阅读说明：本技术 地衣芽孢杆菌LCCC10150在生产β-葡聚糖酶和烟叶发酵中的应用 (Application of bacillus licheniformis LCCC10150 in production of beta-glucanase and tobacco fermentation ) 是由 郝捷 赵新海 柴颖 池景良 郭春生 钟丽娟 陈晨 杨洪峰 刘海龙 郭荣刚 *** 于 2019-09-25 设计创作，主要内容包括：本发明提供了一种地衣芽孢杆菌(Bacillus licheniformis)LCCC10150在生产β-葡聚糖酶和烟叶发酵中的应用,地衣芽孢杆菌(Bacillus licheniformis)LCCC10150能够产生高酶活性的β-葡聚糖酶,利用地衣芽孢杆菌(Bacillus licheniformis)LCCC10150生产β-葡聚糖酶,为β-葡聚糖酶的生产来源提供了一个新的途径,可以实现β-葡聚糖酶的规模化生产；将具有产高酶活力的β-葡聚糖酶的地衣芽孢杆菌应用到烟叶发酵中,能加速烟叶中葡聚糖的分解,明显缩短烟叶的发酵周期,提升烟叶的品质。(The invention provides an application of Bacillus licheniformis (LCCC 10150) in beta-glucanase production and tobacco fermentation, the Bacillus licheniformis (LCCC 10150) can produce beta-glucanase with high enzymatic activity, the Bacillus licheniformis (LCCC 10150) is used for producing the beta-glucanase, a new way is provided for the production source of the beta-glucanase, and the large-scale production of the beta-glucanase can be realized; the bacillus licheniformis with beta-glucanase with high enzyme activity is applied to the fermentation of tobacco leaves, the decomposition of glucan in the tobacco leaves can be accelerated, the fermentation period of the tobacco leaves is obviously shortened, and the quality of the tobacco leaves is improved.)

1. Use of Bacillus licheniformis (Bacillus licheniformis) LCCC10150 for producing beta-glucanase.

2. A method for producing beta-glucanase by utilizing Bacillus licheniformis (LCCC 10150), which is characterized by comprising the step of inoculating the Bacillus licheniformis (LCCC 10150) into a liquid culture medium for fermentation culture to obtain a fermentation liquid.

3. The method of claim 2, wherein the Bacillus licheniformis (LCCC 10150) is inoculated in an amount of 10⁵～10⁷cfu/ml, wherein the culture is shaking culture, the rotating speed is 100-200 r/min, the temperature is 20-40 ℃, and the time is 1-3 d;

preferably, the inoculum size is 10⁶cfu/ml, the rotating speed is 160r/min, the temperature is 35 ℃, and the time is 2 d;

preferably, the liquid medium contains NaNO₃ 0.2g、K₂HPO₄ 0.1g、KCl 0.05g、MgSO₄·7H₂O 0.05g、FeSO₄·7H₂0.01g of O, 5g of oat flour and 100mL of distilled water, wherein the pH value of the culture medium is 6.5-7.0;

preferably, the pH is 6.7.

4. The method of claim 2, further comprising the step of centrifuging the fermentation broth, and precipitating the supernatant by adding ammonium sulfate to obtain a crude enzyme solution.

5. The method according to claim 4, wherein the rotation speed of the centrifugation is 8000-12000 rpm, and the time is 8-12 min;

preferably, the supernatant is added with 30-50% of saturated ammonium sulfate for full dissolution and then centrifuged, and the supernatant is added with 60-80% of saturated ammonium sulfate and centrifuged to obtain the crude enzyme.

6. The method according to claim 4, further comprising the step of desalting the crude enzyme solution using SephadexG-25 column and purifying the crude enzyme solution using SephadexG 100 column chromatography in this order.

7. The method as claimed in claim 6, wherein, in the desalting of SephadexG-25 column, the elution is carried out with 45-55 mmol/L sodium acetate buffer solution with pH of 5-5.5 at an elution speed of 55-65 ml/h;

and/or eluting with 45-55 mmol/L sodium acetate buffer solution with pH of 5-5.5 at an elution speed of 10-15 mL/h during SephadexG 100 column chromatography.

8. Use of Bacillus licheniformis (LCCC 10150) or the method according to any of claims 2-7 for the fermentation of tobacco leaves.

9. A tobacco leaf fermentation method is characterized in that Bacillus licheniformis (LCCC 10150) is inoculated into tobacco leaves for fermentation.

10. The tobacco fermentation process of claim 9, wherein the inoculation amount of Bacillus licheniformis (LCCC 10150) is 10⁵～10⁷cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 50-70%, and the time is 7-14 d.

Technical Field

The invention relates to application of bacillus licheniformis LCCC10150 in production of beta-glucanase and tobacco fermentation, and belongs to the technical field of microbial application.

Background

The tobacco leaf fermentation is one of the important links in the tobacco leaf processing process, and the tobacco leaf fermentation process promotes the deep change of the physical and chemical properties of the tobacco leaves under certain temperature and humidity conditions, so that the method is a primary processing method for improving the product quality in the cigarette industry.

