A method for preparing functional nitrogen source by multi-bacteria co-fermentation of cassava starch and using the same for low-oxygen production of bacterial cellulose

Abstract

The application discloses a method for preparing functional nitrogen source and producing bacterial cellulose by using cassava starch enzymolysis liquid, and belongs to the technical field of fermentation engineering and biomaterials. Cassava starch is subjected to liquefaction by alpha-amylase, saccharification by saccharifying enzyme and pululanase, so that enzymolysis liquid is prepared, and the dilution of the content of reducing sugar is 20-95 g / L; a three-microbe combination composed of saccharomyces cerevisiae, lactobacillus plantarum and bacillus subtilis, or a two-microbe combination composed of saccharomyces cerevisiae and lactobacillus plantarum, is subjected to aerobic fermentation to obtain functional nitrogen source; the functional nitrogen source completely replaces peptone and yeast extract powder, carbon source and inorganic salt are added to prepare a fermentation medium, and equisetopsis arrhizem HNNB6 is inoculated to perform normal-oxygen or low-oxygen static fermentation to prepare bacterial cellulose. The results show that when the initial pH is 4.0, the functional nitrogen source is matched with 42 g / L fructose glucose syrup F60, and normal-oxygen static fermentation is performed for 4 d, the dry weight of bacterial cellulose reaches 5.49+ / -0.27 g / L; the yield of the two-microbe synergistic functional nitrogen source reaches 6.7 g / L under the condition of low-oxygen static fermentation for 7 d, and the low-oxygen / normal-oxygen yield ratio is 91.8%.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering and biomaterials technology, specifically relating to a method using cassava starch enzymatic hydrolysate as a substrate, fermented with Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Lactobacillus plantarum ( Lactiplantibacillus plantarum ) and Bacillus subtilis ( Bacillus subtilis A three-strain combination consisting of *Saccharomyces cerevisiae* and *Lactobacillus plantarum*, or a two-strain combination consisting of *Saccharomyces cerevisiae* and *Lactobacillus plantarum*, is used to ferment and prepare a functional nitrogen source. This functional nitrogen source completely replaces organic nitrogen sources such as peptone and yeast extract, and is used for *Xylose-bacterium* ( Komagataeibacter nataicola A method for producing bacterial cellulose by fermentation of HNNB6 under normal and low-oxygen static conditions. Background Technology

[0002] Bacterial cellulose (BC) is a high-purity extracellular cellulose synthesized by microorganisms such as Acetic Acid Bacteria and Coprinus. It possesses high crystallinity, high water-holding capacity, good mechanical properties, and biocompatibility, and has broad application prospects in food, pharmaceuticals, cosmetics, and environmental materials. However, the industrial production of bacterial cellulose is still limited by the high cost of culture media and the high energy consumption of fermentation. Xylocarpus xylose fermentation requires strict aerobic conditions: Xylocarpus xylose fermentation is highly dependent on oxygen, and yield drops significantly under limited or low-oxygen conditions. Only FY-07, reported in CN119530317A filed by Nankai University, can produce cellulose under limited / anaerobic conditions, but its strain is highly specific to Enterobacteriaceae, making it difficult to extend to widely used industrial production systems of the Xylocarpus genus. Therefore, it is of great significance to develop a method for preparing fermentation products that can replace traditional organic nitrogen sources from stable and inexpensive raw materials and to make them suitable for the production of bacterial cellulose by *Xylostella spp.* under normoxic and hypoxic conditions. Summary of the Invention

