Method for preparing lactic acid from straw

Abstract

The invention discloses a method for preparing lactic acid by using straws, which comprises the following steps: stirring straws, water and cellulase at 50 DEG C for pre-enzymolysis, inoculating a bacillus coagulans MY5 seed solution, and fermenting; after fermentation, supplementing the enzymatic hydrolysate, and fermenting; or, straw is added after fermentation, a bacillus coagulans MY5 seed solution is inoculated, and fermentation is carried out. According to the method, the problems that an existing process is limited to a single fermentation mode, and efficiency loss is caused by space-time separation of saccharification and fermentation are solved, glucose and xylose can be utilized at the same time by adopting the adaptively evolved bacillus coagulans MY5, an exogenous nitrogen source does not need to be added, and the yield of lactic acid can reach 101.48 g / L through a delayed fermentation strategy of inoculation before secondary feeding.

Description

Technical Field

[0001] This invention relates to a method for preparing lactic acid, specifically a method for preparing lactic acid from straw. Background Technology

[0002] In lignocellulose bioconversion processes, stepwise saccharification-fermentation (SHF) and simultaneous saccharification-co-fermentation (SSCF) are two main technical pathways under batch fermentation models. SHF, by separating hydrolysis and fermentation in independent reactors and optimizing their process conditions separately, can improve unit process efficiency. However, the spatial and temporal separation leads to material loss, and the high concentration of fermentable sugars accumulated during hydrolysis can trigger product feedback inhibition by cellulase, thus limiting the final product yield. In contrast, SSCF integrates saccharification and fermentation in the same reactor, enabling immediate consumption of sugar products and effectively maintaining a low sugar concentration environment within the system, thereby significantly alleviating the enzyme activity inhibition problem. However, the core bottleneck of this process lies in the significant difference between the optimal temperature of most fermenting microorganisms and the temperature required for cellulase hydrolysis (50℃). This thermodynamic parameter mismatch directly leads to a decrease in metabolic rate, significantly restricting the operating efficiency of the SSCF process. Therefore, it is necessary to solve the compatibility problem between enzymatic hydrolysis and fermentation conditions (such as temperature and pH), and to develop simultaneous saccharification-fermentation (SSCF) or staged pretreatment-fermentation coupling processes to reduce costs.

[0003] Microbial contamination is a major challenge restricting production efficiency during microbial fermentation. It not only reduces product concentration and quality but can also lead to complete fermentation failure in severe cases. To effectively control contamination risks, bioreactor systems must undergo high-temperature, high-pressure, and moist heat sterilization before startup, and in continuous fermentation modes, the feeding and replenishment processes must be conducted in a sterile environment. However, sterilization, being a typical high-energy-consuming process, along with the human resources, energy consumption, and specialized equipment required to maintain the sterility of the continuous culture system, significantly increases production and operating costs and process control precision. Therefore, developing open fermentation technology that does not require high-temperature sterilization has significant application value. This model not only reduces equipment investment costs but also helps enhance the market competitiveness of industrial biotechnology. Extreme environment fermentation is an important pathway for realizing open processes. For example, studies using heat-resistant strains such as Bacillus coagulans and Bacillus licheniformis to conduct open LA fermentation at temperatures above 50°C show that although high temperatures can effectively inhibit contamination, the toxic substances produced during material pretreatment become more active at high temperatures, which in turn inhibits the metabolic activity of the production strains, making it difficult to achieve the final LA concentration (<100 g / L) to meet industrial standards. Previous studies have confirmed that *Pediococcus acidilactici*, through its biosynthesized antimicrobial peptides, can effectively inhibit the proliferation of contaminating bacteria in open systems, thereby maintaining high-yield L-lactic acid fermentation based on cellulose substrates. Another study used the salt-tolerant strain *H. campaniensis* LS21 to conduct open continuous fermentation in a seawater substrate, successfully maintaining a 65-day contamination-free record. However, the negative effects of high-salt environments, such as equipment corrosion and increased difficulty in product separation, still require further investigation.

