CN109266719B - Method for evaluating hydrolysis capacity of malt starch - Google Patents
Method for evaluating hydrolysis capacity of malt starch Download PDFInfo
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- CN109266719B CN109266719B CN201811204926.7A CN201811204926A CN109266719B CN 109266719 B CN109266719 B CN 109266719B CN 201811204926 A CN201811204926 A CN 201811204926A CN 109266719 B CN109266719 B CN 109266719B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
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- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
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- General Engineering & Computer Science (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention provides a method for evaluating the hydrolysis capacity of malt starch, belongs to the technical field of beer, and can solve the technical problem that the saccharification capacity evaluation index cannot accurately reflect the hydrolysis capacity of the malt starch. The technical scheme comprises the following steps: measuring the activity of alpha-amylase, beta-amylase and limit dextrin enzyme; establishing a relation between the alpha-amylase activity, the beta-amylase activity, the limit dextrin enzyme activity and the proportion of fermentable sugar through regression analysis, and establishing a wort fermentable sugar proportion formula; establishing a malt starch hydrolysis capacity coefficient calculation formula by combining the coefficients of alpha-amylase, beta-amylase and limit dextrinase in the wort fermentable sugar proportion formula; and judging the hydrolysis capacity of the malt starch by using the coefficient of the hydrolysis capacity of the malt starch and the activity of the limiting dextrinase. The method can more accurately evaluate the starch hydrolysis capacity of the malt so as to guide a brewer to adjust the formula and the process according to the real starch hydrolysis capacity, and improve the consistency of the product flavor.
Description
Technical Field
The invention belongs to the technical field of beer, and particularly relates to a method for evaluating the hydrolysis capacity of malt starch.
Background
Barley is the main raw material for brewing beer, various hydrolase activities are activated through malting, and water-insoluble substances in the malt are gradually hydrolyzed into micromolecular water-soluble nutrient substances in the saccharification process for yeast fermentation to generate alcohol and various flavor substances. In order to increase the efficiency of malt utilization, 60% of the barley is starch and the alcohol in beer is fermented from sugar, it is necessary to hydrolyze as much starch as possible completely into fermentable sugars (maltose, glucose, maltotriose, fructose and sucrose) that can be absorbed and utilized by yeast and non-fermentable sugar dextrin that does not cause iodine stain. Therefore, starch hydrolysis ability is one of the important indicators for evaluating malt quality.
As agricultural products, malt is easily fluctuated by the influence of years, planting areas, climate and varieties, the physiological state of yeast and the brewing performance in the fermentation process and the flavor consistency of final products are directly influenced, and the variety and the quality of the malt also influence the utilization rate of raw materials and determine the economy of the brewing process. Therefore, an accurate method for evaluating the hydrolysis capacity of the malt starch is established, and a brewing engineer can be better guided to adjust the process and the formula so as to ensure the consistency of the components of the wort and the stability of the flavor of the product. At present, the saccharifying power of malt is taken as an evaluation index of the hydrolyzing capability of malt starch, but more and more researches show that the hydrolyzing degrees of the starch of the malt with the same saccharifying power are different, the contents of generated fermentable sugars are inconsistent, and the hydrolyzing capability of the malt starch cannot be accurately reflected.
Disclosure of Invention
The invention provides an evaluation method capable of accurately evaluating the hydrolysis capacity of malt starch aiming at the technical problem that the saccharification ability evaluation index cannot accurately reflect the hydrolysis capacity of the malt starch.
In order to achieve the above object, the present invention provides a method for evaluating the hydrolysis ability of malt starch, comprising the steps of:
measuring the activity of alpha-amylase, beta-amylase and limit dextrin enzyme;
establishing a relation between the alpha-amylase activity, the beta-amylase activity, the limit dextrin enzyme activity and the proportion of fermentable sugar through regression analysis, and establishing a wort fermentable sugar proportion formula;
establishing a malt starch hydrolysis capacity coefficient calculation formula by combining the coefficients of alpha-amylase, beta-amylase and limit dextrinase in the wort fermentable sugar proportion formula;
and judging the hydrolysis capacity of the malt starch by using the coefficient of the hydrolysis capacity of the malt starch and the activity of the limiting dextrinase.
