CN113980944B

CN113980944B - High-activity beer complex enzyme and preparation method and application thereof

Info

Publication number: CN113980944B
Application number: CN202111607029.2A
Authority: CN
Inventors: 刘苹; 张毅; 单守水; 孙胜男; 于飞
Original assignee: Zhongzhi Guochuang Biotechnology Shandong Co ltd
Current assignee: Zhongzhi Guochuang Biotechnology Shandong Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-04-15
Anticipated expiration: 2041-12-27
Also published as: CN113980944A

Abstract

The invention discloses a production method of a high-activity beer compound enzyme, which comprises the following steps: (1) mixing and leaching raw malt flour with water; (2) performing ultrafiltration concentration on the leaching liquor by using an ultrafiltration membrane with the cut-off molecular weight of 50000 Da-60000 Da; (3) heating the membrane material obtained in the step (2), and removing thermal condensate to obtain a primary membrane material enzyme solution; (4) passing the membrane substrate obtained in the step (2) through an ultrafiltration membrane with the molecular weight cutoff of 10000 Da-20000 Da, and taking the membrane substrate to obtain a secondary membrane substrate enzyme solution; (5) and (3) enabling the enzyme solution on the primary membrane and the enzyme solution on the secondary membrane to be mixed according to the enzyme activity ratio of the protease to the limit dextrinase (15-30): 1 and mixing. The invention also discloses the beer complex enzyme obtained by the method and application thereof in beer preparation. The beer complex enzyme completely meets the beer brewing requirement, and overcomes the defect that a microbial fermentation enzyme preparation is added due to insufficient malt saccharification force.

Description

High-activity beer complex enzyme and preparation method and application thereof

Technical Field

The invention belongs to the field of biological enzyme preparations, and particularly relates to a high-activity beer complex enzyme, and a preparation method and application thereof.

Background

The beer brewing mainly uses malt (i.e. products of barley or wheat after germination, drying and baking) as main raw materials, carbohydrates, proteins and the like are hydrolyzed by enzyme activity contained in the malt to prepare wort, and the wort is obtained through beer fermentation. However, in order to prevent the green malt (i.e., barley or wheat from sprouting, dried at low temperature and not baked at high temperature) from bringing a "green taste" when drinking and increase the "roasted flavor" of the beer malt, the germinated malt needs to be baked at 80 ℃ to 100 ℃, so that most of enzymes generated in the germination process of barley or wheat are inactivated due to high temperature, and the enzymes are not fully utilized, resulting in the following three problems: firstly, the enzyme activity is insufficient during the preparation of the wort, so that the utilization rate of malt raw materials is reduced, particularly the utilization rate of protein in the malt is only about 20 percent, and most of the protein is precipitated and filtered out in the heating process; secondly, the malt contains one kind of beta-glucan, especially the malt with poor quality has higher content, the beta-glucan can be combined with protein to form precipitate, the yeast is caused to precipitate prematurely, the fermentation and the reduction of diacetyl are influenced, the quality of the finished beer product is unstable, and the non-biological turbidity is easy to generate; thirdly, the dosage and the yield of the auxiliary materials are influenced: at present, more than 30% of rice or starch is added during wort preparation, the process requirements are met only by depending on enzyme in malt for hydrolysis, and the use amount and yield of auxiliary materials are influenced due to insufficient enzyme activity in the malt.

In order to solve the problems, various microbial fermentation enzyme preparations and enzyme preparation products compounded by the microbial fermentation enzyme preparations are added during wort preparation at present, wherein the enzyme preparation products comprise alpha-amylase, saccharifying enzyme, protease and the like. The alpha-amylase is a fermentation product, is used as an additional microbial fermentation enzyme preparation and has a difference with the original enzyme of the malt, and the peculiar smell of the fermentation enzyme preparation is increased, so that the beer quality is influenced.

In recent years, the beer brewing technology is continuously improved, and the specialization division is more detailed. Wherein, the saccharification of the rice and the starch auxiliary materials is provided by special starch sugar enterprises, and the beer brewing process is greatly simplified. However, enzyme preparations derived from microbial fermentation, such as alpha-amylase, saccharifying enzyme, protease, etc., are used in the reprocessing of beer syrup by starch sugar companies. Because the saccharification enzyme and the beer wort are saccharified by the difference of enzyme systems in the malt, the components of the syrup after saccharification are also changed and can not be completely matched with the malt saccharification, and the beer quality is also influenced to a certain extent.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a high-activity beer complex enzyme, and a preparation method and application thereof.

