A method for culturing aspergillus niger spores by using high air permeability puffed rice hull-bran composite substrate

Abstract

This invention belongs to the field of microbiology, specifically relating to a method for cultivating Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate. The method employs a composite substrate formed by combining expanded modified rice husk with wheat bran, overcoming the inherent limitations of a single wheat bran substrate. A fibrous skeletal structure is constructed through high-temperature expansion pretreatment of crushed rice husk, followed by precise blending with wheat bran, significantly improving the substrate's permeability and heat dissipation, and optimizing temperature uniformity. Furthermore, only two turning operations are required to complete the cultivation of Aspergillus niger spores, significantly reducing labor intensity and the risk of contamination. The addition of specific nutrients optimizes the culture medium, achieving increased spore yield and viability while shortening the cultivation cycle, thus balancing practicality and economy. This method can be directly applied to the large-scale cultivation of Aspergillus niger in citric acid fermentation, meeting the needs of industrial-scale applications.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate. Background Technology

[0002] Aspergillus niger is a core microorganism in the citric acid fermentation industry, and its cultivation effect directly affects the fermentation conversion rate and product yield. Current technologies mostly use wheat bran as the culture medium. However, wheat bran as a single substrate has inherent drawbacks: high bulk density, low porosity, and poor aeration. During cultivation, wheat bran easily absorbs moisture and clumps, hindering ventilation and heat dissipation, thus reducing spore quality and yield. Simultaneously, wheat bran has high thermal conductivity, easily causing localized temperature accumulation in the culture system, resulting in poor temperature uniformity and further affecting spore viability. To alleviate these problems, the substrate needs to be frequently turned over during cultivation, requiring 6-7 times per cultivation cycle. This increases labor intensity, raises the risk of contamination, and frequent turning causes mycelial breakage, leading to long cultivation cycles and low spore germination rates, making it difficult to meet the high-efficiency requirements of large-scale industrial-scale microbial cultivation.

[0003] Therefore, developing a method for cultivating Aspergillus niger spores that optimizes the substrate structure, improves ventilation and heat dissipation performance, and reduces operational difficulty has become a pressing technical problem to be solved in this field. Summary of the Invention

[0004] To address the numerous shortcomings of existing methods for cultivating Aspergillus niger spores, this invention discloses a method for cultivating Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate. First, rice husks undergo high-temperature expansion pretreatment, and are then mixed with wheat bran in an appropriate ratio to form the composite substrate. Specific nutrients are added to the composite substrate, and water is added to adjust the moisture content to a suitable level. After inoculating with Aspergillus niger spores at the conventional inoculation rate, the substrate is cultivated under suitable temperature and humidity conditions, requiring only two turnings during cultivation. This invention, through the precise blending of expanded rice husks and wheat bran, significantly improves substrate permeability, optimizes heat dissipation and temperature uniformity, effectively reduces the number of turnings, and lowers labor intensity and the risk of contamination. Synergistic nutrient optimization of the culture medium significantly increases spore yield and germination rate, shortens the cultivation cycle, and can be directly applied to the large-scale cultivation of citric acid fermentation strains, greatly improving economic benefits.

[0005] The specific technical solution of this application is as follows: A method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate, the specific steps of which are as follows: (1) Rice husk pretreatment: The rice husk is crushed to a particle size of 1-3 mm by a crusher, and then processed by a high-temperature extrusion process using a screw extruder. The process parameters are as follows: 120-130℃, 0.5-0.6MPa, heat preservation and pressure holding for 30-50s, and then cooled to 20-30℃ to obtain extruded rice husk. First, the rice husks are crushed and then processed through a high-temperature puffing process, which can improve the looseness and air permeability of the rice husks, laying the foundation for the subsequent construction of an air-permeable framework. The preferred rice husk crushing particle size is 2.0-2.2mm, and the high-temperature puffing parameters are controlled at 123℃, 0.56MPa, and heat preservation and pressure holding for 45s.

[0006] (2) Preparation of composite matrix: According to the dry basis mass percentage, the puffed rice husk and wheat bran are mixed evenly according to the ratio of wheat bran accounting for 80-85 wt% and puffed rice husk accounting for 15-20 wt% in the composite matrix to obtain the basic composite matrix; the preferred composite matrix has wheat bran of 81 wt% and puffed rice husk of 19 wt%.

