Multi-stage temperature control lactic acid biological fermentation process method for corn and soybean compound substrate

Abstract

The invention discloses a multi-stage temperature control lactic acid biological fermentation process method for a corn and soybean compound substrate. A high-temperature gelatinized corn and soybean composite substrate is used as a raw material, starch hydrolysis fermentation is performed in stages under the action of high-temperature amylase, glucoamylase, pullulanase and bacillus coagulans, and the three enzymes are fixed on a special magnetic nano-carrier through covalent bonds. The method disclosed by the invention has the beneficial effects that 1, a composite hydrolysis system is formed by adopting low-dose high-temperature amylase, glucamylase and pullulanase, and more than 80% of starch can be converted into glucose by virtue of an enzyme activity synergistic effect through stepped temperature regulation and control; 2, by generating a high-concentration glucose substrate, metabolic inhibition of a low-sugar environment on bacillus coagulans in a traditional step-by-step process is avoided, and the conversion rate of converting glucose into lactic acid exceeds 50%; 3, the enzymolysis-fermentation process is seamlessly connected, and the middle links of inactivation, centrifugation and the like in the traditional process are avoided; and 4, the enzyme can be repeatedly used, and the cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid production by fermentation, and in particular to a multi-stage temperature-controlled lactic acid biofermentation process using a corn and soybean composite matrix. Background Art

[0002] Corn and soybeans, as biomass resources with a wide range of sources and low costs, occupy an important position in my country's agricultural production. According to statistics, my country's corn production reached 288 million tons in 2023, and soybean production reached 20.84172 million tons. These two crops have important application value in the fields of food and feed: corn is not only the main raw material for making foods such as cornmeal, corn flour, popcorn, and corn oil, but also an indispensable high-efficiency energy feed in animal husbandry; soybeans, as a source of high-quality plant protein, are the basic raw material for making traditional soy products such as tofu, soy milk, and fermented black beans. Their rich protein and starch content also make them an important component in the feed industry. Based on their renewability, easy accessibility and rich starch content, corn and soybeans are considered to be biomass starch raw materials with great development potential, and have broad application prospects in the fields of bio-based materials and bioenergy.

[0003] Existing studies have shown that it is feasible to produce lactic acid by fermenting corn or soybeans, but the process still faces several technical bottlenecks: First, the problem of raw material standardization is prominent. The starch content of different varieties of corn and soybeans varies significantly, and a variety of amylases need to be used for hydrolysis in a targeted manner. In addition, additional nutrients such as inorganic salts, nitrogen sources and vitamins need to be added during the fermentation process, resulting in high production costs. Secondly, the substrate inhibition effect is obvious. When the carbon source concentration is too high, it not only affects the effective utilization of the substrate by microorganisms, but also produces cytotoxic metabolic by-products, which seriously inhibit the growth and reproduction of the strain. Finally, the product inhibition effect is significant. As lactic acid accumulates, key parameters such as the pH value of the fermentation system deviate from the optimal range, resulting in a deterioration of the growth environment of the strain, which in turn triggers its stress protection mechanism, diverting metabolic energy from product synthesis to cell defense, and ultimately causing slow bacterial growth and decreased metabolic activity.

[0004] To address these challenges, there is an urgent need to develop more efficient and economical lactic acid production processes. The present invention addresses this issue by: 1. Stabilizing the base composition of a corn and soybean mixture (CS) through artificial blending. 2. Optimizing the raw material pretreatment process to increase starch utilization and select strains with greater tolerance. These technological improvements will help enhance the economic efficiency and industrial feasibility of CS fermentation for lactic acid production.

[0005] CS, formulated in specific proportions, serves as a basic nutrient matrix. Its primary components include carbohydrates, proteins, and other nutrients, and it is also rich in trace nutrients such as vitamins and minerals. Carbohydrates, primarily in the form of starch, are key substrates for microbial fermentation, accounting for approximately 25% of the total composition. These carbohydrates can be converted through microbial fermentation into a variety of high-value-added bioproducts, such as fuel ethanol, ethanol gasoline, and organic acids such as lactic acid. Notably, CS not only provides the carbon source (starch) required for fermentation, but also contains proteins that can be broken down into nitrogen sources such as amino acids. Furthermore, its rich trace elements provide essential nutritional support for microbial growth. This unique nutrient profile makes CS an ideal fermentation matrix, particularly suitable for the production of biochemicals such as lactic acid, with significant economic value and development potential.

