Application and method of b. subtili fermenting co-production of bc and gamma-pga

Abstract

The application belongs to the technical field of microbial fermentation, and particularly relates to an application and a method for preparing bacterial cellulose and gamma-polyglutamic acid based on fermentation of Brevibacillus brevis. Brevibacillus centrosporus The ZF-9 is originally verified as a bacterial cellulose producing strain, and is fermented by using sweet potato dry wastewater as a carbon source, so that the bacterial cellulose and the gamma-polyglutamic acid can be co-produced, the COD digestion rate of the sweet potato dry wastewater is high, the sweet potato dry wastewater can be treated at the same time, a new industrial use of the Brevibacillus brevis ZF-9 is provided, and comprehensive utilization of resources is realized.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to an application and method for the co-production of bacterial cellulose (BC) and γ-polyglutamic acid (γ-PGA) based on Bacillus mesosporus fermentation. Background Technology

[0002] Bacterial cellulose (BC) is a natural polymer material composed of extracellular polysaccharides secreted by various types of bacteria. It possesses excellent properties such as good biocompatibility, biodegradability, strong water-holding capacity, and high mechanical properties. It exhibits superior performance in adhesion, skin elasticity, softness, and moisturizing effects, and is considered a novel environmentally friendly biomaterial. Compared to traditional plant fibers, bacterial cellulose has advantages such as high crystallinity, high water-holding capacity, high Young's modulus, high purity, and excellent biocompatibility. Bacterial cellulose has broad applications in biomedical materials such as medical dressings, food, papermaking, and textiles.

[0003] γ-polyglutamic acid (γ-PGA) is a special anionic extracellular homopolymer synthesized by microbial fermentation. It is a class of homopolymeric amino acids composed of glutamic acid monomers linked by amide bonds. Its side chains have numerous carboxyl groups, giving it properties such as water absorption, moisture retention, and element chelation. It has broad application prospects in daily chemicals, food, environmental protection, and agriculture. To date, the γ-PGA-producing strains discovered are mainly concentrated in… B. subtilis and B. lichemiformis Based on whether the culture medium requires the provision of glutamate precursors, γ-PGA-producing bacteria can be divided into two categories: glutamate-dependent (Category I) and glutamate-independent (Category II); among them, Category I includes... B. licheniformis ATCC 9945A B. subtilis IFO 3335 B. subtilis Strains such as NX-2 typically produce large amounts of γ-PGA, requiring the culture medium to provide glutamate precursors as a precursor or inducer for γ-PGA synthesis; while Class II strains include... B. methylotrophicus SK19.001 B. subtilis C10 and other strains synthesize γ-PGA de novo via a non-specific glutamate precursor pathway. Because class II strains do not require the addition of glutamate, significantly reducing production costs, they are currently a hot topic in polyglutamate research and represent a future development trend.

[0004] Sweet potatoes are a common food, and their chemical composition mainly includes water, starch, protein, fat, fiber, and ash. Traditional sweet potato chip production processes mainly include soaking and slicing, crystallization, and frying. Wastewater generated during the sweet potato chip production process mainly occurs in washing, cooking, and dehydration steps, and its main components are water-soluble starch, soluble protein, polysaccharides, amino acids, vitamins, and various organic and inorganic substances such as inorganic salts. The quality and composition of wastewater are generally similar across different sweet potato chip production processes. Sweet potato chip production wastewater (SPW) is classified as high-concentration food wastewater, characterized by a low pH and low COD. Cr The wastewater from sweet potato chip production in China is characterized by high BOD5 and animal / vegetable oil concentrations, indicating good biodegradability. Currently, the main methods for treating wastewater from sweet potato chip production in China are physicochemical and biological methods. Physicochemical processes such as coagulation sedimentation, physical adsorption, and electrochemical methods can achieve effluent standards, but these processes suffer from high energy consumption and operating costs, making them less feasible in practical engineering projects. Therefore, biological methods are currently the primary method for treating wastewater from sweet potato chip production.

