CN114836516A - Method for rapidly detecting titer of non-starch polysaccharide enzyme for feed - Google Patents
Method for rapidly detecting titer of non-starch polysaccharide enzyme for feed Download PDFInfo
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- 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
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- 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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- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- G01N2333/928—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
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Abstract
The application relates to the field of quality evaluation of feed complex enzyme, and particularly discloses a method for quickly detecting titer of non-starch polysaccharide enzyme for feed, which comprises the following steps: s1, drawing a standard curve: firstly, drawing a standard curve of a glucose light absorption value and a glucose concentration; s2, feed de-starchy: mixing the feed with water according to the weight ratio of 1 (2-5), adding alpha-amylase to react for 3 hours, adding diastase to react for 4 hours, standing for 2-3 hours, centrifuging to obtain precipitate, and obtaining the feed with the precipitate removed and pulverized; s3, in vitro digestion stage: carrying out in-vitro enzymolysis digestion on the feed subjected to precipitation powder removal and an enzyme solution to be detected to obtain a sample to be detected, and detecting the absorbance of the sample to be detected and a blank sample; s4, determination of reducing sugar: and calculating the content of the reducing sugar according to the standard curve in the S1 and the increment of the reducing sugar according to a formula by using the absorbance measured in the S3, wherein the method has the advantage of improving the accuracy of the evaluation of the effect of the non-starch polysaccharase in the feed on the feed.
Description
Technical Field
The application relates to the field of quality evaluation of feed complex enzyme, in particular to a method for rapidly detecting titer of non-starch polysaccharide enzyme for feed.
Background
The enzyme preparation is a safe and feasible measure to supplement in the daily ration of livestock and poultry, the non-starch polysaccharide enzyme is more and more widely applied in the production of poultry, is one of the most main feeding enzyme preparations, and is added in various daily rations to eliminate the anti-nutritional effect of the non-starch polysaccharide. The non-starch polysaccharide enzyme is used as a feed additive, can hydrolyze non-starch polysaccharide in feed into reducing sugar containing aldehyde group, and is a potential available nutrient for animals, so that the nutritional value of feed raw materials can be obviously improved, and the feed additive has the effects of improving the feed conversion utilization rate, expanding the feed raw material application range, reducing the feed cost, improving the environment and the like.
For the analytical determination of non-starch polysaccharidase, at present, the non-starch polysaccharidase titer in the feed is evaluated in a laboratory by measuring the release amount of reducing sugar in the feed raw materials by using an in vitro enzymolysis method. When the composition of the product to be tested contains amylase, the amylase can also generate reducing sugar on the starch in the feed, so that the measurement result is interfered, and the accurate evaluation of the effect of the non-starch polysaccharidase on the feed is influenced.
Disclosure of Invention
In order to improve the accuracy of the evaluation of the effect of the non-starch polysaccharide enzyme for the feed on the feed, the application provides a method for rapidly detecting the titer of the non-starch polysaccharide enzyme for the feed.
The method for rapidly detecting the titer of the non-starch polysaccharide enzyme for the feed adopts the following technical scheme: a method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed comprises the following steps:
s1, drawing a standard curve: firstly, drawing a standard curve of glucose light absorption luminosity value and glucose concentration;
s2, feed de-starchy: mixing the feed with water according to the weight ratio of 1 (2-5), adding alpha-amylase to react for 3 hours, adding diastase to react for 4 hours, standing for 2-3 hours, centrifuging to obtain precipitate, and obtaining the feed with the precipitate removed and pulverized;
s3, in vitro digestion stage: carrying out in-vitro enzymolysis digestion on the feed subjected to precipitation powder removal and an enzyme solution to be detected to obtain a sample to be detected, and determining the absorbance of the sample to be detected and a blank sample;
s4, determination of reducing sugar: calculating the content of reducing sugar according to the absorbance measured in S3 and a standard curve in S1, and calculating the increment of the reducing sugar according to a formula;
calculating the formula: w ═ C × V/m-C 0 *V 0 /m 0
In the formula, C is the reducing sugar content of the sample to be detected, mg/mL;
v is the volume of the sample to be detected, mL;
m is the mass of the feed after starch removal and pulverization, g;
C 0 the content of reducing sugar in the blank sample is mg/mL;
V 0 volume of blank sample, mL;
m 0 feed mass for the blank sample, g.
