CN112779296A

CN112779296A - Enzyme preparation adding process for promoting fermentation of starch grains

Info

Publication number: CN112779296A
Application number: CN202011233296.3A
Authority: CN
Inventors: 许宏贤; 王乐盛; 林洁; 郑斐; 白挨玺
Original assignee: Nanjing Bestzyme Bio Engineering Co ltd
Current assignee: Nanjing Bestzyme Bio Engineering Co ltd
Priority date: 2019-11-06
Filing date: 2020-11-06
Publication date: 2021-05-11

Abstract

The invention provides an enzyme preparation adding process for promoting fermentation of starchy grains, relates to the field of enzyme preparation application, particularly relates to an enzyme preparation adding process for promoting fermentation of starchy grains, and provides a process for promoting fermentation of corn flour to produce ethanol.

Description

Enzyme preparation adding process for promoting fermentation of starch grains

Technical Field

The invention relates to the field of enzyme preparation application, in particular to application of an adding process of a related enzyme preparation in a deep processing process of starch grains, and more particularly relates to application of enzymes such as alpha-glucosidase, xylanase, trehalase, arabinofuranosidase, cellulase, beta-glucosidase and the like in fermentation of starch grains to produce biofuel ethanol.

Background

The deep processing of crops such as corn and the like can optimize the industrial structure, prolong the industrial chain and increase the added value of products, and is an important measure for solving the problems of three agricultural crops. For example, ethanol (fuel and food), platform chemical acids (e.g., citric acid, lactic acid, amino acids), alcohols (1,3 propylene glycol, butanol), ketones, esters, and the like may be produced. In recent years, with the continuous advance of stock removal of corns and the like in China, the deep processing industry as an important means for stock removal is confronted with new development opportunities, and the production capacity is rapidly increased. Ethanol, citric acid, amino acids, etc. are generally produced by fermentation. For example, fuel ethanol is mainly produced by a fermentation method, and the general production process comprises the following steps: 1) pretreating raw materials, and hydrolyzing starch into a yeast available sugar component by processes such as liquefaction and the like; 2) fermentation: producing ethanol from fermentable sugars by adding yeast; 3) and (3) purification: the fermentation liquor is distilled, rectified and dehydrated to obtain the finished product fuel ethanol.

In addition to the fermentation sugar, a part of non-fermentation sugar remains in the fermentation liquor during the fermentation process, so that the fermentation liquor can not be fully utilized by the saccharomyces cerevisiae. This results in a reduced starch conversion during fermentation, which affects liquor yield. If these non-fermentable sugars could be hydrolyzed using glycoside hydrolases to make more sugar available for alcohol synthesis, the profit of the alcohol manufacturing enterprise could be significantly increased. At the same time, there are also a large number of non-starchy polysaccharides in corn, i.e. non-fermentable sugars, such as cellulose, the main polysaccharide in the primary cell wall of biomass, and hemicellulose, the second most abundant. Xylan in hemicellulose is a representative main component, the structure of the hemicellulose is very complex, a main chain is formed by connecting D-xylopyranose through beta-1, 4 glycosidic bonds, and different substituents such as arabinose, ferulate, glucuronic acid, acetyl and the like are connected on a side chain. The xylanase is used as a main enzyme to act on a main chain of xylan in an endo mode, and the arabinofuranosidase is a side chain degrading enzyme and plays an important role in promoting the degradation of the main chain. Cellulose and hemicellulose are abundant in grains, and increasing their biodegradation can provide more substrates for fermentation, which can significantly increase the profits of alcohol production enterprises.

Due to the characteristics of different enzyme preparations, the requirements on conditions such as pH, temperature, substrate content, reaction time and the like in the catalysis process are strict, and the catalysis conditions of different enzyme preparations are not consistent. When the external catalytic conditions are not suitable for the catalytic mechanism, the catalytic efficiency is low or reverse reaction occurs. In different stages of raw material treatment, fermentation and the like in the alcohol production process, conditions such as composition, pH, temperature and the like of substances in a fermentation system are always changed. The method for adding the required enzyme preparation to the fermentation system in the initial stage of fermentation in the prior art cannot enable different enzyme preparations to achieve the highest catalytic efficiency.

Therefore, it is important to select the appropriate enzyme preparation and to determine the addition time of each enzyme preparation during the alcohol production process.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a method for producing alcohol by fermenting a grain raw material, comprising the steps of: a. liquefying the grain raw material; b. saccharifying; c. fermenting; wherein the step c comprises a step of adding a non-starch polysaccharide enzyme.

In some embodiments of the invention, the method of fermenting a cereal material to produce alcohol comprises the steps of: a. liquefying the grain raw material; b. saccharifying; c. fermenting; wherein the step c comprises a step of adding a non-starch polysaccharide enzyme; and the step b and the step c are carried out simultaneously.

In some embodiments of the invention, the step of adding the non-starch polysaccharide enzyme is within the range of 0-60h after the start of fermentation, preferably 0-48h after the start of fermentation, further preferably 3-48h after the start of fermentation, further preferably 16-48h after the start of fermentation, more preferably 16-40h after the start of fermentation.

In one embodiment, the step of adding the non-starch polysaccharide enzyme is selected from 0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h, 14h, 14.5h, 15h, 15.5h, 16h, 16.5h, 17h, 17.5h, 18h, 18.5h, 19h, 19.5h, 20h, 20.5h, 21h, 21.5h, 22h, 22.5h, 23h, 23.5h, 24h, 24.5h, 25h, 25.5h, 19.5h, 20.5h, 21h, 21.5h, 22h, 22.5h, 23h, 23.5h, 30h, 34h, 30.5h, 34h, 30.5h, 30h, 34h, 30.5h, 34h, 30.5h, 32h, 30.5h, 34h, 38h, 38.5h, 39h, 39.5h, 40h, 40.5h, 41h, 41.5h, 42h, 42.5h, 43h, 43.5h, 44h, 44.5h, 45h, 45.5h, 46h, 46.5h, 47h, 47.5h, or 48 h.

In one embodiment, the step of adding the non-starch based polysaccharidase is 0h after the start of fermentation.

In one embodiment, the step of adding the non-starch based polysaccharidase is 3 hours after the start of fermentation.

In one embodiment, the step of adding the non-starch based polysaccharidase is 16h after the start of fermentation.

In one embodiment, the step of adding the non-starch based polysaccharidase is 24 hours after the start of fermentation.

In one embodiment, the step of adding the non-starch based polysaccharidase is 40h after the start of the fermentation.

In one embodiment, the step of adding the non-starch based polysaccharidase is 48h after the start of fermentation.

In some embodiments of the invention, the method further comprises a yeast propagation step, preferably the yeast propagation step is before the c step.

The non-starch polysaccharide enzyme provided by the invention comprises but is not limited to alpha-glucosidase, trehalase, xylanase, arabinofuranosidase, cellulase, beta-glucosidase, feruloyl esterase, pectinase and mannanase. In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding an alpha-glucosidase, trehalase, xylanase, arabinofuranosidase, cellulase, beta-glucosidase, feruloyl esterase, pectinase, mannanase or any combination thereof, preferably an alpha-glucosidase.

In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding an alpha-glucosidase, trehalase, xylanase, arabinofuranosidase, cellulase, beta-glucosidase or any combination thereof, preferably an alpha-glucosidase.