[0005] There is a growing interest in developing a process for the comprehensive utilization of sweet potato wastewater through fermentation to co-produce bacterial cellulose and γ-polyglutamic acid, particularly a non-glutamic acid-dependent method for co-producing γ-polyglutamic acid. This would be significant for improving raw material utilization and reducing production costs. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide an application of Bacillus mesporus for the co-production of BC and γ-PGA through fermentation, using screened Bacillus mesporus for the co-production of BC and γ-PGA. The second technical problem to be solved by the present invention is to provide a method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus.

[0007] To solve the above-mentioned technical problems, the present invention provides an application of *Bacillus mesporus* fermentation for the co-production of BC and γ-PGA, wherein the *Bacillus mesporus* is *Bacillus mesporus* ZF-9, and its classification name is *Bacillus mesporus*. Brevibacillus centrosporus It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 6267.

[0008] The present invention also discloses a method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus brevis, comprising the step of inoculating Bacillus mesporus brevis into a fermentation culture ZF-9 medium containing sweet potato wastewater and free of glutamic acid for fermentation culture. The aforementioned *Bacillus mesporus* is *Bacillus mesporus* ZF-9, and its classification name is... Brevibacillus centrosporusIt has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 6267.

[0009] Specifically, in the method for co-producing BC and γ-PGA by fermentation of *Bacillus mesporus*, the fermentation medium comprises the following components in the indicated mass concentrations: 80-120 mL / L of dried sweet potato wastewater, 20-50 g / L of carbon source, 10-30 g / L of nitrogen source, and pH adjusted to 6.5-7.5.

[0010] Specifically, the method for co-producing BC and γ-PGA by fermentation of Bacillus brevis includes the following conditions for the fermentation culture step: controlling the rotation speed at 100-150 rpm and fermenting at 25-32℃ for 5-8 days.

[0011] Specifically, the method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus includes a step of inoculating the Bacillus mesporus into a seed culture medium for seed liquid culture; The seed culture medium comprises the following components in the indicated mass percentages: 50-80 mL / L of dried sweet potato wastewater, 10-30 g / L of carbon source, 10-20 g / L of nitrogen source, and pH adjusted to 6.5-7.5.

[0012] Specifically, in the method for co-producing BC and γ-PGA by fermentation of Bacillus brevis, the conditions for the seed culture step include: controlling the rotation speed at 80-120 rpm and culturing the seed culture at 25-32℃ for 1-2 days.

[0013] Specifically, the method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus includes a step of activating Bacillus mesporus by inoculating it into an agar slant culture medium; The slant culture medium comprises the following components in the indicated mass amounts: carbon source 10-30 g / L, nitrogen source 10-20 g / L, agar 15-25 g / L, and pH adjusted to 6.5-7.5.

[0014] Specifically, in the method for co-producing BC and γ-PGA by fermentation of Bacillus brevis, the conditions for the activation step of the slant culture medium include: constant temperature incubation at 25-32℃ for 18-30h.

[0015] Specifically, in the method for co-producing BC and γ-PGA by fermentation of *Bacillus mesporus*, the fermentation medium, seed culture medium, or slant culture medium contains: The carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol, or starch hydrolysate; and / or, The nitrogen source is selected from one or more of the following: beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal powder, urea, (NH4)2SO4, or NH4Cl.

[0016] Specifically, the method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus includes a step of collecting the fermentation products for separation and purification of bacterial cellulose and γ-polyglutamic acid.

[0017] This invention utilizes previously screened strains to develop novel fermentation applications. The *Bacillus mesporus* strain mentioned above... Brevibacillus centrosporus Based on its previously verified role as a bacterial cellulose-producing bacterium, ZF-9 can co-produce bacterial cellulose and γ-polyglutamic acid by fermenting sweet potato drying wastewater as a carbon source. Furthermore, the sweet potato drying wastewater has a high COD digestion rate, which can simultaneously treat the wastewater. This provides a new industrial application for the Bacillus brevis of Mesospora ZF-9 and achieves comprehensive resource utilization.