[0003] A method for preparing a functional nitrogen source and producing bacterial cellulose using cassava starch enzymatic hydrolysate includes the following steps: Step (1) Preparation of cassava starch enzymatic hydrolysate Cassava starch is mixed with water to form a slurry, liquefied by α-amylase, and then saccharified by saccharifying enzymes and pullulanase to obtain a cassava starch enzymatic hydrolysate. The cassava starch enzymatic hydrolysate is diluted to a reducing sugar content of 20–95 g / L. Step (2) Preparation of functional nitrogen source The cassava starch enzymatic hydrolysate is inoculated with a microbial community for aerobic fermentation to obtain a functional nitrogen source; the microbial community is any one of the following two combinations: (i) A combination of three bacteria: Saccharomyces cerevisiae, Lactobacillus plantarum, and Bacillus subtilis; (ii) A combination of two bacteria, Saccharomyces cerevisiae and Lactobacillus plantarum. The functional nitrogen source is a fermentation broth containing microbial-derived usable nitrogen components. Among them, the three-strain combination is more conducive to increasing the content of free amino acids and total protein, while the two-strain combination performs better in the production of bacterial cellulose during low-oxygen static fermentation. Step (3) Preparation of bacterial cellulose culture medium The functional nitrogen source obtained in step (2) was used to completely replace peptone and yeast extract as organic nitrogen source, and carbon source and inorganic salt were added to prepare bacterial cellulose fermentation medium. Step (4) Production of bacterial cellulose by normoxic or hypoxic static fermentation xylomycin ( Komagataeibacter nataicola HNNB6 was inoculated into the culture medium obtained in step (3) and fermented under normal or low oxygen static conditions to obtain bacterial cellulose membrane. Step (5) Bacterial cellulose purification The obtained bacterial cellulose membrane was treated with an alkaline solution to remove bacteria and impurities, washed until neutral, and dried to obtain a dry bacterial cellulose membrane. Compared with the prior art, the present invention has the following advantages: 1. Using cassava starch as a stable, readily available, and low-cost raw material, a combination of enzymatic hydrolysis and microbial fermentation is used to transform it from a traditional carbon source into a functional nitrogen source that can be used for bacterial cellulose fermentation. 2. The functional nitrogen source can replace traditional organic nitrogen sources such as peptone and yeast extract, reducing the cost of bacterial cellulose culture medium. 3. The functional nitrogen source can not only meet the nitrogen supply required for the fermentation of bacterial cellulose by Xylospora xylose, but also improve its cellulose production performance under low-oxygen static conditions. 4. This invention establishes a process route for deriving key nutrient components of the culture medium from a single stable raw material, which is beneficial for the standardization and industrialization of bacterial cellulose fermentation. Attached Figure Description

[0004] Appendix Figure 1 This is a comparison chart of the free amino acid content and total protein content in the functional nitrogen sources obtained from fermentation of different strain combinations in Example 2. The horizontal axis represents the strain combination groups, where BG represents the Bacillus subtilis single-strain group, LG represents the Lactobacillus plantarum single-strain group, SG represents the Saccharomyces cerevisiae single-strain group, LBG represents the Lactobacillus plantarum and Bacillus subtilis double-strain group, SBG represents the Saccharomyces cerevisiae and Bacillus subtilis double-strain group, SLG represents the Saccharomyces cerevisiae and Lactobacillus plantarum double-strain group, and SLBG represents the Saccharomyces cerevisiae, Lactobacillus plantarum and Bacillus subtilis triple-strain group; Appendix Figure 1 The vertical axis A represents the free amino acid content (g / L). Figure 1 The ordinate B represents the total protein content (g / L). Appendix Figure 2 The graph shows the optimization results of the three-strain fermentation conditions in Example 3. Figures A through C represent the changes in total protein content under different conditions, and figures D through F represent the changes in free amino acid content under the corresponding conditions. Figures A and D correspond to optimized fermentation time, B and E to optimized initial pH, and C and F to optimized rotation speed. The horizontal axis represents fermentation time (h), initial pH, and rotation speed (r / min), respectively; the vertical axis represents total protein content and free amino acid content, respectively. Appendix Figure 3 The graph shows the effect of different initial pH functional nitrogen sources on the cellulose yield of bacteria in normoxic static fermentation in Example 4. A represents the dry weight yield of bacterial cellulose when glucose is used as the carbon source, and B represents the dry weight yield of bacterial cellulose when fructose syrup F60 is used as the carbon source. The horizontal axis represents the groups, with G1, G2, G3, G4, and G5 corresponding to functional nitrogen source groups with initial pH values ​​of 3.66, 4, 5, 6, and 7, respectively. CG represents the control group. Appendix Figure 4 This is a comparative graph showing the effects of different functional nitrogen sources from different strains on bacterial cellulose yield under hypoxic fermentation conditions in Example 5. The horizontal axis represents different nitrogen source groups, and the vertical axis represents bacterial cellulose dry weight yield (g / L). Detailed Implementation