The specific technical scheme is as follows:

a production method of a high-activity beer compound enzyme comprises the following steps:

(1) low-temperature leaching: mixing raw malt flour with water, uniformly stirring, and leaching for 1-2 hours at 10-25 ℃;

(2) primary ultrafiltration: carrying out ultrafiltration concentration on the enzyme liquid obtained by leaching by using an ultrafiltration membrane with the cut-off molecular weight of 50000-60000 Da, and taking the substance on the membrane and the substance under the membrane;

(3) heat exchange: heating the membrane material obtained in the step (2) to 30-40 ℃, keeping the temperature for 0.5-1 h, and removing the thermal condensate in the enzyme solution to obtain a primary membrane material enzyme solution;

(4) secondary ultrafiltration: performing ultrafiltration concentration on the membrane substrate obtained in the step (2) through an ultrafiltration membrane with the molecular weight cutoff of 10000-20000 Da, and taking the membrane substrate to obtain a secondary membrane substrate enzyme solution;

(5) blending: and (3) enabling the enzyme solution on the primary membrane and the enzyme solution on the secondary membrane to be mixed according to the enzyme activity ratio of the protease to the limit dextrinase (15-30): 1, mixing to obtain the high-activity beer complex enzyme.

The invention selects malt (which can be called as raw malt) which is rich in enzyme activity, high in enzyme activity and low in temperature drying, and mixes enzymes with different molecular weights through a series of biotechnological processes such as leaching, ultrafiltration and refining to obtain a concentrate containing all enzymes in the malt, and completely retains the inherent enzyme system of the malt, including beta-amylase, alpha-amylase, saccharifying enzyme, limit dextrinase, cellulase, beta-glucanase, protease and the like. On the basis, the enzyme system function can be further strengthened by adding protease, beta-glucanase and the like from malt so as to meet the aim of malt and auxiliary material hydrolysis. If the preparation is not carried out, the enzyme activity ratio of the protease to the limit dextrinase in the original leaching liquor is 5:1, and the defects are that the content of the protease is low and the utilization rate of the protein in the raw material is low; the introduction of protease preparations fermented by genetically engineered bacteria results in the change of the flavor of the traditional beer.

The enzyme preparation is derived from malt, can be used for baking malt and saccharifying auxiliary materials, can be used for producing beer syrup by rice or starch, can completely meet the original brewing requirement of beer, and overcomes the defects of additional microorganism fermentation enzyme preparation due to insufficient saccharifying power of the malt.

Further, before blending in the step (5), adding 8-15 wt% of sodium chloride, 20-30 wt% of sorbitol, 0.05-0.3 wt% of sodium benzoate and 0.05-0.1 wt% of sodium methylparaben into the primary membrane enzyme solution and the secondary membrane enzyme solution respectively for stability treatment.

Further, in the step (1), the mass ratio of the malt flour to the water is 1: (3-5).

Further, after the low-temperature leaching in the step (1), clarifying a leaching system, specifically: diatomaceous earth was added to the leaching system and filtered to obtain a clear enzyme solution.

Still further, 1-6 wt% of 300# diatomite and 0.3-2 wt% of 10# diatomite by mass are added into the leaching system obtained in the step (1).

And further, filtering by using a plate and frame filter, washing the plate and frame with water, collecting the washing liquid, and mixing the washing liquid with the enzyme liquid obtained by filtering. The amount of the washing water is preferably 1 to 2 times of the leaching water.

Further, adding diatomite into the enzyme liquid obtained after heat exchange in the step (3) for fine filtration to obtain clear enzyme liquid, and then carrying out secondary ultrafiltration. The addition amount and the addition method of the diatomite refer to the clarification of a leaching system.

Further, the malt is wheat malt or barley malt, preferably raw barley malt.

The invention also provides a high-activity beer complex enzyme obtained by using the production method.

The invention also provides the application of the high-activity beer complex enzyme in beer production.

When the high-activity beer compound enzyme is used, the high-activity beer compound enzyme is added in a saccharification process, and the original process is not changed.

