阅读说明：本技术 微生物法生产纳米细菌纤维素的方法 (Method for producing nano bacterial cellulose by microbiological method ) 是由 杨加志 李文静 李锦坤 李雪菲 于 2018-07-27 设计创作，主要内容包括：本发明公开了一种微生物法生产纳米细菌纤维素的方法。所述方法使用木醋杆菌作为产细菌纤维素的菌种,分别使用芽孢杆菌、木霉菌、曲霉菌或青霉菌作为产纤维素酶的菌种,在木醋杆菌动态培养过程中与产纤维素酶的菌株共培养,制得纳米细菌纤维素。本发明方法简单,可以一步制备出纳米细菌纤维素,制备工艺绿色环保。(The invention discloses a method for producing nano bacterial cellulose by a microbiological method. The method uses acetobacter xylinum as a bacterial strain for producing bacterial cellulose, uses bacillus, trichoderma, aspergillus or penicillium as a bacterial strain for producing cellulase, and co-cultures the bacillus, the trichoderma, the aspergillus or the penicillium with the bacterial strain for producing cellulase in the dynamic culture process of the acetobacter xylinum to prepare the nano bacterial cellulose. The method is simple, the nano bacterial cellulose can be prepared in one step, and the preparation process is environment-friendly.)

1. The method for producing the nano bacterial cellulose by the microbiological method is characterized by comprising the following steps:

step 1, inoculating acetobacter xylinum seed liquid into fermentation liquor, and dynamically culturing to obtain flocculent bacterial cellulose;

and 2, inoculating a strain seed solution for producing cellulase into the fermentation liquor, dynamically culturing, removing residual cells and the fermentation liquor after the fermentation is finished, and washing to be neutral to obtain the nano bacterial cellulose.

2. The method according to claim 1, wherein the inoculation amount of the acetobacter xylinum seed solution in step 1 is 8-12%.

3. The method according to claim 1, wherein in step 1, the dynamic culture is performed at a stirring speed of 150 to 180 rpm.

4. The method according to claim 1, wherein in step 2, the cellulase producing strain is selected from the group consisting of bacillus, trichoderma, aspergillus, and penicillium.

5. The method according to claim 1, wherein in the step 2, when the cellulase-producing strain is bacillus, the inoculation amount is 3-5%, the dynamic culture temperature after mixing is 27-30 ℃, the co-culture pH is 5.5-6.5, and the co-culture time is 24-48 h.

6. The method according to claim 4, wherein in the step 2, when the cellulase-producing strain is Trichoderma, the inoculation amount is 10-15%, the dynamic culture temperature after mixing is 28-30 ℃, the co-culture pH is 4.0-5.0, and the co-culture time is 72-96 h.

7. The method according to claim 4, wherein in the step 2, when the cellulase-producing strain is Aspergillus, the inoculation amount is 5-10%, the dynamic culture temperature after mixing is 30-35 ℃, the co-culture pH is 5.5-6.0, and the co-culture time is 72-96 h.

8. The method according to claim 4, wherein in the step 2, when the strain producing the cellulase is penicillium, the inoculation amount is 3-5%, the dynamic culture temperature after mixing is 30-32 ℃, the co-culture pH is 5.5-6.5, and the co-culture time is 72-96 h.

9. The method of claim 1, wherein the step 2, the step of removing residual cells and fermentation broth comprises using NaOH with a concentration of 1-5 g/L and H with a concentration of 1-5 g/L₂O₂Water bath is carried out for 0.5-3.0 h under the condition of 70-100 ℃.

Technical Field

The invention relates to a method for producing nano bacterial cellulose by a microbiological method, belonging to the technical field of biological materials.

Background

Bacterial Cellulose (BC for short) is an extracellular polysaccharide secreted by a small number of microorganisms represented by acetobacter xylinum, and is a naturally-formed fiber. Because of high crystallinity, high water holding capacity, good three-dimensional structure and good biocompatibility, the method is widely applied to many fields.

The bacterial cellulose is further degraded to obtain the nano-scale bacterial cellulose, and the length is generally within the range of 200-900 nm. The nano bacterial cellulose not only has the basic structure and performance of the bacterial cellulose, but also has the characteristics of nano particles such as extremely small size, huge specific surface area and the like, can obviously improve the uniformity of chemical reaction of the bacterial cellulose, and can be widely applied to cosmetic matrixes or drug carriers such as dispersing agents, strength enhancers, skin creams and the like.