Preferably, the alpha-amylase activity, the beta-amylase activity and the limit dextrin enzyme activity are obtained by the following methods:
respectively extracting alpha-amylase, beta-amylase and limit dextrinase crude extract by using alpha-amylase, beta-amylase and limit dextrinase extraction buffer;
respectively adding an alpha-amylase substrate, a beta-amylase substrate and a limit dextrinase substrate into the alpha-amylase, beta-amylase and limit dextrinase crude extract, and respectively adding an alpha-amylase termination solution, a beta-amylase termination solution and a limit dextrinase termination solution to terminate the reaction after the reaction;
and respectively measuring the absorbance values of the mixed solutions after the reaction is terminated to obtain the activities of the alpha-amylase, the beta-amylase and the limit dextrinase.
Preferably, the formula for establishing the proportion of fermentable sugars in wort is as follows:
fermentable sugar ratio (%) (69.2 + 0.0101) limit dextrin enzyme activity +0.00347 · α -amylase activity +0.000286 · β -amylase activity).
Preferably, the calculation formula of the malt starch hydrolysis capacity coefficient is as follows:
the malt starch hydrolysis ability coefficient (1.01 × + limit dextrinase activity +0.347 ×. alpha-amylase activity +0.0286 ×. beta-amylase activity)/100.
Preferably, the method for judging the hydrolysis capacity of the malt starch by using the coefficient of the hydrolysis capacity of the malt starch and the limiting dextrinase activity comprises the following steps:
when the malt starch hydrolysis capacity coefficient is less than or equal to 3 and the limit dextrinase activity is less than or equal to 200mU/g, the proportion of fermentable sugar is less than 72 percent, and the malt has low fermentation degree and poor hydrolysis effect when used;
when the malt starch hydrolysis capacity coefficient is more than or equal to 4.5 and the limit dextrinase activity is more than or equal to 370mU/g, the proportion of fermentable sugar is more than 74 percent, the fermentation degree is high when the malt is used, and the hydrolysis effect is good;
when the hydrolysis capacity coefficient of the malt starch is 3-4.5 and the limit dextrin enzyme activity is 200-.
Compared with the prior art, the method has the advantages and positive effects that the starch hydrolysis capability of the malt can be more accurately evaluated by establishing the malt starch hydrolysis capability evaluation method taking the activities of three amylases as the core, so that a brewer can be guided to adjust the formula and the process according to the real starch hydrolysis capability, and the consistency of the product flavor is improved.
Drawings
FIG. 1 is a schematic plot of the fermentable sugar ratio of wort versus the limiting dextrinase activity according to an embodiment of the present invention;
FIG. 2 is a schematic view showing the relationship between the ratio of fermentable sugars and the activity of alpha-amylase in wort according to an embodiment of the present invention;
FIG. 3 is a schematic scatter plot of the relationship between the fermentable sugar ratio of wort and beta-amylase activity according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides a method for evaluating the hydrolysis capacity of malt starch, which comprises the following steps:
s1: and (3) measuring the activity of the alpha-amylase, the activity of the beta-amylase and the activity of the limit dextrin enzyme of the malt.
In the step, the activity of the malt alpha-amylase, the activity of the malt beta-amylase and the activity of the ultimate dextrinase are determined, because the alpha-amylase, the beta-amylase and the ultimate dextrinase are three main enzymes which influence the proportion of fermentable sugars of wort, and the three enzymes synergistically hydrolyze starch. Wherein 70% of starch in the malt is amylopectin, the limit dextrinase is the amylase which has the lowest content in the malt and is only used for decomposing alpha-1, 6-glycosidic bonds, and the alpha-amylase and the beta-amylase can be continuously hydrolyzed only after the limit dextrinase hydrolyzes the alpha-1, 6-glycosidic bonds of the starch, so the limit dextrinase is the rate-limiting enzyme of starch hydrolysis; alpha-amylase randomly cleaves alpha-1, 4-glycosidic bonds, providing a substrate for further hydrolysis by the limiting dextrinase and beta-amylase; beta-amylase is the most abundant enzyme that cleaves alpha-1, 4-glucosidic bonds to produce maltose. Therefore, the proper proportion of the three enzymes is the key for ensuring the complete hydrolysis of the starch.
S2: establishing the relationship between the alpha-amylase activity, the beta-amylase activity, the limit dextrin enzyme activity and the proportion of the fermentable sugar through regression analysis, and establishing a wort fermentable sugar proportion formula.
