A method for improving the yield of aspergillus oryzae and a nanocomposite

Abstract

The application discloses a method for improving the yield of aspergillus oryzae and a nanocomposite. The method for improving the yield of aspergillus oryzae is to improve the growth speed of aspergillus oryzae by adding ZnO / ZIF-8 nanocomposite into a culture medium. The application utilizes the synergistic effect of ZnO and ZIF-8 materials, and the promoting effect of the ZnO / ZIF-8 nanocomposite on the growth of aspergillus oryzae is more obvious. The method has the advantages of simple process, common and low-cost required equipment and high efficiency, and can widen the application of aspergillus oryzae in the biological field.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to a method for increasing the yield of Aspergillus oryzae and a nanocomposite. Background Technology

[0002] Aspergillus oryzae is a common fungus in the genus Aspergillus with high enzyme secretion capabilities. Belonging to the subphylum Deuteromycetes, it possesses strong protein and starch-degrading abilities, making it widely applicable in the food industry. Furthermore, Aspergillus oryzae is a recognized food safety production strain. Through solid-state fermentation, it produces large quantities of enzymes (such as proteases and amylases) and organic acids. These products are used in traditional fermentation industries to produce soy sauce and fermented black beans, and are also frequently used to produce primary and secondary metabolites and enzymes. They are also ideal subjects for gene expression and protein secretion research. Therefore, research on increasing Aspergillus oryzae yield is highly valued and is one of the key technical challenges in expanding its application.

[0003] For example, Chinese invention patent CN106867910A discloses a culture medium for Aspergillus oryzae, which comprises the following components by weight: 8 parts peptone, 6 parts fish meal, 2 parts sweet potato flour, 1 part serine, 1 part Scutellaria baicalensis, 1 part Angelica dahurica, and 1 part pectin. Specific amounts of Fritillaria thunbergii, magnesium sulfate, or Polygonum hydropiper can be further added to this medium. This formula, by adding certain traditional Chinese medicinal herbs to the commonly used materials of the culture medium, unexpectedly achieved outstanding culture results, especially suitable for the culture of Aspergillus oryzae, and has good application prospects.

[0004] For example, Chinese invention patent CN102382772A discloses an industrial production method for Aspergillus oryzae strains used in fermentation. This method involves activating and culturing the Aspergillus oryzae strain (specifically, the Hu Niang 3.042 strain), then expanding the culture in Erlenmeyer flasks, and finally culturing it in a culture chamber. The culture medium formula uses a wheat bran:soybean meal ratio of 4:6, with 48-52 catties of water added to every 100 catties of the mixture. The culture medium is steamed in a steamer for 30 minutes, then cooled to 35-40℃ before inoculation at a ratio of 0.3%. The inoculated material is then placed in a winnowing tray in the culture chamber, with a thickness of 2.5-3 cm, and cultured for 7 days. This method allows for the large-scale production of Aspergillus oryzae strains with high enzyme activity, capable of rapidly fermenting soybean paste, fermented black beans, and soy sauce.

[0005] For example, Chinese invention patent CN103923872B discloses a Shanghai-brewed 3.042 Aspergillus oryzae activity enhancer and its cultivation method. The enhancer, added at the following amounts by weight of the culture medium, consists of 0.1‰–2‰ konjac oligosaccharide, 0.05‰–2‰ taurine, 0.02‰–0.05‰ ferrous sulfate, and 0.002‰–0.005‰ sodium selenite. After 72 hours of fermentation, the Aspergillus oryzae protease activity in the fermentation koji material can reach 20 kU / g, significantly higher than that of koji material without the added compound enhancer or with a single enhancer.

[0006] However, there have been no reports on adding nanocomposites to the culture medium to improve the growth rate of Aspergillus oryzae. Summary of the Invention

[0007] To better promote the growth of Aspergillus oryzae, this invention provides a method for increasing Aspergillus oryzae yield and a nanocomposite.

