CN110714037A - A kind of preparation method of xylanase and its application in beer production - Google Patents

Abstract

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.

Description

A kind of preparation method of xylanase and its application in beer production

technical field

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

Background technique

In the production of beer saccharification, the saccharified mash is separated by a filter tank to obtain a transparent and clear wort, and the separation speed of the wort in the saccharified mash is the filtration speed. The filtration speed of barley malt has an important influence on the beer production efficiency and the quality of the finished beer: the filtration speed is slow, and the viscosity of the mash is high, which is not conducive to the contact and degradation of enzymes and substrates, resulting in non-starch polysaccharides, proteins and starches. The macromolecular substances cannot be fully degraded, and the yield of the extract is low, which prolongs the production time of a single batch of beer and increases the production cost.

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

The complete degradation of arabinoxylan requires a series of enzymes, which mainly include: endo-β-1,4-xylanase (EC 3.2.1.8), β-1,4-xylosidase ( EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55) and ferulic acid esterase (EC 3.1.1.6), the action sites of these enzymes are shown in Figure 1. The A site is β-1,4-xylanase, which acts on the β-1,4-xylanase bond in the main chain of arabinoxylan that is not substituted by arabinofuranosyl groups in an endoscopic manner to generate a degree of polymerization Different xylo-oligosaccharides and a small amount of xylose; where the B site is alpha-L-arabinofuranosidase. Alpha-L-arabinofuranosidase is capable of cleaving arabinoxylan side chain groups. The C site is ferulic acid esterase, which acts on the ferulic acid linked to the arabinofuranosyl group by an ester bond at the O-5 position to release ferulic acid; the D site is β-xylosidase, which acts on The hydrolyzed product of xylanase, xylo-oligosaccharide, further degrades xylo-oligosaccharide from the non-reducing end to form β-xylose, which plays an important role in the complete degradation of arabinoxylan to xylose.

Endoxylanase is the key enzyme for degrading arabinoxylan, and it is also the most studied and thorough arabinoxylan degrading enzyme. Due to the substrate specificity of xylanase from different sources, the steric hindrance effect caused by the molecular structure of arabinoxylan, the affinity of xylanase for arabinoxylan and whether it has side chain hydrolysis activity, etc. , resulting in differences in the ability of microbial xylanase to degrade different arabinoxylans. At present, no xylanase has been found that can degrade high molecular weight arabinoxylan in malt barley malt, which affects the Application of some barley malts with high arabinoxylan content in the beer industry.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides a xylanase for use in beer production.

The technical scheme of the present invention is as follows:

The present invention provides a method for degrading high-molecular-weight arabinoxylan in wort. The method is to add xylanase whose amino acid sequence is shown in SEQ ID NO. 1 in the process of barley maltosification.

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

In one embodiment of the present invention, the method comprises the following steps: ① put the malt and the xylanase into water together, and keep the temperature at 40～50℃ for 60～120min to prepare mash; ② put the mash The liquid is heated up to 50～60°C at a rate of 0.5～1.5°C/min, and kept at 50～60°C for 35～45min; 3. The mash is heated to 70～75°C at a rate of 0.5～1.5°C/min, until starch Completely decomposed.

The invention provides the 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. Trichoderma reesei CICC41495 is used as fermentation strain, and corncob and bran are used as carbon sources for enzyme production and fermentation.

In one embodiment of the present invention, the Trichoderma reesei is CICC41495, which was purchased from the China Industrial Microorganism Collection and Management Center, and the preservation address is Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, and the preservation number is CICC41495.

In an embodiment of the present invention, the ratio of the corncob to the bran is 1-5:1.

In an embodiment of the present invention, the fermentation is culturing at 28-35° C. for 150-200 h.

In one embodiment of the present invention, the fermentation liquid after fermentation is separated, and the separation includes the following steps: (1) precipitating with ammonium sulfate to obtain a crude enzyme liquid; (2) passing the crude enzyme liquid obtained in step (1) through The solution collected after the desalting column is passed through the ion exchange column to collect the components with xylan activity, which are embedded and concentrated; (3) the concentrated enzyme solution is further purified by gel filtration chromatography column, and the components with xylan activity are collected. components of enzymatic activity.