By adopting the technical scheme, the alpha-amylase and the saccharifying enzyme are added into the mixed solution of the feed and the water, so that the starch in the feed can be fully hydrolyzed into the glucose and removed, the interference on the test determination caused by the hydrolysis of the starch in the feed into the glucose in the digestion process is reduced, and the accurate evaluation of the non-starch polysaccharase on the feed effect is improved; adding an enzyme solution to be detected into the feed subjected to starch removal, taking a blank sample as a control, respectively determining the content of reducing sugar according to a drawn standard curve, and calculating the reducing sugar increment of the feed subjected to starch removal compared with the blank sample according to a formula, thereby obtaining the effect of the non-starch polysaccharidase in the enzyme solution to be detected on the feed.
Preferably, the alpha-amylase in step S2 is a high temperature resistant alpha-amylase, and the reaction is performed for 1-2 hours after the alpha-amylase is added.
By adopting the technical scheme, the high-temperature-resistant alpha-amylase has better temperature resistance, improves the adaptability of the alpha-amylase, has strong stability and high efficiency under a high-temperature condition, and can better promote the hydrolysis of starch and improve the hydrolysis efficiency of the starch.
Preferably, in step S2, the feed with the weight ratio of 1 (2-5) is mixed with water, the pH of the mixed solution is adjusted to 5.0-6.0, then high temperature resistant alpha-amylase is added, the enzymolysis reaction is carried out for 1-2h at the temperature of 80-90 ℃, after the solution is cooled to room temperature, the pH of the solution after the enzymolysis by the high temperature resistant alpha-amylase is adjusted to 4.5-5.5, then diastase is added, the oscillation reaction is carried out for 4h at the temperature of 60-68 ℃, the solution is kept stand for 2-3h, and the precipitate is centrifuged to obtain the precipitate, so that the de-precipitated powdered feed is obtained.
By adopting the technical scheme, the pH is adjusted to 5.0-6.0 before the high-temperature resistant alpha-amylase is added so as to achieve the optimal pH of the high-temperature resistant alpha-amylase, and the mixed solution is subjected to enzymolysis reaction at 80-90 ℃ after the high-temperature resistant alpha-amylase is added, so that the enzyme activity of the high-temperature resistant alpha-amylase is improved, the hydrolysis efficiency of the high-temperature resistant alpha-amylase is improved, and the reaction time is shortened; before adding the saccharifying enzyme, the pH is adjusted to 4.5-5.5, and after adding the saccharifying enzyme, the reaction is carried out at 60-68 ℃ to adapt to the optimal reaction condition of the saccharifying enzyme, improve the hydrolysis efficiency of the saccharifying enzyme and further shorten the reaction time.
Preferably, in step S2, after the alpha-amylase is added, the high-voltage pulse electric field treatment is performed, and then the enzymolysis reaction is performed for 0.5-1.5h, and after the glucoamylase is added, the high-voltage pulse electric field treatment is performed, and then the oscillation reaction is performed for 2-3.5 h.
By adopting the technical scheme, the high-voltage pulse electric field treatment is respectively carried out on the alpha-amylase and the saccharifying enzyme, so that the enzymatic activities of the alpha-amylase and the saccharifying enzyme can be improved, the hydrolysis efficiency of the alpha-amylase and the saccharifying enzyme pair is improved, the efficiency of removing starch powder of the feed to be detected is improved, and the purpose of quickly detecting the feed is realized.