In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding an alpha-glucosidase. In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding a trehalase. In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding a xylanase. In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding an arabinofuranosidase. In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding a cellulase. In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding a beta-glucosidase. In one embodiment, the step of adding a non-starch based polysaccharidase comprises adding a feruloyl esterase.

In one embodiment, the alpha-glucosidase is added in an amount of 0.1-20U/g DS, preferably 0.5-10U/g DS, more preferably 2U/g DS. In another embodiment, the alpha-glucosidase is added in an amount of 2U/g DS.

In one embodiment, the amount of alpha-glucosidase added is selected from 0.5U/g DS, 1U/g DS, 1.5U/g DS, 2U/g DS, 2.5U/g DS, 3U/g DS, 3.5U/g DS, 4U/g DS, 4.5U/g DS, 5U/g DS, 5.5U/g DS, 6U/g DS, 6.5U/g DS, 7U/g DS, 7.5U/g DS, 8U/g DS, 8.5U/g DS, 9U/g DS, 9.5U/g DS, or 10U/g DS.

In one embodiment, the amino acid sequence of the α -glucosidase is as set forth in SEQ ID No. 2.

In one embodiment, the nucleotide sequence encoding the α -glucosidase is set forth in SEQ ID No. 1.

In one embodiment, the trehalase is added in an amount of 0.01-10U/g DS, preferably 0.1-5U/g DS, more preferably 0.5U/g DS. In another embodiment, the trehalase is added in an amount of 0.5U/g DS.

In one embodiment, the trehalase is added in an amount selected from the group consisting of 0.1U/g DS, 0.2U/g DS, 0.3U/g DS, 0.4U/g DS, 0.5U/g DS, 0.6U/g DS, 0.7U/g DS, 0.8U/g DS, 0.9U/g DS, 1U/g DS, 1.5U/g DS, 2U/g DS, 2.5U/g DS, 3U/g DS, 3.5U/g DS, 4U/g DS, 4.5U/g DS and 5U/g DS.

In one embodiment, the xylanase is added in an amount of 0.1-100U/g DS, preferably 0.5-50U/g DS, more preferably 10U/g DS. In another embodiment, the xylanase is added in an amount of 10U/g DS.

In one embodiment, the xylanase is added in an amount selected from the group consisting of 0.5U/g DS, 1U/g DS, 1.5U/g DS, 2U/g DS, 2.5U/g DS, 3U/g DS, 3.5U/g DS, 4U/g DS, 4.5U/g DS, 5U/g DS, 5.5U/g DS, 6U/g DS, 6.5U/g DS, 7U/g DS, 7.5U/g DS, 8U/g DS, 8.5U/g DS, 9U/g DS, 9.5U/g DS, 10U/g DS, 10.5U/g DS, 11U/g DS, 11.5U/g DS, 12U/g DS, 12.5U/g DS, 13U/g DS, 13.5U/g DS, 14U/g DS, 14.5U/g DS, 15U/g DS, 15.5U/g DS, 16U/g DS, 16.5U/g DS, 17U/g DS, 17.5U/g DS, 18U/g DS, 18.5U/g DS, 19U/g DS, 19.5U/g DS, or 20U/g DS.

In one embodiment, the arabinofuranosidase is added in an amount of 0.1-100U/g DS, preferably 0.5-50U/g DS, more preferably 10U/g DS. In another embodiment, the arabinofuranosidase is added in an amount of 10U/g DS.

In one embodiment, the arabinofuranosidase is added in an amount selected from the group consisting of 0.5U/g DS, 1U/g DS, 1.5U/g DS, 2U/g DS, 2.5U/g DS, 3U/g DS, 3.5U/g DS, 4U/g DS, 4.5U/g DS, 5U/g DS, 5.5U/g DS, 6U/g DS, 6.5U/g DS, 7U/g DS, 7.5U/g DS, 8U/g DS, 8.5U/g DS, 9U/g DS, 9.5U/g DS, 10U/g DS, 10.5U/g DS, 11U/g DS, 11.5U/g DS, 12U/g DS, 12.5U/g DS, 13U/g DS, 13.5U/g DS, 14U/g DS, 14.5U/g DS, 15U/g DS, 15.5U/g DS, 16U/g DS, 16.5U/g DS, 17U/g DS, 17.5U/g DS, 18U/g DS, 18.5U/g DS, 19U/g DS, 19.5U/g DS or 20U/g DS.

In one embodiment, the amino acid sequence of the arabinofuranosidase is shown in SEQ ID No. 4.

In one embodiment, the nucleotide sequence encoding the arabinofuranosidase is shown in SEQ ID NO. 3.

In one embodiment, the cellulase is added in an amount of 0.1 to 100U/g DS, preferably 0.5 to 50U/g DS, more preferably 10U/g DS. In another embodiment, the cellulase is added in an amount of 10U/g DS.

In one embodiment, the cellulase is added in an amount selected from the group consisting of 0.5U/g DS, 1U/g DS, 1.5U/g DS, 2U/g DS, 2.5U/g DS, 3U/g DS, 3.5U/g DS, 4U/g DS, 4.5U/g DS, 5U/g DS, 5.5U/g DS, 6U/g DS, 6.5U/g DS, 7U/g DS, 7.5U/g DS, 8U/g DS, 8.5U/g DS, 9U/g DS, 9.5U/g DS, 10U/g DS, 10.5U/g DS, 11U/g DS, 11.5U/g DS, 12U/g DS, 12.5U/g DS, 13U/g DS, 13.5U/g DS, 14U/g DS, 14.5U/g DS, 15U/g DS, 15.5U/g DS, 16U/g DS, 16.5U/g DS, 17U/g DS, 17.5U/g DS, 18U/g DS, 18.5U/g DS, 19U/g DS, 19.5U/g DS, or 20U/g DS.

In one embodiment, the beta-glucosidase is added in an amount of 0.1-100U/g DS, preferably 0.5-50U/g DS, more preferably 10U/g DS. In another embodiment, the amount of β -glucosidase added is 10U/g DS.

In one embodiment, the amount of β -glucosidase added is selected from 0.5U/g DS, 1U/g DS, 1.5U/g DS, 2U/g DS, 2.5U/g DS, 3U/g DS, 3.5U/g DS, 4U/g DS, 4.5U/g DS, 5U/g DS, 5.5U/g DS, 6U/g DS, 6.5U/g DS, 7U/g DS, 7.5U/g DS, 8U/g DS, 8.5U/g DS, 9U/g DS, 9.5U/g DS, 10U/g DS, 10.5U/g DS, 11U/g DS, 11.5U/g DS, 12U/g DS, 12.5U/g DS, 13U/g DS, 13.5U/g DS, 14U/g DS, 14.5U/g DS, 15U/g DS, 15.5U/g DS, 16U/g DS, 16.5U/g DS, 17U/g DS, 17.5U/g DS, 18U/g DS, 18.5U/g DS, 19U/g DS, 19.5U/g DS or 20U/g DS.

In one embodiment, the feruloyl esterase is added in an amount of 0.1 to 100U/g DS, preferably 0.5 to 50U/g DS, more preferably 10U/g DS. In another embodiment, the feruloyl esterase is added in an amount of 10U/g DS.