In one embodiment of the present invention, the step of separating is specifically:

(1) Use 70-80% ammonium sulfate to precipitate the protein in the crude enzyme solution, centrifuge at 10,000-12,000 × g for 10-20 min, discard the supernatant, and use 15-25 mmol/L Tris-HCl buffer solution (pH7. 5～8.0) dissolving to obtain enzyme liquid;

(2) After desalting the above enzyme solution with Sephadex G-25 column, load the sample on DEAE-Sepharose Fast Flow ion exchange column, and use 350-410 mL of 15-25 mmol/L Tris-HCl buffer solution containing 0-0.50 mol/L NaCl ( pH8.0) gradient elution, the flow rate is 80～120mL/h. The fractions with xylanase activity were collected, embedded and concentrated with PEG20000;

(3) Take the concentrated enzyme liquid, further purify using Sephacryl S-100 gel filtration chromatographic column, the mobile phase is acetic acid-sodium acetate buffer solution of pH5.0～5.5, 80～150mmol/L, and the flow rate is 18～25mL/ h, collect fractions with xylanase activity

The invention provides a method for degrading high-molecular-weight arabinoxylan in wort for application in beverages.

In one embodiment of the present invention, the beverages include cooked beer, draft beer, fresh beer, dry beer, cold beer, low-alcohol beer, non-alcohol beer, wheat beer, cloudy beer, fruit and vegetable juice beer, fruit and vegetable flavored beer beer.

Beneficial effects: In the present invention, when feeding in saccharification, the xylanase III prepared by the present invention is added to the saccharified mash of barley malt, which promotes the degradation of arabinoxylan in wort, and makes high molecular weight arabinoxylan The content decreased from 301mg/L to 38mg/L, a decrease of 87.4%; the viscosity of the wort was reduced, the filtration speed was increased, and the filtration speed was increased from 5.0mL/min to 9.0mL/min, an increase of 80%; improved It is of great significance to the production of malt and beer.

Description of drawings

Figure 1 shows the cleavage sites of arabinoxylan degrading enzymes, A indicates β-1,4-xylanase, B indicates α-L-arabinofuranosidase, C indicates ferulic acid esterase, and D indicates beta-xylosidase.

Figure 2 is the SDS-PAGE pattern of the purification process of xylanase III. Lane 1: Xylanase III purified by Sephacryl S-100; Lane 2: Xylanase III separated by DEAE Sepharose Fast Flow; Lane 3: Fermentation broth; M, marker.

Figure 3 is a mass spectrum of xylanase III.

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

Detailed ways

The determination of endo-xylanase activity took 10 mg/mL oat xylan as the substrate. Mix 2 mL of substrate solution with 2 mL of appropriately diluted enzyme solution, react at 37 °C for 30 min, add 5.0 mL of DNS reagent to stop the reaction, heat in a boiling water bath for 5 min, measure the OD value at 540 nm, and calculate xylanase according to the standard curve vitality. One unit of enzyme activity (U) refers to the amount of enzyme required to release 1 μmol of xylose per minute under assay conditions.

Example 1 Preparation of Trichoderma reesei CICC41495 fermentation broth

The seed medium was prepared as follows: ammonium sulfate 1.4g/L, glucose 10g/L, potassium dihydrogen phosphate 2.0g/L, yeast powder 1.0g/L, calcium chloride 0.3g/L, magnesium sulfate 0.3g/L, chlorine Cobalt 2.0mg/L, ferrous sulfate 5.0mg/L, zinc sulfate 1.4mg/L, manganese sulfate 1.6mg/L, pH is natural.

Preparation of enzyme production medium: replace the carbon source---glucose in the seed medium with corn and bran (30g/L of corn and 10g/L of bran), and the rest of the ingredients remain unchanged.

Trichoderma reesei CICC41495 was stored on a potato---glucose---agar slant, and 0.9% NaCl solution was used to collect spores for inoculation during use. A 250 mL conical flask was filled with 25 mL of the above-mentioned seed culture medium, and the spore liquid was inserted into it.

25 mL of enzyme production medium was placed in a 250 mL conical flask, and 2.5 mL of seed solution was inserted. The fermentation broth was centrifuged at 10,000 × g for 15 min, and after freeze-drying, the protein in the whole culture broth of Trichoderma reesei CICC41495 was obtained and stored at 4°C for future use.

Example 2 Purification of Endoxylanase III

The purification steps of endo-xylanase III are:

(1) Use 75% saturated ammonium sulfate to precipitate the protein in the crude enzyme solution, centrifuge at 10,000 × g for 15 min, discard the supernatant, and dissolve the precipitate with 20 mmol/L Tris-HCl buffer solution (pH 8.0);

(2) After desalting the above enzyme solution with SephadexG-25 column, load the sample on DEAE-Sepharose Fast Flow ion exchange column, and use 400mL of 20mmol/L Tris-HCl buffer solution (pH8.0) containing 0～0.50mol/L NaCl Gradient elution with a flow rate of 100 mL/h. The fractions with xylanase activity were collected, embedded and concentrated with PEG20000;

(3) get the concentrated enzyme liquid, adopt Sephacryl S-100 gel filtration chromatographic column to further purify, the mobile phase is the acetic acid-sodium acetate buffer solution of pH5.5, 100mmol/L, flow velocity 20mL/h, collect and have xylem polymers The components of carbohydrase activity, the results of SDS-PAGE showed that the purified xylanase reached electrophoresis purity, and the results are shown in Figure 2.

