Aspergillus zonatus ZJUBE2010 and application thereof

Abstract

This invention discloses a marine Aspergillus sp. ZJUBE 2010 and its applications. The marine Aspergillus sp. ZJUBE 2010 of this invention was deposited at the China Center for Type Culture Collection (CCTCC) on June 2, 2010, with accession number CCTCC NO: M2010132. The marine Aspergillus sp. ZJUBE 2010 of this invention can be used for the fermentation production of cellulase, with high yield and good enzyme activity. Furthermore, the marine Aspergillus sp. ZJUBE 2010 can also be used for the fermentation degradation of carbon sources, with high monosaccharide conversion rate. The fermentation process does not require high-temperature and high-pressure sterilization of the raw material fermentation medium or the equipment itself, and the fermentation effect is completely unaffected, effectively reducing equipment manufacturing costs, production costs, and energy consumption.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and particularly relates to a marine Aspergillus niger ZJUBE 2010 and its applications. Background Technology

[0002] Aspergillus niger produces cellulase in the presence of cellulose as an inducer, which is used to degrade cellulose into usable monosaccharides. The most commonly used fermentation substrates in Aspergillus niger fermentation are wheat bran, corn stalks, and rice straw powder, which can serve as both the main carbon source and the inducer.

[0003] The two most common fermentation methods for Aspergillus niger are liquid fermentation and solid-state fermentation. Among them, solid-state fermentation contains almost no free water, which is closer to the natural growth state of microorganisms, resulting in higher enzyme activity, simpler subsequent treatment, less pollution, and no wastewater discharge. Furthermore, solid-state fermentation is more extensive, does not require a strictly sterile environment, and is easier to control for contamination by other microorganisms.

[0004] However, solid-state fermentation presents challenges in sterilization, requiring large amounts of steam and placing higher demands on equipment. Therefore, if the fermentation substrate is not sterilized and raw material fermentation is used directly, it can simplify fermentation equipment, save energy, and reduce production costs. Summary of the Invention

[0005] The purpose of this invention is to provide a marine Aspergillus niger ZJUBE 2010 and its application, aiming to solve the problems of high manufacturing cost, high production cost and high energy consumption in the existing solid-state fermentation process, which requires sterilization of the fermentation substrate.

[0006] This invention is achieved as follows: a marine Aspergillus sp. ZJUBE 2010, which was deposited at the China Center for Type Culture Collection on June 2, 2010, with accession number CCTCCNO: M2010132.

[0007] This invention further discloses the application of the above-mentioned marine Aspergillus niger ZJUBE 2010 in the fermentation production of cellulase.

[0008] This invention further discloses the application of marine Aspergillus niger ZJUBE 2010 in the degradation of carbon sources into monosaccharides through fermentation.

[0009] Preferably, the fermentation method includes the following steps:

[0010] (1) Marine Aspergillus niger ZJUBE 2010 strain was inoculated onto a spore culture medium to culture spores;

[0011] (2) Inoculate the spore suspension from step (1) into the seed culture medium to cultivate the seed solution;

[0012] (3) Inoculate the seed liquid from step (2) into the raw material fermentation medium for raw material solid fermentation; wherein, the raw material solid fermentation specifically refers to: at a temperature of 35 to 40°C, directly inoculate 20 to 50% of the inoculum into the raw material fermentation medium that has not been treated with high temperature and high pressure and culture for 3 to 5 days.

[0013] Preferably, in step (1), the spore culture medium is potato dextrose agar medium, the spore culture temperature is 30-35℃, and the culture time is 3-5 days.

[0014] Preferably, in step (2), the seed culture medium contains 1-5% by mass of available carbon source and 0.25-3% by mass of organic nitrogen source, the seed culture medium is subjected to routine sterilization treatment before inoculation, and the seed liquid culture temperature is 30°C and the culture time is 2 days.

[0015] Preferably, the available carbon source includes a slow-utilizing carbon source; the organic nitrogen source includes peptone.

[0016] Preferably, in step (3), the raw material solid-state fermentation is carried out in a shallow dish, and the thickness of the raw material fermentation medium is 3 to 5 cm.

[0017] 100ml of the raw material fermentation medium comprises: 5-12g sodium chloride, 1-6g of rapidly utilized carbon source, 1-25g of slowly utilized carbon source, and 5-25g of cellulase inducer; the material-to-water ratio of the raw material fermentation medium is 3:5.