[0006] At the same time, in order to overcome the low activity retention rate of the immobilized enzyme and the unsatisfactory enzyme loading capacity in the present invention, the present invention has specially developed an enzyme carrier magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles, to which the three enzymes in the present invention can be connected by covalent bonds. Summary of the Invention

[0007] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process method, which solves the following related problems: 1. The soybean-corn composite matrix requires a variety of amylases and additional nutrients such as inorganic salts, nitrogen sources and vitamins for starch fermentation to produce lactic acid, resulting in high production costs; 2. The substrate inhibition effect is obvious. When the carbon source concentration is too high, it not only affects the effective utilization of the substrate by microorganisms, but also produces cytotoxic metabolic by-products, which seriously inhibit the growth and reproduction of the strain; 3. The product inhibition effect is significant. As lactic acid accumulates, key parameters such as the pH value of the fermentation system deviate from the optimal range, resulting in the deterioration of the strain growth environment, thereby triggering its stress protection mechanism, diverting metabolic energy from product synthesis to cell defense, and ultimately causing slow bacterial growth and decreased metabolic activity; 4. The immobilized enzyme activity retention rate is low and the enzyme loading capacity is not ideal.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A corn and soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process, characterized by comprising the following specific steps:

[0010] S1. The soybean and corn mixture was mixed in a mass ratio of (1-3): (1-5), water was added, and the solid-liquid ratio was adjusted to (1-3): (7-10), and the mixture was continued to be steamed for a certain time to obtain a high-temperature gelatinized substrate;

[0011] S2. The high-temperature gelatinized substrate obtained in step S1 above is added with a high-temperature amylase in an amount of 0.1-0.2% (w / w) of a soybean-corn composite matrix solution, and hydrolyzed at 70-85 degrees Celsius and a pH of 5.0-6.0 for 1-2 hours to obtain a liquefied substrate;

[0012] S3. To the liquefied substrate of step S2 above, glucoamylase is added in an amount of 0.1-0.2% (w / w) corn-soybean composite matrix and pullulanase is added in an amount of 0.05-0.1% (w / w) corn-soybean composite matrix liquid, and hydrolyzed at 50-60 degrees Celsius and a pH of 4-6 for 5-7 hours to obtain a saccharified substrate;

[0013] S4. Add 13-18% (w / w) of Bacillus coagulans to the saccharification substrate prepared in step S3 above, and ferment at 50-60 degrees Celsius and pH 4-6 for 65-80 hours to obtain a fermentation product, namely lactic acid.

[0014] Preferably, the mass ratio of soybean to corn in the above step S1 is 1:4;

[0015] Preferably, in the above step S1, the solid-liquid ratio is adjusted to 1:9;

[0016] Preferably, the high temperature cooking time in the above step S1 is 30 minutes;

[0017] Preferably, in the above step S2, the amount of high-temperature amylase added is 0.15% (w / w) of the soybean-corn composite matrix liquid; the temperature is 80 degrees Celsius, the pH is 5.5, and the hydrolysis time is 1 hour to obtain a liquefied substrate.

[0018] Preferably, in step S3, 0.1% (w / w) corn-soybean composite matrix liquid plus glucoamylase and 0.05% (w / w) corn-soybean composite matrix liquid plus pullulanase are added, and the mixture is hydrolyzed at 55 degrees Celsius and a pH of 4.5 for 6 hours to obtain a saccharified substrate;

[0019] Preferably, in step S4, the saccharification substrate prepared in step S3 is inoculated with 15% (w / w) Bacillus coagulans, and fermented at 55 degrees Celsius and a pH of 4.5 for 72 hours to obtain a fermentation product;

[0020] Preferably, the soybean-corn composite matrix in step S1 is also subjected to microwave pretreatment before steaming, with the treatment time being 3 minutes and the power being 800 watts;

[0021] Preferably, after the soybeans are crushed in the above step S1, ultrasonic assisted pretreatment is added, the pH is 8, the ultrasonic frequency is 40 kHz, and the ultrasonic time is 20 minutes;

[0022] The present invention also discloses a method for preparing magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles, comprising the following steps:

[0023] Step 1. Add 0.1-0.2 mol of succinic anhydride and 0.1-0.2 mol of 3-aminopropyltriethoxysilane to 200-400 ml of N,N-dimethylformamide and stir at 28-32 degrees Celsius for 2.5-3.5 hours;

[0024] Step 2. Add 0.1-0.2 mol of Fe3O4@SiO2 to a mixture of 200-400 ml of N,N-dimethylformamide and 50-100 ml of water and ultrasonically disperse it. Add the mixture to the solution in step 1 and continue stirring for 11-13 hours. Separate the mixture under an external magnetic field to obtain the black product—carboxyl-modified Fe3O4@SiO2 nanospheres. Wash the mixture several times with deionized water and ethanol and dry it at 50-60 degrees Celsius for 10-18 hours.

[0025] Step 2. 0.1-0.2 mol of zinc nitrate hexahydrate and 0.1-0.2 mol of cobalt nitrate hexahydrate are mixed and dissolved in 200-600 ml of methanol, and then the carboxyl-modified Fe3O4@SiO2 synthesized in step (1) is added, and ultrasonic dispersion is continued for 10-20 minutes. A methanol solution containing 0.8-1.6 mol of 2-methylimidazole is added, and ultrasonic dispersion is continued for 20-40 minutes at an ultrasonic frequency of 40 kHz. The product Fe3O4@SiO2@ZnCo-ZIF is separated and washed under an external magnetic field, and dried to obtain magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles.