[0007] (3) Addition of nutrients: Add 0.5-1.0% yeast powder, 2.0-3.0% sorbitol and 1.5-2.0% trehalose by weight percentage to the composite matrix obtained in the previous step, and stir well. The added nutrients have the following effects: Sorbitol has a more gradual metabolic efficiency than glucose, which can continuously provide energy and material basis for the growth and sporulation process of Aspergillus niger, avoid metabolic imbalance caused by rapid carbon source, ensure stable nutrient supply during sporulation, and improve sporulation efficiency; yeast powder can supplement high-quality nitrogen source and growth factors, and strengthen spore attachment ability and vitality; trehalose can maintain the stability of spore cell membrane, and improve germination rate and stress resistance. More preferably, the above-mentioned nutrients are added in the following amounts by weight percentage: 0.5% yeast powder, 2.4% sorbitol, and 1.8% trehalose.

[0008] (4) Adjustment of matrix conditions: Add pure water to the mixed matrix and control the moisture content of the mixed matrix at 60-65 wt%; preferably 65 wt%.

[0009] (5) Bottling and sterilization: The culture medium after the above treatment is put into Erlenmeyer flasks and sterilized at 121℃ for 30 min, and then cooled to 20℃-30℃ for use.

[0010] (6) Inoculation procedure: 4.5-5.0 × 10 5 An inoculum of *Aspergillus niger* spores per gram of oven-dry substrate is inoculated into the composite substrate under sterile conditions and mixed thoroughly; the preferred inoculum amount is 4.6 × 10⁶ spores per gram of oven-dry substrate. 5 1 spore / gram of dry substrate.

[0011] (7) Spore culture: Place the inoculated culture medium in a culture environment with an ambient temperature of 28-30℃ and a humidity of 45-55% for a total culture period of 110-115h. (8) Cultivation process: Turn the food over once at 22-24 hours and once at 30-32 hours, and maintain the above fermentation conditions until the end of the cultivation cycle; Preferably, in steps (7) and (8), the spore culture environment temperature is 30℃ and the humidity is 50%. The substrate is turned over once at 23h and 31h respectively, and the culture is completed at 110h.

[0012] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: 1. Innovative matrix structure, breaking through performance bottlenecks: This invention is the first to modify rice husks through "crushing-high temperature puffing" and then combine them with wheat bran to construct a "puffed rice husk-wheat bran" composite matrix system. Through the porous fiber skeleton structure formed by puffed rice husks, the matrix porosity is increased by 70%-86% compared with wheat bran alone. This solves the core technical problems of poor air permeability and uneven heat dissipation of wheat bran matrix, and achieves continuous oxygen supply and rapid carbon dioxide discharge, creating the optimal gaseous environment for spore growth.

[0013] 2. Process optimization reduces costs and improves operational safety: Based on the excellent ventilation and heat dissipation performance of the composite substrate, the number of times the substrate is turned over is reduced from 6-7 times to 2 times, which greatly reduces the labor intensity and reduces the risk of contamination by miscellaneous bacteria caused by frequent operation. It also avoids growth inhibition caused by mycelial breakage and ensures the stability and safety of the cultivation process.

[0014] 3. Synergistic nutritional enhancement to improve strain quality: Targeted addition of sorbitol, yeast powder, and trehalose to construct a synergistic nutritional system, which improves spore performance from three dimensions: energy supply, growth promotion, and structural protection. This results in a 38%-58% increase in spore yield, a maximum 4.5% increase in germination rate, and a 50%-61% reduction in strain mutation rate, effectively solving the problems of low spore yield, insufficient activity, and high risk of degradation in existing technologies.

[0015] 4. Shorter cycle and higher efficiency, adaptable to industrial needs: The cultivation cycle is shortened by 5.8%-11.3%, which greatly improves industrial production efficiency while ensuring the quality of the strain. At the same time, the raw material rice husk is widely available and inexpensive. When combined with wheat bran, it does not significantly increase the raw material cost. It takes into account both technological advancement and economic practicality. It can be directly applied to the strain expansion cultivation stage of citric acid fermentation, and has broad industrial application prospects and significant economic benefits. Detailed Implementation

[0016] The following detailed embodiments further illustrate the above-described content of the present invention, but do not limit the present invention in any way. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Unless otherwise specified, the following embodiments are all carried out using conventional prior art.