[0018] Preferably, the rapidly utilized carbon source includes glucose, and the slowly utilized carbon source includes corn stalk powder and rice straw powder; the cellulase inducer is wheat bran.

[0019] This invention overcomes the shortcomings of existing technologies and provides a marine Aspergillus sp. ZJUBE 2010 and its applications. The marine Aspergillus sp. ZJUBE 2010 or marine Aspergillus niger. ZJUBE 2010 of this invention was deposited at the China Center for Type Culture Collection (CCTCC) on June 2, 2010, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2010132. The marine Aspergillus sp. ZJUBE 2010 of this invention can be used for solid-state fermentation to degrade monosaccharides as a carbon source.

[0020] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects:

[0021] (1) The marine Aspergillus niger ZJUBE 2010 of the present invention can be used for fermentation to produce cellulase with high yield and good enzyme activity;

[0022] (2) The marine Aspergillus niger ZJUBE 2010 of the present invention is suitable for solid-state fermentation of raw materials, and has a high conversion rate of cellulose fermentation degradation into monosaccharides;

[0023] (3) In the process of using marine Aspergillus niger ZJUBE 2010 for raw material solid fermentation, there is no need to sterilize the raw material fermentation culture medium and the equipment itself under high temperature and high pressure. The fermentation effect is completely unaffected, effectively reducing the equipment manufacturing cost, production cost and energy consumption. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] Example 1

[0026] (1) Preparation of spore culture:

[0027] Spore culture medium: Use potato dextrose agar medium (PDA) (200g potato, 20g glucose, 20g agar powder, distilled water added to 1 liter). Dispense the medium into test tubes in 5ml increments, sterilize at 121℃ for 20 minutes, and then spread the test tubes into slants until cooled and solidified.

[0028] Marine Aspergillus niger ZJUBE 2010 was inoculated onto spore culture medium using an inoculation loop and cultured at 30°C for 5 days.

[0029] Rinse the marine Aspergillus niger ZJUBE 2010 spores with 50ml of sterile distilled water and transfer them to a 150ml Erlenmeyer flask containing glass beads. Shake on a shaker at 180-200rpm for 1 hour to disperse the spores evenly.

[0030] (2) Seed preparation

[0031] Seed culture medium: 1% glucose, 0.25% yeast extract, 0.25% peptone, 1% available carbon source and 0.25% organic nitrogen source by mass, natural pH.

[0032] Inoculation: Adjust the spore suspension concentration to 10. 6 The inoculation amount is 3%, that is, 3 ml of spore suspension is added to every 100 ml of seed culture medium.

[0033] Culture: Seed culture was carried out using the deep liquid immersion culture method in a 1000 ml Erlenmeyer flask (containing 500 ml of liquid), with a shaking speed of 200 rpm and a culture temperature of 30℃ for 48 hours.

[0034] (3) Raw material solid-state fermentation

[0035] Preparation of fermentation medium: 20g wheat bran, 5g corn stalks, 5g rice straw powder, 5g glucose, 0.6g NH4Cl, 0.3g KH2PO4, 0.1g FeSO4·7H2O, 5g NaCl, 50ml deionized water; after mixing the medium evenly, pour it into a shallow dish to maintain a thickness of about 3cm.

[0036] Inoculation: Seed culture with a seed age of 48 hours was inoculated into the fermentation medium at a volume of 30 ml.

[0037] After mixing the seed culture with the culture medium evenly, seal the container with eight layers of gauze.

[0038] Cultivation: Place the shallow dish in a constant temperature incubator and ferment at 37℃ for 5 days until the end of the fermentation process.

[0039] Enzyme activity assay: After fermentation, the culture medium and tap water were mixed at a ratio of 1:10, shaken on a shaker for 1 hour, and centrifuged to remove the culture medium and bacterial spores to obtain a fermentation broth containing cellulase. The enzyme activity on the filter paper was measured to be 14.32 U / g.

[0040] Example 2

[0041] (1) Preparation of spore culture:

[0042] Spore culture medium: Use potato dextrose agar medium (PDA) (200g potato, 20g glucose, 20g agar powder, distilled water added to 1 liter). Dispense the medium into test tubes in 5ml increments, sterilize at 121℃ for 20 minutes, and then spread the test tubes into slants until cooled and solidified.