[0026] The ZnCo-ZIF coating step is completed under the synergistic effect of 500-800 watt microwave radiation and 40 kHz pulse ultrasound, and the reaction time is ≤30 minutes.

[0027] High-temperature gelatinization of CS destroys its original crystalline structure, converting it to an amorphous state, significantly increasing the contact area between enzymes. High-temperature amylase, glucoamylase, and pullulanase are then added to completely decompose the gelatinized CS and its gelatinization products into glucose. High-temperature amylase randomly cleaves α-1,4 glycosidic bonds, rapidly reducing the viscosity of the system; glucoamylase continuously hydrolyzes α-1,4 bonds to form glucose monomers; and pullulanase specifically cleaves α-1,6 glycosidic bonds, completely breaking down the β-limit dextrins of amylopectin, completing the branched structure gaps that the above two amylases cannot hydrolyze. Finally, the high-purity glucose substrate in the enzymatic hydrolyzate is anaerobic fermented with Bacillus coagulans to achieve efficient biosynthesis of lactic acid.

[0028] The addition of pullulanase during the decomposition stage is one of the innovations of this invention. Among industrial-grade amylase preparations, pullulanase offers significant cost-effectiveness: compared to isoamylase, another amylopectin hydrolyzing enzyme, it is relatively inexpensive. Furthermore, the added dosage is lower than that of the other two amylases, improving the decomposition effect.

[0029] The specific steps of the reaction in a fixed reactor under the action of high temperature amylase, glucoamylase, pullulanase and Bacillus coagulans are as follows:

[0030] (1) Adding 0.15% (w / w) thermoamylase to thermogelatinized CS, hydrolyzing at 70-80 degrees Celsius and pH 5.5-6.0 for 1-1.5 hours to obtain a liquefied substrate;

[0031] (2) adding glucoamylase to the liquefied substrate in an amount of 0.1% (w / w) and pullulanase to the hydrolyzed solution in an amount of 0.05% (w / w), and hydrolyzing the liquefied substrate at 55-60 degrees Celsius and a pH of 4.5-5.0 for 6-6.5 hours to obtain a saccharified substrate;

[0032] (3) adding Bacillus coagulans seed solution to the prepared saccharification substrate at an inoculum amount of 5-10%, and fermenting at 55-60 degrees Celsius and a pH of 4.5-5.0 for 72-96 hours to obtain a fermentation product, namely lactic acid.

[0033] The technical effects and advantages of the corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process of the present invention are as follows:

[0034] 1. The invention adopts a low-dose high-temperature amylase 0.15% (w / w), glucoamylase 0.1% (w / w) and pullulanase 0.05% (w / w) to form a composite hydrolysis system, and realizes the synergistic effect of enzyme activity through step-by-step temperature control (80°C→55°C), in which more than 80% of the starch is converted into glucose (detected by enzyme electrode method).

[0035] 2. This invention avoids the metabolic inhibition of Bacillus coagulans caused by the low-sugar environment in the traditional step-by-step process by generating a high-concentration glucose substrate (initial concentration >20g / L), thereby advancing the bacterial growth period by 6 hours and achieving a conversion rate of glucose to lactic acid exceeding 50%.

[0036] 3. This invention uses a seamless connection between the enzymatic hydrolysis and fermentation processes, without the intermediate steps of inactivation and centrifugation in traditional processes, making the operation simple, low-cost, clean and environmentally friendly.

[0037] 4. This invention greatly improves the enzyme loading capacity and the activity retention rate of the immobilized enzyme; the enzyme can be reused, reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a flow chart of a corn and soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process proposed by the present invention;

[0039] Figure 2 This is a schematic diagram comparing the lactic acid yields of fermentation product 1 of Examples 1-3 and Comparative Examples 1-2 in a corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process proposed by the present invention;

[0040] Figure 3 This is a schematic diagram comparing the glucose-lactic acid conversion rates in the fermentation product 1 of Examples 1-3 and Comparative Examples 1-2 in a corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process proposed by the present invention;

[0041] Figure 4 This is a schematic diagram comparing the lactic acid production rates of Examples 1-3 and Comparative Examples 1-2 in a corn-soybean composite matrix multi-stage temperature-controlled lactic acid bio-fermentation process proposed by the present invention; DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0043] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus. In the absence of further restrictions, the elements defined by the sentence "include..." do not exclude the existence of other identical elements in the process, method, article or apparatus that includes the elements.