In the step, a formula of the proportion of fermentable sugars in the wort is established, because in order to ensure the sufficient nutrient substances of the yeast and the taste of the beer in the fermentation process, on one hand, enough fermentable sugars which can be absorbed and utilized by the yeast need to be ensured, and on the other hand, a part of non-fermentable sugars (polysaccharides which are greater than or equal to tetrasaccharide) which cannot be absorbed and utilized by the yeast also need to be remained in the beer, so that the taste of the beer is ensured. Therefore, depending on the kind of yeast and beer, the fermentation degree of wort, i.e., the ratio of fermentable sugars, must be controlled during production. The alpha-amylase, the beta-amylase and the limit dextrinase are three main enzymes which influence the proportion of fermentable sugar in the wort, and a regression equation formula of the proportion of the fermentable sugar in the wort and the three amylase activities is established by establishing the relationship among the alpha-amylase activity, the beta-amylase activity, the limit dextrinase activity and the proportion of the fermentable sugar, so that the aim of predicting the proportion of the fermentable sugar in the wort according to the alpha-amylase activity, the beta-amylase activity and the limit dextrinase activity is fulfilled, and a basis is provided for further establishing a method for accurately evaluating the starch hydrolysis capacity.
S3: and establishing a malt starch hydrolysis capacity coefficient calculation formula by combining the coefficients of alpha-amylase, beta-amylase and limit dextrinase in the wort fermentable sugar ratio formula.
In the step, a calculation formula of the malt starch hydrolysis capacity coefficient is established through the coefficients of alpha-amylase, beta-amylase and limit dextrinase in a wort fermentable sugar proportion formula, the reason is that the wort fermentable sugar proportion is an important parameter for reflecting the malt starch hydrolysis capacity, and the fermentable sugar in the wort is mainly obtained by gradually hydrolyzing starch in malt under the synergistic action of an amylase system. The starch hydrolyzing enzymes in malt mainly comprise alpha-amylase, beta-amylase and limit dextrinase, and the content and the proportion of the three enzymes in the malt are different, so that the proportion of fermentable sugar in the wort is different. Thus, all three enzymes affect the degree of starch hydrolysis, which is related to the starch hydrolyzing ability of malt, but in different degrees. By establishing a regression equation of the ratio of the three enzyme activities to the fermentable sugar, the influence degree and the importance of the three enzymes on the ratio of the fermentable sugar can be seen in the coefficient of the regression equation.
S4: and judging the hydrolysis capacity of the malt starch by using the coefficient of the hydrolysis capacity of the malt starch and the activity of the limiting dextrinase.
By analyzing the activity of a large number of malt amylase systems of different varieties, producing areas, years and malting processes, a scatter diagram of the ratio of the three amylase activities to the fermentable sugar of the wort is drawn, as shown in fig. 1, 2 and 3, the scatter diagram shows that the correlation between the ratio of the limiting dextrinase and the fermentable sugar is strongest, the correlation between the limiting dextrinase and the fermentable sugar is weak, and the correlation between the limiting dextrinase and the fermentable sugar is weak. In the step, the hydrolysis capacity of the malt starch is judged by utilizing the hydrolysis capacity coefficient of the malt starch and the activity of the limit dextrinase, and the method has the advantages that the saccharification force is mainly taken as an index for evaluating the hydrolysis capacity of the starch in the conventional index at present, but the saccharification force mainly reflects the activity of the beta-amylase in the malt, and the correlation with the limit dextrinase and the alpha-amylase is weak. However, in practice, the fermentable sugars of wort are affected most by the limiting dextrinase enzyme, and secondly by the alpha-amylase, which has the least effect. Therefore, in the actual production, there was no difference in the saccharifying ability of malt, but the starch hydrolyzing ability of malt was very different (as in example 2). Therefore, the present invention can effectively reflect the actual starch hydrolyzing ability of malt.
In a preferred embodiment, the α -amylase activity, β -amylase activity and limit dextrin enzyme activity are obtained by:
respectively extracting alpha-amylase, beta-amylase and limit dextrinase crude extract by using alpha-amylase, beta-amylase and limit dextrinase extraction buffer;
respectively adding an alpha-amylase substrate, a beta-amylase substrate and a limit dextrinase substrate into the alpha-amylase, beta-amylase and limit dextrinase crude extract, and respectively adding an alpha-amylase termination solution, a beta-amylase termination solution and a limit dextrinase termination solution to terminate the reaction after the reaction;
and respectively measuring the absorbance values of the mixed solutions after the reaction is terminated to obtain the activities of the alpha-amylase, the beta-amylase and the limit dextrinase.