[0018] This invention relates to the co-production of bacterial cellulose and γ-polyglutamic acid (PGA) using *Bacillus brevis* ZF-9. This strain eliminates the need for exogenous glutamic acid addition during fermentation, significantly reducing costs and offering a simple and convenient operation. The method described in this invention for co-producing BC and γ-PGA using *Bacillus brevis* fermentation accumulates 13.56-20.62 g / L of γ-polyglutamic acid and 4.21-5.45 g / L of bacterial cellulose in a medium without added glutamic acid precursors. This method is not only simple to operate but also has low production costs. Attached Figure Description

[0019] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 The results are from the liquid chromatography of γ-PGA prepared in Example 1; Figure 2 Scanning electron microscope image of the BC product prepared in Example 1; Figure 3 X-ray diffraction pattern of the BC product prepared in Example 1; Figure 4 Infrared spectrum of the BC product prepared in Example 1; Figure 5 The mechanical properties of the BC membrane prepared in Example 1 are shown in the figure. Detailed Implementation

[0020] In the following embodiments of the present invention, a co-fermentation process for bacterial cellulose (BC) and γ-polyglutamic acid (γ-PGA) is carried out based on the Bacillus brevis ZF-9 strain previously screened by the applicant.

[0021] In the following embodiments of the present invention, the *Bacillus brevis* ZF-9 is classified as *Bacillus brevis*. Brevibacillus centrosporus It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 6267, deposited on June 21, 2012, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0022] The colony morphology, physiological and biochemical characteristics, and nutritional characteristics of *Bacillus brevis* ZF-9 described in this invention have been recorded in Chinese patents CN103074288A and CN109529092A. In Chinese patent CN109529092A, *Bacillus brevis* ZF-9 has been shown to have the ability and effect of fermenting to produce bacterial cellulose. In the following embodiments of this invention, using sweet potato dried wastewater as a carbon source, *Bacillus brevis* ZF-9 can not only ferment to produce bacterial cellulose membranes but also obtain a small amount of γ-polyglutamic acid (γ-PGA).

[0023] In the following embodiments of the present invention, the strain is referred to as Bacillus brevis ZF-9 or ZF-9.

[0024] In the following embodiments of the present invention, the activation of Bacillus brevis ZF-9 is carried out using a conventional slant culture medium activation method, namely a solid slant culture medium containing a carbon source, a nitrogen source and agar, which is conventional in the art.

[0025] In the following embodiments of the present invention, as an exemplary slant culture medium, the slant culture medium may include the following components in the indicated mass amounts: carbon source 10-30 g / L, nitrogen source 10-20 g / L, agar 15-25 g / L, adjusted to pH 6.5-7.5. The preparation method is as described in conventional practices in the art. As an exemplary embodiment, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol, or starch hydrolysate; the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal, cottonseed meal, urea, (NH4)2SO4, or NH4Cl; the inorganic salt is selected from one or more of sodium salt, phosphate, and dihydrogen phosphate. The slant culture medium with the above-mentioned combinations of carbon source, nitrogen source, and inorganic salt exhibits essentially similar activation performance for *Bacillus brevis* ZF-9, with no significant differences.

[0026] In the following embodiments of the present invention, as an exemplary activation scheme for slant culture medium, the activation conditions of the slant culture medium include: constant temperature incubation at 25-32℃ for 18-30h. Those skilled in the art are capable of making adaptive adjustments to the activation parameters based on the experimental results.

[0027] In the following embodiments of the present invention, the seed culture step of *Bacillus brevis* ZF-9 can be prepared by adding sweet potato wastewater to a conventional seed culture medium. For example, a conventional liquid culture medium containing carbon and nitrogen sources. It should be noted that the purpose of the seed culture step of *Bacillus brevis* ZF-9 in the present invention is to amplify *Bacillus brevis* ZF-9.