[0043] Marine Aspergillus niger ZJUBE 2010 was inoculated onto spore culture medium using an inoculation loop and cultured at 35°C for 3 days.

[0044] Rinse the marine Aspergillus niger ZJUBE 2010 spores with 50ml of sterile distilled water and transfer them to a 150ml Erlenmeyer flask containing glass beads. Shake on a shaker at 180-200rpm for 1 hour to disperse the spores evenly.

[0045] (2) Seed preparation

[0046] Seed culture medium: 1% glucose, 0.25% yeast extract, 0.25% peptone, 5% available carbon source and 3% organic nitrogen source by mass, natural pH.

[0047] Inoculation: Adjust the spore suspension concentration to 10. 6 The inoculation amount is 3%, that is, 3 ml of spore suspension is added to every 100 ml of seed culture medium.

[0048] Culture: Seed culture was carried out using the deep liquid immersion culture method in a 1000 ml Erlenmeyer flask (containing 500 ml of liquid), with a shaking speed of 200 rpm and a culture temperature of 30℃ for 48 hours.

[0049] (3) Raw material solid-state fermentation

[0050] Preparation of fermentation medium: 1g corn stalk powder, 5g wheat bran, 1g glucose, 0.6g NH4Cl, 0.3g KH2PO4, 0.1g FeSO4·7H2O, 10g NaCl, 60ml deionized water; after mixing the medium evenly, pour it into a shallow dish to maintain a thickness of about 5cm.

[0051] Inoculation: Seed culture with a seed age of 48 hours was inoculated into the fermentation medium at a volume of 30 ml.

[0052] After mixing the seed culture with the culture medium evenly, seal the container with eight layers of gauze.

[0053] Cultivation: Place the shallow dish in a constant temperature incubator and ferment at 37℃ for 5 days until the end of the fermentation process.

[0054] Enzyme activity assay: After fermentation, the culture medium and tap water were mixed at a ratio of 1:10, shaken on a shaker for 1 hour, and centrifuged to remove the culture medium and bacterial spores to obtain a fermentation broth containing cellulase. The enzyme activity on the filter paper was measured to be 12.32 U / g.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of marine Aspergillus niger ZJUBE 2010 in the fermentation production of cellulase, wherein, The marine Aspergillus niger ZJUBE 2010 ( Aspergillus sp. ZJUBE 2010) was deposited at the China Center for Type Culture Collection on June 2, 2010, with accession number CCTCC NO: M2010132; The fermentation method includes the following steps: (1) Marine Aspergillus niger ZJUBE 2010 strain was inoculated onto a spore culture medium to culture spores; (2) Prepare the spores from step (1) into a suspension, and inoculate the spore suspension into the seed culture medium to cultivate the seed liquid; (3) The seed liquid from step (2) is inoculated into a raw material fermentation medium for solid-state fermentation. Specifically, the solid-state fermentation involves inoculating 20-50% of the inoculum directly into a raw material fermentation medium that has not undergone high-temperature and high-pressure treatment at 35-40°C for 3-5 days. 100ml of the raw material fermentation medium includes 5-12g of sodium chloride, 1-6g of rapidly utilized carbon source, 1-25g of slowly utilized carbon source, and 5-25g of cellulase inducer. The material-to-water ratio of the raw material fermentation medium is 3:

5. The rapidly utilized carbon source is glucose, and the slowly utilized carbon source is selected from corn stalk powder and rice straw powder. The cellulase inducer is wheat bran. In step (1), the spore culture medium is potato dextrose agar medium, the spore culture temperature is 30-35℃, and the culture time is 3-5 days; In step (2), the seed culture medium contains 1-5% by mass of available carbon source and 0.25-3% by mass of organic nitrogen source. The seed culture medium is subjected to routine sterilization treatment before inoculation. The seed culture temperature is 30°C and the culture time is 2 days.

2. The application as described in claim 1, characterized in that, The available carbon source includes a slow-utilizing carbon source; the organic nitrogen source includes peptone.

3. The application as described in claim 1, characterized in that, In step (3), the raw material solid-state fermentation is carried out in a shallow dish, and the thickness of the raw material fermentation medium is 3 to 5 cm.