[0004] In LA fermentation systems using lignocellulose as a raw material, the system solids content needs to be increased to approximately 30% (w / w) to obtain fermentable sugars and LA concentrations with practical application value. However, increasing the solids content significantly increases the proportion of water-insoluble substances (WIS), leading to problems such as increased system viscosity and impurity enrichment. Existing processes mostly employ simultaneous saccharification and co-fermentation (SSCF) mode, but this strategy has an inherent flaw—the difference in optimal reaction conditions between cellulase and microorganisms makes it difficult to simultaneously optimize the hydrolysis and fermentation processes in a single reactor. Current research on continuous lignocellulose fermentation systems mainly focuses on staged saccharification-fermentation (SHF) processes and their improved processes (PSSF). The technical approach involves constructing an external pre-saccharification reactor to complete substrate pretreatment, followed by continuous fermentation using a substrate gradient addition strategy. In contrast, the empirical research data and theoretical modeling system for continuous processes using lignocellulose raw materials for direct simultaneous saccharification-co-fermentation (SSCF) still lack sufficient support. Compared to other substrate systems, continuous fermentation of lignocellulose faces multiple technical bottlenecks in the absence of an external saccharification tank: the high-solids system imposes stringent requirements on solid-liquid mixing efficiency and substrate homogeneity, directly affecting the stability of fermentation broth transport; thermodynamic mismatch (insufficient synergy between substrate saccharification at 50℃ and lactic acid (LA) fermentation at 42-50℃) leads to extended process cycles; the semi-open material replacement operation requires the construction of an engineered strain to enhance stress resistance; and the LA yield of a single-tank continuous fermentation system remains lower than the baseline value of traditional batch processes due to limitations in the hydrolysis efficiency of cellulase systems. Notably, the high compatibility between cellulose LA fermentation strains and cellulases in terms of temperature (42℃~50℃ vs 50℃) and pH (4.8~5.5) parameters provides theoretical feasibility for constructing a continuous lignocellulose SSCF system. Key technologies to be overcome in this system include: developing a reactor configuration that balances substrate saccharification and anaerobic fermentation requirements, enhancing the mixing and mass transfer efficiency of the high-solids system, and improving the strains' resistance to contamination in open operation. Studies have shown that using heat-resistant B. coagulans in a steam-pretreated corn stalk system (15% w / w solids content) to implement single-stage continuous fermentation has verified the technical feasibility, but it has not yet achieved a technological breakthrough due to problems such as low LA yield (35.2 g / L) and insufficient system stability.

[0005] Optimizing the fermentation process is key to improving LA production efficiency. In liquid fermentation, dynamic feeding strategies and real-time pH control can maintain optimal strain activity. However, in LA fermentation using lignocellulose as a raw material, to achieve meaningful levels of fermentable sugars and LA concentrations, the total enzymatic hydrolysis and solid content of the fermentation system need to reach approximately 30%, thus requiring solid-state fermentation. However, existing processes are mostly limited to a single fermentation mode, and the spatial and temporal separation of saccharification and fermentation leads to efficiency losses. Therefore, there is an urgent need to develop an integrated process of staged pretreatment-simultaneous saccharification co-fermentation (SSCF). Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing lactic acid using straw, which solves the problem that existing processes are limited to a single fermentation mode and that the separation of saccharification and fermentation in time and space leads to efficiency loss. The method of pre-saccharification and simultaneous saccharification and co-fermentation of straw raw materials can not only realize the simultaneous enzymatic hydrolysis and saccharification of straw and lactic acid fermentation, but also eliminates the need for separate detoxification and the addition of exogenous nutrients. The highest lactic acid content in a single tank is 101.48 g / L.

[0007] To achieve the above objectives, the present invention provides a method for preparing lactic acid from straw, the method comprising: (1) Straw, water, and cellulase were stirred at 50°C for pre-enzymatic hydrolysis, and then inoculated with Bacillus coagulans (Bacillus). Bacillus coagulans MY5 seed liquid is fermented; (2) After fermentation in step (1), add enzyme hydrolysate and continue fermentation; or, after fermentation in step (1), add straw and inoculate with Bacillus coagulans (Bacillus subtilis). Bacillus coagulans MY5 seed liquid is fermented; The Bacillus coagulans ( Bacillus coagulans The accession number of MY5 is GDMCC No:66445. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is May 30, 2025. The content of liquefiable solids in the solution after adding the supplemental enzymatic hydrolysate or straw is 20% to 40%.

[0008] Preferably, in step (1), the mass-to-volume ratio of straw to water is (550~800) g : (1500~1850) mL, and the content of liquefiable solids in the solution after adding the enzyme hydrolysate or adding straw is 35%.

[0009] Preferably, in step (1), the cellulase loading is 30 FPU / g, and the stirring rate is 180 rpm.

[0010] Preferably, in step (1), the inoculated Bacillus coagulans ( Bacillus coagulansThe total volume ratio of MY5 seed liquid to pre-enzymatic hydrolysis solution is 10%.