During the purification process, the protein concentration of the samples was determined by the Coomassie brilliant blue method, and the purity of the inhibitory protein was analyzed by SDS-PAGE. The specific method is:

(1) Mix the sample with 4 times the volume of loading buffer solution (2% SDS, 0.1% bromophenol blue, 10% glycerol), and bath in boiling water for 5 minutes;

(2) Take 30 μL of sample (the separation gel concentration of SDS-PAGE is 12.5%, the concentration of the stacking gel is 5%), and the voltage is 60V until the bromophenol blue indicator band reaches the bottom of the stacking gel and forms a straight line → 80V voltage until the bromophenol blue The indicator tape reaches the bottom of the separating gel;

(3) Fix with fixative solution (ratio of methanol:acetic acid:water 5:1:4) for 30min, and stain with 0.25% Coomassie brilliant blue R-250 solution for 1h;

(4) Decolorize with decolorizing solution (methanol:acetic acid:water ratio is 1:1:8) until the background is clear.

The purified xylanase was identified as endo-β-1,4-xylanase III (EC 3.2.1.8) by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (Figure 3). The theoretical molecular weight of the enzyme is 38.0kDa, and the theoretical pI is 6.97. It belongs to the GHF10 family of glycoside hydrolase. The molecular weight of the xylanase determined by SDS-PAGE is 32.0kDa, and the pI value is about 9.0.

Table 1 Mass spectrometry identification results of xylanase

Example 3 Application of purified endo-xylanase III in barley maltosaccharification process

(1) The saccharification process is:

The preparation method of the saccharified wort simulated industrial production is:

① 25.0kg of finely pulverized malt and 100L of tap water at 46°C were put into the saccharification pot (the volume of the saccharification pot was 200L), and kept at 45°C for 90 minutes to make the degradation of barley malt high molecular weight arabinoxylan by xylanase more effective. full;

② The mash is heated to 65°C at a rate of 1°C/min, and kept at 65°C for 40 minutes;

3. The mash is heated to 72°C at a rate of 1°C/min, and an iodine test is carried out every 5 minutes until color development is complete;

④Pump the malt mash into the filter tank and let it stand for 30min;

⑤Use the sieve plate at the bottom of the filter tank to filter the wort, collect the clear wort, and calculate the volume of the wort collected per unit time. Filtration velocity (V) is expressed as the volume of wort collected per unit time (unit: mL/min).

After the saccharification, the arabinoxylan content, viscosity and filtration rate in the wort were measured, and the control was the saccharified wort without enzymes to reflect the improvement effect of xylanase on the saccharification and filtration indexes.

(2) Determination of arabinoxylan content by phloroglucinol method

Preparation of color developer: 0.5g of phloroglucinol was dissolved in 1mL of absolute ethanol, then 1mL of concentrated hydrochloric acid, 0.5mL of 17.5g/L glucose solution and 55mL of glacial acetic acid were added respectively, mixed well, and stored in a brown bottle.

Preparation of standard curve: preparation of 20, 40, 60, 80 and 100 mg/L series of xylose working solutions respectively. Take 2 mL of the working solutions of each concentration, add 10 mL of color-developing reagent to each test tube, replace the control with 2 mL of distilled water, after mixing, accurately react in a boiling water bath for 25 min, cool to room temperature, measure the absorbance value at 552 nm, and draw a standard curve.

Determination of high molecular weight arabinoxylan (HMW-AX) content

Take 2 mL of wort and mix with 3 mL of absolute ethanol and precipitate at 4°C overnight, centrifuge at 10,000 × g for 15 min, discard the supernatant, reconstitute the precipitate with 2 mL of distilled water, take 0.1 mL of the reconstituted solution, add 1.9 mL of distilled water, and follow The method for preparing the standard curve adopts the phloroglucinol method to determine the content of arabinoxylan.

(3) Filtration speed (V): In a 32 cm funnel, neutral filter paper is used as the medium, and the volume of wort collected per unit time is expressed; the viscosity is measured with a HAAKE falling ball viscometer.

Figure 4 shows the effects of the addition of purified xylanase III with different activities on the degradation, viscosity and filtration rate of arabinoxylan in the saccharified wort.

The results in Figure 4 show that the addition of xylanase III greatly improved the filtration performance of the mash. When 25U/g malt xylanase III was added to the mash, the content of polymerized arabinoxylan in the agreed wort decreased from 301mg/L to 38mg/L, and the degradation rate was 80%; the viscosity decreased from 1.508mPa· s decreased to 1.4mPa·s, a decrease of 7.2%. The filtration rate was increased from 5.0 mL/min to 9.0 mL/min, and the filtration rate was increased by 80%.

