阅读说明：本技术 一种葡糖酸醋杆菌及其应用 (Acetobacter gluconicum and application thereof ) 是由 张婷婷 冯颖杰 杨宗灿 刘文召 张展 于 2021-06-16 设计创作，主要内容包括：本发明公开了一种葡糖酸醋杆菌及其应用,葡糖酸醋杆菌ZT-01的保藏编号为CGMCC NO.21544。本公开的葡糖酸醋杆菌可以耐受的烟碱浓度达到3.5mg/mL,无需对烟草培养基进行降烟碱处理,便可直接对烟草培养基进行发酵。不但细菌纤维素产量高,葡糖酸醋杆菌能够更直接更充分的利用烟草培养基底物,所得细菌纤维素含有烟草组分可部分或全部代替卷烟纸中木质纤维、再造烟叶粘合剂等,降低卷烟中杂气,在烟草废料综合利用方面具有较好的经济效益。(The invention discloses gluconacetobacter gluconicum with the preservation number of CGMCC NO.21544 and application thereof. The concentration of nicotine that the gluconacetobacter of this disclosure can tolerate reaches 3.5mg/mL, need not to carry out the nicotine reduction processing to the tobacco culture medium, alright directly ferment the tobacco culture medium. The yield of the bacterial cellulose is high, the acetobacter gluconicum can more directly and fully utilize the tobacco culture substrate, the obtained bacterial cellulose contains tobacco components, and the tobacco components can partially or completely replace wood fibers, reconstituted tobacco binders and the like in cigarette paper, reduce miscellaneous gas in cigarettes and have better economic benefit in the aspect of comprehensive utilization of tobacco waste.)

1. Acetobacter gluconicum (Gluconacetobacter sp.) ZT-01 has a preservation number of CGMCC NO. 21544.

2. A microbial preparation comprising the acetobacter gluconicum ZT-01 according to claim 1.

3. Use of the acetobacter gluconae ZT-01 according to claim 1 or the microbial preparation according to claim 2 in the field of preparation of bacterial cellulose.

4. A preparation method of bacterial cellulose is characterized in that firstly, acetobacter gluconicum (Gluconacetobacter sp.) ZT-01 with the preservation number of CGMCC NO.21544 is prepared into seed liquid, and then the seed liquid is inoculated into a culture medium prepared by tobacco raw materials for fermentation.

5. The method for preparing bacterial cellulose according to claim 4, wherein the tobacco raw material is tobacco powder and/or tobacco stems.

6. The method for preparing bacterial cellulose according to claim 4 or 5, characterized by comprising the steps of:

step (1): preparing an aqueous solution containing glucose, peptone, yeast powder and citric acid monohydrate, adjusting pH, sterilizing, inoculating acetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and performing shake culture at constant temperature for a period of time to obtain a seed solution;

step (2): taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and culturing for a period of time.

7. The method for preparing bacterial cellulose according to claim 6, wherein the step (1) is as follows:

preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, adjusting the pH to 4.8-5, sterilizing at 121 ℃ for 15min, inoculating acetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and then performing constant-temperature shaking culture at 25-30 ℃ under the condition of 110-150rmp to obtain a seed solution.

8. The method for preparing bacterial cellulose according to claim 6, wherein the step (2) is as follows:

adding deionized water into waste tobacco powder and/or waste tobacco stems of a cigarette factory according to the material-liquid ratio of 1 (5-15), heating and extracting at the constant temperature of 50-90 ℃ for 0.5-2 h, filtering to obtain an extracting solution, performing high-temperature moist heat sterilization at the temperature of 121 ℃ for 15min to serve as a tobacco culture medium, inoculating the seed solution into the tobacco culture medium according to the volume percentage content of 6-15%, and culturing for a period of time.

9. The method for preparing bacterial cellulose according to claim 6, wherein the step (2) is as follows:

diluting the concentrated solution of the sheet factory to 10-25% by volume, performing high temperature moist heat sterilization at 121 deg.C for 15min to obtain tobacco culture medium, inoculating the seed solution to the tobacco culture medium according to 6-15% by volume, and culturing for a certain period of time.