[0008] In a first aspect, the present invention provides a method for increasing the yield of Aspergillus oryzae, wherein the method is to increase the growth rate of Aspergillus oryzae by adding a ZnO / ZIF-8 nanocomposite to the culture medium.

[0009] In this invention, the concentration of the ZnO / ZIF-8 nanocomposite in the culture medium is greater than 75 μg / mL.

[0010] In this invention, the mass ratio of ZnO to ZIF-8 in the ZnO / ZIF-8 nanocomposite is (1~4):(1~4).

[0011] In this invention, the ZnO / ZIF-8 nanocomposite is prepared by the following method: according to the mass ratio of ZnO to ZIF-8, the corresponding weight is weighed, placed in a mortar, ethanol is added, ground, and finally dried to obtain the ZnO / ZIF-8 nanocomposite.

[0012] In this invention, the ZnO is prepared by the following method: Zn(CH3COO)2•2H2O and NaOH are dissolved in water respectively, the two solutions are mixed together and stirred at room temperature; then they are centrifuged with water and ethanol respectively, and then dried in a drying oven; after being taken out, they are ground and then dried in an oven to finally obtain ZnO.

[0013] In this invention, ZIF-8 is prepared by the following method: Zn(NO3)2•6H2O is dissolved in methanol and dissolved by sonication; 2-methylimidazole is dissolved in methanol and dissolved by sonication; the 2-methylimidazole solution is slowly added dropwise to the Zn(NO3)2•6H2O solution at room temperature, and the mixture is magnetically stirred, allowed to stand, centrifuged with ethanol, and finally dried to obtain ZIF-8.

[0014] In this invention, the dripping rate is 1 drop / 2 seconds.

[0015] Secondly, a nanocomposite for increasing Aspergillus oryzae yield, wherein the nanocomposite is a ZnO / ZIF-8 nanocomposite.

[0016] In this invention, the mass ratio of ZnO to ZIF-8 in the ZnO / ZIF-8 nanocomposite is (1~4):(1~4).

[0017] In this invention, the ZnO / ZIF-8 nanocomposite is prepared by the following method: (1) Zn(CH3COO)2•2H2O and NaOH are dissolved in water respectively, and the two solutions are mixed together and stirred at room temperature; then they are centrifuged with water and ethanol respectively, and placed in a drying oven to dry; after being taken out, they are ground and then placed in an oven to dry, finally obtaining ZnO; (2) The ZIF-8 is prepared by the following method: Zn(NO3)2•6H2O is dissolved in methanol and dissolved by sonication; 2-methylimidazole is dissolved in methanol and dissolved by sonication; at room temperature, 2-methylimidazole solution is slowly added dropwise to Zn(NO3)2•6H2O solution and stirred magnetically, allowed to stand, centrifuged with ethanol, and finally dried to obtain ZIF-8; (3) According to the mass ratio of ZnO to ZIF-8, the corresponding weight is weighed, placed in a mortar, ethanol is added, ground, and finally dried to obtain ZnO / ZIF-8 nanocomposite.

[0018] In summary, the present invention has the following beneficial effects:

[0019] This invention utilizes the synergistic effect of ZnO and ZIF-8 materials, making the ZnO / ZIF-8 nanocomposite more effective in promoting the growth of Aspergillus oryzae. This method has the advantages of simple process, common and inexpensive equipment, and high efficiency, which can broaden the application of Aspergillus oryzae in the biological field. Attached Figure Description

[0020] Figure 1 X-ray diffraction patterns of ZnO, ZIF-8 and ZnO / ZIF-8 nanocomposites prepared in this invention;

[0021] Figure 2a and Figure 2b The infrared spectra of the samples (ZnO and ZIF-8) and (ZnO / ZIF-8 nanocomposite) prepared in this invention are shown respectively.

