阅读说明：本技术 地衣芽孢杆菌LCCC10161在生产α-淀粉酶和烟叶发酵中的应用 (Application of bacillus licheniformis LCCC10161 in production of alpha-amylase and tobacco fermentation ) 是由 王胜利 关艳丽 叶亚军 池景良 张永岗 赵新海 于宏男 刘月明 张宝 *** 李 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)LCCC10161在生产α-淀粉酶和烟叶发酵中的应用,地衣芽孢杆菌(Bacillus licheniformis)LCCC10161能够产生高酶活性的α-淀粉酶,利用地衣芽孢杆菌(Bacillus licheniformis)LCCC10161生产α-淀粉酶,为α-淀粉酶的生产来源提供了一个新的途径,可以实现α-淀粉酶的规模化生产；将具有产高酶活力的α-淀粉酶的地衣芽孢杆菌应用到烟叶发酵中,能加速烟叶中淀粉的降解,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 10161) in producing alpha-amylase and tobacco fermentation, the Bacillus licheniformis (LCCC 10161) can produce alpha-amylase with high enzymatic activity, the Bacillus licheniformis (LCCC 10161) is used for producing the alpha-amylase, a new way is provided for the production source of the alpha-amylase, and the large-scale production of the alpha-amylase can be realized; the bacillus licheniformis with the alpha-amylase producing high enzyme activity is applied to the fermentation of the tobacco leaves, so that the degradation of starch 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) LCCC10161 for producing alpha-amylase.

2. A method for producing alpha-amylase by utilizing Bacillus licheniformis (LCCC 10161), which is characterized in that the method comprises the steps of inoculating the activated Bacillus licheniformis (LCCC 10161) into a liquid culture medium for fermentation culture, and centrifuging the fermentation liquid to obtain a supernatant.

3. The method of claim 2, wherein the Bacillus licheniformis (LCCC 10161) is inoculated in an amount of 10⁵～10⁷cfu/ml, wherein the culture is shaking culture, the rotating speed is 150-250 r/min, the temperature is 20-45 ℃, and the time is 3-5 d;

preferably, the inoculum size is 10⁶cfu/ml, the rotating speed is 200r/min, the temperature is 36 ℃, and the time is 4 d;

preferably, the liquid medium contains: 0.8g of bean cake powder, 2.0g of corn flour, 1.0g of NaCl and K₂HPO₄ 0.01g、KH₂PO₄ 0.01g、CaCl₂0.3g of distilled water and 100mL of distilled water, wherein the pH value is 7.5-8.5, and the content of a liquid culture medium is 8-15%;

preferably, the pH is 8.0, and the filling amount of the liquid culture medium is 10%;

preferably, Bacillus licheniformis (Bacillus licheniformis) LCCC10161 is subjected to activated culture for 15-25 days and then inoculated;

preferably, the culture medium used for activating the Bacillus licheniformis (Bacillus licheniformis) LCCC10161 is LB culture medium;

preferably, the LB medium contains 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride;

preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 8000-12000 rpm, and the time is 20-40 min.

4. The method of claim 2, further comprising the steps of adding ammonium sulfate to the supernatant for precipitation, and desalting by dialysis to obtain a crude enzyme solution.

5. The method of claim 4, wherein the saturation of ammonium sulfate is 40-60%;

preferably, the supernatant is dissolved by adding ammonium sulfate, centrifuged, and precipitated with ddH₂Dissolving O, and desalting by dialysis with an acetic acid buffer solution of 15-25 mmol/L, pH 5.8.8-6.2.

6. The method according to claim 4, further comprising the step of purifying the crude enzyme solution by sequentially using a DEAE Sepharose Fast Flow anion exchange chromatography column and a Sephadex G-75 gel filtration chromatography column.

7. The method according to claim 6, wherein the specific steps of the purification are as follows:

(1) loading the crude enzyme solution to DEAE Sepharose Fast Flow anion exchange chromatography column, and eluting with acetic acid buffer solution A to A₂₈₀After the solution is not changed, eluting the solution by using an acetic acid buffer solution containing 0.1mol/L NaCl at the flow rate of 0.8-1.2 mL/min to obtain a collection solution;

(2) and (3) loading the collected liquid to a Sephadex G-75 gel filtration chromatographic column, eluting with an acetic acid buffer solution A containing 0.2mol/L NaCl at the flow rate of 0.2-0.4 mL/min, and concentrating the collected active components to the density of 1.1-1.2G/mL.

8. Use of Bacillus licheniformis (LCCC 10161) 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 10161) is inoculated into tobacco leaves for fermentation.

10. The tobacco fermentation process of claim 9, wherein the inoculation amount of Bacillus licheniformis (LCCC 10161) is 10⁵～10⁷cfu/g, the fermentation temperature is 20-45 ℃, the humidity is 50-70%, and the time is 5-10 d.

Technical Field

The invention relates to application of bacillus licheniformis LCCC10161 in tobacco fermentation for producing alpha-amylase, belonging 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.