[0017] In the following examples, the bran raw material is derived from a by-product of a wheat flour processing plant, with a particle size of 0.8-2.0 mm; the rice husk raw material is derived from a major by-product generated during rice processing, with a particle size of 4-8 mm; and the Aspergillus niger used has the preservation number CGMCC No. 5751.

[0018] Example 1: A method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate, the specific steps of which are as follows: 1. Rice husk pretreatment: Crush the rice husks to a particle size of 2.2-2.4mm using a hammer mill, and process them using a high-temperature extrusion process with screw extruder. The parameters are controlled at 125℃, 0.55MPa, and heat and pressure holding for 43s. After cooling to 25℃, they are ready for use.

[0019] 2. Preparation of composite matrix: Mix 83% wheat bran and 17% puffed rice husk evenly according to the dry basis mass ratio (w / w) to obtain the basic composite matrix.

[0020] 3. Adding nutrients: Add 0.7% yeast powder, 2.8% sorbitol and 1.6% trehalose by weight to the composite matrix obtained in the previous step, and stir until the components are evenly distributed.

[0021] 4. Matrix condition adjustment: Add pure water to the mixed matrix to control the moisture content of the mixed matrix at 64wt%. After testing, the porosity of the composite matrix at this time is 64.9%.

[0022] 5. Bottling and sterilization: Pour the culture medium into Erlenmeyer flasks, plug the mouth of the flask with a breathable cotton plug, sterilize at 121℃ for 30 minutes, and cool to 25℃ for later use.

[0023] 6. Vaccination procedure: Administer approximately 4.6 × 10 5 The addition amount is 1 spore / gram of oven-dry substrate. Under sterile conditions, Aspergillus niger spores are inoculated into the composite substrate and mixed thoroughly.

[0024] 7. Spore culture: Place the inoculated culture medium in a culture environment and control the ambient temperature at 29℃ and humidity at 47% for culture.

[0025] 8. Cultivation process: The substrate was turned over once at 24h and 32h to ensure the substrate's air permeability and heat dissipation. At 112h, the culture substrate was completely covered with black spores, and the cultivation was completed.

[0026] Experimental results: After the culture was completed, the spore yield was approximately 1.9 × 10⁻¹¹ per gram of culture medium. 10 Count / g; spore germination rate 99.2%; strain variation rate 0.32%.

[0027] Example 2: A method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate, the specific steps of which are as follows: 1. Rice husk pretreatment: Crush the rice husks to a particle size of 1.9-2.1mm using a hammer mill, and process them using a high-temperature extrusion process with screw extruder. The parameters are controlled at 124℃, 0.53MPa, and heat and pressure holding for 37s. After cooling to 25℃, they are ready for use.

[0028] 2. Preparation of composite matrix: Mix 82% wheat bran and 18% puffed rice husk evenly according to the dry basis mass ratio (w / w) to obtain the basic composite matrix.

[0029] 3. Addition of nutrients: Add 0.8% yeast powder, 2.5% sorbitol and 1.5% trehalose to the composite matrix by weight percentage, and stir until the components are evenly distributed.

[0030] 4. Matrix condition adjustment: Add pure water to the mixed matrix to control the moisture content of the mixed matrix at 63wt%. After testing, the porosity of the composite matrix at this time is 63.6%.

[0031] 5. Bottling and sterilization: Pour the culture medium into Erlenmeyer flasks, plug the mouth of the flask with a breathable cotton plug, sterilize at 121℃ for 30 minutes, and cool to 24℃ for later use.

[0032] 6. Vaccination procedure: administer approximately 4.5 × 10 5 The addition amount is 1 spore / gram of oven-dry substrate. Under sterile conditions, Aspergillus niger spores are inoculated into the composite substrate and mixed thoroughly.

[0033] 7. Spore culture: Place the inoculated culture medium in a culture environment and control the ambient temperature at 28℃ and humidity at 49% for culture.

[0034] 8. Cultivation process: The substrate was turned over once at 23h and 31h to ensure the substrate's air permeability and heat dissipation. The cultivation was completed when the substrate was fully covered with black spores at 110h.

[0035] Experimental results: After the culture was completed, the spore yield was approximately 1.8 × 10⁶ spores per gram of culture medium. 10 Count / g; spore germination rate 99.0%; strain variation rate 0.35%.

[0036] Example 3: A method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate, the specific steps of which are as follows: 1. Rice husk pretreatment: Crush the rice husks to a particle size of 1.7-1.9mm using a hammer mill, and process them using a high-temperature extrusion process with screw extruder. The parameters are controlled at 125℃, 0.52MPa, and heat and pressure holding for 42s. After cooling to 25℃, they are ready for use.