10. The method for preparing bacterial cellulose according to claim 6, wherein the step (2) is as follows:

taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and standing and culturing for 7-14 days; alternatively, the first and second electrodes may be,

taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and performing shake culture for 5-8 days under 50-150 rmp.

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to acetobacter gluconicum and application thereof.

Background

The yield of tobacco leaves in China is 450-ten thousand tons every year, and the tobacco wastes such as low-grade tobacco leaves, stems, branch tobaccos and the like generated in the tobacco planting and processing process can reach 25 percent of the total yield of the tobacco leaves. At present, the comprehensive utilization of waste and inferior tobacco leaf products at home and abroad mainly comprises the extraction of components such as solanesol, nicotine, protein and the like, and the preparation of organic fertilizer and chicken feed. But still a great deal of waste and inferior tobacco products are discarded or incinerated as wastes, which causes waste of natural resources and great environmental pollution.

Bacterial Cellulose (BC) has a chemical composition similar to plant cellulose, but has higher purity, water-holding capacity, degree of polymerization, degree of crystallinity, as well as better biocompatibility and higher mechanical strength. Based on its unique excellent properties, bacterial cellulose has received extensive attention from science and industry as a novel nano biomaterial, and has been widely studied and applied in various fields such as food industry, biomedicine, paper making, acoustic devices, textile and the like. The cost of the raw materials of the culture medium in the production of the bacterial cellulose is high, particularly the cost of the carbon source is high, and the large-scale production and the commercialization process of the bacterial cellulose are restricted.

The tobacco is rich in components such as sugar, amino acid, inorganic salt and the like, and the only literature reports at present are that the tobacco waste is fermented by utilizing acetobacter xylinum to generate bacterial cellulose. However, the growth of acetobacter xylinum used for fermentation is inhibited after the nicotine concentration is 1.6mg/mL, the yield is reduced, the tobacco culture medium needs to be subjected to nicotine reduction treatment, and the treatment method is complex in process and high in cost.

Therefore, how to provide a microbial strain capable of producing bacterial cellulose by fermentation using tobacco raw materials as a culture medium becomes a technical problem to be solved urgently in the field.

Disclosure of Invention

The invention aims to provide a novel technical scheme of acetobacter gluconicum ZT-01 which can utilize tobacco raw materials as a culture medium to produce bacterial cellulose by fermentation.

According to a first aspect of the present invention, there is provided a strain of Acetobacter gluconicum ZT-01.

The preservation number of the Gluconacetobacter sp ZT-01 is CGMCC NO. 21544.

According to a second aspect of the present invention, there is provided a microbial preparation.

The microbial preparation comprises acetobacter gluconicum ZT-01 disclosed by the disclosure.

According to a third aspect of the invention, the application of the acetobacter gluconicum ZT-01 or the microbial preparation disclosed in the disclosure in the field of preparing bacterial cellulose is provided.

According to a fourth aspect of the present invention, a method for preparing bacterial cellulose is provided.

The preparation method of the bacterial cellulose comprises the steps of preparing a seed solution from acetobacter gluconicum (Gluconacetobacter sp.) ZT-01 with the preservation number of CGMCC NO.21544, and inoculating the seed solution into a culture medium prepared from tobacco raw materials for fermentation.

Optionally, the tobacco material is tobacco powder and/or tobacco stems.

Optionally, the method comprises the following steps:

step (1): preparing an aqueous solution containing glucose, peptone, yeast powder and citric acid monohydrate, adjusting pH, sterilizing, inoculating acetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and performing shake culture at constant temperature for a period of time to obtain a seed solution;

step (2): taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and culturing for a period of time.

Optionally, the step (1) is specifically as follows:

preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, adjusting the pH to 4.8-5, sterilizing at 121 ℃ for 15min, inoculating acetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and then performing constant-temperature shaking culture at 25-30 ℃ under the condition of 110-150rmp to obtain a seed solution.