In this embodiment, the methods for measuring the α -amylase activity, the β -amylase activity, and the limit dextrin enzyme activity are specifically defined, and it is understood that the methods for measuring the α -amylase activity, the β -amylase activity, and the limit dextrin enzyme activity in the embodiments are not limited to those listed in the above embodiments, and may be other methods reasonably selected and adjusted in the art by those skilled in the art according to common knowledge.
In a preferred embodiment, the formula for establishing the fermentable sugar ratio of wort is as follows:
fermentable sugar ratio (%) (69.2 + 0.0101) limit dextrin enzyme activity +0.00347 · α -amylase activity +0.000286 · β -amylase activity).
In this example, a regression equation of wort fermentable sugar ratio and three enzyme activities can be established in combination with the measured α -amylase activity, β -amylase activity, and limiting dextrinase activity, and the fermentable sugar ratio of wort. Meanwhile, the coefficients of three different amylases in the calculation formula of the ratio of the fermentable sugars of the wort can reflect the influence degree of the three amylases on the ratio of the fermentable sugars of the wort.
In a preferred embodiment, the calculation formula of the maltostarch hydrolysis capacity coefficient is as follows:
the malt starch hydrolysis ability coefficient (1.01 × + limit dextrinase activity +0.347 ×. alpha-amylase activity +0.0286 ×. beta-amylase activity)/100.
In this embodiment, a calculation formula of the hydrolysis capacity coefficient of the malt starch is established by combining the coefficients of the alpha-amylase, the beta-amylase and the limit dextrinase in the formula of the fermentable sugar ratio of the malt juice, it should be noted that the coefficients of the alpha-amylase, the beta-amylase and the limit dextrinase in the formula of the fermentable sugar ratio of the malt juice reflect the degree of influence of the three amylases on the ratio of the fermentable sugar of the malt juice, the ratio of the fermentable sugar of the malt juice is an important parameter reflecting the hydrolysis capacity of the malt starch, and the calculation formula of the hydrolysis capacity coefficient of the malt starch is established by using the coefficients of the alpha-amylase, the beta-amylase and the limit dextrinase in the formula of the fermentable sugar ratio, so that the hydrolysis capacity coefficient of the malt starch can be more accurately calculated by using the activities of the three amylases.
In a preferred embodiment, the method for determining the hydrolysis capacity of the malt starch by using the coefficient of the hydrolysis capacity of the malt starch and the limiting dextrinase activity comprises the following specific steps:
when the hydrolysis capacity coefficient of the malt starch is less than or equal to 3 and the limit dextrinase activity is less than or equal to 200mU/g, the proportion of fermentable sugar is less than 72 percent, the malt has low fermentation degree and poor hydrolysis effect when being used;
when the malt starch hydrolysis capacity coefficient is more than or equal to 4.5 and the limit dextrinase activity is more than or equal to 370mU/g, the proportion of fermentable sugar is more than 74 percent, the fermentation degree is high when the malt is used, and the hydrolysis effect is good;
when the hydrolysis capacity coefficient of the malt starch is 3-4.5 and the limit dextrin enzyme activity is 200-.
In the embodiment, a method for judging the hydrolysis capacity of the malt starch is specifically provided, and it should be noted that the ratio of fermentable sugars is obtained by combining the coefficient of the hydrolysis capacity of the malt starch and the activity of the limiting dextrinase, so that the hydrolysis capacity of the starch is judged. In addition, the judgment method of the embodiment is obtained by counting the amylase system, the starch hydrolysis capacity and the ratio of fermentable sugars of the wort of more than 60 batches of different malts, so that the accuracy of the judgment method is ensured.
In order to more clearly describe the method for evaluating the hydrolysis ability of malt starch provided in the examples of the present invention in detail, the following description will be given with reference to the specific examples.