In one embodiment, the ferulic acid esterase is added in an amount selected from the group consisting of 0.5U/g DS, 1U/g DS, 1.5U/g DS, 2U/g DS, 2.5U/g DS, 3U/g DS, 3.5U/g DS, 4U/g DS, 4.5U/g DS, 5U/g DS, 5.5U/g DS, 6U/g DS, 6.5U/g DS, 7U/g DS, 7.5U/g DS, 8U/g DS, 8.5U/g DS, 9U/g DS, 9.5U/g DS, 10U/g DS, 10.5U/g DS, 11U/g DS, 11.5U/g DS, 12U/g DS, 12.5U/g DS, 13U/g DS, 13.5U/g DS, 14U/g DS, 14.5U/g DS, 15U/g DS, 15.5U/g DS, 16U/g DS, 16.5U/g DS, 17U/g DS, 17.5U/g DS, 18U/g DS, 18.5U/g DS, 19U/g DS, 19.5U/g DS, or 20U/g DS.

In some embodiments, step a comprises preparing a cereal material feed solution with a suitable feed-to-water ratio, and adding amylase to the feed solution; adding saccharifying enzyme in the step b; and c, adding a fermenting organism and a nitrogen source.

In some embodiments, the adding of amylase in step a is adding high temperature amylase; adding saccharifying enzyme in the step b; and c, adding yeast and nitrogen source urea.

In one embodiment, the ratio of formulation water in step a is 1: 0.5-10.0 of grain raw material liquid, wherein the amylase is high-temperature amylase, and the addition amount is 1-200U/g DS; in the step b, the adding amount of the saccharifying enzyme is 20-600U/g DS; the yeast added in the step c is yeast obtained after 0.5g to 5g of dry yeast is added for propagation; and c, adding the nitrogen source urea in the step c in an amount of 0-1000 ppm.

In one embodiment, the cereal raw material is formulated in the step a in a ratio of 1: 0.5 to 10.0, preferably 1: 1.0-5.0, more preferably 1: 2.0-3.0.

In one embodiment, the amylase in the step a is high-temperature amylase, and the addition amount of the amylase is 1-200U/g DS, preferably 10-100U/g DS.

In one embodiment, the saccharifying enzyme is added in step b in an amount of 20 to 600U/g DS, preferably 50 to 500U/g DS.

In one embodiment, the yeast after propagation in step c is yeast obtained after propagation of 0.5g to 5g of dry yeast, preferably yeast obtained after propagation of 1g of dry yeast.

In one embodiment, the amount of urea as nitrogen source added in step c is between 0 and 1000ppm, preferably 400 ppm.

In one embodiment, said step a comprises formulating a water 1: 2.0-3.0 of grain raw material liquid, adjusting the pH value to 4.5-6.0, and adding high-temperature amylase for high-temperature liquefaction. In another embodiment, the step b and the step c are carried out simultaneously, and the method comprises the steps of adjusting the pH value of the liquefied feed liquid to about 4.3, timely cooling to room temperature, uniformly subpackaging the mixture into a shake flask, adding 50-500U/g DS saccharifying enzyme, adding 1g of dry yeast and 400ppm nitrogen source urea, and fermenting at the temperature of 28-36 ℃ for 48-96 hours.

In one embodiment, said step a comprises formulating a water 1: 2.0-3.0 of grain raw material liquid, adjusting the pH value to 4.5-6.0, and adding high-temperature amylase for high-temperature liquefaction; and c, simultaneously performing the steps b and c, regulating the pH of the liquefied feed liquid to about 4.3, timely cooling to room temperature, uniformly subpackaging into a shake flask, adding 50-500U/g DS saccharifying enzyme, adding 1g of yeast obtained after dry yeast propagation and 400ppm of nitrogen source urea, and fermenting at the temperature of 28-36 ℃ for 48-96 hours.

The cereal raw material in the present invention specifically includes but is not limited to corn, sorghum, wheat, barley, oat, rye, rice, cassava, sweet potato, etc., all containing cellulose and/or hemicellulose. In one embodiment, the cereal material is selected from corn, sorghum, wheat, barley, oats, rye, rice, tapioca or sweet potato, in particular corn, more in particular whole-milled corn flour.

The non-starch polysaccharide enzymes of the present invention may further include related agents, such as one or more preservatives and/or antimicrobial agents (e.g., bacteriostatic agents), including but not limited to: sorbitol, sodium chloride, glycerin, potassium sorbate, and other preservatives and/or antimicrobials known in the art. In one embodiment, the step of adding the non-starch based polysaccharidase is a non-starch based polysaccharidase preparation comprising the addition of a mixture with one or more preservatives and/or antimicrobial agents.

The noun explains:

α -glucosidase: also known as alpha-D-glucoside hydrolase, which cleaves the alpha-1, 4 glycosidic bond from the non-reducing end of an oligosaccharide substrate to release glucose; under the conditions of 40 ℃ and pH5.0, 1mL of enzyme sample reacts with a substrate alpha-methyl-D-glucoside, and 1 mu g of glucose is generated in 60min, namely 1 enzyme activity unit expressed by U/mL or U/g.

The trehalase is an enzyme capable of directly hydrolyzing trehalose into 2 molecules of glucose, the enzyme activity of the trehalase is defined as 1g of solid enzyme powder (or 1mL of liquid enzyme), and the trehalase can hydrolyze 1mg of trehalose per minute at 50 ℃ and pH4.2, namely 1 enzyme activity unit expressed as U/g (U/mL).

The xylanase is an enzyme system for degrading xylan, comprises beta-1, 4-endoxylanase, beta-xylosidase, alpha-L-arabinosidase, alpha-D-glucuronidase, acetyl xylanase and phenolic acid esterase, and can degrade xylan hemicellulose which exists in the nature in a large amount. In the xylanase system, beta-1, 4-endoxylanase is the most critical hydrolase, which hydrolyzes xylan into xylooligosaccharides such as small oligosaccharides and xylobiose, and small amounts of xylose and arabinose by hydrolyzing the beta-1, 4-glycosidic bond of xylan molecule. The enzyme activity refers to the enzyme activity which can accurately react with xylan for 30min at the temperature of 50 ℃ and the pH value of the xylanase at 5.5, and generates xylose reducing power equivalent to 1 mu mol per minute, and the enzyme activity is an enzyme activity unit and is expressed by U/g (U/mL). The xylanase involved in the invention includes but is not limited to xylanase with the activity.

Arabinofuranosidase refers to a enzyme that hydrolyzes the terminal non-reducing alpha-L-arabinofuranoside residue in alpha-L-arabinoside and is classified as EC3.2.1.55. The enzyme activity refers to the enzyme amount required for accurately reacting at 45 ℃ for 4min at pH4.2 and hydrolyzing the p-nitrophenyl-L-arabinofuranoside to generate 1mmol of p-nitrophenol per minute, and is an enzyme activity unit. The arabinofuranosidase involved in the invention includes but is not limited to arabinofuranosidase with the activity. The arabinofuranosidase removes the arabinoside chain on xylan, promotes the degradation of xylan, can hydrolyze the arabinoside chain into short-chain soluble substances, and increases the clarity of fermented mash.