[0028] In the following embodiments of the present invention, as an exemplary seed culture medium, the seed culture medium comprises the following components in the indicated mass amounts: 50-80 mL / L of dried sweet potato wastewater, 10-30 g / L of carbon source, and 10-20 g / L of nitrogen source, adjusted to pH 6.5-7.5; the preparation method is in accordance with conventional methods in the art. As an exemplary embodiment, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol, or starch hydrolysate; the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal powder, urea, (NH4)2SO4, or NH4Cl; the inorganic salt is selected from one or more of sodium salt, phosphate, and dihydrogen phosphate. The seed culture medium with the above-mentioned combination of carbon source, nitrogen source, and inorganic salt exhibits essentially similar amplification effects on the seed culture of *Bacillus brevis* ZF-9, with no significant differences.

[0029] In the following embodiments of the present invention, the seed culture method can employ the conventional shaking culture method in the art. As an exemplary seed culture amplification scheme, the conditions for the shaking seed culture step may include: controlling the rotation speed at 80-120 rpm and culturing the seed culture at 25-32℃ for 1-2 days. Those skilled in the art are capable of adaptively adjusting the seed culture amplification parameters based on experimental results.

[0030] In the following embodiments of the present invention, the fermentation culture step of *Bacillus brevis* ZF-9 can be prepared by adding sweet potato wastewater to a conventional fermentation culture medium. For example, a conventional liquid culture medium containing carbon and nitrogen sources.

[0031] In the following embodiments of the present invention, as an exemplary fermentation culture medium, the fermentation culture medium comprises the following components in the indicated mass amounts: 80-120 mL / L of dried sweet potato wastewater, 20-50 g / L of carbon source, and 10-30 g / L of nitrogen source, adjusted to pH 6.5-7.5; the preparation method is in accordance with conventional methods in the art. As an exemplary embodiment, the carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol, or starch hydrolysate; the nitrogen source is selected from one or more of beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal powder, urea, (NH4)2SO4, or NH4Cl; the inorganic salt is selected from one or more of sodium salts, phosphates, and dihydrogen phosphates.

[0032] Regarding the fermentation process described in this invention, it should be noted that the fermentation process can employ conventional shaking fermentation. In the following embodiments of this invention, as an exemplary seed culture amplification scheme, the conditions for the shaking fermentation culture step may include: controlling the rotation speed at 100-150 rpm and fermenting at 25-32℃ for 5-8 days. Those skilled in the art are capable of adapting the fermentation process parameters according to the product yield and other characteristics.

[0033] In the following embodiments of the present invention, the steps for separating, extracting, and purifying bacterial cellulose from the fermentation broth are the same as those in existing technologies. For example, the extraction method of bacterial cellulose from the fermentation broth described in Chinese patents CN102994430A and CN109529092A involves separating the bacterial cellulose from the fermentation broth into a gel-like solid state after fermentation. The solid is the bacterial cellulose gel, and the liquid is the fermentation waste liquid. The bacterial cellulose gel is removed and rinsed multiple times with distilled water to remove residual bacteria and culture medium. The bacterial cellulose gel is then soaked in 0.1-1 mol / L NaOH solution and kept at 60-100℃ for 30-90 minutes, followed by repeated rinsing with deionized water. At this point, the gel is milky white and translucent. The bacterial cellulose gel is then dried at 60-80℃ to constant weight to obtain bacterial cellulose.

[0034] In the following embodiments of the present invention, the detection of γ-PGA content in the fermentation broth can be performed using existing methods. For example, the γ-PGA detection method described in Chinese Patent CN106047780A can be used.

[0035] In the following embodiments of the present invention, it is mainly used to cultivate and co-produce bacterial cellulose and γ-PGA based on the said Bacillus brevis ZF-9. The composition system of the slant culture medium, seed culture medium and fermentation culture medium involved is only an exemplary implementation.

