阅读说明：本技术 一种木聚糖酶的制备方法及其在啤酒生产中的应用 (Preparation method of xylanase and application of xylanase in beer production ) 是由 孙军勇 陆健 田甜甜 王茂章 颜义勇 商曰玲 于 2019-11-29 设计创作，主要内容包括：本发明公开了一种木聚糖酶的制备方法及其在啤酒生产中的应用,属于啤酒生产技术领域。本发明将木聚糖酶添加到大麦麦芽的糖化醪液中,能够使麦汁中的聚合态阿拉伯木聚糖含量最高下降80％,麦汁的粘度降低7.2％,过滤速度提高80％。对于改善啤酒品质、提高啤酒产量具有重要意义。(The invention discloses a preparation method of xylanase and application of the xylanase in beer production, and belongs to the technical field of beer production. The xylanase is added into the mash of barley malt, so that the content of polymerized arabinoxylan in the wort can be reduced by 80 percent at most, the viscosity of the wort is reduced by 7.2 percent, and the filtering speed is improved by 80 percent. Has important significance for improving the quality of the beer and increasing the yield of the beer.)

1. A method for degrading high molecular weight arabinoxylan in wort is characterized in that xylanase with an amino acid sequence shown as SEQ ID NO.1 is added in the malt treatment process.

2. The method according to claim 1, wherein the xylanase is added together with malt at the start of mashing at an addition level of 15-30U/g malt.

3. The method of claim 1, comprising the steps of ① putting malt and the xylanase into water, keeping the temperature at 40-50 ℃ for 60-120 min to obtain mash, ② raising the temperature of the mash to 50-60 ℃ at a rate of 0.5-1.5 ℃/min, keeping the temperature at 50-60 ℃ for 35-45 min, and ③ raising the temperature of the mash to 70-75 ℃ at a rate of 0.5-1.5 ℃/min until the starch is completely decomposed.

4. Use of the method according to claims 1 to 3 for increasing the filtration rate of wort.

5. A microbial preparation method of xylanase is characterized in that Trichoderma reesei CICC41495 is taken as a fermentation strain, and corncobs and bran are taken as carbon sources for enzyme production and fermentation.

6. The preparation method according to claim 5, wherein the ratio of the corncobs to the bran is 1-5: 1.

7. The method according to claim 5, wherein the culture is carried out at 28 to 35 ℃ for 150 to 200 hours.

8. The method according to claim 5, wherein the fermentation broth after fermentation is subjected to separation, and the separation comprises the steps of: (1) decolorizing with decolorizing solution, precipitating with ammonium sulfate to obtain crude enzyme solution; (2) passing the crude enzyme solution obtained in the step (1) through a desalting column, then passing the collected solution through an ion exchange column, collecting components with xylan activity, and embedding and concentrating the components; (3) the concentrated enzyme solution was further purified by gel filtration chromatography and fractions having xylanase activity were collected.

9. Use of the method according to any one of claims 1 to 3 in the preparation of a beverage.

10. The use according to claim 9, wherein the beverage comprises a draft beer, dry beer, ice beer, low alcohol beer, non-alcohol beer, wheat beer, cloudy beer, fruit and vegetable juice type beer, fruit and vegetable flavored type beer.

Technical Field

The invention relates to a preparation method of xylanase and application of the xylanase in beer production, and belongs to the technical field of beer production.

Background

In the beer saccharification production, the mash is separated by a filter tank to obtain transparent and clear wort, and the separation speed of the wort in the mash is the filtration speed. The filtration rate of barley malt has an important influence on both the beer production efficiency and the quality of the finished beer: the filtering speed is slow, the viscosity of the mash is high, the contact and the degradation of enzyme and substrate are not facilitated, the macromolecular substances such as non-starch polysaccharide, protein, starch and the like can not be fully degraded, the extract yield is low, the production time of a single batch of beer is prolonged, and the production cost is increased.