Cellulase is a general term for a group of enzymes that degrade the beta-1, 4-glucosidic bonds of cellulose to convert cellulose to cellobiose and glucose, and is a synergistic multicomponent enzyme system. The main components of the cellulase are endo-beta-1, 4-glucosidase, exoglucanase and beta-glucosidase. The first two enzymes dissolve mainly the cellulose, the latter enzyme converts cellobiose to glucose, and the degradation of cellulose is achieved when the activity ratios of the three main components are appropriate. The enzyme activity definition refers to that the enzyme quantity required for degrading and releasing 1 mu mol of reducing sugar from a sodium carboxymethylcellulose solution with the concentration of 5mg/mL is one activity unit (U) every minute after the accurate reaction is carried out for 15min at the temperature of 50 ℃ at the pH value of 4.8, and the reducing sugar is equal to the glucose. The cellulase to which the present invention relates includes, but is not limited to, cellulase having the activity.

Beta-glucosidase can hydrolyze beta-glucosidic bonds and is one of the important components of cellulase systems. The enzyme activity refers to that the enzyme amount required for degrading 1 mu mol of p-nitrophenol in p-nitrophenol beta-D-glucoside hydrolyzed solution per minute is one activity unit (U) after the accurate reaction is carried out for 15min at the temperature of 50 ℃ and the pH value is 4.8.

The amylase is capable of hydrolyzing alpha-1, 4 glucosidic bonds in a starch molecular chain, cutting the starch chain into short-chain dextrin, a small amount of maltose and glucose, and rapidly reducing the viscosity of the starch, wherein the enzyme activity is defined as 1g of solid enzyme powder (or 1mL of liquid enzyme), 1mg of soluble starch is liquefied in 1min at 70 ℃ and pH6.0, namely 1 enzyme activity unit, and is expressed by U/g (U/mL). The amylase involved in the invention includes but is not limited to amylase with the activity.

The saccharifying enzyme is amyloglucosidase which takes starch as a substrate and hydrolyzes alpha-1, 4, alpha-1, 6 and alpha-1, 3 glucosidic bonds from the non-reducing end of the starch to generate glucose under a certain condition, wherein the enzyme activity of the saccharifying enzyme defines that 1mL of enzyme solution or 1g of enzyme powder hydrolyzes soluble starch for 1 hour to generate 1mg of glucose under the conditions of 40 ℃ and pH4.6, namely one enzyme activity unit is expressed by U/mL (or U/g). The saccharifying enzyme involved in the invention includes but is not limited to amylase with the activity.

The term "dry solids content (DS)" refers to the total solids of the slurry in dry weight percent.

The term "about" refers to ± 10% of the referenced value.

Advantageous effects of the invention

During the alcoholic fermentation, the conditions of composition, pH, temperature, etc. of the substances at different stages are constantly changing. The alpha-glucosidase, the trehalase, the xylanase, the arabinofuranosidase, the cellulase, the beta-glucosidase and the ferulic acid esterase are added into an alcohol fermentation system within a specific time period after the start of fermentation, and the enzymatic reaction efficiency can be remarkably improved by optimizing the time for adding the enzymes. The enzyme preparation adding process provided by the invention can hydrolyze non-fermented sugar and reduce the content of residual sugar, thereby improving the yield of alcohol; on the other hand, the hemicellulose and the cellulose in the substrate are cracked or separated, so that the plant cell walls are quickly and effectively decomposed or collapsed, the starch and the protein wrapped by the hemicellulose and the cellulose are completely released, the substrate utilization rate is improved, the final alcohol content is improved, the hydrophilicity of the hemicellulose and the cellulose can be reduced to the maximum extent, the water content of the wet-based DDG can be greatly reduced, the concentration and drying efficiency in the DDGS production process is improved, the steam consumption is reduced, and the energy consumption is further saved.

Drawings

FIG. 1 shows a graph of the analysis of ethanol content in mash at different times of fermentation with alpha-glucosidase addition in example 2;

FIG. 2 shows a graph of ethanol content in mash at different times of fermentation addition of alpha-glucosidase (Tianye) in example 3;

FIG. 3 shows a graph of the analysis of the ethanol content in the mash with trehalase addition at different times of fermentation in example 4;

FIG. 4 shows a graph of the analysis of ethanol content in mash at different times of addition of xylanase in fermentation in example 5;

FIG. 5 shows a graph of ethanol content in mash when arabinofuranosidase was not added at the same time as fermentation in example 6;

FIG. 6 shows a graph of ethanol content in mash at different times of cellulase addition in fermentation in example 7;

FIG. 7 shows a graph of the analysis of ethanol content in mash at different times of fermentation with β -glucosidase addition in example 8.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention will be described in detail with reference to the following embodiments. It should be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1 Effect of alpha-glucosidase addition at different times on alcohol fermentation

1. Experimental methods of the invention

Raw material liquefaction: a certain amount of whole-ground corn flour (purchased from a certain alcohol factory A) is taken to prepare a mixture with a water ratio of 1: 2.0-3.0 of feed liquid. Adjusting pH to 4.5-6.0, and adding appropriate amount of high temperature amylase (such as amylase)

X5 in an amount of 10 to 100U/g DS). Liquefaction conditions: the temperature is 95 ℃ and the time is 120 min.

Expanding culture of yeast: adding 1g dry yeast (from Angel Yeast Co., Ltd., Angel super brewing high activity yeast) into 50ml centrifuge tube, adding 9g distilled water, mixing, placing in 38 deg.C water bath, and activating for 60 min.

Fermentation: cooling the liquefied material liquid to room temperature in time, adjusting pH to 4.3, subpackaging into shake flask, and adding appropriate amount of diastase (such as

A1, 50-500U/g DS), active yeast (yeast at the end of propagation) and nitrogen source urea (added in an amount of 400 ppm). Adding the mixture during fermentation for 0h and 40h and yeast propagationAdding 0.2U/g DS and 2U/g DS alpha-glucosidase (the amino acid sequence is SEQ ID NO.2, and the nucleotide sequence for coding the amino acid is SEQ ID NO. 1). Fermentation conditions are as follows: the temperature is 32 ℃ and the time is 72 h.

Adjusting the pH of mash: the pH was adjusted with 1mol/L hydrochloric acid or 3mol/L sodium hydroxide solution.

And (4) detecting a result: centrifuging part of the fermented feed liquid to obtain supernatant for high performance liquid chromatography analysis, residual reducing sugar analysis and total sugar filtration analysis; another portion of the feed was taken directly for total sugar measurement.

2. Control group experiment method

X5 in an amount of 10 to 100U/g DS). Liquefaction conditions: the temperature is 95 ℃ for 120 min.

Expanding culture of yeast: adding 1g dry yeast into 50ml centrifuge tube, adding 9g distilled water, mixing, placing in 38 deg.C water bath, and activating for 60 min.

Fermentation: cooling the liquefied material liquid to room temperature in time, adjusting pH to 4.3, packaging into shake flask, and adding appropriate amount of saccharifying enzyme (such as amylase)

A1, 50-500U/g DS), active yeast (obtained from Angel Yeast Co., Ltd., yeast at the end of propagation) and nitrogen source urea. Fermentation conditions are as follows: the temperature is 32 ℃ and the time is 72 h.

