Application of aspergillus niger LCCC30112 in production of glucoamylase and fermentation of tobacco leaves
阅读说明：本技术 黑曲霉lccc30112在生产葡萄糖淀粉酶和烟叶发酵中的应用 (Application of aspergillus niger LCCC30112 in production of glucoamylase and fermentation of tobacco leaves ) 是由 纪旭东 钟丽娟 王胜利 赵新海 温源 张庆华 赵赛月 王伟华 张宝 *** 李力 于 2019-09-25 设计创作，主要内容包括：本发明提供了一种黑曲霉(Aspergillus niger)LCCC30112在生产葡萄糖淀粉酶和烟叶发酵中的应用,黑曲霉(Aspergillus niger)LCCC30112能够产生高酶活性的葡萄糖淀粉酶,利用黑曲霉(Aspergillus niger)LCCC30112生产葡萄糖淀粉酶,为葡萄糖淀粉酶的生产来源提供了一个新的途径,可以实现葡萄糖淀粉酶的规模化生产；将具有产高酶活力的葡萄糖淀粉酶的黑曲霉应用到烟叶发酵中,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 Aspergillus niger (Aspergillus niger) LCCC30112 in production of glucoamylase and fermentation of tobacco leaves, wherein the Aspergillus niger (Aspergillus niger) LCCC30112 can produce glucoamylase with high enzyme activity, and the Aspergillus niger (Aspergillus niger) LCCC30112 is used for producing glucoamylase, so that a new way is provided for a production source of the glucoamylase, and large-scale production of the glucoamylase can be realized; the Aspergillus niger with glucoamylase 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 Aspergillus niger LCCC30112 for the production of glucoamylase.
2. A method for producing glucoamylase by using Aspergillus niger (LCCC 30112), which is characterized in that the method comprises the steps of inoculating activated Aspergillus niger (LCCC 30112) in a liquid culture medium for fermentation culture, and centrifuging the fermentation liquid to obtain a supernatant.
3. The method of claim 2, wherein the Aspergillus niger (Aspergillus niger) LCCC30112 is inoculated in an amount of 105～107cfu/ml, wherein the culture is aeration culture, and the aeration quantity is 1: 0.5-1.5V/V.min, the temperature is 20-40 ℃, and the time is 2-4 d;
preferably, the inoculum size is 106cfu/ml, ventilation 1: 1.0V/V.min, the temperature is 30 ℃, and the time is 3 d;
preferably, the liquid medium contains: 2g/L of cane sugar, 4g/L of corn starch, 1g/L of soybean meal, 0.5g/L of sodium nitrate and 0.05g/L, pH of monopotassium phosphate are 5.0-6.0, and the loading amount of the liquid culture medium is 60-80%;
preferably, the pH is 5.5, and the filling amount of the liquid culture medium is 75%;
preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 5000-7000 r/min, and the time is 10-20 min;
preferably, Aspergillus niger (LCCC 30112) is activated and cultured in a PDA culture medium for 2-5 days and then inoculated;
preferably, the PDA medium contains: 200g/L of potato, 20g/L of glucose and 15g/L of agar.
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 crude enzyme.
5. The method of claim 4, wherein the saturation of ammonium sulfate is 75-85%;
preferably, after the supernatant is added with ammonium sulfate for dissolution, 7000-9000 r/min is centrifuged for 20-40 min, and the precipitate is dialyzed by deionized water.
6. The method according to claim 4, wherein the method further comprises a step of purifying the crude enzyme using a Sepharose Fast Flow anion exchange chromatography column and a Sephadex G-50 gel filtration chromatography column in this order.
7. The method according to claim 6, wherein the specific steps of the purification are as follows:
(1) loading the crude enzyme to a Sepharose Fast Flow anion exchange chromatography column, eluting with Tris-HCl buffer solution at the Flow rate of 1.5-2.5 mL/min, collecting active components, dialyzing with deionized water, and freeze-drying to obtain enzyme powder;
(2) dissolving the enzyme powder by using a citric acid-disodium hydrogen phosphate buffer solution, loading the enzyme powder to a Sephadex G-50 gel filtration chromatography column, eluting by using a Tris-HCl buffer solution at the flow rate of 1.5-2.5 mL/min, collecting active components, dialyzing by using deionized water, and freeze-drying to obtain the purified enzyme powder.
8. Use of Aspergillus niger (Aspergillus niger) LCCC30112 or the method according to any of claims 2 to 7 for the fermentation of tobacco leaves.
9. A tobacco leaf fermentation method is characterized in that Aspergillus niger (LCCC 30112) is inoculated in tobacco leaves for fermentation.
10. The tobacco leaf fermentation method according to claim 9, wherein the amount of Aspergillus niger (Aspergillus niger) LCCC30112 inoculated is 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 40-70%, and the time is 5-10 d.
Technical Field
The invention relates to application of Aspergillus niger LCCC30112 in production of glucoamylase tobacco fermentation, and belongs to the technical field of microbial application.