[0022] Figure 3Phenotypic images of Aspergillus oryzae under the influence of different concentrations of ZnO / ZIF-8 nanocomposites prepared in this invention: (a) 100 mL water, (b) 90 mL water + 10 mL ZnO / ZIF-8 solution, (c) 80 mL water + 20 mL ZnO / ZIF-8 solution, (d) 70 mL water + 30 mL ZnO / ZIF-8 solution, (e) 60 mL water + 40 mL ZnO / ZIF-8 solution, (f) 50 mL water + 50 mL ZnO / ZIF-8 solution. Detailed Implementation

[0023] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0024] Unless otherwise specified, the terms used in this specification have the same meaning as those commonly understood by those skilled in the art; however, in the event of any conflict, the definitions in this specification shall prevail.

[0025] The terms “comprising,” “including,” “containing,” “having,” “comprising,” or other variations thereof are intended to cover non-closed inclusion, and no distinction is made between these terms. The term “comprising” means that other steps and ingredients may be added without affecting the final result. The term “comprising” also includes the terms “consisting of” and “substantially consisting of”. The compositions and methods / processes of the present invention comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0026] All numerical values ​​or expressions relating to component amounts, process conditions, etc., used in the specification and claims are to be understood to be modified with “about” in all cases. All ranges relating to the same component or property include endpoints that can be independently combined. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range referenced in this application is intended to include all subranges within that range.

[0027] Unless otherwise specified, the experimental methods described in the various embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available.

[0028] The present invention will be further described in detail below with reference to the embodiments.

[0029] Preparation of ZnS / ZIF-8 nanocomposite

[0030] The ZnS / ZIF-8 nanocomposite was prepared according to the following steps:

[0031] (1) Dissolve 2.7246 g (0.0124 mol) of Zn(CH3COO)2•2H2O and 0.5120 g (0.0128 mol) of NaOH in 100 mL of water respectively. Mix the two solutions together at room temperature and stir for 30 min. Then centrifuge once with water and twice with ethanol, and dry in a drying oven at 60 °C for 5-8 h. After taking it out, grind it and dry it in a 200 °C oven for 1 h to finally obtain ZnO.

[0032] (2) Dissolve 1.7846 g of Zn(NO3)2•6H2O in 60 mL of methanol and sonicate to dissolve; dissolve 1.968 g of 2-methylimidazole in 20 mL of methanol and sonicate to dissolve; slowly add the 2-methylimidazole solution dropwise (1 drop / 2 s) to the Zn(NO3)2•6H2O solution at room temperature and stir magnetically for 30 min. After standing for 24 h, centrifuge three times with ethanol and finally dry at 60 °C for 5-8 h to obtain ZIF-8.

[0033] (3) The ZnO obtained in (1) and the ZIF-8 obtained in (2) were weighed according to the mass ratio of ZnO to ZIF-8 of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4 respectively. They were placed in a mortar, 20 mL of ethanol was added, and the mixture was ground for 20 min. Finally, it was dried at 60 °C for 5-8 h to obtain ZnO / ZIF-8 nanocomposites. The corresponding sample numbers are shown in Table 1.

[0034] Table 1 Sample Number and Composition

[0035] sample serial number ZnO(g) ZIF-8(g) m(ZnO):m(ZIF-8) Example 1 C1 0.4 0.1 4:1 Example 2 C2 0.3 0.1 3:1 Example 3 C3 0.4 0.2 2:1 Example 4 C4 0.2 0.2 1:1 Example 5 C5 0.2 0.4 1:2 Example 6 C6 0.1 0.3 1:3 Example 7 C7 0.1 0.4 1:4

[0036] like Figure 1 As shown, by Figure 1 The X-ray diffraction (XRD) patterns clearly show that the diffraction peaks of ZnO and ZIF-8 match the standard diffraction peaks, and there are no other extraneous peaks. Samples C1-C7 show ZnO diffraction peaks at approximately 31.72°, 34.34°, 36.2°, 47.48°, 56.50°, 62.8°, 66.16°, 67.84°, 69.02°, and 76.88° at 2θ, respectively. New peaks appear at 22.1°, 24.48°, 25.48°, and 26.62°, which coincide with the ZIF-8 peaks. This proves that samples 1-7 not only contain ZnO peaks but also exhibit ZIF-8 peaks, indicating that the synthesized samples (C1-C7) are a nanocomposite composed of ZIF-8 and ZnO.