For the fermentation of tobacco leaves, natural fermentation and artificial fermentation are mostly adopted at present. The natural fermentation is mainly carried out by means of the change of natural climate, and is mainly characterized in that the temperature rise in spring of one year is utilized to promote the activity of enzymes in the tobacco leaves during the storage period of the tobacco leaves, so that the tobacco leaves are subjected to a slow fermentation process to achieve the purpose of improving the quality of the tobacco leaves. The artificial fermentation is a method for accelerating the change of the tobacco leaves by utilizing artificial conditions (namely suitable temperature and humidity) suitable for the change of the internal quality of the tobacco leaves. However, both methods have the disadvantage that the fermentation progresses slowly.

The beta-glucanase is a general name of a plurality of enzymes for catalyzing and hydrolyzing beta-glucan, and is one of the main enzymes used in tobacco leaf fermentation. The most commonly used enzyme-producing strains for the international industrial production of glucan: bacillus licheniformis can be used for producing enzyme preparation of food. Therefore, the screening of the bacillus licheniformis with high enzyme activity has important significance.

Disclosure of Invention

The invention aims to provide application of Bacillus licheniformis (LCCC 10150) in production of beta-glucanase and tobacco fermentation, wherein the Bacillus licheniformis (LCCC 10150) can produce the beta-glucanase with high yield and is very suitable for production of the beta-glucanase and tobacco fermentation.

In one aspect, the invention provides an application of Bacillus licheniformis (LCCC 10150) in producing beta-glucanase. The Bacillus licheniformis (Bacillus licheniformis) LCCC10150 can produce beta-glucanase with high enzymatic activity, and provides a new source for producing the beta-glucanase.

In another aspect, the present invention also provides a method for producing beta-glucanase by using Bacillus licheniformis (LCCC 10150), which comprises the step of inoculating the Bacillus licheniformis (LCCC 10150) into a liquid medium for fermentation culture to obtain a fermentation broth.

Inoculation amount of Bacillus licheniformis (Bacillus licheniformis) LCCC10150 10⁵～10⁷cfu/ml, the culture is shaking culture, and the rotating speed is100-200 r/min, 20-40 ℃ and 1-3 d;

preferably, the inoculum size is 10⁶cfu/ml, the rotating speed is 160r/min, the temperature is 35 ℃, and the time is 2 d;

preferably, the liquid medium contains NaNO₃ 0.2g、K₂HPO₄ 0.1g、KCl 0.05g、MgSO₄·7H₂O 0.05g、FeSO₄·7H₂0.01g of O, 5g of oat flour and 100mL of distilled water, wherein the pH value of the culture medium is 6.5-7.0;

preferably, the pH is 6.7.

Further, the method also comprises the steps of centrifuging the fermentation liquor, and adding ammonium sulfate into the supernatant for precipitation to obtain a crude enzyme solution.

Preferably, the rotating speed of the centrifugation is 8000-12000 rpm, and the time is 8-12 min;

preferably, the supernatant is added with 30-50% of saturated ammonium sulfate for full dissolution and then centrifuged, and the supernatant is added with 60-80% of saturated ammonium sulfate and centrifuged to obtain the crude enzyme.

The method also comprises the step of desalting the crude enzyme solution by using a SephadexG-25 column and purifying by using a SephadexG 100 column chromatography in sequence.

Preferably, when desalting is carried out by a SephadexG-25 column, eluting with 45-55 mmol/L sodium acetate buffer solution with the pH of 5-5.5 at the elution speed of 55-65 ml/h;

and/or eluting with 45-55 mmol/L sodium acetate buffer solution with pH of 5-5.5 at an elution speed of 10-15 mL/h during SephadexG 100 column chromatography.

In another aspect, the invention provides a tobacco leaf fermentation method, wherein Bacillus licheniformis (LCCC 10150) is inoculated into tobacco leaves for fermentation.

Preferably, the inoculation amount of Bacillus licheniformis (LCCC 10150) is 10⁵～10⁷cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 50-70%, and the time is 7-14 d.

The invention has the beneficial effects that:

the invention discloses Bacillus licheniformis (LCCC 10150) capable of producing beta-glucanase with high enzyme activity, provides a new way for the production source of the beta-glucanase, and applies the Bacillus licheniformis capable of producing the beta-glucanase with high enzyme activity to tobacco leaf fermentation, so that the decomposition of glucan in tobacco leaves can be accelerated, the fermentation period of the tobacco leaves is obviously shortened, and the quality of the tobacco leaves is improved.

Detailed Description

The present invention is described in detail with reference to specific examples, which are provided to facilitate the understanding of the technical solutions of the present invention by those skilled in the art, and the implementation or use of the present invention is not limited by the description of the present invention.

In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, if not specified. Bacillus licheniformis (Bacillus licheniformis) used in the invention is purchased from Liaoning province microorganism strain preservation center and is numbered LCCC 10150.