[0037] 2. Preparation of composite matrix: Mix 83% wheat bran and 7% puffed rice husk evenly according to the dry basis mass ratio (w / w) to obtain the basic composite matrix.

[0038] 3. Addition of nutrients: Add 0.6% yeast powder, 2.6% sorbitol and 1.7% trehalose to the composite matrix and stir until the components are evenly distributed.

[0039] 4. Matrix condition adjustment: Add pure water to the mixed matrix to control the moisture content of the mixed matrix at 62wt%. After testing, the porosity of the composite matrix at this time is 62.5%.

[0040] 5. Bottling and sterilization: Pour the culture medium into Erlenmeyer flasks, plug the mouth of the flask with a breathable cotton plug, sterilize at 121℃ for 30 minutes, and cool to 25℃ for later use.

[0041] 6. Vaccination procedure: Administer approximately 4.7 × 10 5 The addition amount is 1 spore / gram of oven-dry substrate. Under sterile conditions, Aspergillus niger spores are inoculated into the composite substrate and mixed thoroughly.

[0042] 7. Spore culture: Place the inoculated culture medium in a culture environment and control the ambient temperature at 29℃ and humidity at 46% for culture.

[0043] 8. Cultivation process: The substrate was turned over once at 24h and 30h to ensure the substrate's air permeability and heat dissipation. The cultivation was completed when the substrate was fully covered with black spores at 113h.

[0044] Experimental results: After the culture was completed, the spore yield was approximately 1.9 × 10⁻¹¹ per gram of culture medium. 10 Count / g; spore germination rate 99.5%; strain variation rate 0.30%.

[0045] Example 4: A method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate, the specific steps of which are as follows: 1. Rice husk pretreatment: Crush the rice husks to a particle size of 2.0-2.2mm using a hammer mill, and process them using a high-temperature extrusion process with screw extruder. The parameters are controlled at 123℃, 0.56MPa, and heat and pressure holding for 45s. After cooling to 23℃, they are ready for use.

[0046] 2. Preparation of composite matrix: Mix 81% wheat bran and 19% puffed rice husk evenly according to the dry basis mass ratio (w / w) to obtain the basic composite matrix.

[0047] 3. Addition of nutrients: Add 0.5% yeast powder, 2.4% sorbitol and 1.8% trehalose to the composite matrix by weight percentage, and stir until the components are evenly distributed.

[0048] 4. Matrix condition adjustment: Add pure water to the mixed matrix to control the moisture content of the mixed matrix at 65 wt%. After testing, the porosity of the composite matrix at this time is 65.2%.

[0049] 5. Bottling and sterilization: Pour the culture medium into Erlenmeyer flasks, plug the mouth of the flask with a breathable cotton plug, sterilize at 121℃ for 30 minutes, and cool to 23℃ for later use.

[0050] 6. Vaccination procedure: Administer approximately 4.6 × 10 5 The addition amount is 1 spore / gram of oven-dry substrate. Under sterile conditions, Aspergillus niger spores are inoculated into the composite substrate and mixed thoroughly.

[0051] 7. Spore culture: Place the inoculated culture medium in a culture environment and control the ambient temperature at 30℃ and humidity at 50% for culture.

[0052] 8. Cultivation process: The substrate was turned over once at 23h and 31h to ensure the substrate's air permeability and heat dissipation. The cultivation was completed when the substrate was fully covered with black spores at 110h.

[0053] Experimental results: After the culture was completed, the spore yield was approximately 1.9 × 10⁻¹¹ per gram of culture medium. 10 Count / g; spore germination rate 99.3%; strain variation rate 0.29%.

[0054] Example 5: A method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate, the specific steps of which are as follows: 1. Rice husk pretreatment: The rice husks are crushed to a particle size of 2.1-2.3mm using a hammer mill, and then processed using a screw extruder for high-temperature puffing. The parameters are controlled at 126℃, 0.54MPa, and heat and pressure holding for 41s. After cooling to 24℃, they are ready for use.

[0055] 2. Preparation of composite matrix: Mix 82% wheat bran and 18% puffed rice husk evenly according to the dry basis mass ratio (w / w) to obtain the basic composite matrix.