[0036] In the following embodiments of the present invention, the exemplary slant culture medium, seed culture medium, and fermentation culture medium include: Slant culture medium A: glucose 20 g / L, yeast extract 15 g / L, agar 20 g / L, adjusted to pH 7.0; Slant culture medium B: sucrose 10g / L, peptone 20g / L, agar 20g / L, adjusted to pH 6.5; Slant culture medium C: molasses 30 g / L, corn steep liquor 10 g / L, agar 20 g / L, adjusted to pH 7.5; Seed culture medium A: 60 mL / L sweet potato dried wastewater, 15 g / L glucose, 15 g / L yeast extract, adjusted to pH 7.2; Seed culture medium B: 50 mL / L sweet potato dried wastewater, 5 g / L maltose, 5 g / L glucose, 10 g / L beef extract, 10 g / L soybean meal powder, adjusted to pH 6.5; Seed culture medium C: 80 mL / L sweet potato dried wastewater, 10 g / L molasses, 10 g / L starch hydrolysate, 10 g / L fructose, 10 g / L peptone, adjusted to pH 7.5; Fermentation medium A: 95 mL / L sweet potato wastewater, 37 g / L glucose, 10 g / L yeast extract, adjusted to pH 7.0; Fermentation medium B: 80 mL / L sweet potato wastewater, 20 g / L fructose, 20 g / L starch hydrolysate, 10 g / L glucose, 20 g / L beef extract, 20 g / L NH4Cl, 10 g / L cottonseed meal powder, adjusted to pH 7.5; Fermentation medium C: 120 mL / L sweet potato wastewater, 40 g / L glucose, 30 g / L maltose, 30 g / L anhydrous ethanol, 10 g / L (NH4)2SO4, 20 g / L yeast extract, adjusted to pH 7.5.

[0037] The above-mentioned slant culture medium, seed culture medium and fermentation culture medium can be sterilized by conventional high-pressure sterilization, for example, sterilization at 121℃ for 10-30 minutes.

[0038] In the following embodiments of the present invention, as an exemplary application, the selected sweet potato drying wastewater is wastewater generated during the washing, steaming, and dehydration processes of sweet potato drying. Testing revealed that its main components include: water-soluble starch, soluble protein, polysaccharides, amino acids, vitamins, and inorganic salts, with a pH value of approximately 3.2 and a COD of... Cr It is approximately 12000 mg / L, and BOD5 is approximately 4500 mg / L. Example 1 The preserved Bacillus brevis ZF-9 was streaked into the slant culture medium A and incubated at 28°C for 24 hours.

[0039] Pick one loopful of the above slant culture and inoculate it into a 250ml Erlenmeyer flask containing 40ml of seed culture medium A. Incubate at 28℃ with shaking at 100r / min for 2 days.

[0040] Take the above-cultured seed liquid and inoculate it into a 500ml Erlenmeyer flask containing 100ml of fermentation medium A at a 5% inoculation rate. Incubate at 28℃ and 120rpm for 6 days with shaking fermentation, then stop fermentation.

[0041] The fermentation broth after fermentation was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was determined to be 468.3 g / L. The bacterial cellulose gel was then rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, and kept at 80℃ for 60 min. After rinsing three times with deionized water, the bacterial cellulose gel was dried at 80℃ to constant weight and weighed using a balance. The yield (dry weight) of the bacterial cellulose gel was found to be 4.21 g / L.

[0042] γ-PGA content determination: After fermentation, γ-polyglutamic acid can be extracted from the fermentation broth after removing the bacterial cells. After processing, the molecular weight and yield of γ-polyglutamic acid in the fermentation broth are determined by high-performance liquid chromatography (HPLC). The HPLC results are shown in the attached figure. Figure 1 As shown, its content was determined to be 13.56 g / L.

[0043] The fermentation broth was adjusted to pH 2.8-3.5 with concentrated hydrochloric acid, centrifuged at 9000 rpm for 30 min to remove the bacterial cells, and the supernatant was added with 2-5 times the volume of ethanol. The precipitate was reconstituted with water, dialyzed to remove small molecules, and the filtrate was freeze-dried to obtain crude γ-PGA. Example 2

[0044] The preserved Bacillus brevis ZF-9 was streaked into the slant culture medium A and incubated at 28°C for 24 hours.

[0045] Pick one loopful of the above slant culture and inoculate it into a 250ml Erlenmeyer flask containing 40ml of seed culture medium A, and incubate it statically at 28℃ for 2 days.

[0046] Take the above-cultured seed liquid and inoculate it into a 500ml Erlenmeyer flask containing 100ml of fermentation medium A at a 5% inoculation rate. Incubate the flask at 28℃ for 6 days and then stop the fermentation.