3. The analysis method comprises the following steps:

the high performance liquid chromatography analysis method comprises the following steps: the instrument comprises the following steps: shimadzu LC-20A, column: aminex HPX-87H column, 5mmol/L sulfuric acid as mobile phase, 0.6ml/min flow rate, RID-20A as detector.

Determination of residual carbohydrates (residual reducing sugars, residual dextrins, residual starches): film sugar determination method

(1) Reducing sugar assay

Measuring 50ml of fermentation liquor to reach a constant volume of 250 ml, filtering by absorbent cotton, taking 10 ml of filtrate, adding the filtrate into a flask containing 5 ml of each of the first solution and the second solution of the Fehling and 20 ml of water, measuring sugar by a general sugar determination method, and performing a blank test by using 0.25% glucose under the same conditions.

And (4) calculating a result:

in the formula: a-number of ml of glucose solution used for dropping 10 ml of Fehling's solution

B-number of ml of glucose solution consumed after titration with 10 ml of test solution

50-sample uptake in ml

(2) Determination of Total sugar

50ml of fermentation liquor is measured, 40 ml of water is added, 10 ml of 20% hydrochloric acid is added, a rubber plug with a 1.0-meter long glass tube is plugged, the fermentation liquor is converted for 60 minutes in a boiling water bath, the fermentation liquor is taken out and cooled, the fermentation liquor is neutralized to be slightly acidic by 20% sodium hydroxide, a 250 ml volumetric flask is transferred to be added with water to a scale, after shaking up, the fermentation liquor is filtered by absorbent cotton, 10 ml of filtrate is absorbed and added into triangular flasks containing 5 ml of each of the Feilin A solution, the second solution and 20 ml of water, titration is carried out by 0.25% glucose solution, and then a blank test is carried out by titrating 10 ml of the Feilin solution by 0.25% glucose.

And (4) calculating a result:

in the formula: number of ml of glucose solution for A-blank test

B-consumption of glucose solution in ml after titration with 10 ml of test solution

(3) Determination of Total sugar filtered

100 ml of a beer diluted filtrate for measuring reducing sugar is taken, 10 ml of 20% hydrochloric acid is added, a rubber plug with a 1.0-meter long glass tube at a plug port is converted for 60 minutes in a boiling water bath, the beer is taken out and cooled, the beer is neutralized to be slightly acidic by 20% sodium hydroxide, a 250 ml volumetric flask is transferred to be added with water to a scale, after shaking up, the beer is filtered by absorbent cotton, 10 ml of the filtrate is absorbed and added into triangular flasks containing 5 ml of each of the Feilin A solution and the Feilin B solution and 20 ml of water, titration is carried out by 0.25% glucose solution, and then 10 ml of the Feilin solution is titrated by 0.25% glucose solution to be used as a blank test.

(4) Calculation of dextrin and residual starch

Residual dextrin (filtered total sugar-reducing sugar) × 0.9 g dextrin per 100 ml

Residual starch (residual total sugar-filtered total sugar) × 0.9 g dextrin/100 ml

4. Results of the experiment

0.2U/g DS and 2U/g DS alpha-glucosidase are added when the alcoholic fermentation is carried out for 0h and 40h and the yeast is expanded. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, and detecting residual reducing sugar, filtered total sugar and residual total sugar in the mash. The results are shown in tables 1 and 2 below. According to the analysis of experimental results, the residual sugar content of mash can be obviously reduced only by adding 2U/g DS alpha-glucosidase during fermentation for 40h, and simultaneously the ethanol content in the feed liquid is improved after the fermentation is finished. On the contrary, the alpha-glucosidase is added during fermentation for 0h and yeast propagation, which only has the effect of reducing blood sugar, and the ethanol content in the feed liquid is not improved after the fermentation is finished.

TABLE 1 results of HPLC analysis with addition of alpha-glucosidase

TABLE 2 results of mash residual reducing sugar, filtered total sugar, residual total sugar with alpha-glucosidase added

Example 2 Effect of alpha-glucosidase addition at various times during fermentation on alcohol fermentation

Referring to the experimental method, the control group and the analytical method of the present invention in example 1, after subpackaging the shake flasks, 2U/g DS α -glucosidase (amino acid sequence is SEQ ID NO.2, nucleotide sequence encoding the amino acid is SEQ ID NO.1) was added at 0h, 3h, 16h, 24h, 40h, 48h and 60h of alcoholic fermentation, respectively. Changing corn flour to corn flour from alcohol plant B

The experimental results are as follows:

2U/g DS alpha-glucosidase is added in 0h, 3h, 16h, 24h, 40h, 48h and 60h of alcoholic fermentation. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, detecting residual reducing sugar in the mash, and filtering total sugar and residual total sugar. The results are shown in table 3, table 4 and fig. 1 below. According to the analysis of experimental results, 2U/g DS alpha-glucosidase added in 16-48h of fermentation can obviously reduce the residual sugar content of mash, and simultaneously the ethanol content in the mash is improved by 0.9-1.87% after the fermentation is finished. However, when 2U/g DS alpha-glucosidase was added at fermentation times of 0h, 3h and 60h, the residual sugar and alcohol content in the mash at the end of fermentation did not increase.

It should be noted that, in general, the ethanol yield per ton of corn can be increased by about 7L for every 0.1% increase of the ethanol yield value in industry, and at this time, it can be considered that there is a significant difference between the two ethanol yield values. The calculation is carried out on the scale of a winery with 10 ten thousand tons of annual output, the yield value of the ethanol is improved by 0.1 percent, and the annual ethanol yield of the winery is increased by 7 multiplied by 10⁵L, annual income of winery can be increased by 3.3X 10⁶And (5) Yuan. According to the result data of the embodiment, the ethanol yield can be effectively improved by the enzyme preparation adding process, and further, the significant economic benefit is brought to a winery.

TABLE 3 high performance liquid chromatography results of alpha-glucosidase addition

TABLE 4 results of mash residual reducing sugar, filtered total sugar, residual total sugar with alpha-glucosidase added

Example 3 Effect of addition of alpha-glucosidase from Ganshina products on alcohol fermentation at different fermentation times

Referring to the experimental method, control group and analytical method of the present invention in example 1, the enzyme added after the shake flask was dispensed was replaced with 2U/g of α -glucosidase (trade name: transglucosidase L, "Tianye") from DS Tianye enzyme preparation.

Corn meal was replaced with corn meal purchased from a certain alcohol plant B.

The experimental results are as follows:

2U/g DS alpha-glucosidase from Ganshina products company is added in 0h, 3h, 16h, 24h, 40h, 48h and 60h of alcoholic fermentation. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, detecting residual reducing sugar in the mash, and filtering total sugar and residual total sugar. The results are shown in table 5, table 6 and fig. 2 below. According to the analysis of experimental results, 2U/g DS alpha-glucosidase of Ganshira enzyme products company can be added when the fermentation time is 16h-48h, so that the residual sugar content of the mash can be reduced, and the ethanol content in the mash is improved by 1.2% -1.5% at the end of fermentation.

TABLE 5 high Performance liquid chromatography analysis results of alpha-glucosidase (Tianye) addition

TABLE 6 results of mash residual reducing sugar, total sugar filtration, and residual total sugar by adding alpha-glucosidase (Tianye)

Example 4 Effect of trehalase addition on alcoholic fermentation at various times during fermentation

Referring to the experimental, control and analytical methods of the present invention of example 1, only the enzyme added after the shake flask was dispensed was replaced with trehalase (Spirizyme Ultra T, Novoxil) at 0.5U/g DS. Corn meal was replaced with corn meal purchased from a certain alcohol plant B.