[0005] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. Example 1: Preparation of cassava starch enzymatic hydrolysate Cassava starch and water were gelatinized at a mass ratio of 1:10. The mixture was cooled to 70℃, and 0.01% (v / v) α-amylase was added for liquefaction for 30 min. The mixture was then cooled to 55℃, the pH was adjusted to 5.0, and 0.015% (v / v) saccharifying enzyme and 0.05 wt% pullulanase were added for synergistic saccharification. The mixture was stirred for 3 h to obtain cassava starch enzymatic hydrolysate. The reducing sugar content of the hydrolysate was determined using the DNS method (3,5-dinitrosalicylic acid method). The reducing sugar content was diluted to 80 g / L with deionized water and used as the base substrate for subsequent preparation of functional nitrogen sources. Example 2: Screening of nitrogen sources with single strain, dual-strain synergy, and tri-strain synergy Saccharomyces cerevisiae, Lactobacillus plantarum, and Bacillus subtilis were activated and passaged twice, and cultured in a shaker at 200 r / min for 24 h to prepare seed culture. Using cassava starch hydrolysate with 80 g / L reducing sugar obtained in Example 1 as the fermentation substrate, the total inoculum was 6% (v / v), and aerobic fermentation was carried out at 30℃ and 200 r / min for 24 h. The abbreviations for each group are defined as follows: BG for Bacillus subtilis single-strain group, LG for Lactobacillus plantarum single-strain group, SG for Saccharomyces cerevisiae single-strain group, LBG for Lactobacillus plantarum and Bacillus subtilis double-strain group, SBG for Saccharomyces cerevisiae and Bacillus subtilis double-strain group, SLG for Saccharomyces cerevisiae and Lactobacillus plantarum double-strain group, and SLBG for Saccharomyces cerevisiae, Lactobacillus plantarum, and Bacillus subtilis triple-strain group. Experimental results are attached. Figure 1 As shown. The results showed that the free amino acid and total protein contents of the functional nitrogen source obtained by the three-strain co-fermentation group (SLBG) were higher than those of the single-strain group and the two-strain group, indicating that three-strain co-fermentation is more conducive to improving the content of available nitrogen components in the functional nitrogen source. Therefore, subsequent optimization will be based on the three-strain co-fermentation (1:1:1) process. Example 3: Optimization of three-strain synergistic fermentation conditions Based on the synergistic combination of three microorganisms determined in Example 2 (a mixture of *Saccharomyces cerevisiae*, *Lactobacillus plantarum*, and *Bacillus subtilis* in a volume ratio of 1:1:1, with a total inoculum of 6% (v / v)), single-factor optimization was performed on fermentation time, initial pH, and rotation speed, using free amino acid content and total protein content as evaluation indicators. To maintain consistency with the attached... Figure 2 The order of description is consistent; this embodiment describes the process in the order of "fermentation time—initial pH—rotation speed," and the results correspond to the appendix. Figure 2 A and D, B and E, C and F. (1) Fermentation time optimization (corresponding appendix) Figure 2 (A and D): With a fixed temperature of 30℃, 200 r / min, and initial pH of 6.0, the fermentation effects were investigated at 12, 24, 36, 48, and 60 h. The results showed that the total protein content and free amino acid content reached optimal levels at 60 h of fermentation. Further extending the fermentation time resulted in a slight decrease in these indicators due to nutrient consumption and the accumulation of metabolic byproducts, and also produced unpleasant odors. Therefore, 60 h was determined to be the optimal fermentation