[0037] like Figure 2a As shown, the infrared (IR) spectrum of ZnO is located at approximately 500, 3417, 1400, and 1552 cm⁻¹.-1 The absorption peaks originate from the stretching vibrations of Zn-O, water molecules, and hydroxyl groups or chemically adsorbed water, respectively. In the IR spectrum of ZIF-8, these peaks are located at approximately 3135, 2929, and 1587 cm⁻¹. -1 The absorption peaks at 1434 and 1311 cm⁻¹ are attributed, in turn, to the stretching vibrations of the aromatic CH₄ on the imidazole ring, the aliphatic CH₄ on the methyl group, and the C=N stretching vibration, respectively. -1 The peak at 1145 cm corresponds to the tensile vibration of the entire ring. -1 The peaks originate from the tensile vibration of aromatic CN, at 993 and 766 cm⁻¹. -1 The absorption peak at 686 cm⁻¹ originates from the bending signal of the imidazole ring; similarly, the absorption peak at 686 cm⁻¹... -1 The peak at 500 cm⁻¹ is obtained by the out-of-plane bending of the 2-methylimidazolium ring; -1 Zn-N vibrations were detected on both sides, which is due to the coordination of zinc ions with the binder through nitrogen atoms in ZIF-8. Figure 2b As shown, the synthesized samples (C1~C7) are at 516 cm⁻¹ -1 The peak, and the Zn-O in ZnO at 500 cm⁻¹ -1 The consistent tensile vibration at the 685~1587cm³ location confirms the presence of ZnO. -1 The absorption peaks within the range correspond to ZIF-8, indicating that the structure of ZIF-8 is also preserved in the nanocomposite, which is consistent with the XRD pattern ( Figure 1 The conclusions drawn are consistent with those in ( ).

[0038] Methods to increase Aspergillus oryzae yield

[0039] The method to increase the yield of Aspergillus oryzae is to add the above-mentioned ZnO / ZIF-8 nanocomposite to the culture medium to increase the growth rate of Aspergillus oryzae.

[0040] Zinc is a fundamental element in all life forms because it plays an important role in many cellular functions, such as catalysts, cofactors, and protein structure stabilizers. Among the many Zn-based nanomaterials, zinc oxide (ZnO) nanoparticles are particularly noteworthy because: (1) ZnO has an exciton binding energy as high as 60 meV and a band gap of 3.37 eV at room temperature, which theoretically gives it stable emission capabilities from ultraviolet to visible light; (2) This material has excellent chemical stability and strong resistance to radiation damage; (3) Zinc is an important cofactor in many mammalian and fungal enzymes, which can regulate nucleic acid synthesis and various metabolic pathways; (4) It does not contain any toxic elements, the preparation method is simple, and the raw materials are inexpensive and readily available. ZIF-8 material has the following advantages: (1) It not only has a large specific surface area, adjustable pore size, and diverse structure, but also has unsaturated metal active centers, thus endowing porous ZIF-8 material with superior adsorption performance; (2) The surface of ZIF-8 material has a rich microporous structure, which facilitates its combination with active substances and reactants, making it an ideal adsorption-catalysis material; (3) It has high hydrothermal and chemical stability. The inventors unexpectedly discovered that by utilizing the synergistic effect of ZIF-8 and ZnO nanocomposite, the effective contact area between the nanocomposite and Aspergillus oryzae can be significantly expanded, thereby promoting the growth of Aspergillus oryzae.