[0056] 3. Addition of nutrients: Add 0.7% yeast powder, 2.7% sorbitol and 1.5% trehalose to the composite matrix by weight percentage, and stir until the components are evenly distributed.

[0057] 4. Matrix condition adjustment: Add pure water to the mixed matrix to control the moisture content of the mixed matrix at 62wt%. After testing, the porosity of the composite matrix at this time is 64.7%.

[0058] 5. Bottling and sterilization: Pour the culture medium into Erlenmeyer flasks, plug the mouth of the flask with a breathable cotton plug, sterilize at 121℃ for 30 minutes, and cool to 25℃ for later use.

[0059] 6. Vaccination procedure: Administer approximately 4.6 × 10 5 The addition amount is 1 spore / gram of oven-dry substrate. Under sterile conditions, Aspergillus niger spores are inoculated into the composite substrate and mixed thoroughly.

[0060] 7. Spore culture: Place the inoculated culture medium in a culture environment and control the ambient temperature at 28℃ and humidity at 51% for culture.

[0061] 8. Cultivation process: The substrate was turned over once at 22h and 32h to ensure the substrate's air permeability and heat dissipation. The cultivation was completed when the substrate was fully covered with black spores at 112h.

[0062] Experimental results: After the culture was completed, the spore yield was approximately 1.8 × 10⁶ spores per gram of culture medium. 10 Count / g; spore germination rate 99.2%; strain variation rate 0.31%.

[0063] Comparative Example 1: The method for culturing Aspergillus niger spores on a single bran substrate, the specific steps of which are as follows: 1. Culture substrate preparation: Pure wheat bran was used as the culture substrate, and water was added to control the moisture content at 64%. The test showed that the porosity of the substrate was 35.2%.

[0064] 2. Bottling and sterilization: Pour the culture medium into Erlenmeyer flasks, plug the mouth of the flask with a breathable cotton plug, sterilize at 121℃ for 30 minutes, and cool to 25℃ for later use.

[0065] 3. Vaccination procedure: Administer approximately 4.8 × 10 5 The amount of spores per gram of dry substrate was determined by introducing Aspergillus niger spores into the composite substrate under sterile conditions and then mixing thoroughly.

[0066] 4. Spore culture: Place the inoculated culture medium in a culture environment and control the ambient temperature at 29℃ and humidity at 47% for culture.

[0067] 5. Cultivation process: The substrate was turned over once at 16h, 24h, 32h, 40h, 48h and 56h to ensure the substrate's air permeability and heat dissipation. The cultivation was completed when the substrate was fully covered with black spores at 124h.

[0068] After the culture was completed, the spore yield was measured to be approximately 1.2 × 10⁻⁶. 10 Count / g; spore germination rate 95.2%; strain variation rate 0.70%.

[0069] Comparative Example 2: The method for culturing Aspergillus niger spores in a wheat bran-straw substrate is as follows: 1. Preparation of composite substrate: Mix 83% wheat bran and 17% straw evenly according to the dry weight ratio (w / w) to obtain the composite substrate.

[0070] 2. Matrix condition adjustment: Water was added to the mixed matrix to control the moisture content at 63%, and the test showed that the matrix porosity was 37.6%.

[0071] 3. Bottling and sterilization: Pour the culture medium into Erlenmeyer flasks, plug the mouth of the flask with a breathable cotton plug, sterilize at 121℃ for 30 minutes, and cool to 24℃ for later use.

[0072] 4. Vaccination procedure: Administer approximately 4.7 × 10 5 The amount of spores per gram of dry substrate was determined by introducing Aspergillus niger spores into the composite substrate under sterile conditions and then mixing thoroughly.

[0073] 5. Spore culture: Place the inoculated culture medium in a culture environment and control the ambient temperature at 30℃ and humidity at 51% for culture.

[0074] 6. Cultivation process: The substrate was turned over once at 16h, 24h, 32h, 40h, 48h and 56h to ensure the substrate's air permeability and heat dissipation. After 120h of cultivation, the substrate was fully covered with black spores and the cultivation was completed.

[0075] After the culture was completed, the spore yield was measured to be 1.3 × 10⁻⁶. 10 Count / g; spore germination rate 96.1%; strain variation rate 0.75%.

[0076] Table 1 below shows a comparison of the matrix porosity of the comparative examples and embodiments. Table 1. Comparison of Porosity between Comparative Examples and Embodiments It is evident that the culture media of Comparative Examples 1 and 2 have low porosity and poor aeration. During the culture process, hypoxia and excessively high local temperatures lead to a high rate of bacterial mutation.