Example 1
Relationship between different varieties, different batches of maltogenic amylase systems and proportion of fermentable sugars
1. Sample source: 55 malts of different factories and different varieties
2. Malt Amylase Activity analysis
1) 300mg (. + -. 25mg) of malt powder was weighed into a 10mL EP tube and the exact powder mass was recorded. Weighing 2 parts of each sample, one part for measuring alpha/beta-amylase activity (mass M1) and one part for measuring limit dextrinase activity (mass M2);
2) 5mL of an alpha/beta-amylase extraction buffer (100mM maleic acid, pH 5.5) was added to 1 part, and a limiting dextrinase extraction buffer (100mM maleic acid, 25mM DTT, pH 5.5) was added to 1 part, followed by mixing, placing in a metal water bath, and shaking at 20 ℃ for 16 hours. Centrifuging at 12000rpm for 10min, and collecting supernatant as alpha/beta-amylase extractive solution and limit dextrinase extractive solution.
3) The crude alpha/beta-amylase extract was diluted 250-fold with alpha-amylase dilution buffer (50mM malic acid, 3.5g/L sodium hydroxide, 3g/L sodium chloride, 2.95g calcium chloride, pH 5.4). Adding 50 mu L of the alpha-amylase substrate into a 2mL centrifuge tube, carrying out warm bath at 40 ℃ for 2min, adding 50 mu L of the alpha-amylase substrate (Megazyme), reacting at 40 ℃ for 10min, adding 750 mu L of alpha-amylase termination solution (1% trisodium phosphate, pH 11), uniformly mixing, and measuring the absorbance at 405nm (OD405), namely alpha-amylase O.D.
α -amylase activity (U/g absolute malt) ═ α -amylase o.d. 17 dilution factor 100 × 5000/181/(100-moisture)/M1.
4) The crude alpha/beta-amylase extract was diluted 25-fold with beta-amylase dilution buffer (11.6g/L maleic acid, 0.37g/L EDTA, 1g/L BSA, pH 6.2). Adding 50 mu L of the mixture into a 2mL centrifuge tube, carrying out warm bath at 40 ℃ for 2min, adding 50 mu L of beta-amylase substrate, reacting at 40 ℃ for 10min, adding 750 mu L of beta-amylase termination solution (1% Trizma), uniformly mixing, and measuring the light absorption value (OD405) at 405nm to obtain the beta-amylase O.D.
Beta-amylase activity (U/g absolute malt) ═ beta-amylase o.d. 17 dilution factor 5860 × 5000/181/(100-moisture)/M1.
5) Putting 11.5-12.5mg into a 2mL centrifuge tube, carrying out water bath at 40 ℃ for 2min, accurately taking 100 mu L of limit dextrinase crude extract, adding the limit dextrinase crude extract into the centrifuge tube filled with a substrate, carrying out accurate reaction at 40 ℃ for 10min, adding 1mL of limit dextrinase stop solution (1% Trizma), carrying out normal-temperature centrifugation at 12000rpm for 40min, sucking out the supernatant, and measuring the absorbance value (OD595) at 595nm to obtain the limit dextrinase O.D.
Limit dextrinase (mU/g absolute malt) 11.2 limit dextrinase o.d. 500000 2 5/(100-moisture)/M2.
3. Analysis of proportion of fermentable sugar in wort
Protocol preparation method of wort: a50.0 g malt flour sample is accurately weighed into a saccharifying cup with known weight, 200mL water with 45 ℃ is added, and the temperature is kept for 30min in a water bath with 45 ℃ under continuous stirring. Heating the mash to 70 ℃ within 25min by heating the water bath at the speed of 1 ℃/min, adding 100mL of water at 70 ℃ into the cup, keeping the mash at 70 ℃ for 1h, and rapidly cooling to room temperature within 10-15 min. The stirrer was rinsed with water, the outer wall of the mashing cup was wiped dry, and water was added to accurately weigh 450.0g of its contents. The mash was agitated with a glass rod and filtered through a medium speed filter paper, and about 100mL of the initially collected filtrate was returned to the refilter and the filtrate was collected in a dry beaker.
Sugar spectrum analysis: and (3) separating and quantitatively detecting the contents of glucose, maltose, maltotriose, maltotetraose and the above sugars in the wort by adopting size exclusion chromatography and combining an ion exchange mode.
The fermentable sugar ratio is (monosaccharide + disaccharide + trisaccharide) × 100/(monosaccharide + disaccharide + trisaccharide + tetrasaccharide and above).