time. (2) Initial pH optimization (corresponding to the appendix) Figure 2 (B and E): Fermentation was carried out at a fixed temperature of 30℃, 200 r / min, and for 60 h, with initial pH values ​​of 4.0, 5.0, 6.0, 7.0, and 8.0. The results showed that the initial pH of 7.0 resulted in the highest content of free amino acids and total protein in the functional nitrogen source. Therefore, pH 7.0 was determined to be the optimal initial pH for the co-fermentation of three microorganisms to prepare a functional nitrogen source. (3) Speed ​​optimization (corresponding appendix) Figure 2 (C and F): With a fixed temperature of 30℃, initial pH of 5.0, and fermentation time of 48 h, the effects of 100, 150, and 200 r / min on the fermentation efficiency were investigated. The results showed that the highest contents of free amino acids and total protein were obtained at a rotation speed of 200 r / min, therefore 200 r / min was determined to be the optimal rotation speed. To meet the needs of different subsequent application scenarios, this embodiment further prepared functional nitrogen source products with initial pH values ​​of 3.66, 4.0, 5.0, 6.0 and 7.0 for subsequent bacterial cellulose fermentation experiments. Example 4: Effects of different pH-functionalized nitrogen sources on normoxic BC fermentation yield Functional nitrogen sources with different initial pH values ​​(3.66, 4.0, 5.0, 6.0, 7.0) prepared in Example 3 were diluted 5 times and used as nitrogen source components. The culture medium formulation was as follows: functional nitrogen source diluted 5 times, glucose 30 g / L or fructose syrup F60 42 g / L, potassium dihydrogen phosphate 1 g / L, citric acid 0.5 g / L, magnesium sulfate 1.5 g / L, and ammonium sulfate 6 g / L. After mixing the above components, a corresponding carbon source control group CG was set up. The pH of the culture medium was adjusted to 3.75 and sterilized at 121℃ for 20 min. The functional nitrogen sources were divided into 6 groups according to their initial pH values: G1 (pH 3.66), G2 (pH 4.0), G3 (pH 5.0), G4 (pH 6.0), G5 (pH 7.0), and CG. Inoculate with activated *Bacillus xylostella* seed culture at a 5% (v / v) inoculation rate. Komagataeibacter nataicola HNNB6 was fermented aerobically in a breathable conical flask at 30℃ for 4 days. After the culture was completed, the bacterial cellulose membrane was removed, soaked in 0.5 wt% NaOH solution at 80℃ for 90 min, rinsed with deionized water until neutral, dried to constant weight, and the dry weight yield was measured. Figure 3 B represents the dry weight yield of bacterial cellulose when fructose syrup F60 is used as the carbon source. The results show: (1) When the functional nitrogen source with an initial pH of 4.0 (G2 group) is used as the nitrogen source and the fructose syrup F60 (42 g / L) is used as the carbon source, the dry weight yield of bacterial cellulose reaches 5.49±0.27 g / L, indicating that the functional nitrogen source can be used to replace the traditional organic nitrogen source. (2) When using high fructose corn syrup F60 as the carbon source (see attached) Figure 3 (B), bacterial cellulose production is slightly higher than that when glucose is used as the carbon source (see appendix). Figure 3 A). (3) Within the pH gradient range (3.66 to 7.0) examined in this embodiment, both excessively low and excessively high initial pH of the functional nitrogen source are not conducive to bacterial cellulose synthesis. Among them, the yield of functional nitrogen source with an initial pH of 4.0 (G2 group) is relatively high. Example 5: Comparison of functional nitrogen sources from different strains under hypoxic fermentation conditions The effects of functional nitrogen sources from different bacterial strains on bacterial cellulose production by *Bacillus xylose* during hypoxic fermentation were investigated. The single-strain functional nitrogen sources (SG, LG, BG) and the dual-strain synergistic functional nitrogen source (SLG) prepared in Example 2, as well as the triple-strain synergistic functional nitrogen source (SLBG, initial pH 5.0) prepared in Example 3, were used as nitrogen sources. The reason for choosing the triple-strain functional nitrogen source with an initial pH of 5.0 is that the overall hypoxic fermentation medium is acidic (pH 3.75), and using a functional nitrogen source with a pH of 5.0 is beneficial for pH transition and acidity adjustment. The above-mentioned functional nitrogen sources were diluted 4 times to prepare fermentation medium. The formula was: 4-fold diluted functional nitrogen source, 40 g / L glucose, 1 g / L potassium dihydrogen phosphate, 0.5 g / L citric acid, 1.5 g / L magnesium sulfate, and 6 g / L ammonium sulfate. The pH was adjusted to 3.75, and the medium was sterilized at 121℃ for 20 min. *Bacillus xylostella* was inoculated at a 5% (v / v) inoculum. Komagataeibacter nataicola HNNB6 seed culture was placed in a sealed, vented bag, the gas inside was expelled, and the bag was sealed. It was then incubated at 30°C under low-oxygen conditions for 7 days. A control group (CG) was prepared using traditional peptone + yeast extract. Results are attached. Figure 4 As shown. It should be noted that in this embodiment, single-strain functional nitrogen sources (SG, LG, BG) and three-strain functional nitrogen sources (SLBG) are used as comparative research items to evaluate the impact of functional nitrogen sources from different bacterial groups on the performance of low-oxygen fermentation; among them, the dual-strain synergistic functional nitrogen source (SLG, i.e., Saccharomyces cerevisiae + Lactobacillus plantarum) corresponds to the scheme defined in claim 3, which is the low-oxygen fermentation application scheme that is the focus of protection of this invention. The results show that: (1) The peptone + yeast extract control group (CG) produced only 39.0% of the bacterial cellulose under hypoxic conditions compared to normoxic conditions, indicating that the ability of xylose-producing bacteria to produce cellulose was significantly reduced under hypoxic conditions. (2) The single-strain functional nitrogen sources (SG, LG, BG) have limited effect on improving the performance of low-oxygen fermentation. Among them, the functional nitrogen sources of Saccharomyces cerevisiae (SG) and Lactobacillus plantarum (LG) showed a certain promoting effect. (3) The dual-strain synergistic nitrogen source (SLG) showed better performance in cellulose production under low oxygen conditions. (4) The synergistic functional nitrogen source (SLG) of Saccharomyces cerevisiae and Lactobacillus plantarum reached a bacterial cellulose dry weight yield of 6.7 g / L under hypoxic conditions, with a hypoxic / noroxic yield ratio of 91.8%, which was higher than that of the peptone + yeast extract control group (CG). This phenomenon indicates that the functional nitrogen source obtained by synergistic fermentation of the two bacteria is beneficial to improving the cellulose production performance of Xylose-like bacteria under hypoxic conditions, and its mechanism of action needs further investigation. In summary, the three-strain synergistic nitrogen source of the present invention can not only completely replace expensive organic nitrogen sources, but the two-strain (Saccharomyces cerevisiae + Lactobacillus plantarum) synergistic nitrogen source can improve the cellulose production performance of Xylose-like bacteria under low-oxygen conditions, providing a brand-new technical path for the low-energy consumption and low-cost industrial production of bacterial cellulose.