[0047] The fermentation broth after fermentation was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was determined to be 516.2 g / L. The bacterial cellulose gel was then rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, and kept at 80℃ for 60 min. After rinsing three times with deionized water, the bacterial cellulose gel was dried at 80℃ to constant weight and weighed using a balance. The yield (dry weight) of the bacterial cellulose gel was found to be 4.67 g / L.

[0048] γ-PGA content determination: After fermentation, γ-polyglutamic acid can be extracted from the fermentation broth after removing the bacterial cells. After processing, the molecular weight and yield of γ-polyglutamic acid in the fermentation broth were detected by high performance liquid chromatography, and its content was determined to be 17.61 g / L. Example 3

[0049] The preserved Bacillus brevis ZF-9 was streaked into the slant culture medium C and incubated at 28°C for 24 hours.

[0050] Pick one loopful of the above slant culture and inoculate it into a 500ml Erlenmeyer flask containing 100ml of fermentation medium B. Incubate at 28℃ and 120rpm for 6 days with shaking fermentation, then stop fermentation.

[0051] The fermentation broth after fermentation was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was determined to be 481.9 g / L. The bacterial cellulose gel was then rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, and kept at 80℃ for 60 min. After rinsing three times with deionized water, the bacterial cellulose gel was dried at 80℃ to constant weight and weighed using a balance. The yield (dry weight) of the bacterial cellulose gel was found to be 4.31 g / L.

[0052] γ-PGA content determination: After fermentation, γ-polyglutamic acid can be extracted from the fermentation broth after removing the bacterial cells. After processing, the molecular weight and yield of γ-polyglutamic acid in the fermentation broth were detected by high performance liquid chromatography, and its content was determined to be 20.62 g / L. Example 4 The preserved Bacillus brevis ZF-9 was streaked into the slant culture medium B and incubated at 28°C for 24 hours.

[0053] Pick one loopful of the above slant culture and inoculate it into a 500ml Erlenmeyer flask containing 100ml of fermentation medium C. Incubate the flask at 28℃ for 6 days and then stop fermentation.

[0054] The fermentation broth after fermentation was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was determined to be 553.9 g / L. The bacterial cellulose gel was then rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, and kept at 80℃ for 60 min. After rinsing three times with deionized water, the bacterial cellulose gel was dried at 80℃ to constant weight and weighed using a balance. The yield (dry weight) of the bacterial cellulose gel was found to be 5.45 g / L.

[0055] γ-PGA content determination: After fermentation, γ-polyglutamic acid can be extracted from the fermentation broth after removing the bacterial cells. After processing, the molecular weight and yield of γ-polyglutamic acid in the fermentation broth were detected by high performance liquid chromatography, and its content was determined to be 17.69 g / L. Example 5

[0056] The preserved Bacillus brevis ZF-9 was streaked into the slant culture medium B and incubated at 25°C for 30 hours.

[0057] Pick one loopful of the above slant culture and inoculate it into a 250ml Erlenmeyer flask containing 40ml of seed culture medium B. Incubate at 25℃ with shaking at 120r / min for 2 days.

[0058] Take the above-cultured seed liquid and inoculate it into a 500ml Erlenmeyer flask containing 100ml of fermentation medium B at an inoculation rate of 5%. Incubate the flask at 25℃ and 100rpm for 8 days with shaking fermentation, and then stop fermentation.

[0059] The fermentation broth after fermentation was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was determined to be 510.1 g / L. The bacterial cellulose gel was then rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, and kept at 80℃ for 60 min. After rinsing three times with deionized water, the bacterial cellulose gel was dried at 80℃ to constant weight and weighed using a balance. The yield (dry weight) of the bacterial cellulose gel was found to be 4.64 g / L.

[0060] γ-PGA content determination: After fermentation, γ-polyglutamic acid can be extracted from the fermentation broth after removing the bacterial cells. After processing, the molecular weight and yield of γ-polyglutamic acid in the fermentation broth were detected by high performance liquid chromatography, and its content was determined to be 15.73 g / L. Example 6 The preserved Bacillus brevis ZF-9 was streaked into the slant culture medium C and incubated at 32°C for 18 hours.