4. Relationship and regression equation of activity of fermentable sugar and amylase in wort
Regression analysis is carried out on the ratio of the alpha-amylase activity, the beta-amylase activity and the limit dextrin enzyme activity obtained by the method to the fermentable sugar in the wort, and a regression equation is obtained by the regression analysis as follows:
fermentable sugar ratio (%) (69.2 + 0.0101) limit dextrin enzyme activity +0.00347 · α -amylase activity +0.000286 · β -amylase activity
5. Hydrolysis coefficient of malt starch
The principle of starch hydrolysis capacity coefficient is formulated as follows: and (3) taking the coefficient of the proportion of each amylase to the fermentable sugar in the regression equation as the influence degree of each amylase on the starch hydrolysis capacity. Thus, the malt starch hydrolysis capacity factor (1.01 × (limit) dextrinase +0.347 × (α -amylase + 0.0286) × (β -amylase)/100
Standard formulation principle: when the proportion of fermentable sugar is less than 72 percent, the hydrolysis capacity coefficients of the maltostarch are all less than or equal to 3, and the activity of the limit dextrin enzyme is less than or equal to 200 mU/g; when the proportion of fermentable sugar is more than or equal to 74 percent, the hydrolysis capacity coefficients of the malt starch are all more than or equal to 4.5, and the activity of the limiting dextrinase is more than or equal to 370 mU/g.
Example 2
Evaluation of hydrolysis Capacity of malt starch of the same variety and different manufacturers
1. Sample source: malt samples from different suppliers of the same cultivar, Canadian malt Copeland.
2. Conventional index of starch hydrolysis capacity: analysis of glycation power
A20.0 g sample of fine malt was weighed into a mashing cup of known weight for use. 450-. Cooling to 20 + -0.5 deg.C, adding 20 + -0.5 deg.C distilled water to make the content weight of the saccharifying cup reach 520.0g, and filtering with double-layer medium speed filter paper.
100mL of starch solution is respectively added into a 200mL volumetric flask, then 5mL of acetic acid-sodium acetate buffer solution is added, and the mixture is placed into a water bath with the temperature of 20 +/-0.1 ℃ for heat preservation for 20 min. Adding 5mL of malt extract, keeping the temperature in a water bath at 20 +/-0.1 ℃ for 30min, adding 4mL of sodium hydroxide solution, shaking up, fixing the volume to the scale with distilled water, and shaking up. 2.35mL of sodium hydroxide solution is firstly added into the blank sample, then 5.00mL of malt extract is added, the mixture is shaken up, and the volume is determined to the scale by distilled water.
The solution in each 50mL volumetric flask was pipetted into 4 250mL iodine vials, and 3mL sodium hydroxide solution and 25mL iodine standard solution were added, respectively, and the vials were put in the dark with a lid for 15 min. After the dark reaction was complete, 4.5mL of sulfuric acid solution was added to each vial and immediately titrated with sodium thiosulfate standard solution until the blue color disappeared. The glycation power was calculated according to the following formula.
Wherein D is the saccharifying power of 100g of anhydrous malt, WK;
V1-mL consumption of sodium thiosulfate standard solution by blank titration;
v2-sample titration consumed milliliters, mL, of standard solution of sodium thiosulfate;
concentration of N-sodium thiosulfate standard solution, mol/L;
wc-mass fraction of sample water,%;
342-20 g of malt sample, 0.171X 200/50X 500/5X 100/20, where 0.171 is the mass of maltose, g, equivalent to 1mL of 0.05mol/L iodine solution.
3. Malt amylase series analysis
1) 300mg (. + -. 25mg) of malt powder was weighed into a 10mL EP tube and the exact powder mass was recorded. Weighing 2 parts of each sample, one part for measuring alpha/beta-amylase activity (mass M1) and one part for measuring limit dextrinase activity (mass M2);
2) 5mL of an alpha/beta-amylase extraction buffer (100mM maleic acid, pH 5.5) was added to 1 part, and a limiting dextrinase extraction buffer (100mM maleic acid, 25mM DTT, pH 5.5) was added to 1 part, followed by mixing, placing in a metal water bath, and shaking at 20 ℃ for 16 hours. Centrifuging at 12000rpm for 10min, and collecting supernatant as alpha/beta-amylase extractive solution and limit dextrinase extractive solution.