Preferably, in step S2, the mass ratio of the alpha-amylase to the feed is 1:900-1300, and the mass ratio of the saccharifying enzyme to the feed is 1: 150-250.
By adopting the technical scheme, the proper mass ratio of the alpha-amylase, the saccharifying enzyme and the feed is favorable for fully hydrolyzing the starch in the feed, and the accuracy of the titer of the non-starch polysaccharide enzyme for the feed to be detected is improved; when the mass ratio of the alpha-amylase to the feed exceeds 1:1300 or the mass ratio of the saccharifying enzyme to the feed exceeds 1:250, the alpha-amylase or the saccharifying enzyme is excessive, waste is caused, and cost is increased, and when the mass ratio of the alpha-amylase to the feed is lower than 1:900 or the mass ratio of the saccharifying enzyme to the feed is lower than 1:150, starch in the feed is not hydrolyzed sufficiently, so that the accuracy of measuring the titer of the non-starch polysaccharide enzyme is influenced.
Preferably, in step S2, after the precipitate is centrifuged, distilled water is added for centrifugation again, and the centrifugation with distilled water is repeated 3 to 5 times.
By adopting the technical scheme, the sediment is washed by distilled water and then centrifuged, and the reducing sugar hydrolyzed by high-temperature resistant alpha-amylase and glucoamylase in the feed to be tested can be sufficiently eluted after 3-5 times, so that the titer accuracy of the non-starch polysaccharide enzyme for the feed to be tested is further improved; and (3) repeatedly centrifuging for 3-5 times, such as 3 times, 4 times and 5 times, wherein too many times of centrifugation easily reduce the detection efficiency, and too few times of centrifugation easily cause insufficient elution of reducing sugar hydrolyzed by high temperature resistant alpha-amylase and saccharifying enzyme in the feed to be detected, thus affecting the accuracy of non-starch polysaccharide enzyme titer evaluation.
Preferably, in step S2, the de-precipitated feed is dried at 60-67 deg.C for 3-5h, cooled to room temperature, and then refrigerated at 0-4 deg.C.
By adopting the technical scheme, in order to keep the de-precipitated feed in a dry state and make the de-precipitated feed not easy to dilute, the de-precipitated feed is dried at 60-67 ℃ for 3-5h, such as 60 ℃ for 5h, 67 ℃ for 3h and 65 ℃ for 4h, so as to measure the data accuracy in the follow-up measurement. If the temperature is too high and the drying time is too long, the water content of the de-starchy feed is lost too much and the de-starchy feed is not easy to be dissolved subsequently.
Preferably, the in vitro digestion stage of step S3 includes the steps of:
a. simulating gastric digestion stage: respectively weighing 2-5g of two equal-mass feed samples without precipitation powder, adding one of the two equal-mass feed samples into 25-35mL of acetic acid-sodium acetate buffer solution, adding 1-3mL of enzyme solution to be detected to serve as a sample group to be detected, adding the other equal-mass feed sample into acetic acid-sodium acetate buffer solution to serve as a blank sample group, wherein the volume of the acetic acid-sodium acetate buffer solution in the blank sample group is the sum of the volumes of the acetic acid-sodium acetate buffer solution in the sample group to be detected and the enzyme solution to be detected, and placing the sample group to be detected and the blank sample group into a water bath shaker at 40-45 ℃ for incubation for 4-5 hours to obtain an incubation solution;
b. simulating the small intestine digestion stage: respectively and slowly adding 0.1-0.3mL of 5mol/L sodium hydroxide solution into the hatching solution prepared from the sample group to be detected and the blank sample group, adjusting the pH of the hatching solution to 7.0-8.0 by using the sodium hydroxide solution, respectively adding 1-3mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and putting the hatching solution into a water bath shaker at the temperature of 40-45 ℃ for digestion for 7-9h to obtain a digestive juice;
c. and (3) color development stage: standing the digestive juice prepared from the sample group to be detected and the blank sample group for 20-30min, respectively taking 0.1-0.2mL of the supernatant after standing, sequentially adding 0.5-0.8mL of acetic acid-sodium acetate buffer solution and 0.3-0.7mL of the LDNS reagent, heating in a boiling water bath for 3-5min, cooling to room temperature, respectively adding 1.0-2.0mL of deionized water, and carrying out color comparison at 540 nm.