The experimental results are as follows:

0.5U/g DS trehalase is added when the alcohol is fermented for 0h, 3h, 16h, 24h, 40h, 48h and 60h respectively. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, and detecting residual reducing sugar, filtered total sugar and residual total sugar in the mash. The results are shown in table 7, table 8 and fig. 3 below. According to the analysis of experimental results, the result of adding 0.5U/g DS trehalase is basically consistent when the fermentation is carried out for 0-60h, and the ethanol content in mash at the end of fermentation is improved by about 0.65%.

TABLE 7 HPLC analysis results of trehalase addition

TABLE 8 results of residual reducing sugars, total sugars filtered, and residual total sugars from mash to which trehalase was added

Example 5 Effect of xylanase addition on alcoholic fermentation at different times of fermentation

Referring to the experimental, control and analytical methods of the invention of example 1, only the enzymes added after the shake flask had been dispensed were exchanged for xylanase at 10U/g DS (Novoxil X2753). Corn meal was replaced with corn meal purchased from a certain alcohol plant B.

The experimental results are as follows:

adding xylanase with DS 10U/g when fermenting alcohol for 0h, 3h, 16h, 24h, 40h, 48h and 60h respectively. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, and detecting residual reducing sugar, filtered total sugar and residual total sugar in the mash. The results are shown in table 9, table 10 and fig. 4 below. According to the analysis of experimental results, the xylanase added with 10U/g DS is basically consistent when the fermentation is carried out for 0-48h, the residual sugar content can be reduced, and the ethanol content in mash is improved by 0.94-1.07% when the fermentation is finished.

TABLE 9 high Performance liquid chromatography results of xylanase addition

TABLE 10 results of residual reducing sugars, total sugars filtered, residual total sugars of mash with xylanase added

Example 6 Effect of addition of arabinofuranosidase at various times during fermentation on alcohol fermentation

Referring to the experimental method, control group and analytical method of the present invention of example 1, only the enzyme added after the flask was filled separately was replaced with 10U/g DS arabinofuranosidase (amino acid sequence SEQ ID NO.4, nucleotide sequence encoding the amino acid SEQ ID NO. 3). Corn meal was replaced with corn meal purchased from a certain alcohol plant B.

The experimental results are as follows:

adding 10U/g DS arabinosidase when fermenting alcohol for 0h, 3h, 16h, 24h, 40h, 48h and 60 h. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, and detecting residual reducing sugar, filtered total sugar and residual total sugar in the mash. The results are shown in table 11, table 12 and fig. 5 below. According to the analysis of experimental results, when the arabinofuranosidase with the DS of 10U/g is added during the fermentation of 0-48h, the residual sugar content can be reduced, and the ethanol content in mash is improved by about 0.65% at the end of the fermentation.

TABLE 11 high Performance liquid chromatography results of arabinosidase addition

TABLE 12 results of residual reducing sugars, total sugars filtration, and residual total sugars of mash to which arabinofuranosidase was added

Example 7 Effect of cellulase addition at various times during fermentation on alcohol fermentation

Referring to the experimental, control and analytical methods of the present invention of example 1, the enzymes added after shake flask split were replaced with 10U/g DS cellulase (Novoxil 476). The corn flour is replaced by the corn flour purchased from a certain alcohol plant B.

The experimental results are as follows:

adding 10U/g DS cellulase when fermenting alcohol for 0h, 3h, 16h, 24h, 40h, 48h and 60h respectively. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, and detecting residual reducing sugar, filtered total sugar and residual total sugar in the mash. The results are shown in table 13, table 14 and fig. 6 below. According to the analysis of experimental results, the ethanol content in the mash is improved by 1.01-4.80% when 10U/g DS cellulase is added in 0-48h, the effect of adding 10U/g DS cellulase in 0h is the best, and the improvement of the ethanol content is better than that of adding 10U/g DS cellulase in 40 h.

TABLE 13 results of HPLC analysis with cellulase addition

TABLE 14 results of residual reducing sugars, total sugars filtered, and residual total sugars of the mash with cellulase addition

Example 8 Effect of beta-glucosidase addition on alcohol fermentation at various times during fermentation

Referring to the experimental, control and analytical methods of the present invention of example 1, the enzyme added after shake flask split was changed to 10U/g DS beta-glucosidase (Novexin 188L). Corn meal was replaced with corn meal purchased from a certain alcohol plant B.

The experimental results are as follows:

adding 10U/g DS beta-glucosidase during alcoholic fermentation for 0h, 3h, 16h, 24h, 40h, 48h and 60h respectively. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, and detecting residual reducing sugar, filtered total sugar and residual total sugar in the mash. The results are shown in table 15, table 16 and fig. 7 below. According to the analysis of experimental results, the ethanol content in mash is improved by 1.30-2.60% after the fermentation is finished by adding 10U/g DS beta-glucosidase in 0-48h, the best result is obtained by adding 10U/g DS beta-glucosidase in 0h of fermentation, and the effect of improving the alcohol content and reducing the residual sugar is obviously better than that of adding 10U/g DS beta-glucosidase in 40h of fermentation.

TABLE 15 results of HPLC analysis with addition of beta-glucosidase

TABLE 16 results of mash residual reducing sugar, total sugar filtration, and residual total sugar with beta-glucosidase added

Example 9 Effect of alpha-glucosidase addition on alcoholic fermentation 40h fermentation of corn meal from different sources

Referring to the experimental method, control group and analytical method of the present invention of example 1, only corn flour was changed to corn flour purchased from a certain alcohol plant B.

The experimental results are as follows:

2U/g DS alpha-glucosidase is added when alcohol is fermented for 40 h. After fermentation, analyzing the components in the fermentation liquor by using high performance liquid chromatography, and detecting residual reducing sugar, filtered total sugar and residual total sugar in the mash. The results are shown in tables 17 and 18 below. According to the analysis of experimental results, when the corn flour of a certain alcohol plant B is used as a raw material, 2U/g DS alpha-glucosidase is added during fermentation for 40 hours, and the ethanol content in mash at the end of fermentation can be improved by 2.96% while the residual sugar content of the mash is reduced.