The invention has the following beneficial effects:

the invention extracts all enzyme preparations in the malt, completely reserves the inherent enzyme system of the malt, and matches the enzymes with different molecular weights to obtain a concentrate containing all the enzymes in the malt, thereby increasing the content of fermentable sugar, improving the utilization rate of raw materials and the fermentation degree of the malt juice, improving the protein conversion utilization rate, reducing the production cost, simplifying the saccharification process of beer brewing and improving the flavor and the quality of beer. The high-activity beer complex enzyme obtained by the invention can improve the utilization rate of malt by more than 20 percent and improve the utilization rate of protein in the malt by 30 to 45 percent. When the high-activity beer complex enzyme is used, the high-activity beer complex enzyme is added in a saccharification process, the original process is not changed, special equipment is not needed, the use is convenient, and the brewing requirement of beer is met.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Example 1

(1) low-temperature leaching: mixing raw barley malt powder and water according to a mass ratio of 1:3, mixing, uniformly stirring, and leaching for 1h under the stirring condition at the temperature of 15-20 ℃; after leaching, adding 5wt% of 300# diatomite and 1wt% of 10# diatomite into a leaching system, uniformly stirring, and filtering by using a plate and frame filter press to obtain a clear enzyme solution; the plate frame was washed with twice the water used for extraction, and the wash was collected and mixed with the clear enzyme solution described above.

(2) Primary ultrafiltration: and (2) performing ultrafiltration concentration on the enzyme liquid obtained in the step (1) by using an ultrafiltration membrane with the molecular weight cutoff of 60000Da, and taking the membrane upper substance and the membrane lower substance.

(3) Heat exchange: heating the membrane material obtained in the step (2) to 38 ℃, preserving heat for 0.7h, and removing the thermal condensate in the enzyme solution; then, 1wt% of 300# diatomaceous earth and 2wt% of 10# diatomaceous earth by mass were added to the membrane supernatant, and filtered with a plate and frame filter press to obtain a clear enzyme solution; and (3) cleaning the plate frame by using water twice as much as the leaching water, respectively collecting filtrate and cleaning liquid, directly mixing the filtrate with the clear enzyme solution, and using the cleaning liquid for next feeding. And (4) obtaining the enzyme solution on the primary membrane through the treatment.

(4) Secondary ultrafiltration: and (3) performing ultrafiltration concentration on the membrane lower substance obtained in the step (2) through an ultrafiltration membrane with the molecular weight cutoff of 10000Da to obtain a membrane upper substance, so as to obtain a secondary membrane upper substance enzyme solution.

And (3) respectively adding 10wt% of sodium chloride, 20wt% of sorbitol, 0.05wt% of sodium benzoate and 0.05wt% of sodium methyl hydroxybenzoate into the primary membrane substrate enzyme liquid obtained in the step (3) and the secondary membrane substrate enzyme liquid obtained in the step (4) for stability treatment, and detecting the protease activity of the primary membrane substrate enzyme liquid subjected to the stability treatment and the limit dextrinase activity of the secondary membrane substrate enzyme liquid.

(5) Blending: and (3) carrying out stability treatment on the primary membrane substrate enzyme solution and the secondary membrane substrate enzyme solution according to the enzyme activity ratio of the protease to the limit dextrinase of 30: 1, mixing to obtain the high-activity beer complex enzyme.

Through detection, in the obtained high-activity beer complex enzyme, 10 ten thousand u/mL of beta-amylase enzyme activity, 300u/mL of alpha-amylase enzyme activity, 6 ten thousand u/mL of carbohydrase enzyme activity, 1000u/mL of limit dextrinase enzyme activity, 600u/mL of cellulase enzyme activity, 200u/mL of beta-glucanase enzyme activity and 3 ten thousand u/mL of protease enzyme activity are obtained.

Example 2

The difference from the example 1 is that in the step (4), 15wt% of sodium chloride, 25wt% of sorbitol, 0.1wt% of sodium benzoate and 0.05wt% of sodium methylparaben are added to the primary membrane enzyme solution and the secondary membrane enzyme solution respectively to carry out the stabilization treatment;

the remaining technical features are the same as those of example 1.

Through detection, in the obtained high-activity beer complex enzyme, 11 ten thousand u/mL of beta-amylase enzyme activity, 250u/mL of alpha-amylase enzyme activity, 7 ten thousand u/mL of carbohydrase enzyme activity, 1200u/mL of limit dextrinase enzyme activity, 700u/mL of cellulase enzyme activity, 200u/mL of beta-glucanase enzyme activity and 3.6 ten thousand u/mL of protease enzyme activity are obtained.