[0044] The present invention adopts the following method to detect the content of each component:

[0045] (1) The starch content was determined by the enzymatic hydrolysis method in accordance with the national standard GB 5009.9-2016. After heating and gelatinization, the starch was hydrolyzed into small molecular sugars using Gaofeng's amylase, and finally hydrolyzed into monosaccharides using hydrochloric acid. The reducing sugar content was then determined and converted into starch content.

[0046] (2) Glucose and lactic acid concentrations were determined using an enzyme electrode method using a SIEMAN M-900 biochemical process analyzer. The detection conditions were pH 6-8 and the detection range was 0.3-9 g / L for glucose and 0.03-2 g / L for lactic acid.

[0047] Glucose-lactic acid conversion rate = actual glucose-lactic acid production / theoretical glucose-lactic acid production × 100% (based on mass)

[0048] Lactic acid production rate = lactic acid concentration g / (L·h) produced per hour;

[0049] Air-dried corn and soybeans from rural Northeast China were crushed into a powder (moisture content of 10%-20%). Tap water was then added to adjust the solid-liquid weight ratio to 1:9. The mixture was steamed at 100°C for 30 minutes. 20-30g of the high-temperature-treated corn-soybean composite matrix was then oven-dried at 105°C for 24 hours to obtain a high-temperature gelatinized corn-soybean matrix. Testing revealed a starch content of 24.6% in the high-temperature gelatinized corn-soybean matrix.

[0050] The lactic acid bacteria used were commercially available Bacillus coagulans, and the thermoamylase, glucoamylase, and pullulanase were commercially available enzymes.

[0051] The nutrient composition was the following per liter of deionized water: 10 g peptone, 5 g yeast extract, 2 g (NH4)2SO4, 2 g KH2PO4, and 0.58 g MgSO4·7H2O.

[0052] Example 1

[0053] This embodiment 1 provides a corn and soybean composite matrix multi-stage temperature-controlled lactic acid bio-fermentation process, the specific implementation steps include:

[0054] (1) Corn and soybeans collected from rural areas in Northeast China were crushed and mixed to a solid-liquid ratio of 1:9 with 80 g corn and 20 g soybeans, and then steamed in an electric rice cooker for 30 min to obtain a high-temperature gelatinized substrate.

[0055] (2) adding high-temperature amylase to the high-temperature gelatinized substrate of step (1) at an addition amount of 0.15% (w / w) corn-soybean composite matrix liquid, and hydrolyzing at 80° C. and pH 5.5 for 1 hour to obtain a liquefied substrate;

[0056] (3) adding 0.1% (w / w) corn-soybean composite matrix liquid of glucoamylase and 0.05% (w / w) corn-soybean composite matrix liquid of pullulanase to the liquefied substrate of step (2), and hydrolyzing the mixture at 55° C. and pH 4.5 for 6 hours to obtain a saccharified substrate;

[0057] (4) adding 15% (w / w) Bacillus coagulans to the saccharification substrate prepared in step (3), and fermenting at 55° C. and pH 4.5 for 72 hours to obtain a fermentation product.

[0058] The high-temperature amylase, glucoamylase, and pullulanase added in steps S2 and S3 are fixed on the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles through chemical bonds;

[0059] The preparation steps of the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles are as follows:

[0060] Step 1. Add 0.2 mol of succinic anhydride and 0.2 mol of 3-aminopropyltriethoxysilane to 400 ml of N,N-dimethylformamide and stir at 30 degrees Celsius for 3 hours;

[0061] Step 2. Ultrasonic dispersion of 0.2 mol of Fe₃O₄@SiO₂ was added to a mixture of 400 ml of N,N-dimethylformamide and 100 ml of water. The mixture was then added to the solution in Step 1 and stirred for 12 hours. The black product, carboxyl-modified Fe₃O₄@SiO₂ nanospheres, was separated under an external magnetic field. The resulting product was washed several times with deionized water and ethanol and dried at 60°C for 15 hours.

[0062] Step 2. 0.2 mol of zinc nitrate hexahydrate and 0.2 mol of cobalt nitrate hexahydrate were mixed and dissolved in 500 ml of methanol, and then the carboxyl-modified Fe3O4@SiO2 synthesized in step (1) was added, and ultrasonic dispersion was continued for 15 minutes. A methanol solution containing 1.6 mol of 2-methylimidazole was added, and ultrasonic dispersion was continued for 30 minutes at an ultrasonic frequency of 40 kHz. The product Fe3O4@SiO2@ZnCo-ZIF was separated and washed under an external magnetic field, and dried to obtain magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles.

[0063] Example 2

[0064] This embodiment 2 provides a corn and soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process, the specific implementation steps include:

[0065] (1) Corn and soybeans collected from rural areas in Northeast China were crushed and mixed to a solid-liquid ratio of 1:9 with 80 g corn and 20 g soybeans, and then steamed in an electric rice cooker for 30 min to obtain a high-temperature gelatinized substrate.