Optionally, the step (2) is specifically as follows:

adding deionized water into waste tobacco powder and/or waste tobacco stems of a cigarette factory according to the material-liquid ratio of 1 (5-15), heating and extracting at the constant temperature of 50-90 ℃ for 0.5-2 h, filtering to obtain an extracting solution, performing high-temperature moist heat sterilization at the temperature of 121 ℃ for 15min to serve as a tobacco culture medium, inoculating the seed solution into the tobacco culture medium according to the volume percentage content of 6-15%, and culturing for a period of time.

Optionally, the step (2) is specifically as follows:

diluting the concentrated solution of the sheet factory to 10-25% by volume, performing high temperature moist heat sterilization at 121 deg.C for 15min to obtain tobacco culture medium, inoculating the seed solution to the tobacco culture medium according to 6-15% by volume, and culturing for a certain period of time.

Optionally, the step (2) is specifically as follows:

taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and standing and culturing for 7-14 days; alternatively, the first and second electrodes may be,

taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and performing shake culture for 5-8 days under 50-150 rmp.

The concentration of nicotine that the gluconacetobacter of this disclosure can tolerate reaches 3.5mg/mL, need not to carry out the nicotine reduction processing to the tobacco culture medium, alright directly ferment the tobacco culture medium. The yield of the bacterial cellulose is high, the acetobacter gluconicum can more directly and fully utilize the tobacco culture substrate, the obtained bacterial cellulose contains tobacco components, and the tobacco components can partially or completely replace wood fibers, reconstituted tobacco binders and the like in cigarette paper, reduce miscellaneous gas in cigarettes and have better economic benefit in the aspect of comprehensive utilization of tobacco waste.

Other features of the present invention and advantages thereof will become apparent from the following detailed description of exemplary embodiments thereof, which proceeds with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.

FIG. 1 is an electron micrograph of a strain of Acetobacter gluconicum ZT-01 of the present disclosure.

FIG. 2 is a tree evolved from Acetobacter gluconicum ZT-01.

FIG. 3 is an electron micrograph of the bacterial cellulose prepared in example 1.

Fig. 4 is an XRD pattern of the bacterial cellulose prepared in example 1.

Detailed Description

Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.

The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.

Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.

In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values.

The disclosure provides an acetobacter gluconicum (Gluconaceobacter sp.) ZT-01, which is deposited in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.21544, the strain name is acetobacter gluconicum, the strain number is ZT-01, the classification name is acetobacter gluconicum, the preservation time is 2020, 12 and 23 days, and the preservation address is West Lu No. 1 Hospital 3 of the sunward area of Beijing.

An electron micrograph of Acetobacter gluconicum ZT-01 is shown in FIG. 1, and an evolutionary tree is shown in FIG. 2.

The present disclosure also provides a microbial preparation comprising acetobacter gluconicum ZT-01.

The disclosure also provides an application of the acetobacter gluconicum ZT-01 or a microbial preparation in the field of preparation of bacterial cellulose.

The disclosure also provides a preparation method of the bacterial cellulose, which comprises the steps of preparing a seed solution from the Gluconacetobacter sp ZT-01 with the preservation number of CGMCC NO.21544, and inoculating the seed solution into a culture medium prepared from tobacco raw materials for fermentation.

The tobacco material may be tobacco powder and/or tobacco stem.

The preparation method of the bacterial cellulose disclosed by the invention can comprise the following steps of:

step (1): preparing water solution containing glucose, peptone, yeast powder and citric acid monohydrate, adjusting pH, sterilizing, inoculating Acetobacter gluconicum ZT-01 with preservation number of CGMCC NO.21544, and shake culturing at constant temperature for a period of time to obtain seed solution.

The step (1) may be specifically as follows:

preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, adjusting the pH to 4.8-5, sterilizing at 121 ℃ for 15min, inoculating acetobacter gluconicum ZT-01 with the preservation number of CGMCC NO.21544, and then performing constant-temperature shaking culture at 25-30 ℃ under the condition of 110-150rmp to obtain a seed solution.

Step (2): taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and culturing for a period of time.