Alpha-amylase is one of the main enzymes used in tobacco leaf fermentation by hydrolyzing alpha-1, 4-glucosidic bonds in a starch molecular chain to cut the starch chain into short-chain dextrin, oligosaccharide and a small amount of maltose and glucose. The alpha-amylase can be produced by microbial fermentation, and can also be extracted from plants and animals. At present, the alpha-amylase is produced on a large scale by a microbial fermentation method in industrial production. Useful α -amylase producing bacteria are: bacillus subtilis, bacillus licheniformis, bacillus stearothermophilus, bacillus coagulans, bacillus amyloliquefaciens, aspergillus oryzae, aspergillus niger and the like.

Disclosure of Invention

The invention aims to provide application of Bacillus licheniformis LCCC10161 in production of alpha-amylase and tobacco fermentation, wherein the Bacillus licheniformis LCCC10161 can produce the alpha-amylase at high yield and is very suitable for production of the alpha-amylase and tobacco fermentation.

In one aspect, the invention provides the application of the bacillus licheniformis LCCC10161 in the production of alpha-amylase, and the bacillus licheniformis LCCC10161 can produce the alpha-amylase with high enzyme activity, thereby providing a new source for the production source of the alpha-amylase.

In another aspect, the present invention provides a method for producing alpha-amylase by using Bacillus licheniformis (LCCC 10161), which comprises the steps of inoculating the activated Bacillus licheniformis (LCCC 10161) into a liquid culture medium for fermentation culture, and centrifuging the fermentation liquid to obtain a supernatant.

Preferably, the inoculation amount of Bacillus licheniformis (LCCC 10161) is 10⁵～10⁷cfu/ml, wherein the culture is shaking culture, the rotating speed is 150-250 r/min, the temperature is 20-45 ℃, and the time is 3-5 d;

preference is given toOf 10 (a)⁶cfu/ml, the rotating speed is 200r/min, the temperature is 36 ℃, and the time is 4 d;

preferably, the liquid medium contains: 0.8g of bean cake powder, 2.0g of corn flour, 1.0g of NaCl and K₂HPO₄0.01g、KH₂PO₄ 0.01g、CaCl₂0.3g of distilled water and 100mL of distilled water, wherein the pH value is 7.5-8.5, and the content of a liquid culture medium is 8-15%;

preferably, the pH is 8.0, and the filling amount of the liquid culture medium is 10%;

preferably, Bacillus licheniformis (Bacillus licheniformis) LCCC10161 is subjected to activated culture for 15-25 days and then inoculated;

preferably, the culture medium used for activating the Bacillus licheniformis (Bacillus licheniformis) LCCC10161 is LB culture medium;

preferably, the LB medium contains 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride;

preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 8000-12000 rpm, and the time is 20-40 min.

Further, the method also comprises the steps of adding ammonium sulfate into the supernatant for precipitation, and obtaining crude enzyme liquid after dialysis and desalination;

preferably, the saturation degree of the ammonium sulfate is 40-60%;

preferably, the supernatant is dissolved by adding the ammonium sulfate, centrifuged, and precipitated with ddH₂Dissolving O, and desalting by dialysis with an acetic acid buffer solution of 15-25 mmol/L, pH 5.8.8-6.2.

Further, the method comprises a step of purifying the crude enzyme solution by using a DEAE Sepharose Fast Flow anion exchange chromatography column and a Sephadex G-75 gel filtration chromatography column.

Preferably, the specific steps of the crude enzyme solution purification are as follows:

(1) loading the crude enzyme solution to a Sepharose Fast Flow anion exchange chromatography column, eluting with an acetic acid buffer solution A until A280 is unchanged, and then eluting with an acetic acid buffer solution containing 0.1mol/L NaCl at the Flow rate of 0.8-1.2 mL/min to obtain a collection solution;

(2) and (3) loading the collected liquid to a Sephadex G-75 gel filtration chromatographic column, eluting with an acetic acid buffer solution A containing 0.2mol/L NaCl at the flow rate of 0.2-0.4 mL/min, and concentrating the collected active components to the density of 1.1-1.2G/mL.

On the other hand, the invention also provides application of the Bacillus licheniformis (Bacillus licheniformis) LCCC10161 or the method in tobacco leaf fermentation.

On the other hand, the invention also provides a tobacco leaf fermentation method, which is to inoculate Bacillus licheniformis (LCCC 10161) in tobacco leaves for fermentation.

Preferably, the inoculation amount of Bacillus licheniformis (LCCC 10161) is 10⁵～10⁷cfu/g, the fermentation temperature is 20-45 ℃, the humidity is 50-70%, and the time is 5-10 d.

The invention has the beneficial effects that:

the invention discloses Bacillus licheniformis (Bacillus licheniformis) LCCC10161 capable of producing alpha-amylase with high enzymatic activity, which provides a new way for the production source of the alpha-amylase and can realize the large-scale production of the alpha-amylase; the bacillus licheniformis with the alpha-amylase producing high enzyme activity is applied to the fermentation of the tobacco leaves, so that the degradation of starch 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.

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. The Bacillus licheniformis (Bacillus licheniformis) strain used by the invention is purchased from Liaoning province microorganism strain preservation center, and the serial numbers are as follows: LCCC 10161.