[0077] Experimental Example The oxygen content in the culture media of the examples and comparative examples was detected using a smart handheld pump-type oxygen detector, the carbon dioxide content was detected using a portable carbon dioxide detector, and the temperature of the culture media was measured using a platinum resistance thermometer. The results are shown below: Table 2 Comparison of oxygen content in the culture medium during the culture processes of the examples and comparative examples (%) Comparing the above table: (1) In the embodiment of the present invention, the oxygen concentration was stable at 15.8%-20.1% throughout the culture process without obvious troughs, while the oxygen concentration in the comparative example was 10.3%-20.1% throughout the culture process, with a trough in oxygen concentration at 32-40h. The dissolved oxygen was lowest at 32h. Overall, the oxygen content in the embodiment was 47.6%-56.3% higher than that in the comparative example at 32h, which ensured the oxygen demand for spore growth.

[0078] (2) The culture of the example can be completed in 110-113h, which is 11-14h earlier than Comparative Example 1 (124h) and 7-10h earlier than Comparative Example 2 (120h), and the culture efficiency is significantly improved.

[0079] In summary, the puffed rice husk in this application can significantly improve the porosity and air permeability of the substrate, providing a continuous and sufficient oxygen environment for the growth of Aspergillus niger spores, thereby shortening the culture cycle, ensuring the uniformity of spore growth, and comprehensively improving culture efficiency and quality.

[0080] Table 3 Comparison of carbon dioxide content in the culture medium during the culture processes of the examples and comparative examples (%) By comparing the above table: (1) The peak carbon dioxide concentration in the entire culture process of the example was 7.36%, which was much lower than the peak of 14.01 in the comparative example, and the peak concentration was reduced by 47.4%.

[0081] (2) The carbon dioxide concentration of the control group reached its peak at 32 h of culture and then gradually decreased thereafter, while the peak of the control group occurred at 24 h, entering the concentration decline stage 8 h earlier than the control group.

[0082] In summary, puffed rice husks significantly increase the porosity of the substrate, which can accelerate the carbon dioxide expulsion rate, avoid local carbon dioxide enrichment during the cultivation process, create a better gaseous environment for Aspergillus niger spore growth, and thus shorten the cultivation cycle and improve the quality of spore cultivation.

[0083] Table 4 Comparison of culture substrate temperature during the cultivation process of the examples and comparative examples The results in the table above show that the temperature rise within the substrate of the examples was slightly lower than that of the comparative examples compared to the ambient temperature. During the 32-hour peak temperature period, the comparative example 1 in the 29℃ environment group exceeded the ambient temperature by 2.2℃, while Examples 1 and 3 exceeded by only 2.0℃ and 1.9℃, respectively. In the 30℃ environment group, the comparative example 2 exceeded the ambient temperature by 2.1℃, while Example 4 exceeded by only 2.0℃. The temperature rise of Examples 2 and 5 in the 28℃ environment group was also relatively low. Furthermore, the examples only required two photographs during the cultivation process, while the comparative examples required six photographs. However, the heat dissipation effect was consistent, with both achieving a stable temperature drop from the peak to near the ambient temperature. The examples completed cultivation earlier, indicating that the "expanded rice husk + bran" substrate of this invention has superior heat dissipation performance with fewer photographs.

[0084] The core data of Aspergillus niger spores from the combined examples and comparative examples are shown in the table below: Table 5 Comparison of Aspergillus niger spore culture indices between the examples and comparative examples Based on the comparison of the core cultivation indicators in the table above, the technical advantages of this invention are as follows: (1) Porosity: The porosity of the composite matrix of the present invention reaches 62.5%-65.2%, which is much higher than 35.2% of Comparative Example 1 and 37.6% of Comparative Example 2, and is 66.2%-85.2% higher than the comparative examples, providing a solid structural basis for gas exchange and heat dissipation; (2) Number of times of photo taking: The number of times of photo taking during the cultivation process has been reduced from 6 times in the existing technology to 2 times, a reduction of 66.6%, which greatly reduces the intensity of manual labor and reduces the risk of contamination by miscellaneous bacteria caused by frequent operation; (3) Culture cycle: The culture cycle was shortened to 110-113h, which is 11-14h shorter than Comparative Example 1 (124h) and 7-10h shorter than Comparative Example 2 (120h), with a shortening rate of 5.8%-11.2%, which significantly improved industrial production efficiency. (4) Spore yield: Spore yield reached 1.8-1.9×10 10 The number of cells / g was increased by 50.0%-58.3% compared to Comparative Example 1 and by 38.4%-46.1% compared to Comparative Example 2, which significantly improved the strain production efficiency; (5) Spore germination rate: The spore germination rate was stable at 99.0%-99.5%, which was 3.9%-4.5% higher than that of control sample 1 (95.2%) and 3.0%-3.5% higher than that of control sample 2 (96.1%), ensuring the high activity of the strain; (6) Strain variation rate: The strain variation rate is as low as 0.29%-0.35%, which is 50.0%-58.5% lower than that of control sample 1 (0.70%) and 53.3%-61.3% lower than that of control sample 2 (0.75%), effectively reducing the risk of strain degradation and ensuring the stability of fermentation production.