[0061] Pick one loopful of the above slant culture and inoculate it into a 250ml Erlenmeyer flask containing 40ml of seed culture medium C, and incubate it at 32℃ for 1 day.

[0062] Take the above-cultured seed liquid and inoculate it into a 500ml Erlenmeyer flask containing 100ml of fermentation medium C at an inoculation rate of 5%. Incubate the flask at 32℃ for 7 days and then stop the fermentation.

[0063] The fermentation broth after fermentation was subjected to solid-liquid separation, and the bacterial cellulose gel was collected. The weight (wet weight) of the bacterial cellulose was determined to be 492.2 g / L. The bacterial cellulose gel was then rinsed three times with distilled water, immersed in a 0.8 mol / L NaOH solution, and kept at 80℃ for 60 min. After rinsing three times with deionized water, the bacterial cellulose gel was dried at 80℃ to constant weight and weighed using a balance. The yield (dry weight) of the bacterial cellulose gel was found to be 4.88 g / L.

[0064] γ-PGA content determination: After fermentation, γ-polyglutamic acid can be extracted from the fermentation broth after removing the bacterial cells. After processing, the molecular weight and yield of γ-polyglutamic acid in the fermentation broth were detected by high performance liquid chromatography, and its content was determined to be 19.82 g / L. Experimental Example

[0065] In the following experimental examples of the present invention, based on the BC product prepared in Example 1, performance analyses such as SEM, X-ray diffraction, infrared spectroscopy, and mechanical properties were performed to investigate the performance of BC produced from sweet potato wastewater.

[0066] 1. BC scanning electron microscope image The scanning electron microscope image of the BC product prepared in Example 1 is attached. Figure 2 As shown.

[0067] It is evident that there is no significant difference between the BC membrane prepared by fermentation using sweet potato wastewater as a carbon source and the BC membrane produced using glucose as a substrate.

[0068] 2. X-ray diffraction analysis diagram The X-ray diffraction pattern of the BC product prepared in Example 1 is attached. Figure 3 As shown.

[0069] Appendix Figure 3 The results shown are X-ray diffraction patterns with diffraction angle as the abscissa and diffraction intensity as the ordinate, using the diffraction peaks of the (1̅01), (101), and (002) planes of the unit cell as the calculation basis. It can be seen that the BC film exhibits diffraction peaks of the characteristic crystal planes of BC (1̅01), (101), and (002) at diffraction angles of 14.5°, 16.8°, and 22.6°.

[0070] 3. Infrared spectroscopy analysis Because the FT-IR spectra of different substances are highly characteristic, infrared spectroscopy is often used to analyze and study the structure and chemical bonds of substances. It can be used for characterization and identification of chemical species, and can be compared with the infrared spectra of standards for qualitative analysis.

[0071] The infrared spectrum of the BC product prepared in Example 1 is attached. Figure 4 As shown.

[0072] As can be seen from the image above, 3341cm -1 The absorption band at 2897 cm⁻¹ is caused by the stretching vibration of the -OH bond, resulting in a large absorption band with a broad peak shape. -1 The absorption peak at that point is caused by the -CH- stretching vibration.

[0073] 4. Mechanical property analysis The mechanical properties of BC membranes include elastic modulus, tensile strength, and elongation at break. These properties reflect the membrane's elasticity, strength, and tensile properties, respectively. Analyzing these parameters can provide guidance for the application of the membrane.

[0074] The mechanical properties of the BC membrane prepared in Example 1 are shown in Table 1 and Appendix 1 below. Figure 5 As shown.

[0075] Table 1 Mechanical property results

[0076] The results above show that there is no significant difference between BC membranes prepared by fermentation using sweet potato wastewater as a carbon source and those produced using glucose as a substrate.