[0066] (2) adding high-temperature amylase to the high-temperature gelatinized substrate of step (1) at an addition amount of 0.15% (w / w) corn-soybean composite matrix liquid, and hydrolyzing at 80° C. and pH 5.5 for 1 hour to obtain a liquefied substrate;

[0067] (3) adding 0.1% (w / w) corn-soybean composite matrix liquid of glucoamylase and 0.05% (w / w) corn-soybean composite matrix liquid of pullulanase to the liquefied substrate of step (2), and hydrolyzing the mixture at 55° C. and pH 4.5 for 6 hours to obtain a saccharified substrate;

[0068] (4) Adding 20% (w / w) of Bacillus coagulans to the saccharification substrate prepared in step (3), and fermenting at 55° C. and pH 4.5 for 72 hours to obtain a fermentation product.

[0069] The high-temperature amylase, glucoamylase, and pullulanase added in steps S2 and S3 are fixed on the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles through chemical bonds;

[0070] Example 3

[0071] This embodiment 3 provides a corn and soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process, the specific implementation steps include:

[0072] (1) Corn and soybeans collected from rural areas in Northeast China were crushed and mixed to a solid-liquid ratio of 1:9 with 80 g corn and 20 g soybeans, and then steamed in an electric rice cooker for 30 min to obtain a high-temperature gelatinized substrate.

[0073] (2) adding high-temperature amylase to the high-temperature gelatinized substrate of step (1) at an addition amount of 0.15% (w / w) corn-soybean composite matrix liquid, and hydrolyzing at 80° C. and pH 5.5 for 1 hour to obtain a liquefied substrate;

[0074] (3) adding 0.1% (w / w) corn-soybean composite matrix liquid of glucoamylase and 0.05% (w / w) corn-soybean composite matrix liquid of pullulanase to the liquefied substrate of step (2), and hydrolyzing the mixture at 55° C. and pH 4.5 for 6 hours to obtain a saccharified substrate;

[0075] (4) adding Bacillus coagulans at an inoculum amount of 25% (w / w) to the saccharification substrate prepared in step (3), and fermenting at 55° C. and pH 4.5 for 72 hours to obtain a fermentation product.

[0076] The high-temperature amylase, glucoamylase, and pullulanase added in steps S2 and S3 are fixed on the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles through chemical bonds;

[0077] Comparative Example 1

[0078] Comparative Example 1 provides a corn and soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process, the specific implementation steps include:

[0079] (1) Corn and soybeans collected from rural areas in Northeast China were crushed and mixed to a solid-liquid ratio of 1:9 with 80 g corn and 20 g soybeans, and then steamed in an electric rice cooker for 30 min to obtain a high-temperature gelatinized substrate.

[0080] (2) adding high-temperature amylase to the high-temperature gelatinized substrate of step (1) at an addition amount of 0.15% (w / w) corn-soybean composite matrix liquid, and hydrolyzing at 80° C. and pH 5.5 for 1 hour to obtain a liquefied substrate;

[0081] (3) adding 0.1% (w / w) corn-soybean composite matrix liquid of glucoamylase, adding 0.05% (w / w) corn-soybean composite matrix liquid of pullulanase, and inoculating 25% (w / w) Bacillus coagulans to the liquefied substrate of step (2), and hydrolyzing and fermenting the resulting mixture at 55° C. and pH 4.5 for 72 hours to obtain a fermentation substrate;

[0082] The high-temperature amylase, glucoamylase, and pullulanase added in steps S2 and S3 are fixed on the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles through chemical bonds;

[0083] Comparative Example 2

[0084] Comparative Example 2 provides a corn and soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process, the specific implementation steps of which include:

[0085] (1) Corn and soybeans collected from rural areas in Northeast China were crushed and mixed to a solid-liquid ratio of 1:9 with 80 g corn and 20 g soybeans, and then steamed in an electric rice cooker for 30 min to obtain a high-temperature gelatinized substrate.

[0086] (2) adding high-temperature amylase to the high-temperature gelatinized substrate of step (1) at an addition amount of 0.15% (w / w) corn-soybean composite matrix liquid, and hydrolyzing at 80° C. and pH 5.5 for 1 hour to obtain a liquefied substrate;

[0087] (3) adding 0.1% (w / w) corn-soybean composite matrix liquid of glucoamylase and 0.05% (w / w) corn-soybean composite matrix liquid of pullulanase to the liquefied substrate of step (2), and hydrolyzing the mixture at 55° C. and pH 4.5 for 6 hours to obtain a saccharified substrate;

[0088] (4) Adding 25% (w / w) of Bacillus coagulans and nutrients to the saccharification substrate prepared in step (3), and fermenting at 55° C. and pH 4.5 for 72 hours to obtain a fermentation product.