TABLE 17 high Performance liquid chromatography analysis results of addition of alpha-glucosidase

TABLE 18 results of mash residual reducing sugar, filtered total sugar, residual total sugar with alpha-glucosidase added

Sequence listing

<110> Nanjing Baismig bioengineering GmbH

<120> enzyme preparation adding process for promoting fermentation of starch grains

<160> 4

<170> SIPOSequenceListing 1.0

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Ile Gly Pro Arg Ala Asn Ser Gln Ser Cys Pro Gly Tyr Lys Ala Ser

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Asn Val Gln Lys Gln Ala Arg Ser Leu Thr Ala Asp Leu Thr Leu Ala

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Gly Thr Pro Cys Asn Ser Tyr Gly Lys Asp Leu Glu Asp Leu Lys Leu

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Leu Val Glu Tyr Gln Thr Asp Glu Arg Leu His Val Met Ile Tyr Asp

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Ala Asp Glu Glu Val Tyr Gln Val Pro Glu Ser Val Leu Pro Arg Val

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Gly Ser Asp Glu Asp Ser Glu Asp Ser Val Leu Glu Phe Asp Tyr Val

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Glu Glu Pro Phe Ser Phe Thr Ile Ser Lys Gly Asp Glu Val Leu Phe

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Asp Ser Ser Ala Ser Pro Leu Val Phe Gln Ser Gln Tyr Val Asn Leu

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Arg Thr Trp Leu Pro Asp Asp Pro Tyr Val Tyr Gly Leu Gly Glu His

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Ser Asp Pro Met Arg Leu Pro Thr Tyr Asn Tyr Thr Arg Thr Leu Trp

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Asn Arg Asp Ala Tyr Gly Thr Pro Asn Asn Thr Asn Leu Tyr Gly Ser

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Asp Gly Lys Gln Tyr Leu Glu Tyr Asn Leu Leu Gly Gly Val Leu Asp

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Phe Tyr Phe Phe Tyr Gly Glu Asp Pro Lys Gln Ala Ser Met Glu Tyr

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Ser Lys Ile Val Gly Leu Pro Ala Met Gln Ser Tyr Trp Thr Phe Gly

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Val Cys Pro Pro Pro Pro Asn Pro Ile Thr Val Arg Val Val Val Tyr

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

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Asp Tyr Met Asp Lys Arg Arg Val Phe Thr Leu Asp Pro Gln Arg Phe

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Pro Leu Glu Lys Met Arg Glu Leu Val Thr Tyr Leu His Asn His Asp

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Gln His Tyr Ile Val Met Val Asp Pro Ala Val Ser Val Ser Asn Asn

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Asn Gly Ser Leu Tyr Glu Gly Ala Val Trp Pro Gly Val Thr Val Phe

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Pro Asp Trp Phe Asn Glu Gly Thr Gln Asp Tyr Trp Thr Ala Gln Phe

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Gln Gln Phe Phe Asp Pro Lys Ser Gly Val Asp Ile Asp Ala Leu Trp

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Ile Asp Met Asn Glu Ala Ser Asn Phe Cys Pro Tyr Pro Cys Leu Asp

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

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Gln Pro Ser Ser Lys Arg Ser Val Lys Arg Ala Gln Gly Asp Lys Gly

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Leu Ile His Ala Gly Glu Gly Tyr Ala Glu Tyr Asp Thr His Asn Leu

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Tyr Gly Thr Arg Leu Val Met Ser Ser Ala Ser Arg Thr Ala Met Gln

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Ala Arg Arg Pro Asp Val Arg Pro Leu Val Ile Thr Arg Ser Thr Phe

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Ala Gly Ala Gly Ala His Val Gly His Trp Leu Gly Asp Asn Phe Ser

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Asp Trp Val His Tyr Arg Ile Ser Ile Ala Gln Ile Leu Ser Phe Ala

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Ser Met Phe Gln Ile Pro Met Val Gly Ala Asp Val Cys Gly Phe Gly

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Ser Asn Thr Thr Glu Glu Leu Cys Ala Arg Trp Ala Ser Leu Gly Ala

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Phe Tyr Thr Phe Tyr Arg Asn His Asn Glu Leu Gly Asp Ile Ser Gln

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Gln Ser Gln Thr Gly Glu Pro Phe Leu Gln Pro Gln Phe Tyr Leu Tyr

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Pro Glu Asp Ser Asn Thr Phe Ala Asn Asp Arg Gln Phe Phe Tyr Gly

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Asp Ala Leu Leu Val Ser Pro Val Leu Asn Glu Gly Ser Thr Ser Val

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Asp Ala Tyr Phe Pro Asp Asp Ile Phe Tyr Asp Trp Tyr Thr Gly Ala

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Val Val Arg Gly His Gly Glu Asn Ile Thr Leu Ser Asn Ile Asn Ile

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Phe Thr Tyr Ser Lys Gly Glu Leu His Val Lys Gly Thr Phe Gly Gln

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tggtcgagat tggcaatgaa gatatgctgg gaggcggatg cgagtcctac gccgagcgtt 1440

ttactgcctt ctatgatgca atccatgcgg cttacccgga tctcatcctt attgctagca 1500

ccagcgaggc ggattgcttg cccgagtcga tgcccgaggg tagctgggtc gactaccacg 1560

actacagcac gcccgatgga ctggtgggcc agttcaacta tttcgacaac ctggaccgct 1620

ctgtgccata cttcatcggc gagtattcgc gctgggagat cgactggccc aacatgaagg 1680

ggtcggtttc cgaggctgtt ttcatgatcg ggttcgagag gaacagcgat gtggtcaaga 1740

tggcggcgta tgcgccattg ctccagctgg tgaactcgac tcagtggacg gtaagtcact 1800

gttgcgcagc ggtattcgac ggcatggatg agctaacgag tggtagccgg acctgatcgg 1860

atacacccag tcacccgatg acattttcct ctcgaccagc tactacgtgc aggaaatgtt 1920

ctcgcgcaac cgtggtgata ccatcaagga ggtgacctct gacagcgact ttggaccgtt 1980

gtactgggtt gcgtcgagcg ccggcgactc gtactacgtg aagctggcca actacggctc 2040

ggagacgcaa gaccttaccg tgagcatccc aggaacgagc acaggcaagt tgacggtgct 2100

ggcggacaat gaccccgacg cgtacaactc tgacacccag acgctggtca cgccgagcga 2160

atcgacggtg caggcgagca atggtacctt tacctttagt ttgccggcat gggcggtggc 2220

tgtcctggct gcgaactag 2239

<210> 4

<211> 628

<212> PRT

<213> Aspergillus niger (Aspergillus niger)