[0085] In summary, this invention, through the modification of puffed rice husks and the precise compounding of wheat bran, combined with the synergistic optimization of nutrient components, achieves a comprehensive improvement in air permeability, heat dissipation, operability, yield, activity, and stability during the cultivation of Aspergillus niger spores. The overall technical effect is significantly better than that of existing technologies.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any equivalent substitutions or modifications made by those skilled in the art within the principles of the present invention without any creative effort should be included within the protection scope of the present invention.

Claims

1. A method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate, characterized in that, The specific steps are as follows: (1) Rice husk pretreatment: The rice husk is crushed to a particle size of 1-3 mm by a crusher, and then processed by a high-temperature extrusion process using a screw extruder. The process parameters are as follows: 120-130℃, 0.5-0.6MPa, heat preservation and pressure holding for 30-50s, and then cooled to 20-30℃ to obtain extruded rice husk. (2) Preparation of composite matrix: According to the dry basis mass percentage, the puffed rice husk and wheat bran are mixed evenly according to the ratio of wheat bran accounting for 80-85% and puffed rice husk accounting for 15-20% in the composite matrix to obtain the basic composite matrix; (3) Addition of nutrients: Add 0.5-1.0% yeast powder, 2.0-3.0% sorbitol and 1.5-2.0% trehalose by weight percentage to the composite matrix obtained in the previous step, and stir well. (4) Adjustment of substrate conditions: Add purified water to the mixed substrate to control the moisture content of the mixed substrate at 60-65 wt%; (5) Bottling and sterilization: The culture medium after the above treatment is put into Erlenmeyer flasks and sterilized at 121℃ for 30 min, and then cooled to 20℃-30℃ for use. (6) Inoculation procedure: 4.5-5.0 × 10 5 The inoculation rate was 1 spore / gram of oven-dry substrate. Aseptically, Aspergillus niger spores were introduced into the composite substrate and mixed thoroughly. (7) Spore culture: Place the inoculated culture medium in a culture environment with an ambient temperature of 28-30℃ and a humidity of 45-55% for a total culture period of 110-115h. (8) Cultivation process: Turn the food over once at 22-24h and once at 30-32h, and maintain the above fermentation conditions until the end of the cultivation cycle.

2. The method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate according to claim 1, characterized in that, In step (1), the rice husk is crushed to a particle size of 2.0-2.2 mm, and the high-temperature puffing parameters are controlled at 123℃, 0.56MPa, and heat preservation and pressure holding for 45s.

3. The method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate according to claim 1, characterized in that, In step (2), the composite matrix contains 81 wt% wheat bran and 19 wt% expanded rice husk.

4. The method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate according to claim 1, characterized in that, In step (3), the amount of nutrients added is 0.5% yeast powder, 2.4% sorbitol and 1.8% trehalose by weight percentage.

5. The method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate according to claim 1, characterized in that, In step (4), the moisture content of the mixed matrix is ​​controlled at 65 wt%.

6. The method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate according to claim 1, characterized in that, The inoculation amount in step (6) is 4.6 × 10⁻⁶. 5 1 spore / gram of dry substrate.

7. The method for culturing Aspergillus niger spores using a highly permeable expanded rice husk-wheat bran composite substrate according to claim 1, characterized in that, In steps (7) and (8), the spore culture environment temperature is 30℃ and the humidity is 50%. The substrate is turned over once at 23h and 31h respectively, and the culture is completed at 110h.

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