Arabinoxylan is the most important component in the barley endosperm cell wall, belongs to non-starch polysaccharide, and accounts for about 20% of the dry weight of the endosperm cell wall and 4-10% of the total weight of barley seeds. Studies have shown that wort and finished beer still contain higher concentrations of arabinoxylans: in 36 kinds of domestic and foreign beers, the highest concentration of the arabinoxylan reaches 849mg/L, and the viscosity and the filtration speed of wort are seriously influenced. The addition of exogenous microbial enzymes capable of degrading arabinoxylan is an effective solution to this problem.

The complete degradation of arabinoxylan needs a series of enzymes to complete, and the enzyme system mainly comprises: endo-beta-1, 4-xylanase (EC 3.2.1.8), beta-1, 4-xylosidase (EC 3.2.1.37), alpha-L-arabinofuranosidase (EC3.2.1.55) and ferulic acid esterase (EC 3.1.1.6), the sites of action of which are shown in FIG. 1. The A site is beta-1, 4-xylanase which acts on beta-1, 4-xylosidic bonds which are not substituted by arabinofuranose groups in the main chain of the arabinoxylan in an endogenous mode to generate xylo-oligosaccharides with different polymerization degrees and a small amount of xylose; wherein the B site is alpha-L-arabinofuranosidase. The alpha-L-arabinofuranosidase can cleave the arabinoxylan side groups. The C site is feruloyl esterase which acts on ferulic acid which is connected with the arabinofuranose group on the O-5 site by an ester bond to release the ferulic acid; the D site is beta-xylosidase which acts on a hydrolysate of xylanase, namely xylo-oligosaccharide, further degrades the xylo-oligosaccharide from a non-reduction end to generate the beta-xylose, and plays an important role in the process of thoroughly degrading the arabinoxylan into the xylose.

Endoxylanase is a key enzyme for degrading arabinoxylan and is currently the most well studied and best studied arabinoxylan degrading enzyme. Due to factors such as substrate specificity of xylanase from different sources, steric hindrance effect caused by molecular structure of the arabinoxylan, affinity of the xylanase to the arabinoxylan, whether the xylanase has side chain hydrolysis activity and the like, the degradation capability of the microbial xylanase to different arabinoxylans is different, and no xylanase which can be suitable for degrading high molecular weight arabinoxylan in malting barley malt has been found at present, so that the application of part of barley malt with high arabinoxylan content in the beer industry is influenced.

Disclosure of Invention

In order to solve the problems, the invention provides an application of xylanase in beer production.

The technical scheme of the invention is as follows:

the invention provides a method for degrading high-molecular-weight arabinoxylan in wort, which is characterized in that xylanase with an amino acid sequence shown as SEQ ID NO.1 is added in a barley malt saccharification process.

In one embodiment of the present invention, the xylanase is added together with malt at the beginning of saccharification at an amount of 15 to 30U/g malt.

In one embodiment of the invention, the method comprises the following steps of ① putting malt and xylanase into water, preserving heat at 40-50 ℃ for 60-120 min to obtain mash, ② raising the temperature of the mash to 50-60 ℃ at the speed of 0.5-1.5 ℃/min, preserving heat at 50-60 ℃ for 35-45 min, and ③ raising the temperature of the mash to 70-75 ℃ at the speed of 0.5-1.5 ℃/min until starch is completely decomposed.

The invention provides an application of a method for degrading high-molecular-weight arabinoxylan in wort in improving the filtration speed of malt.

The invention provides a microbial preparation method of xylanase, which takes Trichoderma reesei CICC41495 as a fermentation strain and corncobs and bran as carbon sources for enzyme production and fermentation.

In one embodiment of the invention, the trichoderma reesei is CICC41495, purchased from China center for culture collection of industrial microorganisms, and stored at No. 6 Hospital No. 24 of the Zhonghao Luxiaoqiao of the Korean district, Beijing, with the storage number CICC 41495.