Application example 1

The high-activity beer compound enzyme obtained in the example 1 and the commercial compound enzyme are respectively used for preparing beer. Wherein, the commercial complex enzyme is a special microbial fermentation enzyme preparation for beer fermentation. The commercial complex enzyme is mixed with water to make the protease activity of the commercial complex enzyme is the same as that of the high-activity beer complex enzyme in the example 1. And detecting the enzyme activity of the blended commercial complex enzyme, wherein the enzyme activity of beta-amylase is 12 ten thousand u/mL, the enzyme activity of alpha-amylase is 230u/mL, the enzyme activity of carbohydrase is 7 ten thousand u/mL, the enzyme activity of limit dextrinase is 1100u/mL, the enzyme activity of cellulase is 700u/mL, the enzyme activity of beta-glucanase is 230u/mL, and the enzyme activity of protease is 3 ten thousand u/mL.

Taking the preparation of 12 ° P beer as an example, the specific method is as follows:

(1) saccharifying malt powder with a material-water mass ratio of 1:3, adding corresponding complex enzyme (the addition amount is shown in table 1), wherein specific saccharification conditions are as follows: firstly heating the feed liquid to 50-55 ℃, stirring for 1h, slowly heating to 60-65 ℃, saccharifying for 1.5h, heating to 80 ℃, inactivating enzyme, cooling to 70 ℃, and filtering.

(2) Filtering by using a plate frame with the filtering area of 100 square meters.

(3) Collecting filtered wort, adjusting sugar degree to 11.5P, boiling, adding hops, fermenting in a tank, specifically fermenting at 11 ℃, controlling pressure to be 0-0.03 MPa, discharging condensate at the bottom of the tank after fermenting for 24h, measuring indexes such as sugar degree and pH value every day, sealing the tank when the sugar degree is reduced to 4P, controlling pressure to be 0.17-0.2 MPa, and tasting taste every day when the sugar degree is stable and does not basically reduce until the taste is qualified and is cooled to 0-1 ℃.

The total number of 7 experimental groups is set, and the adding condition of the complex enzyme in each group and various indexes of the product and the preparation process are shown in table 1. In Table 1, the malt extraction ratio is the ratio of dry matter extracted from malt and dissolved in water to dry matter of malt, and the alpha-amino nitrogen content represents the protein utilization ratio. The comparison results of the finished beer are shown in Table 2.

TABLE 1 wort comparison after enzymatic hydrolysis

TABLE 2 comparison of finished beer

The compound enzyme with 0.4kg/t dry basis is added in the beer production saccharification, the filtering speed is improved by about 30 percent, the production period is shortened, the raw material utilization rate is improved by 20 percent, and the production cost is reduced; the alpha-amino nitrogen content is high, the yeast propagation is fast, the fermentation period is shortened, the beer flavor is harmonious, no peculiar smell is generated, the foam is rich, the diacetyl content is lower, and the beer taste is mellow and smooth.

Compared with the test of the existing products in the current market, the beta-amylase has equivalent enzyme activity, the effect generated by the complex enzyme is superior to the use effect of a control sample under the condition of the same addition amount, the use effect is equivalent to that of the complex enzyme after the addition amount is increased to 1.5 times, the complex enzyme has a good enzymolysis effect particularly on the malt with defects, and the effect of producing beer by fermenting the high-quality malt can be achieved; the enzyme systems in the complex enzyme are all from malt, are homologous with malt raw materials used in beer, and do not introduce other foreign flavors.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A production method of a high-activity beer compound enzyme is characterized by comprising the following steps:

(5) blending: and (3) enabling the enzyme solution on the primary membrane and the enzyme solution on the secondary membrane to be mixed according to the enzyme activity ratio of the protease to the limit dextrinase (15-30): 1, mixing to obtain high-activity beer complex enzyme;

wherein the malt is barley malt;

in the step (1), the mass ratio of the malt flour to the water is 1: (3-5) after low-temperature leaching, adding diatomite into the leaching system, uniformly stirring, and filtering to obtain a clear enzyme solution;

adding 1-6 wt% of 300# diatomite and 0.3-2 wt% of 10# diatomite by mass into the leaching system obtained in the step (1);

adding diatomite into the enzyme liquid obtained after heat exchange in the step (3) and carrying out fine filtration to obtain clear enzyme liquid;

before blending in the step (5), adding 8-15 wt% of sodium chloride, 20-30 wt% of sorbitol, 0.05-0.3 wt% of sodium benzoate and 0.05-0.1 wt% of sodium methylparaben into the primary membrane enzyme solution and the secondary membrane enzyme solution respectively for stability treatment.

2. The production method according to claim 1, wherein the filtration is carried out using a plate-and-frame filter, and the plate-and-frame is washed with water, and the washing liquid is collected and mixed with the enzyme solution obtained by the filtration.

3. Use of a production method according to claim 1 or 2 in the production of beer.