[0089] The high-temperature amylase, glucoamylase, and pullulanase added in steps S2 and S3 are fixed on the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles through chemical bonds;

[0090] Comparative Example 3

[0091] Comparative Example 3 provides a corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process, the specific implementation steps of which include:

[0092] 1) Corn and soybeans collected from rural areas in Northeast China were ground and then mixed to a solid-liquid ratio of 1:9 (80 g corn and 20 g soybeans), and cooked in an electric rice cooker for 30 min to obtain a high-temperature gelatinized substrate.

[0093] (2) adding high-temperature amylase to the high-temperature gelatinized substrate of step (1) at an addition amount of 0.15% (w / w) corn-soybean composite matrix liquid, and hydrolyzing at 80° C. and pH 5.5 for 1 hour to obtain a liquefied substrate;

[0094] (3) adding 0.1% (w / w) corn-soybean composite matrix liquid of glucoamylase and 0.05% (w / w) corn-soybean composite matrix liquid of pullulanase to the liquefied substrate of step (2), and hydrolyzing the mixture at 55° C. and pH 4.5 for 6 hours to obtain a saccharified substrate;

[0095] (4) adding Bacillus coagulans at an inoculum amount of 25% (w / w) to the saccharification substrate prepared in step (3), and fermenting at 55° C. and pH 4.5 for 72 hours to obtain a fermentation product.

[0096] The high-temperature amylase, glucoamylase, and pullulanase added in steps S2 and S3 above were not fixed to the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles by chemical bonds and were not chemically modified;

[0097] Test Item 1: Lactic Acid Production Test

[0098] Table 1 Lactic acid production test results

[0099] Lactic acid production / (g / L) Example 1 16.4 Example 2 17.3 Example 3 19.4 Comparative Example 1 2.4 Comparative Example 2 3.2

[0100] As can be seen from Table 1 above, in Comparative Examples 1-3, it can be seen that under the constant temperature conditions of pH 5.5 and temperature 55°C, the lactic acid content is increased from 16.4g / L to 19.4g / L, confirming that the addition of high bacterial count effectively promotes the fermentation reaction process by enhancing the substrate conversion efficiency. Comparative Example 3 and Comparative Example 1 show that the former has a higher lactic acid yield, indicating that staged hydrolysis to increase the glucose concentration of the substrate before fermentation is more conducive to maintaining the metabolic activity of the flora and avoiding substrate inhibition. Comparative Example 3 and Comparative Example 2 (exogenously added composite nutrients) found that the introduction of additional carbon sources (C) and nitrogen sources (N) will cause the system C / N ratio to be unbalanced, destroy the inherent carbon and nitrogen distribution, interfere with the steady-state operation of the internal metabolism of the strain, and ultimately lead to a decrease in the yield of lactic acid. Analyzing the above problems, the possible reasons are specifically analyzed as follows: 1. C / N ratio imbalance causes metabolic flow deviation: 1) Excess nitrogen: the bacteria prioritize the use of metabolic energy for biomass synthesis (cell proliferation) rather than lactic acid synthesis; 2) Insufficient carbon: if nitrogen source is added as the main source, carbon is relatively insufficient, which limits the production of lactic acid synthesis precursor (pyruvate). 2. Catabolite repression (Carbon Catabolite Repression, CCR): 1) High concentration of easily available carbon source: If a fast-acting carbon source such as glucose is added exogenously, the CCR effect is triggered: amylase gene expression is inhibited, and the continuous hydrolysis ability of starch in the matrix is reduced; the bacteria preferentially utilize exogenous glucose, resulting in insufficient carbon source supply in the later stage. Experimental evidence: The decrease in lactic acid production in Comparative Example 2 may be accompanied by a sudden drop in glucose concentration in the later stage of fermentation (verifiable by HPLC detection). 3. Osmotic stress and metabolic toxicity: High salt / hypertonic environment: Exogenously added inorganic salts (such as KH2PO4, (NH4)2SO4) may increase the osmotic pressure of the fermentation broth: inhibit the cell membrane function of the bacteria and reduce the efficiency of nutrient absorption; trigger the expression of osmotic pressure regulating genes (such as proU), consume ATP, and reduce the energy for lactate synthesis; accumulation of toxic byproducts: excessive nitrogen sources may lead to the accumulation of ammonia (NH3), change the pH and inhibit the activity of lactate dehydrogenase (LDH); 4. Destruction of bacterial metabolic homeostasis: Metabolic pathway competition: exogenous nutrients induce metabolic diversity, and some bacteria turn to synthesizing other products (such as acetic acid and ethanol): GC-MS detection of comparative example 2 may show an increase in acetic acid concentration; the lactic acid / acetic acid molar ratio is reduced from 10:1 in Example 3 to 3:1 in Comparative Example 2; energy distribution imbalance: the bacteria need to cope with exogenous nutrient metabolism and lactic acid synthesis at the same time, resulting in insufficient ATP supply.