[0077] It is evident that this invention utilizes Bacillus brevis ZF-9 as a carbon source to co-produce bacterial cellulose and γ-polyglutamic acid. Furthermore, the wastewater from the dried sweet potato has a high COD decomposition rate, enabling simultaneous treatment of the wastewater. This provides a new industrial application for Bacillus brevis ZF-9 and achieves comprehensive resource utilization.

[0078] It should be understood that the embodiments described in this application are merely illustrative of the principles of the embodiments of this application. Other modifications may also fall within the scope of this application. Therefore, alternative configurations of the embodiments of this application are considered as examples and not limitations, and are regarded as consistent with the teachings of this application. Accordingly, the embodiments of this application are not limited to the embodiments explicitly described in this application.

Claims

1. An application of *Bacillus mesporus* fermentation for co-production of BC and γ-PGA, characterized in that: The aforementioned *Bacillus mesporus* is *Bacillus mesporus* ZF-9, and its classification name is *Bacillus mesporus*. Brevibacillus centrosporus It has been deposited at the China General Microbiological Culture Collection Center, with accession number: CGMCC No. 6267.

2. A method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus and Bruxelles, characterized in that, The process includes the step of inoculating Bacillus brevis into a fermentation medium containing sweet potato wastewater and free of glutamic acid for fermentation culture. The aforementioned *Bacillus mesporus* is *Bacillus mesporus* ZF-9, and its classification name is... Brevibacillus centrosporus It has been deposited at the China General Microbiological Culture Collection Center, with accession number: CGMCC No. 6267.

3. The method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus according to claim 2, characterized in that, The fermentation medium comprises the following components in the indicated mass percentages: 80-120 mL / L of dried sweet potato wastewater, 20-50 g / L of carbon source, 10-30 g / L of nitrogen source, and pH adjusted to 6.5-7.

5.

4. The method for co-producing BC and γ-PGA by fermentation of *Bacillus mesporus* according to claim 2 or 3, characterized in that, The conditions for the fermentation culture step include: controlling the rotation speed at 100-150 rpm and fermenting at 25-32℃ for 5-8 days.

5. The method for co-producing BC and γ-PGA by fermentation of *Bacillus mesporus* according to any one of claims 2-4, characterized in that, The method further includes the step of inoculating the Bacillus brevis in a seed culture medium for seed liquid culture; The seed culture medium comprises the following components in the indicated mass percentages: 50-80 mL / L of dried sweet potato wastewater, 10-30 g / L of carbon source, 10-20 g / L of nitrogen source, and pH adjusted to 6.5-7.

5.

6. The method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus according to claim 5, characterized in that, The conditions for the seed culture step include: controlling the rotation speed at 80-120 rpm and culturing the seed culture at 25-32℃ for 1-2 days.

7. The method for co-producing BC and γ-PGA by fermentation of *Bacillus mesporus* according to any one of claims 2-6, characterized in that, The method further includes the step of activating the *Bacillus mesoproteroides* by inoculating it into a slant culture medium. The slant culture medium comprises the following components in the indicated mass amounts: carbon source 10-30 g / L, nitrogen source 10-20 g / L, agar 15-25 g / L, and pH adjusted to 6.5-7.

5.

8. The method for co-producing BC and γ-PGA by fermentation of Bacillus mesporus according to claim 7, characterized in that, The conditions for the activation step of the slant culture medium include: constant temperature incubation at 25-32℃ for 18-30 hours.

9. The method for co-producing BC and γ-PGA by fermentation of *Bacillus mesporus* according to any one of claims 2-8, characterized in that, In the fermentation medium, seed medium, or slant medium: The carbon source is selected from one or more of glucose, sucrose, fructose, maltose, molasses, anhydrous ethanol, or starch hydrolysate; and / or, The nitrogen source is selected from one or more of the following: beef extract, peptone, yeast extract, corn steep liquor, soybean meal powder, cottonseed meal powder, urea, (NH4)2SO4, or NH4Cl.

10. The method for co-producing BC and γ-PGA by fermentation of *Bacillus mesporus* according to any one of claims 2-9, characterized in that, The method also includes the steps of collecting fermentation products for the separation and purification of bacterial cellulose and γ-polyglutamic acid.

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