By adopting the technical scheme, the feed without starch powder is subjected to the gastric-simulated digestion stage and the small intestine-simulated digestion stage, so that the measurement of the feed is closer to the process of animal digestion.
Preferably, in the gastric digestion stage, adding acetic acid-sodium acetate buffer solution, and preheating at 40-50 deg.C for 15-25 min.
By adopting the technical scheme, the process that the feed sample without starch powder enters the animal body before simulated gastric digestion is simulated by preheating, the detection deviation caused by inconsistent temperature before reaction is avoided, and the titer of the non-starch polysaccharide enzyme can be objectively reflected.
Based on the consideration of the environment and the process of digestion in the animal body, the preheating is carried out for 15-25min at 40-50 ℃ after adding the acetic acid-sodium acetate buffer solution, for example, the preheating is carried out for 15min at 50 ℃, 25min at 40 ℃ and 20min at 45 ℃, so that the digestion stage before the animal enters the stomach is simulated more objectively.
Preferably, the color development stage comprises standing, centrifuging the supernatant for 6-10min, and collecting the centrifuged supernatant 0.1-0.2 mL.
By adopting the technical scheme, the digestion solution is stood and then centrifuged for 6-10min, so that the reducing sugar obtained after hydrolysis of the non-starch polysaccharide enzyme can be fully dissolved in the supernatant, and the titer evaluation accuracy of the non-starch polysaccharide enzyme in the feed is improved. The centrifugation time is more than 10min, the detection efficiency is reduced, and when the centrifugation time is less than 6min, the reducing sugar obtained after hydrolysis of the non-starch polysaccharide enzyme is insufficiently separated from the supernatant, so that the evaluation accuracy of the titer of the non-starch polysaccharide enzyme is influenced.
Based on the appropriate selection of the amount of material to be tested, 0.1mL to 0.2mL, e.g., 0.1mL, 0.15mL, 0.2mL, of supernatant is taken after centrifugation, which is easily wasted if excess is aspirated.
In summary, the present application has the following beneficial effects:
1. by adding alpha-amylase and glucoamylase into the solution obtained by mixing the feed and water in advance, the starch in the feed can be fully hydrolyzed into glucose and removed, interference on test determination after the starch in the feed is hydrolyzed into glucose in the digestion process is reduced, and accurate evaluation of the effect of the non-starch polysaccharase on the feed is improved.
2. The method has simple operation process and accurate determination result, and is convenient for determining the enzymolysis effect of the non-starch polysaccharide enzyme in the feed in vitro.
3. By setting the gastric-simulated digestion stage and the small intestine-simulated digestion stage, the digestion environment of the feed in an animal body is simulated, and the titer of the non-starch polysaccharidase can be reflected more objectively.