Example 4 Analysis of the product of hydrolysis of high molecular weight arabinoxylan from barley malt by xylanase III

In order to study the reason why xylanase Ⅲ can significantly increase the filtration rate of barley malt mash during saccharification, the degradation products of barley malt high molecular weight arabinoxylan were analyzed by high performance liquid chromatography.

Table 2 Analysis of products of high molecular weight arabinoxylan hydrolyzed by xylanase III

The results in Table 2 show that after xylanase III reacted with high molecular weight arabinoxylan at 37°C for 30 min, the hydrolyzed products of high molecular weight arabinoxylan were mainly xylose and arabinose, and its composition was 41.4% arabinose, Xylose 44.0%, no hydrolyzed products of typical endo-xylanases such as xylobiose and xylotriose. The analysis results of high performance liquid chromatography showed that xylanase Ⅲ has the function of hydrolyzing the side chain arabinose when it acts on the high molecular weight arabinoxylan of malt, which may be because xylanase Ⅲ can significantly degrade the high molecular weight in the mash. Arabinoxylan, the reason for the increased filtration speed.

Comparative Example 1

The specific embodiment is the same as Example 3, except that Aspergillus niger xylanase and Bacillus subtilis xylanase are added, each 25U/g malt, and the content, filtration speed and viscosity of high molecular weight arabinoxylan in the wort are determined.

Table 3 Wort filtration indexes with different xylanases added

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> A kind of preparation method of xylanase and its application in beer production

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 347

<212> PRT

<213> Trichoderma reesei

<400> 1

Met Lys Ala Asn Val Ile Leu Cys Leu Leu Ala Pro Leu Val Ala Ala

1 5 10 15

Leu Pro Thr Glu Thr Ile His Leu Asp Pro Glu Leu Ala Ala Leu Arg

20 25 30

Ala Asn Leu Thr Glu Arg Thr Ala Asp Leu Trp Asp Arg Gln Ala Ser

35 40 45

Gln Ser Ile Asp Gln Leu Ile Lys Arg Lys Gly Lys Leu Tyr Phe Gly

50 55 60

Thr Ala Thr Asp Arg Gly Leu Leu Gln Arg Glu Lys Asn Ala Ala Ile

65 70 75 80

Ile Gln Ala Asp Leu Gly Gln Val Thr Pro Glu Asn Ser Met Lys Trp

85 90 95

Gln Ser Leu Glu Asn Asn Gln Gly Gln Leu Asn Trp Gly Asp Ala Asp

100 105 110

Tyr Leu Val Asn Phe Ala Gln Gln Asn Gly Lys Ser Ile Arg Gly His

115 120 125

Thr Leu Ile Trp His Ser Gln Leu Pro Ala Trp Val Asn Asn Ile Asn

130 135 140

Asn Ala Asp Thr Leu Arg Gln Val Ile Arg Thr His Val Ser Thr Val

145 150 155 160

Val Gly Arg Tyr Lys Gly Lys Ile Arg Ala Trp Asp Val Val Asn Glu

165 170 175

Ile Phe Asn Glu Asp Gly Thr Leu Arg Ser Ser Val Phe Ser Arg Leu

180 185 190

Leu Gly Glu Glu Phe Val Ser Ile Ala Phe Arg Ala Ala Arg Asp Ala

195 200 205

Asp Pro Ser Ala Arg Leu Tyr Ile Asn Asp Tyr Asn Leu Asp Arg Ala

210 215 220

Asn Tyr Gly Lys Val Asn Gly Leu Lys Thr Tyr Val Ser Lys Trp Ile

225 230 235 240

Ser Gln Gly Val Pro Ile Asp Gly Ile Gly Ser Gln Ser His Leu Ser

245 250 255

Gly Gly Gly Gly Ser Gly Thr Leu Gly Ala Leu Gln Gln Leu Ala Thr

260 265 270

Val Pro Val Thr Glu Leu Ala Ile Thr Glu Leu Asp Ile Gln Gly Ala

275 280 285

Pro Thr Thr Asp Tyr Thr Gln Val Val Gln Ala Cys Leu Ser Val Ser

290 295 300

Lys Cys Val Gly Ile Thr Val Trp Gly Ile Ser Asp Lys Asp Ser Trp

305 310 315 320

Arg Ala Ser Thr Asn Pro Leu Leu Phe Asp Ala Asn Phe Asn Pro Lys

325 330 335

Pro Ala Tyr Asn Ser Ile Val Gly Ile Leu Gln

340 345

Claims (10)

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.

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