<400> 4

Met Val Ala Phe Ser Ala Leu Ser Gly Val Ser Ala Leu Ser Leu Leu

1 5 10 15

Leu Cys Leu Val Gln His Ala His Gly Val Ser Leu Lys Val Ser Thr

20 25 30

Gln Gly Gly Asn Ser Ser Ser Pro Ile Leu Tyr Gly Phe Met Phe Glu

35 40 45

Asp Ile Asn His Ser Gly Asp Gly Gly Ile Tyr Gly Gln Leu Leu Gln

50 55 60

Asn Pro Gly Leu Gln Gly Thr Thr Pro Asn Leu Thr Ala Trp Ala Ala

65 70 75 80

Val Gly Asp Ala Thr Ile Ala Ile Asp Gly Asp Ser Pro Leu Thr Ser

85 90 95

Ala Ile Pro Ser Thr Ile Lys Leu Asp Val Ala Asp Asp Ala Thr Gly

100 105 110

Ala Val Gly Leu Thr Asn Glu Gly Tyr Trp Gly Ile Pro Val Asp Gly

115 120 125

Ser Glu Phe Gln Ser Ser Phe Trp Ile Lys Gly Glu Tyr Ser Gly Asp

130 135 140

Ile Thr Val Arg Leu Val Gly Asn Tyr Thr Gly Thr Glu Tyr Gly Ser

145 150 155 160

Ala Thr Ile Thr His Thr Ser Thr Ala Asp Asn Phe Thr Gln Ala Ser

165 170 175

Val Lys Phe Pro Thr Thr Lys Ala Pro Asp Gly Asn Val Leu Tyr Glu

180 185 190

Leu Thr Val Asp Gly Ser Val Ala Ala Gly Ser Ser Leu Asn Phe Gly

195 200 205

Tyr Leu Thr Leu Phe Gly Glu Thr Tyr Lys Ser Arg Glu Asn Gly Leu

210 215 220

Lys Pro Gln Leu Ala Asn Val Leu Ala Asp Met Lys Gly Ser Phe Leu

225 230 235 240

Arg Phe Pro Gly Gly Asn Asn Leu Glu Gly Asn Ser Ala Glu Asn Arg

245 250 255

Trp Lys Trp Asn Glu Thr Ile Gly Asp Leu Trp Asp Arg Pro Gly Arg

260 265 270

Glu Gly Thr Trp Thr Tyr Tyr Asn Thr Asp Gly Leu Gly Leu His Glu

275 280 285

Tyr Phe Tyr Trp Cys Glu Asp Leu Gly Leu Val Pro Val Leu Gly Val

290 295 300

Trp Asp Gly Phe Ala Leu Glu Ser Gly Gly Asn Thr Pro Ile Thr Gly

305 310 315 320

Asp Ala Leu Thr Pro Tyr Ile Asp Asp Val Leu Asn Glu Leu Glu Tyr

325 330 335

Ile Leu Gly Asp Thr Ser Thr Thr Tyr Gly Ala Trp Arg Ala Ala Asn

340 345 350

Gly Gln Glu Glu Pro Trp Asn Leu Thr Met Val Glu Ile Gly Asn Glu

355 360 365

Asp Met Leu Gly Gly Gly Cys Glu Ser Tyr Ala Glu Arg Phe Thr Ala

370 375 380

Phe Tyr Asp Ala Ile His Ala Ala Tyr Pro Asp Leu Ile Leu Ile Ala

385 390 395 400

Ser Thr Ser Glu Ala Asp Cys Leu Pro Glu Ser Met Pro Glu Gly Ser

405 410 415

Trp Val Asp Tyr His Asp Tyr Ser Thr Pro Asp Gly Leu Val Gly Gln

420 425 430

Phe Asn Tyr Phe Asp Asn Leu Asp Arg Ser Val Pro Tyr Phe Ile Gly

435 440 445

Glu Tyr Ser Arg Trp Glu Ile Asp Trp Pro Asn Met Lys Gly Ser Val

450 455 460

Ser Glu Ala Val Phe Met Ile Gly Phe Glu Arg Asn Ser Asp Val Val

465 470 475 480

Lys Met Ala Ala Tyr Ala Pro Leu Leu Gln Leu Val Asn Ser Thr Gln

485 490 495

Trp Thr Pro Asp Leu Ile Gly Tyr Thr Gln Ser Pro Asp Asp Ile Phe

500 505 510

Leu Ser Thr Ser Tyr Tyr Val Gln Glu Met Phe Ser Arg Asn Arg Gly

515 520 525

Asp Thr Ile Lys Glu Val Thr Ser Asp Ser Asp Phe Gly Pro Leu Tyr

530 535 540

Trp Val Ala Ser Ser Ala Gly Asp Ser Tyr Tyr Val Lys Leu Ala Asn

545 550 555 560

Tyr Gly Ser Glu Thr Gln Asp Leu Thr Val Ser Ile Pro Gly Thr Ser

565 570 575

Thr Gly Lys Leu Thr Val Leu Ala Asp Asn Asp Pro Asp Ala Tyr Asn

580 585 590

Ser Asp Thr Gln Thr Leu Val Thr Pro Ser Glu Ser Thr Val Gln Ala

595 600 605

Ser Asn Gly Thr Phe Thr Phe Ser Leu Pro Ala Trp Ala Val Ala Val

610 615 620

Leu Ala Ala Asn

625

Claims

1. A method of fermenting a cereal material to produce alcohol, the method comprising the steps of:

a. liquefying the grain raw material;

b. saccharifying;

c. fermenting;

wherein the step c comprises a step of adding a non-starch polysaccharide enzyme.

2. The method of claim 1, wherein the b step is performed simultaneously with the c step.

3. The process according to claim 1, wherein the step of adding the non-starch polysaccharidase is selected from 0-60h after the start of fermentation, preferably 3-48h after the start of fermentation, more preferably 16-40h after the start of fermentation, most preferably 40h after the start of fermentation.

4. The method according to claim 1, further comprising a yeast propagation step, preferably said yeast propagation step is prior to said c-step.

5. The method according to any one of claims 1 to 4, wherein the step of adding a non-starch polysaccharide enzyme comprises adding a non-starch polysaccharide enzyme selected from the group consisting of α -glucosidase, trehalase, xylanase, arabinofuranosidase, cellulase, β -glucosidase, feruloyl esterase, pectinase, mannanase or any combination thereof, preferably α -glucosidase.

6. The method according to claim 5, wherein the alpha-glucosidase is added in an amount of 0.1-20U/g DS, preferably 0.5-10U/g DS, more preferably 2U/g DS.

7. The method according to claim 5, wherein the trehalase is added in an amount of 0.01-10U/g DS, preferably 0.1-5U/g DS, more preferably 0.5U/g DS.

8. The method according to claim 5, wherein the xylanase is added in an amount of 0.1-100U/g DS, preferably 0.5-50U/g DS, more preferably 10U/g DS.

9. The method according to claim 5, wherein the arabinofuranosidase is added in an amount of 0.1-100U/g DS, preferably 0.5-50U/g DS, more preferably 10U/g DS.

10. The process according to claim 5, wherein the cellulase is added in an amount of 0.1-100U/g DS, preferably 0.5-50U/g DS, more preferably 10U/g DS.

11. The method according to claim 5, wherein the amount of β -glucosidase added is 0.1-100U/g DS, preferably 0.5-50U/g DS, more preferably 10U/g DS.

12. The method according to claim 5, wherein the ferulic acid esterase is added in an amount of 0.1 to 100U/g DS, preferably 0.5 to 50U/g DS, more preferably 10U/g DS.

13. The method according to claim 2, wherein the step a comprises preparing a grain raw material liquid with a proper material-water ratio, and adding amylase in the material liquid; adding saccharifying enzyme in the step b; and c, adding yeast and a nitrogen source in the step c.

14. The method of claim 13, said a step comprising formulating a water ratio of 1: 2.0-3.0 of grain raw material liquid, adjusting the pH value to 4.5-6.0, and adding high-temperature amylase for high-temperature liquefaction; and c, simultaneously performing the steps b and c, regulating the pH of the liquefied feed liquid to about 4.3, timely cooling to room temperature, uniformly subpackaging into a shake flask, adding 50-500U/g DS saccharifying enzyme, adding 1g of yeast obtained after dry yeast propagation and 400ppm of nitrogen source urea, and fermenting at the temperature of 28-36 ℃ for 48-96 hours.

15. The method according to claim 1, the cereal raw material being selected from corn, sorghum, wheat, barley, oats, rye, rice, cassava or sweet potato, in particular corn, more in particular whole-milled corn flour.

16. The method of claim 1, the step of adding a non-starch based polysaccharidase comprising adding a non-starch based polysaccharidase preparation in admixture with one or more preservatives and/or antimicrobial agents.