Claims

1. A method for preparing a functional nitrogen source and producing bacterial cellulose using cassava starch enzymatic hydrolysate, characterized in that, Includes the following steps: (1) Cassava starch and water are mixed at a mass ratio of 1:5 to 1:50 to form a paste, which is then liquefied by α-amylase and saccharified by saccharifying enzyme and pullulanase to obtain cassava starch hydrolysate; (2) The cassava starch hydrolysate is released to a reducing sugar content of 20-95 g / L, and then inoculated with microorganisms for aerobic fermentation to obtain a functional nitrogen source; the microorganisms are any one of the following two combinations: (i) brewer's yeast ( Saccharomyces cerevisiae Lactobacillus plantarum ( Lactiplantibacillus plantarum ) and Bacillus subtilis ( Bacillus subtilis The three bacteria form a tribological combination; (ii) A combination of two bacteria, Saccharomyces cerevisiae and Lactobacillus plantarum; (3) Use the functional nitrogen source to completely replace peptone and yeast extract as organic nitrogen source, add carbon source and inorganic salt, and prepare bacterial cellulose fermentation medium; (4) Inoculation with Xylospora xylose ( Komagataeibacter nataicola HNNB6 was subjected to normoxic or hypoxic static fermentation to obtain bacterial cellulose.

2. The method according to claim 1, characterized in that, The microbial community mentioned in step (2) is a combination of Saccharomyces cerevisiae, Lactobacillus plantarum and Bacillus subtilis, with an inoculation volume ratio of 1:1:1 and a total inoculation amount of 3% to 10% of the volume of the cassava starch hydrolysate, v / v.

3. The method according to claim 1, characterized in that, The microbial community mentioned in step (2) is a combination of Saccharomyces cerevisiae and Lactobacillus plantarum, with an inoculation volume ratio of 1:

1. The total inoculation amount of the microbial community is 3% to 10% of the volume of the cassava starch hydrolysate, v / v.

4. The method according to claim 1, characterized in that, In step (2), the cassava starch hydrolysate is diluted to a reducing sugar content of 80 g / L, the total inoculation amount of the microbial community is 6% of the volume of the hydrolysate, v / v, and the fermentation conditions are: initial pH 4.0-8.0, temperature 28-37℃, rotation speed 100-300 r / min, fermentation time 24-72 h, and the microbial community is the three-strain combination or two-strain combination as defined in claim 1.

5. The method according to claim 2, characterized in that, In step (2), the fermentation temperature is 30℃, the rotation speed is 200 r / min, and the fermentation time is 60 h.

6. The method according to claim 1, characterized in that, The free amino acid content in the functional nitrogen source obtained in step (2) is ≥0.5 g / L, and the total protein content is ≥0.1 g / L.

7. The method according to claim 1, characterized in that, The carbon source in step (3) is at least one of glucose or fructose syrup F60, and the total amount of carbon source added is 20-60 g / L; the inorganic salts include potassium dihydrogen phosphate 0.5-5 g / L, citric acid 0.5-1.5 g / L, magnesium sulfate 0.5-5 g / L and ammonium sulfate 6 g / L; the pH of the culture medium is adjusted to 3.65-4.

00.

8. The method according to claim 7, characterized in that, The carbon source mentioned in step (3) is fructose syrup F60, and the amount added is 42 g / L.

9. The method according to claim 1, characterized in that, In step (4), the inoculum size of *Xylostella spp.* HNNB6 is 3%–10%, v / v, the fermentation temperature is 28–32℃, and the fermentation time is 5–10 days.

10. The method according to claim 3, characterized in that, Step (4) describes normal or low-oxygen static fermentation as low-oxygen static fermentation, specifically: the inoculated culture medium is placed into a ventable sealed container, the gas inside the container is released and then sealed, and fermented at 30°C for 7 days.

11. The method according to claim 1, characterized in that, The process also includes step (5): after rinsing the bacterial cellulose membrane obtained from fermentation with deionized water, soaking it in 0.1 wt% to 1 wt% NaOH solution at 80 to 90°C for 60 to 120 minutes, rinsing it with deionized water until neutral, and drying it to constant weight to obtain a bacterial cellulose dry membrane.

12. The use of the functional nitrogen source prepared by the method of any one of claims 1 to 11 in the production of bacterial cellulose as an alternative to peptone and yeast extract.

13. The application according to claim 12, characterized in that, The functional nitrogen source is used to improve the yield of bacterial cellulose produced by Xylospora xylose HNNB6 under low-oxygen static fermentation conditions.

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