, characterized in that: In step S4, the amount of the cellulase is as follows: Aspergillus glaucus Cellulase and Aspergillus riparianus The total amount of cellulase is 5-15 FPU / g of the coix residue, the amount of β-glucosidase is 10-30 CBU / g of the coix residue; the amount of cellulose mixed enzyme is 10-20 FPU / g of the coix residue; The dosage of the bovine serum albumin and the PEG10000 is 0.05-0.10 g / g of the coix seed residue.
8. Coix straw hydrolyzate prepared by the method according to any one of claims 1 to 7.
9. A method for culturing oleaginous yeast under non-sterile conditions, comprising the following steps: The oil-producing yeast is inoculated into the coix straw hydrolyzate according to claim 8, trace elements are added, and fermentation culture is carried out at 150-250 rpm and 25° C.-30° C.
10. The method according to claim 9, characterized in that: The oleaginous yeast comprises Lipomyces starkeyi ATCC 58680, Rhodosporidium toruloides AS 2.1389, Yarrowia lipolytica AS 2.1398 and Cryptococcus curvatus ATCC 20509.