[0011] Preferably, in step (1), the pre-enzymatic hydrolysis time is (6 ~ 24) h.

[0012] Preferably, in step (2), the inoculated Bacillus coagulans ( Bacillus coagulans The total volume ratio of MY5 seed solution to the solution after adding straw is 10%.

[0013] Preferably, in step (2), the number of times straw is added and the inoculation with Bacillus coagulans (Bacillus) Bacillus coagulans The number of times MY5 seed solution is applied is several.

[0014] Preferably, in steps (1) and (2), the Bacillus coagulans ( Bacillus coagulans All MY5 samples were activated with mMRS medium, and their absorbance values ​​(OD600) were all 1.2.

[0015] More preferably, the mMRS medium consists of peptone, yeast extract, glucose, MnSO4•4H2O, L-glutamate sodium, Tween 80, and distilled water, the pH of the mMRS medium is 5.0, and the activation time is 12 h.

[0016] More preferably, the amounts of peptone, yeast extract, glucose, MnSO4•4H2O, L-glutamate sodium, Tween 80 and distilled water are 12.5 g / L, 12.5 g / L, 50 g / L, 0.05 g / L, (100~500) mM, 1 mL and 1000 mL, respectively.

[0017] This invention provides a method for preparing lactic acid from straw, which solves the problem that existing processes are limited to a single fermentation mode and suffer from efficiency losses due to the spatial and temporal separation of saccharification and fermentation. It has the following advantages: 1. To obtain a high L-LA concentration in the fermentation broth, this invention explored two fermentation modes: stepwise saccharification fermentation and simultaneous saccharification co-fermentation. Subsequently, experiments were conducted on pre-saccharification simultaneous saccharification co-fermentation with added enzymatic hydrolysate and pre-saccharification simultaneous saccharification co-fermentation with added straw. Results showed that simultaneous saccharification co-fermentation, compared to stepwise saccharification fermentation, reduced the inhibitory effect of reducing sugars on cellulase and osmotic pressure through immediate sugar conversion. However, the lactic acid yield of 25% TS hydrolysate SSCF was still lower than the industrial standard. Feeding strategy analysis revealed that strictly controlling the main influencing factors—inoculation time (temperature of the hydrolysate and inoculation after a certain time) and the solid content in the fermenter (TS25%~TS35%)—significantly inhibited the fermentation process due to the accumulation of inhibitors in 35% TS hydrolysate. The experiment with pre-saccharification simultaneous saccharification co-fermentation with added straw (Fed-PSSCF) showed that the delayed fermentation strategy of inoculation before secondary feeding could achieve a lactic acid yield of 101.48 g / L, basically meeting the requirements of industrial lactic acid fermentation.

[0018] 2. The present invention utilizes the adaptively evolved Bacillus coagulans MY5, which can simultaneously utilize glucose and xylose without the need for the addition of exogenous nitrogen sources, significantly reducing production costs. The efficient utilization of residual sugars becomes a key breakthrough in improving the economic efficiency of the process. Attached Figure Description

[0019] Figure 1 The diagram shows the results of different fermentation modes and TS fermentation according to the present invention.

[0020] Figure 2 This diagram shows the results of pre-saccharification, simultaneous saccharification, and co-fermentation of the enzyme hydrolysate in this invention.

[0021] Figure 3 This diagram shows the results of simultaneous saccharification and co-fermentation of straw raw materials according to the present invention.

[0022] Figure 4 This is a flowchart illustrating the simultaneous saccharification and co-fermentation of straw raw materials for this invention. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The strains, culture media, and enzyme hydrolysate involved in the following examples are as follows: The Bacillus coagulans ( Bacillus coagulansThe accession number of MY5 is GDMCC No:66445. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is May 30, 2025.

[0025] TS 35% enzymatic hydrolysate: glucose: 106.6 g / L, xylose: 56.62 g / L and arabinose: 5.67 g / L.

[0026] The mMRS medium consists of 12.5 g / L peptone, 12.5 g / L yeast extract, 50 g / L glucose, 0.05 g / L MnSO4•4H2O, 100~500 mM L-glutamate, 1 mL Tween 80, and 1000 mL distilled water. The pH is adjusted to 5.0 and then autoclaved.