The step (2) may be specifically as follows:

adding deionized water into waste tobacco powder and/or waste tobacco stems of a cigarette factory according to the material-liquid ratio of 1 (5-15), heating and extracting at the constant temperature of 50-90 ℃ for 0.5-2 h, filtering to obtain an extracting solution, performing high-temperature moist heat sterilization at the temperature of 121 ℃ for 15min to serve as a tobacco culture medium, inoculating the seed solution into the tobacco culture medium according to the volume percentage content of 6-15%, and culturing for a period of time.

The step (2) may be specifically as follows:

diluting the concentrated solution of the sheet factory to 10-25% by volume, performing high temperature moist heat sterilization at 121 deg.C for 15min to obtain tobacco culture medium, inoculating the seed solution to the tobacco culture medium according to 6-15% by volume, and culturing for a certain period of time.

The step (2) may be specifically as follows:

taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and standing and culturing for 7-14 days; alternatively, the first and second electrodes may be,

taking the tobacco extract as a tobacco culture medium, inoculating the seed liquid into the tobacco culture medium according to the volume percentage of 6-15%, and performing shake culture for 5-8 days under 50-150 rmp.

The experimental procedures used in the examples below are conventional unless otherwise specified, the materials and reagents used therein are commercially available, and the equipment used in the experiments are well known to those skilled in the art without otherwise specified.

The tobacco material was obtained as follows:

the tobacco sheet concentrated solution is obtained on a certain tobacco sheet production line belonging to tobacco industry Limited liability company in Henan, and is provided by tobacco industry Limited liability company in Henan;

the tobacco powder is obtained from a certain cigarette factory belonging to tobacco industry Limited liability company in Henan, can be compounded according to the formula according to the requirement, and is provided by tobacco industry Limited liability company in Henan.

Example 1

(1) Preparing a seed solution: preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, sterilizing at the temperature of 121 ℃ for 15min at the pH of 4.8, inoculating Acetobacter gluconicum ZT-01, and performing constant-temperature shaking culture at the temperature of 30 ℃ and 120rmp for 48h to form a seed solution.

(2) Preparing a tobacco culture medium: diluting the concentrated solution of the sheet factory to 10%, and performing moist heat sterilization at 121 ℃ for 15min to obtain the tobacco culture medium.

(3) Inoculating the seed liquid into a tobacco culture medium according to the volume ratio of 6%, culturing at 30 ℃ under 110rmp by shaking for 6 days to obtain the bacterial cellulose T1.

And acquiring an electron microscope image and an XRD image of the bacterial cellulose T1.

Example 2

(1) Preparing a seed solution: preparing an aqueous solution containing 20g/L glucose, 5g/L peptone, 5g/L yeast powder and 1g/L citric acid monohydrate, sterilizing at the temperature of 121 ℃ for 15min at the pH of 4.8, inoculating Acetobacter gluconicum ZT-01, and performing constant-temperature shaking culture at the temperature of 30 ℃ and 110rmp for 24h to form a seed solution.

(2) Preparing a tobacco culture medium: adding deionized water into waste tobacco powder of a cigarette factory according to a material-liquid ratio of 1:10, heating and extracting at a constant temperature of 70 ℃ for 2h, filtering to obtain an extracting solution, and performing moist heat sterilization at a high temperature of 121 ℃ for 15min to obtain the tobacco culture medium.

(3) Inoculating the seed solution into a tobacco culture medium according to the volume ratio of 10%, and standing and culturing for 14 days at 25 ℃ to obtain the bacterial cellulose T2.

TABLE 1 bacterial cellulose yield

Serial number	Bacterial cellulose yield (g/L)
		Example 1	4.3
Example 2	5.6

As can be seen from Table 1, the yield of bacterial fiber obtained by directly fermenting a tobacco waste extract or a thin sheet concentrate diluent with Acetobacter gluconicum ZT-01 was high.

As can be seen from FIG. 3, the bacterial cellulose obtained by fermentation has a net-like structure, and the XRD pattern in FIG. 4 is the same as that of typical type I cellulose, and the Crystallinity Index (CI) crystallinity calculated based on the peak intensity using the Segal method is 90.04%, with higher crystallinity.

Although some specific embodiments of the present invention have been described in detail by way of examples, it should be understood by those skilled in the art that the above examples are for illustrative purposes only and are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.