3) The crude alpha/beta-amylase extract was diluted 250-fold with alpha-amylase dilution buffer (50mM malic acid, 3.5g/L sodium hydroxide, 3g/L sodium chloride, 2.95g calcium chloride, pH 5.4). Adding 50 mu L of the alpha-amylase substrate into a 2mL centrifuge tube, carrying out warm bath at 40 ℃ for 2min, adding 50 mu L of the alpha-amylase substrate (Megazyme), reacting at 40 ℃ for 10min, adding 750 mu L of alpha-amylase termination solution (1% trisodium phosphate, pH 11), uniformly mixing, and measuring the absorbance at 405nm (OD405), namely alpha-amylase O.D.
α -amylase activity (U/g absolute malt) ═ α -amylase o.d. 17 dilution factor 100 × 5000/181/(100-moisture)/M1.
4) The crude alpha/beta-amylase extract was diluted 25-fold with beta-amylase dilution buffer (11.6g/L maleic acid, 0.37g/L EDTA, 1g/L BSA, pH 6.2). Adding 50 mu L of the mixture into a 2mL centrifuge tube, carrying out warm bath at 40 ℃ for 2min, adding 50 mu L of beta-amylase substrate, reacting at 40 ℃ for 10min, adding 750 mu L of beta-amylase termination solution (1% Trizma), uniformly mixing, and measuring the light absorption value (OD405) at 405nm to obtain the beta-amylase O.D.
Beta-amylase activity (U/g absolute malt) ═ beta-amylase o.d. 17 dilution factor 5860 × 5000/181/(100-moisture)/M1.
5) Putting 11.5-12.5mg into a 2mL centrifuge tube, carrying out water bath at 40 ℃ for 2min, accurately taking 100 mu L of limit dextrinase crude extract, adding the limit dextrinase crude extract into the centrifuge tube filled with a substrate, carrying out accurate reaction at 40 ℃ for 10min, adding 1mL of limit dextrinase stop solution (1% Trizma), carrying out normal-temperature centrifugation at 12000rpm for 40min, sucking out the supernatant, and measuring the absorbance value (OD595) at 595nm to obtain the limit dextrinase O.D.
Limit dextrinase (mU/g absolute malt) 11.2 limit dextrinase o.d. 500000 2 5/(100-moisture)/M2
4. Analysis of proportion of fermentable sugar in wort
Protocol preparation method of wort: a50.0 g malt flour sample is accurately weighed into a saccharifying cup with known weight, 200mL water with 45 ℃ is added, and the temperature is kept for 30min in a water bath with 45 ℃ under continuous stirring. Heating the mash to 70 ℃ within 25min by heating the water bath at the speed of 1 ℃/min, adding 100mL of water at 70 ℃ into the cup, keeping the mash at 70 ℃ for 1h, and rapidly cooling to room temperature within 10-15 min. The stirrer was rinsed with water, the outer wall of the mashing cup was wiped dry, and water was added to accurately weigh 450.0g of its contents. The mash was agitated with a glass rod and filtered through a medium speed filter paper, and about 100mL of the initially collected filtrate was returned to the refilter and the filtrate was collected in a dry beaker.
Sugar spectrum analysis: and (3) separating and quantitatively detecting the contents of glucose, maltose, maltotriose, maltotetraose and the above sugars in the wort by adopting size exclusion chromatography and combining an ion exchange mode.
Fermentable sugar ratio (monosaccharide + disaccharide + trisaccharide) 100/(monosaccharide + disaccharide + trisaccharide + tetrasaccharide and above)
5. Analysis of malt plant use Effect
The malt to be analyzed is taken, and mass production and material feeding brewing are carried out according to the same formula and process. In the mass production, the malt is 50-100% in proportion, the material and water are crushed and then the crushed material and water are put into the mixture according to the material-water ratio of 1 to (2-5) for saccharification, the saccharification temperature is 60-68 ℃, the saccharification time is 40-80min, the wort is filtered after the saccharification is finished, the same amount of hop is added into the wort for boiling for 30-80min, and the wort is cooled after the boiling is finished, so that the required wort is obtained.
Analysis of fermentation degree of cold wort: 100mL of wort sample is weighed, boiled in water for 10min, transferred to a 500mL triangular flask, added with 20.0g of yeast cake with water absorbed by filter paper, plugged with a cotton plug, placed in a shaking table, at 18 ℃, at 150rpm, and shaken for 16 h. After the fermentation is finished, the fermentation liquor is filtered by double-layer medium-speed filter paper, and the filtered fermentation liquor is used for measuring the ultimate fermentation degree by Anto Parr.