Background
The glucoamylase can hydrolyze alpha-1.4 glycosidic bond and alpha-1.6 glycosidic bond in starch, and gradually hydrolyze glucose from non-reducing end in substrate molecule in hydrolysis process, and can be used for degrading starch in tobacco leaves. The fermentation of tobacco leaves is a process for promoting the physical and chemical properties of the tobacco leaves to be deeply changed under certain temperature and humidity conditions, and 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.
Aspergillus niger (Aspergillus niger) belongs to Aspergillus filamentous fungi, and is widely used in the fermentation industry due to its strong protein secretion ability, and the strains produce the most enzymes, such as amylase, protease, pectinase, cellulase, lactase, phytase, tannase, penicillin, natamycin, vancomycin, and the like. More than 20 kinds of enzyme using strains for food industry are judged by food safety expert committee such as FAO/WHO/JECFA, wherein Aspergillus niger is the first high-living strain with permitted use and safety. In the prior art, reports of Aspergillus niger with high enzyme activity screened from the existing strains and application thereof are not found.
Disclosure of Invention
The invention aims to provide application of Aspergillus niger (LCCC 30112) in production of glucoamylase and tobacco fermentation, wherein the Aspergillus niger (LCCC 30112) can be used for high-yield production of glucoamylase and is very suitable for production of glucoamylase and tobacco fermentation.
In one aspect, the invention provides the use of Aspergillus niger (Aspergillus niger) LCCC30112 for the production of glucoamylase, Aspergillus niger (Aspergillus niger) LCCC30112 is capable of producing glucoamylase with high enzymatic activity, providing a new source for the production source of glucoamylase.
In another aspect, the present invention provides a method for producing glucoamylase using Aspergillus niger (Aspergillus niger) LCCC30112, comprising the steps of inoculating activated Aspergillus niger (Aspergillus niger) LCCC30112 in a liquid medium for fermentation culture, and centrifuging the fermentation broth to obtain a supernatant.
Preferably, the Aspergillus niger LCCC30112 is inoculated in an amount of 105～107cfu/ml, wherein the culture is aeration culture, and the aeration quantity is 1: 0.5-1.5V/V.min, the temperature is 20-40 ℃, and the time is 2-4 d;
preferably, the inoculum size is 106cfu/ml, ventilation 1: 1.0V/V.min, the temperature is 30 ℃, and the time is 3 d;
preferably, the liquid medium contains: 2g/L of cane sugar, 4g/L of corn starch, 1g/L of soybean meal, 0.5g/L of sodium nitrate and 0.05g/L, pH of monopotassium phosphate are 5.0-6.0, and the loading amount of the liquid culture medium is 60-80%;
preferably, the pH is 5.5, and the filling amount of the liquid culture medium is 75%;
preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 5000-7000 r/min, and the time is 10-20 min;
preferably, Aspergillus niger (LCCC 30112) is activated and cultured in a PDA culture medium for 2-5 days and then inoculated;
preferably, the PDA medium contains: 200g/L of potato, 20g/L of glucose and 15g/L of agar.
Further, the method also comprises the steps of adding ammonium sulfate into the supernatant for precipitation, and obtaining crude enzyme after dialysis and desalination.
Preferably, the saturation degree of the ammonium sulfate is 75-85%;
preferably, the saturation of ammonium sulfate is 80%;
preferably, after the supernatant is added with ammonium sulfate for dissolution, 7000-9000 r/min is centrifuged for 20-40 min, and the precipitate is dialyzed by deionized water;
preferably, the centrifugation is carried out for 30min at 8000 r/min.
Further, the method may further comprise a step of purifying the crude enzyme using a Sepharose Fast Flow anion exchange chromatography column and a Sephadex G-50 gel filtration chromatography column.
Preferably, the specific steps of the purification are as follows:
(1) loading the crude enzyme to a Sepharose Fast Flow anion exchange chromatography column, eluting with Tris-HCl buffer solution at the Flow rate of 1.5-2.5 mL/min, collecting active components, dialyzing with deionized water, and freeze-drying to obtain enzyme powder;
(2) dissolving the enzyme powder by using a citric acid-disodium hydrogen phosphate buffer solution, loading the enzyme powder to a Sephadex G-50 gel filtration chromatography column, eluting by using a Tris-HCl buffer solution at the flow rate of 1.5-2.5 mL/min, collecting active components, dialyzing by using deionized water, and freeze-drying to obtain the purified enzyme powder.
In another aspect, the invention also provides the use of Aspergillus niger (Aspergillus niger) LCCC30112 or the method in tobacco leaf fermentation.
In another aspect, the invention also provides a tobacco leaf fermentation method, Aspergillus niger (Aspergillus niger) LCCC30112 is inoculated in tobacco leaves for fermentation.
Preferably, the Aspergillus niger LCCC30112 is inoculated in an amount of 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 40-70%, and the time is 5-10 d.
The invention has the beneficial effects that:
the invention discloses Aspergillus niger LCCC30112 capable of producing glucoamylase with high enzyme activity, which provides a new way for the production source of glucoamylase and can realize the large-scale production of glucoamylase; the Aspergillus niger with glucoamylase 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 Aspergillus niger strain used in the invention is purchased from Liaoning province microorganism strain preservation center, and the number is as follows: LCCC 30112.