[0027] Example 1 Evaluation of different fermentation modes SHF (Stepwise Saccharification and Fermentation) Group: 2 L of TS35% enzymatic hydrolysate was added to a 5 L fermenter. After the temperature was adjusted to 50℃, a quantitative inoculation of Bacillus coagulans MY5 seed culture (OD600=1.2) activated in mMRS medium for 12 hours was performed. The inoculation parameters were set to 10% (v / v) (the volume ratio of Bacillus coagulans MY5 seed culture (OD600=1.2) to TS35% enzymatic hydrolysate was 1:10). The main influencing factors for the SHF group were inoculation time and solids content in the fermenter. The inoculation time was when the enzymatic hydrolysate reached 50℃, and the solids content in the fermenter was TS35%.

[0028] SSCF (Simultaneous Saccharification and Co-fermentation) group: 565 g of straw and 1800 mL of RO water (TS 25%, representing the highest liquefiable solids content from a single enzymatic hydrolysis) were added to a 5 L fermenter for a 12-hour pre-enzymatic hydrolysis stage. The pre-enzymatic hydrolysis parameters were set as follows: cellulase (purchased from Sinoenzyme; enzyme activity (FPA): ≥280 U / g) loading of 30 FPU / g straw, hydrolysis system temperature of 50℃, and stirring speed of 180 rpm. After the 12-hour pre-enzymatic hydrolysis stage, a quantitative inoculation of *Bacillus coagulans* MY5 seed culture (OD600=1.2) activated on 12-hour mMRS medium was performed, maintaining an inoculation volume percentage of 10% (v / v) to ensure the strain was in the logarithmic growth phase for rapid colonization.

[0029] In the experiments of the SHF and SSCF groups, samples were taken every 3 to 6 hours, and the contents of glucose, xylose, arabinose, and LA in the fermentation broth were measured by HPLC.

[0030] like Figure 1 As shown, the results of different fermentation modes and TS fermentation of the present invention are illustrated, where a) is the result of batch fermentation with 35% TS; and b) is the result of simultaneous saccharification and co-fermentation with 25% TS. Figure 1 As shown in a), in the stepwise saccharification fermentation (SHF) experiment, the TS 35% enzymatic hydrolysate began fermentation at 12 h, but essentially stopped fermenting after 18 h, with an LA yield of only 9.3 g / L. This is presumably due to cell osmotic pressure inhibition caused by the initial sugar concentration of 180.6 g / L. Furthermore, from... Figure 1 As shown in b), in the simultaneous saccharification-co-fermentation (SSCF) experiment, the straw substrate exhibited simultaneous accumulation of glucose, xylose, and LA within the initial 36 h. This may be due to the synergistic effect of improved cellulose / hemicellulose enzymatic hydrolysis efficiency and microbial metabolism after pretreatment process optimization. During this process, monosaccharides released during saccharification are dynamically converted into LA, achieving dual regulation: effectively alleviating the product feedback inhibition of cellulase catalytic efficiency by free reducing sugars; and simultaneously avoiding microbial osmotic stress caused by the accumulation of extracellular sugar concentration. It is speculated that the pretreatment method of steam explosion combined with mechanical crushing significantly increased substrate accessibility, enabling cellulase and xylanase to efficiently release fermentable sugars (glucose and xylose) in the initial stage. The production rate (R1) exceeded the microbial consumption rate (R2), leading to a continuous increase in sugar concentration. At this time, strain MY4 preferentially utilizes glucose to rapidly produce acid through the glycolysis pathway (EMP), while xylose metabolism exhibits lag due to transport efficiency or reducing power limitations. The decrease in sugar content after 36 hours was related to substrate consumption, enzyme activity decay, and product inhibition. Cellulose / hemicellulose substrates were gradually depleted, and the thermal inactivation of cellulase or cellobiose accumulation under high solids content inhibited subsequent hydrolysis. Simultaneously, strain MY4 continued to utilize residual sugars for homofermentation, leading to secondary accumulation of LA. This dynamic process reveals the spatiotemporal coupling effect of hydrolysis kinetics and microbial metabolic networks in the SSCF system.