[0041] The ZnO / ZIF-8 nanocomposites from Examples 1-7 were prepared into aqueous solutions with a concentration of 75 μg / mL for later use. Aspergillus oryzae (3.042) was then cultured in pure water and solutions containing different amounts of ZnO / ZIF-8 nanocomposites for 72 h, and the phenotype of Aspergillus oryzae was observed. Figure 3 As shown, Figure 3 Phenotypic diagrams of Aspergillus oryzae from Example 1 with and without different concentrations of ZnO / ZIF-8 nanocomposite are shown. It can be seen that: compared to the culture medium without ZnO / ZIF-8 nanocomposite (…),… Figure 3 Compared to a), the addition of ZnO / ZIF-8 nanocomposite ( Figure 3 (b~f) The ZnO / ZIF-8 nanocomposite significantly promoted the growth of Aspergillus oryzae cells, and the greater the amount of ZnO / ZIF-8 nanocomposite added, the faster the growth rate of Aspergillus oryzae. When the added amount did not exceed 30 mL, after 72 h of incubation, the Aspergillus oryzae turned yellow, indicating that it had matured. When the added amount exceeded 30 mL, the growth rate of Aspergillus oryzae further accelerated, and the biomass increased significantly. After 72 h of incubation, the Aspergillus oryzae was still white, indicating that it was not yet mature and showed a tendency to grow further. It can be seen that the ZnO / ZIF-8 nanocomposite can significantly increase the biomass of Aspergillus oryzae and promote the growth of Aspergillus oryzae cells.

[0042] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for increasing the yield of Aspergillus oryzae, characterized in that, The method described above improves the growth rate of Aspergillus oryzae by adding ZnO / ZIF-8 nanocomposite to the culture medium; the concentration of ZnO / ZIF-8 nanocomposite in the culture medium is above 75 μg / mL. The ZnO / ZIF-8 nanocomposite was prepared by the following method: (1) Zn(CH3COO)2•2H2O and NaOH were dissolved in water respectively, and the two solutions were mixed together and stirred at room temperature; then they were centrifuged with water and ethanol respectively, and then placed in a drying oven to dry; after being taken out, they were ground and then placed in an oven to dry, finally obtaining ZnO; (2) The ZIF-8 was prepared by the following method: Zn(NO3)2•6H2O was dissolved in methanol and sonicated; 2-methylimidazole was dissolved in methanol and sonicated; 2-methylimidazole solution was slowly added dropwise to Zn(NO3)2•6H2O solution at room temperature and magnetically stirred, allowed to stand, centrifuged with ethanol, and finally dried to obtain ZIF-8; (3) According to the mass ratio of ZnO to ZIF-8, the corresponding weight was weighed, put into a mortar, added with ethanol, ground, and finally dried to obtain ZnO / ZIF-8 nanocomposite.

2. The method for increasing Aspergillus oryzae yield according to claim 1, characterized in that, The mass ratio of ZnO to ZIF-8 in the ZnO / ZIF-8 nanocomposite is (1~4):(1~4).

3. The method for increasing Aspergillus oryzae yield according to claim 2, characterized in that, The ZnO / ZIF-8 nanocomposite was prepared by the following method: according to the mass ratio of ZnO to ZIF-8, the corresponding weights were weighed, placed in a mortar, ethanol was added, the mixture was ground, and finally dried to obtain the ZnO / ZIF-8 nanocomposite.

4. The method for increasing Aspergillus oryzae yield according to claim 3, characterized in that, The ZnO was prepared by the following method: Zn(CH3COO)2•2H2O and NaOH were dissolved in water respectively, and the two solutions were mixed together and stirred at room temperature; then the solutions were centrifuged with water and ethanol respectively, and dried in a drying oven; after being taken out, they were ground and then dried in an oven to finally obtain ZnO.

5. The method for increasing Aspergillus oryzae yield according to claim 4, characterized in that, ZIF-8 was prepared by the following method: Zn(NO3)2•6H2O was dissolved in methanol and dissolved by sonication; 2-methylimidazole was dissolved in methanol and dissolved by sonication; the 2-methylimidazole solution was slowly added dropwise to the Zn(NO3)2•6H2O solution at room temperature and stirred magnetically, allowed to stand, centrifuged with ethanol, and finally dried to obtain ZIF-8.

6. The method for increasing Aspergillus oryzae yield according to claim 5, characterized in that, The dripping rate is 1 drop / 2 seconds.

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