In one embodiment of the invention, the ratio of the corncobs to the bran is 1-5: 1.

In one embodiment of the invention, the fermentation is carried out at 28-35 ℃ for 150-200 h.

In one embodiment of the present invention, the fermented liquid is separated after fermentation, and the separation comprises the following steps: (1) precipitating with ammonium sulfate to obtain crude enzyme solution; (2) passing the crude enzyme solution obtained in the step (1) through a desalting column, then passing the collected solution through an ion exchange column, collecting components with xylan activity, and embedding and concentrating the components; (3) the concentrated enzyme solution was further purified by gel filtration chromatography and fractions having xylanase activity were collected.

In one embodiment of the present invention, the separation step specifically comprises:

(1) precipitating protein in the crude enzyme solution by adopting ammonium sulfate with 70-80% saturation, centrifuging for 10-20 min at 10,000-12,000 Xg, discarding supernatant, and dissolving the precipitate by using 15-25 mmol/L Tris-HCl buffer solution (pH 7.5-8.0) to obtain enzyme solution;

(2) desalting the enzyme solution by using a SephadexG-25 column, loading the desalted enzyme solution to a DEAE-Sepharose Fast Flow ion exchange column, and performing gradient elution by using 350-410 mL of 15-25 mmol/L Tris-HCl buffer solution (pH8.0) containing 0-0.50 mol/L NaCl at the Flow rate of 80-120 mL/h. Collecting the fractions with xylanase activity, and embedding and concentrating with PEG 20000;

(3) taking the concentrated enzyme solution, further purifying by a Sephacryl S-100 gel filtration chromatographic column, collecting components with xylanase activity, wherein the mobile phase is acetic acid-sodium acetate buffer solution with the pH of 5.0-5.5 and the mmol/L of 80-150, and the flow rate is 18-25 mL/h

The invention provides a method for degrading high molecular weight arabinoxylan in wort, which is applied to drinks.

In one embodiment of the present invention, the beverage comprises a draft beer, dry beer, ice beer, low alcohol beer, non-alcohol beer, wheat beer, turbid beer, fruit and vegetable juice type beer, fruit and vegetable taste type beer.

Has the advantages that: when the xylanase III is added into the mash of barley malt during saccharification feeding, the degradation of the arabinoxylan in the wort is promoted, so that the content of the high molecular weight arabinoxylan is reduced from 301mg/L to 38mg/L, and is reduced by 87.4%; the viscosity of wort is reduced, the filtering speed is increased from 5.0mL/min to 9.0mL/min, and is increased by 80%; improves the product quality and has important significance for the production of malt and beer.

Drawings

FIG. 1 shows the cleavage site of an arabinoxylan degrading enzyme system, wherein A represents a.beta.1, 4-xylanase, B represents an.alpha. -L-arabinofuranosidase, C represents a feruloyl esterase, and D represents a.beta. -xylosidase.

FIG. 2 is an SDS-PAGE pattern of xylanase III purification. Lane 1: xylanase III purified by Sephacryl S-100; lane 2: DEAE Sepharose Fast Flow separated xylanase III; lane 3: fermentation liquor; m, marker.

FIG. 3 is a mass spectrum of xylanase III.

FIG. 4 shows the effect of xylanase III addition on arabinoxylan content, viscosity and filtration rate in wort.

Detailed Description

The determination of endoxylanase activity takes oat xylan with the concentration of 10mg/mL as a substrate. Mixing 2mL of substrate solution with 2mL of enzyme solution diluted properly, reacting at 37 ℃ for 30min, adding 5.0mL of DNS reagent to terminate the reaction, heating in a boiling water bath for 5min, measuring the OD value at 540nm, and calculating the activity of xylanase according to a standard curve. One enzyme activity unit (U) refers to the amount of enzyme required to release 1. mu. mol xylose per minute under the assay conditions.