[0031] Example 2: Fed-feed enzymatic hydrolysate presaccharification and simultaneous saccharification and fermentation The fed-batch PSCF is a strategy for achieving precise control of high substrate loading and total sugar concentration. Therefore, this invention designed two sets of simultaneous saccharification and fermentation experiments using pre-saccharified fed-batch enzymatic hydrolysates: 565 g of straw and 1800 mL of RO water (TS25%, representing the highest liquefiable solids content from a single enzymatic hydrolysis) were added to a 5 L fermenter for a 24-hour pre-enzymatic hydrolysis stage. The pre-enzymatic hydrolysis parameters were set as follows: cellulase loading 30 FPU / g straw, hydrolysis system temperature 50℃, and stirring speed 180 rpm. After completing the 24-hour pre-enzymatic hydrolysis stage, Bacillus coagulans MY5 seed culture (OD600=1.2) activated on mMRS medium for 12 hours was quantitatively inoculated for fermentation, with the inoculation volume ratio maintained at 10% (v / v). After 48 h of fermentation, 2 L of TS25% enzymatic hydrolysate and 2 L of TS35% enzymatic hydrolysate were added respectively, resulting in final TS contents of 25% and 30%, respectively.

[0032] Samples were taken every 3 to 12 hours in both groups of experiments, and the contents of glucose, xylose, arabinose and LA in the fermentation broth were measured by HPLC.

[0033] like Figure 2 The diagram shows the results of pre-saccharification, simultaneous saccharification, and co-fermentation using supplemented enzyme hydrolysate according to the present invention. Figure a) shows the results of supplementing TS25% with TS25% enzyme hydrolysate; figure b) shows the results of supplementing TS25% with TS35% enzyme hydrolysate. Figure 2 As shown in a), in the TS25% experimental group, the LA content reached 54.95 g / L at 48 h, while the glucose and xylose contents decreased to 9.33 g / L and 21.19 g / L, respectively. After 6 h of feeding, the LA content was diluted and fermentation continued, but it still decreased to 47.56 g / L. Subsequently, it began to rise continuously, reaching 68.09 g / L at the end of fermentation, with glucose and xylose remaining at 25.21 g / L and 20.05 g / L, respectively. In the TS30% experimental group, the LA content reached 70.81 g / L at 48 h, while the glucose and xylose contents decreased to 7.20 g / L and 23.25 g / L, respectively. After 6 h of feeding, the LA content was diluted and fermentation continued, but it still decreased to 52.8 g / L. Subsequently, it began to rise slowly and continuously, reaching only 55.94 g / L at the end of fermentation, with glucose and xylose remaining at 47.76 g / L and 25.2 g / L, respectively. In the TS25% experimental group, fermentation continued after feeding, while in the TS30% experimental group, fermentation essentially stopped after feeding. This is presumably due to the accumulation of high concentrations of enzyme hydrolysate inhibitors, which inhibited fermentation. There were no differences in parameters between the two groups in the first 48 hours, but differences still existed, indicating a significant batch effect in the fermentation experiment.

[0034] Example 3: Pre-saccharification, simultaneous saccharification, and co-fermentation of straw raw materials The total system in the 5 L fermenter consisted of 791.12 g of straw and 1560 mL of RO water. The cellulase dosage was set at 30 FPU / g straw. Figure 4 The flowchart shown in the figure illustrates the simultaneous saccharification and co-fermentation process of this invention using straw as a raw material. The specific experimental procedure is as follows: 341.12 g of straw was first added to the fermenter, along with water and cellulase (precisely measured total substrate) for pre-enzymatic hydrolysis. The pre-enzymatic hydrolysis system was maintained at 50°C and a stirring rate of 180 rpm. After 6 hours of pre-enzymatic hydrolysis, a first feeding was performed, adding 278.57 g of straw to the fermenter. After another 18 hours of pre-enzymatic hydrolysis, a second feeding was performed, adding 171.43 g of straw to the fermenter. In both experiments, Bacillus coagulans activated on mMRS medium for 12 hours was quantitatively inoculated 2 hours before the first feeding (TS 20%), 6 hours before the second feeding (TS 30%), and 12 hours after the second feeding (TS 35%). Bacillus coagulans MY5 seed culture (OD600=1.2) was used, with an inoculation volume percentage of 10% (v / v); the inoculation time was recorded as 0 h. Samples were taken every 12 h in both groups of experiments, and the contents of glucose, xylose, arabinose, and LA in the fermentation broth were measured by HPLC.