[0101] Test Item 2: Glucose-Lactate Conversion Rate Test

[0102] Glucose concentration was determined using an enzyme electrode method using a SIEMAN M-900 biochemical process analyzer. The test conditions were: pH 6-8; the detection range was 0.3-9 g / L glucose.

[0103] Glucose-lactic acid conversion rate = actual glucose-lactic acid production / theoretical glucose-lactic acid production × 100% (based on mass)

[0104] Table 2 Glucose lactate conversion test results

[0105] Glucose lactate conversion rate / % Example 1 90 Example 2 93 Example 3 95 Comparative Example 1 36 Comparative Example 2 60

[0106] As shown in Table 2 above, it can be seen that in Control Examples 1, 2, and 3 (different strain addition amounts), under the constant temperature conditions of pH 5.5 and temperature 55°C, the glucose-lactic acid conversion rate increased from 90% to 95%, confirming that the addition of high bacterial amounts effectively promoted the fermentation reaction process by enhancing substrate conversion efficiency. Comparative Example 3 (distributed fermentation) and Comparative Example 1 (synchronous fermentation) found that the former had a higher glucose-lactic acid conversion rate, indicating that staged hydrolysis to increase the glucose concentration of the substrate before fermentation is more conducive to maintaining bacterial metabolic activity and avoiding substrate inhibition. Control Example 3 and Comparative Example 2 (exogenous addition of composite nutrients) found that the introduction of additional carbon source (C) and nitrogen source (N) can cause the system C / N ratio to be unbalanced, destroy the inherent carbon and nitrogen distribution, interfere with the steady-state operation of the internal metabolism of the strain, and ultimately lead to a decrease in the glucose-lactic acid conversion rate. The possible reasons are: In general, the exogenous addition of complex nutrients destroys the metabolic homeostasis of Bacillus coagulans through multiple mechanisms such as C / N ratio imbalance, catabolism inhibition, osmotic stress, and metabolic pathway competition, resulting in the diversion of energy and carbon sources to non-target products, and ultimately reducing the lactic acid conversion rate.

[0107] Test Item 3: Comparison of Lactic Acid Production Rate

[0108] Lactic acid concentration was determined using an enzyme electrode method using a SIEMAN M-900 biochemical process analyzer. The assay conditions were pH 6-8 and the assay range was 0.03-2 g / L lactic acid.

[0109] Lactic acid production rate = lactic acid concentration g / (L·h) produced per hour;

[0110] Table 3 Lactic acid production rate results

[0111] Lactic acid production rate / (g / (L·h)) Example 1 0.23 Example 2 0.24 Example 3 0.27 Comparative Example 1 0.02 Comparative Example 2 0.03

[0112] As can be seen from Table 3 above, in comparison with Examples 1, 2, and 3 (different amounts of bacterial species added), it was found that under constant temperature conditions of pH 5.5 and temperature 55°C, when the amount of Bacillus coagulans added was increased from 15% to 25%, the lactic acid production rate continued to increase, confirming that the addition of high bacterial amounts effectively promoted the fermentation reaction process by enhancing the substrate conversion efficiency. Comparison of Example 3 (distributed fermentation) with Comparative Example 1 (synchronous fermentation) found that the former had a fast lactic acid production rate, indicating that staged hydrolysis to increase the glucose concentration of the substrate before fermentation is more conducive to maintaining the metabolic activity of the bacterial community and avoiding substrate inhibition; in comparison with Example 3 and Comparative Example 2 (exogenous addition of complex nutrients), it was found that the introduction of additional carbon sources (C) and nitrogen sources (N) would lead to an imbalance in the C / N ratio of the system, destroy the inherent carbon and nitrogen distribution, interfere with the steady-state operation of the internal metabolism of the strain, and ultimately lead to a decrease in the conversion of lactic acid. The possible reasons are:

[0113] Test Item 4: Enzyme loading capacity and activity retention rate test

[0114] Table 4 Enzyme loading capacity and activity retention test results

[0115] Enzyme loading / (mg / g) Activity retention rate / % Example 3 140 230 Comparative Example 3 79 93

[0116] Comparison of Example 3 and Comparative Example 3 shows that connecting the three enzymes to the Fe3O4@SiO2@Zn Co-ZIF composite carrier through chemical bonds can greatly increase the enzyme loading and activity retention rate. The possible reasons are: 1. Co 2+ Doping changes the electron cloud distribution of ZnCo-ZIF, enhances the coordination effect with the active center of pullulanase, and improves the immobilization efficiency; 2. The bimetallic synergistic effect expands the pore size, adapts to the molecular size of pullulanase, and reduces mass transfer resistance.

[0117] The above embodiments may be implemented in whole or in part through software, hardware, firmware or any other combination. When implemented using software, the above embodiments may be implemented in whole or in part in the form of a computer program product.

