CN110714037A - Preparation method of xylanase and application of xylanase in beer production - Google Patents

Preparation method of xylanase and application of xylanase in beer production Download PDF

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CN110714037A
CN110714037A CN201911199189.0A CN201911199189A CN110714037A CN 110714037 A CN110714037 A CN 110714037A CN 201911199189 A CN201911199189 A CN 201911199189A CN 110714037 A CN110714037 A CN 110714037A
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xylanase
beer
malt
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孙军勇
陆健
田甜甜
王茂章
颜义勇
商曰玲
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
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    • C12C7/04—Preparation or treatment of the mash
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12N9/248—Xylanases
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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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

Preparation method of xylanase and application of xylanase in beer production
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.
Example 1 preparation of a Trichoderma reesei CICC41495 fermentation broth
The seed culture medium is prepared as follows: 1.4g/L of ammonium sulfate, 10g/L of glucose, 2.0g/L of monopotassium phosphate, 1.0g/L of yeast powder, 0.3g/L of calcium chloride, 0.3g/L of magnesium sulfate, 2.0mg/L of cobalt chloride, 5.0mg/L of ferrous sulfate, 1.4mg/L of zinc sulfate, 1.6mg/L of manganese sulfate and natural pH.
Preparing an enzyme production culture medium: the carbon source- -glucose in the seed culture medium was replaced by corn and bran (30 g/L corn and 10g/L bran), and the remaining ingredients were unchanged.
Trichoderma reesei CICC41495 was kept on potato-glucose-agar slant and spores were collected for inoculation with 0.9% NaCl solution at the time of use. And (3) filling 25mL of the seed culture medium into a 250mL triangular bottle, inoculating a spore solution, culturing at the temperature of 30 ℃ and the rotating speed of a shaking table of 200r/min for 36-48 h.
25mL of enzyme-producing culture medium is put into a 250mL triangular flask, 2.5mL of seed liquid is inoculated, the culture temperature is 30 ℃, the rotating speed of a shaking table is 200r/min, and the culture is carried out for 168 hours. Centrifuging the fermentation liquor by 10000 Xg for 15min, and freeze-drying to obtain the protein in the trichoderma reesei CICC41495 full culture solution and storing the protein at 4 ℃ for later use.
Example 2 purification of endo-xylanase III
The purification steps of the endo-xylanase III are as follows:
(1) precipitating the protein in the crude enzyme solution with 75% saturation ammonium sulfate, centrifuging at 10,000 Xg for 15min, discarding the supernatant, and dissolving the precipitate with 20mmol/L Tris-HCl buffer solution (pH8.0);
(2) the enzyme solution was desalted using SephadexG-25 column, loaded onto DEAE-Sepharose Fast Flow ion exchange column, and eluted with 400mL of 20mmol/L Tris-HCl buffer solution (pH8.0) containing 0 to 0.50mol/L NaCl at a gradient Flow rate of 100 mL/h. Collecting the fractions with xylanase activity, and embedding and concentrating with PEG 20000;
(3) and (3) taking the concentrated enzyme solution, further purifying by using a Sephacryl S-100 gel filtration chromatographic column, collecting components with xylanase activity at the flow rate of 20mL/h, wherein the mobile phase is acetic acid-sodium acetate buffer solution with the pH value of 5.5 and 100mmol/L, and the SDS-PAGE result shows that the purified xylanase achieves electrophoretic purity, and the result is shown in figure 2.
During the purification, the protein concentration of the sample was determined by Coomassie Brilliant blue method, and the purity of the inhibitory protein was analyzed by SDS-PAGE. The specific method comprises the following steps:
(1) mixing the sample with 4 times volume of loading buffer solution (2% SDS, 0.1% bromophenol blue 10% glycerol), and boiling water bath for 5 min;
(2) taking 30 mu L of sample (the concentration of separation gel of SDS-PAGE is 12.5%, the concentration of concentrated gel is 5%), and applying 60V voltage until the bromophenol blue indicator band reaches the bottom of the concentrated gel to form a straight line → 80V voltage until the bromophenol blue indicator band reaches the bottom of the separation gel;
(3) fixing with fixing solution (methanol: acetic acid: water ratio of 5: 1: 4) for 30min, and dyeing with 0.25% Coomassie brilliant blue R-250 solution for 1 h;
(4) destaining the solution with destaining solution (methanol: acetic acid: water ratio 1: 1: 8) until the background is clear.
The purified xylanase is identified as endo-beta-1, 4-xylanase III (EC 3.2.1.8) by matrix-assisted laser desorption ionization tandem flight time mass spectrum (FIG. 3). The theoretical molecular weight value of the enzyme is 38.0kDa, the theoretical pI is 6.97, and the enzyme belongs to glycoside hydrolase GHF10 family; the xylanase has a molecular weight of 32.0kDa as determined by SDS-PAGE and a pI of about 9.0.
Table 1 Mass Spectrometry identification results of xylanases
Figure BDA0002295441320000051
Example 3 use of purified endo-xylanase III in barley malting Process
(1) The saccharification process comprises the following steps:
the preparation method of the saccharified wort simulating industrial production comprises the following steps:
① 25.0.0 kg of finely ground malt and 100L of tap water at 46 ℃ are put into a saccharifying pot (the volume of the saccharifying pot is 200L), and the temperature is kept at 45 ℃ for 90min, so that the xylanase can degrade the high molecular weight arabinoxylan of the barley malt more fully;
② heating the mash to 65 deg.C at a rate of 1 deg.C/min, and maintaining the temperature at 65 deg.C for 40 min;
③ heating the mash to 72 deg.C at a rate of 1 deg.C/min, and performing iodine test every 5min until color development is complete;
④ pumping malt mash into a filter tank, and standing for 30 min;
⑤ the wort is filtered using a sieve at the bottom of the filter tank, the clear wort is collected and the volume of wort collected per unit time is calculated the filtration rate (V) is expressed as the volume of wort collected per unit time (in mL/min).
After saccharification is finished, the indexes such as the content, viscosity, filtration speed and the like of the arabinoxylan in the wort are measured, and the control is the saccharified wort without enzyme so as to reflect the improvement effect of xylanase on the saccharified filtration index.
(2) Method for determining content of arabinoxylan by phloroglucinol method
Preparing a color developing agent: 0.5g of phloroglucinol is dissolved with 1mL of absolute ethanol, 1mL of concentrated hydrochloric acid, 0.5mL of 17.5g/L glucose solution and 55mL of glacial acetic acid are added respectively, and the mixture is uniformly mixed and stored in a brown bottle.
Preparing a standard curve: preparing 20, 40, 60, 80 and 100mg/L series xylose working solution respectively. Respectively taking 2mL of working solution with each concentration, respectively adding 10mL of color developing agent into each test tube, replacing the contrast with 2mL of distilled water, uniformly mixing, accurately reacting in a boiling water bath for 25min, cooling to room temperature, measuring the absorbance value under 552nm, and drawing a standard curve.
Determination of high molecular weight arabinoxylan (HMW-AX) content
Uniformly mixing 2mL of wort and 3mL of absolute ethyl alcohol, precipitating at 4 ℃ overnight, centrifuging at 10000 Xg for 15min, discarding supernatant, redissolving the precipitate with 2mL of distilled water, taking 0.1mL of redissolved solution, adding 1.9mL of distilled water, and determining the content of the arabinoxylan by adopting a phloroglucinol method according to a method for preparing a standard curve.
(3) Filtration rate (V): expressed as the volume of wort collected per unit time in a 32cm funnel using neutral filter paper as the medium; viscosity was measured using a HAAKE falling ball viscometer.
The effect of the addition of purified xylanase III of different activities on the degradation, viscosity and filtration rate of arabinoxylan in the agreed mash is shown in FIG. 4.
The results in FIG. 4 show that the addition of xylanase III greatly improves the filtration performance of the mash. When 25U/g of malt xylanase III is added into the mash, the content of polymerized arabinoxylan in the wort is reduced from 301mg/L to 38mg/L, and the degradation rate is 80%; the viscosity decreased from 1.508 mPas to 1.4 mPas, which was 7.2%. The filtration speed is increased from 5.0mL/min to 9.0mL/min, and the filtration speed is increased by 80%.
Example 4 product analysis of xylanase III hydrolysis of barley malt high molecular weight arabinoxylan
In order to research the reason that xylanase III can obviously improve the filtration speed of barley malt mash in the saccharification process, a product for degrading high-molecular-weight arabinoxylan of barley malt is analyzed by adopting a high performance liquid chromatography.
TABLE 2 analysis of the products of xylanase III hydrolysis of high molecular weight arabinoxylans
Figure BDA0002295441320000061
The results in Table 2 show that after xylanase III was reacted with high molecular weight arabinoxylans at 37 ℃ for 30min, the high molecular weight arabinoxylan hydrolysates were mainly xylose and arabinose, consisting of 41.4% arabinose, 44.0% xylose, and no hydrolysis products of typical endoxylanases like xylobiose and xylotriose. The analysis result of the high performance liquid chromatography shows that the xylanase III has the function of hydrolyzing the side chain arabinose when acting on the high molecular weight arabinoxylan of the malt, which is probably the reason that the xylanase III can obviously degrade the high molecular weight arabinoxylan in the mash and improve the filtering speed.
Comparative example 1
The specific implementation manner is the same as that in example 3, except that Aspergillus niger xylanase and Bacillus subtilis xylanase are added, and the content, the filtration rate and the viscosity of high molecular weight arabinoxylan in wort are measured by adding 25U/g malt.
TABLE 3 wort filtration index with different xylanase additions
Figure BDA0002295441320000071
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> preparation method of xylanase and application thereof in beer production
<160>1
<170>PatentIn version 3.3
<210>1
<211>347
<212>PRT
<213>Trichoderma reesei
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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 AsnGln 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 LeuGly 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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CN110713998A (en) * 2019-11-29 2020-01-21 江南大学 Preparation method and application of arabinoxylan degrading enzyme system

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