6. Malt Performance evaluation
The data of the performance test are shown in table 1, and according to the results in the table, it can be found that:
1) from the conventional index of the malt, the saccharifying power is more than 300 for 7 malts, and the starch hydrolyzing capability is consistent.
2) According to the starch hydrolysis capacity coefficient and the judgment standard of the ultimate dextrin enzyme activity: no. 1 malt is low starch hydrolyzing ability malt, No. 2-5 malt is medium starch hydrolyzing ability malt; no. 6-7 shows high starch hydrolyzing ability. As a result of sugar spectrum analysis, the proportion of fermentable sugar in No. 1 malt is less than 72%, that of No. 2 malt is 73-74%, and that of No. 5 malt is more than 74%. The starch hydrolysis ability is consistent with the result of sugar spectrum analysis.
3) From the actual plant performance: the fermentation degree of No. 1 malt is low, the fermentation degree of No. 2-5 malt is moderate, and the fermentation degree of No. 6-7 malt is high. In accordance with the new evaluation criteria for starch hydrolyzing ability.
TABLE 1 malt Performance test results
According to the new evaluation method, the brewer can adjust the formula and process according to the real starch hydrolysis capability. For example, the No. 1 malt needs to be matched with the malt with high starch hydrolysis capability, so that the saccharification time is prolonged, and the saccharification temperature is adjusted; no. 6-7 malt can shorten saccharification time and is used together with malt with low starch hydrolysis capability. The evaluation measures powerfully reduce the instability of sugar spectrum and fermentation degree caused by malt fluctuation and improve the consistency of product flavor.
Claims (2)
1. The method for evaluating the hydrolysis capacity of malt starch is characterized by comprising the following steps:
measuring the activity of alpha-amylase, beta-amylase and limit dextrin enzyme;
establishing a relation between the alpha-amylase activity, the beta-amylase activity, the limit dextrin enzyme activity and the proportion of fermentable sugar through regression analysis, and establishing a wort fermentable sugar proportion formula;
establishing a malt starch hydrolysis capacity coefficient calculation formula by combining the coefficients of alpha-amylase, beta-amylase and limit dextrinase in the wort fermentable sugar proportion formula;
the hydrolysis capacity of the malt starch is judged by utilizing the hydrolysis capacity coefficient of the malt starch and the activity of the limiting dextrinase, and the method specifically comprises the following steps:
when the malt starch hydrolysis capacity coefficient is less than or equal to 3 and the limit dextrinase activity is less than or equal to 200mU/g, the proportion of fermentable sugar is less than 72 percent, and the malt has low fermentation degree and poor hydrolysis effect when used;
when the malt starch hydrolysis capacity coefficient is more than or equal to 4.5 and the limit dextrinase activity is more than or equal to 370mU/g, the proportion of fermentable sugar is more than 74 percent, the fermentation degree is high when the malt is used, and the hydrolysis effect is good;
when the hydrolysis capacity coefficient of the malt starch is 3-4.5 and the limit dextrin enzyme activity is 200-;
wherein, the established formula of the proportion of the fermentable sugars of the wort is as follows:
fermentable sugar ratio (%) =69.2+ 0.0101: +0.00347 · α -amylase activity +0.000286 · β -amylase activity;
the established calculation formula of the malt starch hydrolysis capacity coefficient is as follows:
malt starch hydrolysis ability coefficient = (1.01:limitdextrinase activity + 0.347:α -amylase activity + 0.0286:β -amylase activity)/100.
2. The method of claim 1, wherein the α -amylase activity, β -amylase activity and limit dextrin enzyme activity are obtained by:
respectively extracting alpha-amylase, beta-amylase and limit dextrinase crude extract by using alpha-amylase, beta-amylase and limit dextrinase extraction buffer;
respectively adding an alpha-amylase substrate, a beta-amylase substrate and a limit dextrinase substrate into the alpha-amylase, beta-amylase and limit dextrinase crude extract, and respectively adding an alpha-amylase termination solution, a beta-amylase termination solution and a limit dextrinase termination solution to terminate the reaction after the reaction;
and respectively measuring the absorbance values of the mixed solutions after the reaction is terminated to obtain the activities of the alpha-amylase, the beta-amylase and the limit dextrinase.
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