[0118] The above is only a specific implementation method of the present application, but the scope of protection of the present application is not limited to this. Any technician familiar with this technical field can easily think of changes or replacements within the technical scope disclosed in this application, which should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

[0119] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A corn and soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process, characterized in that: The specific steps include: S1. The soybean and corn mixture was mixed in a mass ratio of (1-3): (1-5), water was added, and the solid-liquid ratio was adjusted to (1-3): (7-10), and the mixture was continued to be steamed for a certain time to obtain a high-temperature gelatinized substrate; S2. The high-temperature gelatinized substrate obtained in step S1 above is added with a high-temperature amylase in an amount of 0.1-0.2% (w / w) of a soybean-corn composite matrix solution, and hydrolyzed at 70-85 degrees Celsius and a pH of 5.0-6.0 for 1-2 hours to obtain a liquefied substrate; S3. To the liquefied substrate of step S2 above, glucoamylase is added in an amount of 0.1-0.2% (w / w) corn-soybean composite matrix liquid and pullulanase is added in an amount of 0.05-0.1% (w / w) corn-soybean composite matrix liquid, and hydrolyzed at 50-60 degrees Celsius and a pH of 4-6 for 5-7 hours to obtain a saccharified substrate; S4. Add 13-18% (w / w) of Bacillus coagulans to the saccharification substrate prepared in step S3 above, and ferment at 50-60 degrees Celsius and pH 4-6 for 65-80 hours to obtain a fermentation product, namely lactic acid. The high-temperature amylase, glucoamylase, and pullulanase added in the above steps S2 and S3 are fixed on the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles through chemical bonds.

2. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 1, characterized in that: The initial concentration of the glucose substrate obtained in step S3 is ≥20 g / L.

3. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 1, characterized in that: In the above step S4, the conversion rate of glucose into lactic acid is above 50%.

4. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 1, characterized in that: The above-mentioned lactic acid preparation process seamlessly connects the enzymatic hydrolysis and fermentation steps, without the core links such as fire extinguishing and centrifugation in traditional processes, making the operation simple, low-cost, clean and environmentally friendly.

5. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 1, characterized in that: In step S1, the soybean-corn composite matrix is also subjected to microwave pretreatment before steaming, with a treatment time of 2-3 minutes and a microwave power of 600-1000 watts. After the soybeans are crushed in step S1, ultrasonic-assisted pretreatment is continued, with the pH adjusted to 8-9, the ultrasonic frequency to 40-50 kHz, and the ultrasonic time to 20-30 minutes.

6. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 1, characterized in that: When high-temperature amylase is added for hydrolysis in the above step S2, trehalose is added at the same time as an enzyme stabilizer.

7. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 1, characterized in that: The high-temperature amylase, glucoamylase, and pullulanase added in the above steps S2 and S3 were immobilized on the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles by covalent immobilization.

8. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 7, characterized in that: The particle size of the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles is 30-40 nanometers.

9. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 7, characterized in that: The steps for preparing the magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles are as follows: Step 1. Add 0.1-0.2 mol of succinic anhydride and 0.1-0.2 mol of 3-aminopropyltriethoxysilane to 200-400 ml of N,N-dimethylformamide and stir at 28-32 degrees Celsius for 2.5-3.5 hours; Step 2. Add 0.1-0.2 mol of Fe3O4@SiO2 to a mixture of 200-400 ml of N,N-dimethylformamide and 50-100 ml of water and ultrasonically disperse it. Add the mixture to the solution in step 1 and continue stirring for 11-13 hours. Separate the mixture under an external magnetic field to obtain the black product—carboxyl-modified Fe3O4@SiO2 nanospheres. Wash the mixture several times with deionized water and ethanol and dry it at 50-60 degrees Celsius for 10-18 hours. Step 2. 0.1-0.2 mol of zinc nitrate hexahydrate and 0.1-0.2 mol of cobalt nitrate hexahydrate are mixed and dissolved in 200-600 ml of methanol, and then the carboxyl-modified Fe3O4@SiO2 synthesized in step (1) is added, and ultrasonic dispersion is continued for 10-20 minutes. A methanol solution containing 0.8-1.6 mol of 2-methylimidazole is added, and ultrasonic dispersion is continued for 20-40 minutes at an ultrasonic frequency of 40 kHz. The product Fe3O4@SiO2@ZnCo-ZIF is separated and washed under an external magnetic field, and dried to obtain magnetic Fe3O4@SiO2@ZnCo-ZIF nanoparticles.

10. The corn-soybean composite matrix multi-stage temperature-controlled lactic acid biofermentation process according to claim 9, characterized in that: The ZnCo-ZIF coating step is completed under the synergistic effect of 500-800 watt microwave radiation and 40 kHz pulsed ultrasound, with a reaction time of ≤30 minutes.

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