The preparation method of the nano bacterial cellulose comprises a chemical method, a physical method and a biological method. Chemical and physical methods have many limitations: the chemical method requires hydrolysis with strong acid, has high requirements for reaction equipment, and is difficult to treat residues after reaction (lukylin, huyang, amur joss, etc.. nanocellulose research progress [ J ] Guangzhou chemical industry, 2013,41(20): 1-3.). The physical method requires high pressure and special equipment, and has large energy consumption (leaf courage, preparation of nano-cellulose [ J ] chemical development, 2007,19(10): 1568-. The existing biological method for preparing the nano-scale bacterial cellulose is environment-friendly, but the size adjustment steps are complicated, so that a simpler biological method is necessary to select (the species of zucchini, Liuwen, Liu hong Feng. the preparation and application of the nano-scale bacterial cellulose [ J ] Chinese paper making, 2012,31(6):68-73 ].

Disclosure of Invention

The invention aims to provide a method for producing nano bacterial cellulose by a microbiological method. The method comprises the steps of dynamically culturing acetobacter xylinum in a fermentation culture tank, inoculating a strain for producing cellulase when the acetobacter xylinum can stably produce bacterial cellulose, and co-culturing, wherein the produced cellulase decomposes large-size bacterial cellulose into nano bacterial cellulose.

The technical solution for realizing the purpose of the invention is as follows:

the method for producing the nano bacterial cellulose by the microbiological method comprises the following steps:

step 1, inoculating acetobacter xylinum seed liquid into fermentation liquor, and dynamically culturing to obtain flocculent bacterial cellulose;

and 2, inoculating a strain seed solution for producing cellulase into the fermentation liquor, dynamically culturing, removing residual cells and the fermentation liquor after the fermentation is finished, and washing to be neutral to obtain the nano bacterial cellulose.

Step 1, the inoculation amount of the acetobacter xylinum seed liquid is 8% -12%.

Step 1, dynamically culturing, wherein the stirring speed is 150-180 rpm.

Step 2, the strain producing the cellulase is selected from bacillus, trichoderma, aspergillus or penicillium.

And 2, when the cellulase-producing strain is bacillus, the inoculation amount is 3-5%, the dynamic culture temperature after mixing is 27-30 ℃, the co-culture pH is 5.5-6.5, and the co-culture time is 24-48 h.

And 2, when the cellulase-producing strain is trichoderma, the inoculation amount is 10-15%, the dynamic culture temperature after mixing is 28-30 ℃, the co-culture pH is 4.0-5.0, and the co-culture time is 72-96 h.

And 2, when the cellulase-producing strain is aspergillus, the inoculation amount is 5-10%, the dynamic culture temperature after mixing is 30-35 ℃, the co-culture pH is 5.5-6.0, and the co-culture time is 72-96 h.

And 2, when the strain producing the cellulase is penicillium, the inoculation amount is 3-5%, the dynamic culture temperature after mixing is 30-32 ℃, the co-culture pH is 5.5-6.5, and the co-culture time is 72-96 h.

In the step 2, the method for removing the residual cells and the fermentation liquid uses NaOH with the concentration of 1-5 g/L and H with the concentration of 1-5 g/L₂O₂Water bath is carried out for 0.5-3.0 h under the condition of 70-100 ℃.

Compared with the prior art, the invention has the following remarkable advantages:

(1) the fermentation process is green and environment-friendly, is non-toxic and harmless, and can produce the nano bacterial cellulose in one step in a fermentation tank;

(2) the produced nano bacterial cellulose is easy to post-treat, and the residual cells and fermentation liquor are removed and then washed to be neutral by water.

Drawings

FIG. 1 is a flow chart of a microbiological method for producing nano-bacterial cellulose.

FIG. 2 is a scanning electron micrograph of bacterial cellulose of an unadjusted comparative example size.

FIG. 3 is a transmission electron microscope image of the size-adjusted nano-bacterial cellulose of example 1.

FIG. 4 is a transmission electron microscope image of the size-adjusted nano-bacterial cellulose of example 2.

FIG. 5 is a transmission electron microscope image of the size-adjusted nano-bacterial cellulose of example 5.

FIG. 6 is a transmission electron microscope image of the size-adjusted nano-bacterial cellulose of example 6.

Detailed Description

The present invention will be described in more detail with reference to the following examples and the accompanying drawings.

In the following examples, seed liquid formulations: 22.5g/L glucose, 27.5g/L sucrose, 1g/L ammonium sulfate, 5g/L potassium dihydrogen phosphate, 0.7g/L magnesium sulfate, 0.2g/L calcium lactate, 0.6g/L citric acid, 1.5g/L acetic acid, 10g/L peptone and 7.5g/L yeast extract powder.

The formula of the fermentation liquid is as follows: 22.5g/L glucose, 27.5g/L sucrose, 1g/L ammonium sulfate, 5g/L potassium dihydrogen phosphate, 0.7g/L magnesium sulfate, 0.2g/L calcium lactate, 0.6g/L citric acid, 1.5g/L acetic acid, 10g/L peptone and 7.5g/L yeast extract powder.