[0035] like Figure 3 The diagram shows the results of simultaneous saccharification and co-fermentation of straw raw materials according to the present invention. A) The results of inoculation fermentation before the first feeding (initial TS only 20%); b) The results of inoculation fermentation before the second feeding (initial TS increased to 30%, and the pre-enzymatic sugar content significantly increased); c) The results of inoculation fermentation after the second feeding (initial TS increased to 35%, and the pre-enzymatic sugar content increased by 160.9 g / L). Figure 3It can be seen that there are significant differences among the three experimental groups. In the first pre-feeding inoculation fermentation group, due to the initial total sugar (TS) of only 20% and low sugar content after 4 hours of pre-enzymatic hydrolysis, fermentation was rapid after inoculation. However, in the subsequent two feedings, it is speculated that the enzymatic hydrolysis efficiency decreased due to pH influence, and the liquefaction effect was poor. Under this high-solids environment, the LA content was only 59.36 g / L at the end of fermentation, with residual glucose and xylose contents of 24.96 g / L and 26.9 g / L, respectively. In the second pre-feeding inoculation fermentation group, the initial TS had increased to 30%, and the pre-enzymatic sugar content was significantly increased. A significant lag period occurred after inoculation, with the fermentation rate starting to increase after 120 hours. Fermentation did not stop even at the expected 180 hours, so the fermentation time was extended until the end of fermentation (without neutralizing agent consumption). Finally, at 336 hours, the LA content reached 101.48 g / L, with residual glucose and xylose contents of 17.34 g / L and 14.89 g / L, respectively. This basically met the requirements for industrial lactic acid fermentation, but the fermentation time was as long as 336 hours. After the second feeding, the initial TS of the inoculated fermentation experimental group had increased to 35%, and the pre-enzymatic sugar content increased by 160.9 g / L. A significant lag period also appeared after inoculation. Similarly, the fermentation rate began to increase after 120 h, but fermentation basically stopped after 36 h. At the end of fermentation, the LA content was only 44.58 g / L, and the glucose and xylose contents were 71.5 g / L and 28.23 g / L, respectively.

[0036] Therefore, the pre-saccharification and simultaneous saccharification co-fermentation of straw raw materials (Fed-PSSCF) can achieve simultaneous enzymatic hydrolysis and saccharification of straw and lactic acid fermentation, and can achieve a single tank lactic acid yield of >100g / L without separate detoxification and addition of exogenous nutrients.

[0037] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for preparing lactic acid from straw, characterized in that, The method includes: (1) Straw, water, and cellulase were stirred at 50°C for pre-enzymatic hydrolysis, and then inoculated with Bacillus coagulans (Bacillus). Bacillus coagulans MY5 seed liquid is fermented; (2) After fermentation in step (1), add enzyme hydrolysate and continue fermentation; or, After fermentation in step (1), straw is added and Bacillus coagulans is inoculated ( Bacillus coagulans MY5 seed liquid is fermented; The Bacillus coagulans ( Bacillus coagulans The accession number of MY5 is GDMCC No:66445. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is May 30, 2025. The content of liquefiable solids in the solution after adding the supplemental enzymatic hydrolysate or straw is 20% to 40%.

2. The method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of straw to water is (550~800) g : (1500~1850) mL, and the content of liquefiable solids in the solution after adding the enzyme hydrolysate or adding straw is 35%.

3. The method according to claim 1, characterized in that, In step (1), the cellulase loading is 30 FPU / g, and the stirring rate is 180 rpm.

4. The method according to claim 1, characterized in that, In step (1), the inoculated Bacillus coagulans ( Bacillus coagulans The total volume ratio of MY5 seed liquid to pre-enzymatic hydrolysis solution is 10%.

5. The method according to claim 1, characterized in that, In step (1), the pre-enzymatic hydrolysis time is (6 ~ 24) h.

6. The method according to claim 1, characterized in that, In step (2), the inoculated Bacillus coagulans ( Bacillus coagulans The total volume ratio of MY5 seed solution to the solution after adding straw is 10%.

7. The method according to claim 1, characterized in that, In step (2), the number of times straw is added and the inoculation with Bacillus coagulans (Bacillus) Bacillus coagulans The number of times MY5 seed solution is applied is several.

8. The method according to claim 1, characterized in that, In steps (1) and (2), the Bacillus coagulans ( Bacillus coagulans All MY5 samples were activated with mMRS medium, and their absorbance values ​​(OD600) were all 1.

2.

9. The method according to claim 8, characterized in that, The mMRS medium consists of peptone, yeast extract, glucose, MnSO4 • 4H2O, L-glutamate sodium, Tween 80, and distilled water. The pH of the mMRS medium is 5.0, and the activation time is 12 h.

10. The method according to claim 9, characterized in that, The amounts of peptone, yeast extract, glucose, MnSO4 •4H2O, L-glutamate sodium, Tween 80, and distilled water were 12.5 g / L, 12.5 g / L, 50 g / L, 0.05 g / L, (100~500) mM, 1 mL, and 1000 mL, respectively.

Images