Detailed Description
Preparation of reagents and materials
1. The enzyme activity of the high-temperature resistant alpha-amylase is 10000U/g;
2. the enzyme activity of the saccharifying enzyme is 50000U/g;
3. acetic acid-sodium acetate buffer: weighing 20.52g of anhydrous sodium acetate in a 1000mL beaker, adding 900mL of deionized water, stirring and dissolving, adjusting the pH of the solution to 5.50 by using glacial acetic acid, transferring the solution to a 1000mL volumetric flask, fixing the volume to the scale, and storing the solution at room temperature for 2 months to be effective;
4. disodium hydrogen phosphate-sodium dihydrogen phosphate buffer: separately weighing disodium hydrogen phosphate (Na) 2 HPO 4 ·12H 2 O)60.2g and sodium dihydrogen phosphate (NaH) 2 PO 4 ·2H 2 O)5.0g, adding water to dissolve and fixing the volume to 1000mL, and shaking up;
5.5 mol/L sodium hydroxide solution: weighing 20.0g of sodium hydroxide, adding distilled water for dissolving, fixing the volume to 100ml, and shaking up;
6. DNS reagent, 3.15g (chemical purity) of 3, 5-dinitrosalicylic acid is weighed, 500ml of distilled water is added, the mixture is stirred for 5s, the temperature of the mixture is increased to 45 ℃, then 100ml of 5mol/L sodium hydroxide solution is gradually added, the mixture is continuously stirred until the solution is clear and transparent (note that the temperature of the solution does not exceed 48 ℃ in the process of adding the sodium hydroxide), and 91.0g of potassium sodium tartrate tetrahydrate, 2.50g of phenol and 2.50g of anhydrous sodium sulfite are gradually added; the water bath heating at 45 ℃ was continued while adding 300ml of distilled water, and the stirring was continued until the added substances were completely dissolved. Stopping heating in the water bath, cooling to room temperature, and adding distilled water to a constant volume of 1000 ml; filtering with sintered glass filter, collecting filtrate, storing in brown bottle, storing in dark place, storing at room temperature for 7 days, and allowing use for 6 months;
7. enzyme solution to be tested
The non-starch polysaccharidase mixture [ xylanase (10000U/g) + glucanase (2000U/g) +100g cellulase (500U/g) + mannanase (1000U/g) + amylase 500U/g), the addition amount is 100 g/ton feed to be detected ], hereinafter referred to as NSP enzyme;
a mixture of non-starch polysaccharidase and amylase [ xylanase (10000U/g) + glucanase (2000U/g) + cellulase (500U/g) + mannanase (1000U/g), the addition amount is 100 g/ton of feed to be detected ], which is hereinafter referred to as NSP enzyme + amylase;
according to the addition amount of the feed to be detected in different embodiments, NSP enzyme or NSP enzyme + amylase with corresponding weight is weighed, the weight ratio of the NSP enzyme or NSP enzyme + amylase to the feed to be detected is 1:10000, 100mL of acetic acid-sodium acetate buffer solution is added into the NSP enzyme or NSP enzyme + amylase for dilution, and the mixture is magnetically stirred for 45min to obtain the enzyme solution to be detected.
Drawing a glucose standard curve
Weighing 1.000 g of anhydrous glucose, adding distilled water to dissolve and fix the volume to 100mL, and preparing a glucose stock solution with the concentration of 10.0 mg/mL;
respectively sucking 0.0mL (standard blank sample), 1.0mL, 2.0mL, 5.0mL, 10.0mL and 20.0mL of glucose stock solution, adding distilled water for diluting to constant volume of 100mL, and preparing glucose standard solution with concentration of 0.1-2.0 mg/mL;
respectively sucking 0.1mL of the glucose standard solution (making two parallel solutions), respectively adding 0.7mL of acetic acid-sodium acetate buffer solution and 0.4mL of DNS reagent, oscillating for 3-5s, heating in a boiling water bath for 5min, cooling to room temperature by using tap water, adding 1.3mL of deionized water, adjusting to zero by using a standard blank sample as a control, and measuring an absorbance OD value at 540 nm;
and drawing a standard curve (Y ═ aX + b) by taking the glucose concentration as an axis Y and the absorbance OD value as an axis X, wherein the standard curve needs to be drawn again each time the DNS reagent is newly prepared.
Examples
Example 1
The embodiment of the application discloses a method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed, which comprises the following steps:
s1, drawing a standard curve: firstly, drawing a standard curve of glucose light absorption luminosity value and glucose concentration;
s2, feed de-starchy: grinding the feed, sieving with a sieve with an aperture of 500 mu m, taking 10g of the ground feed, adding 20g of water, mixing, then adding 0.02g of medium temperature alpha-amylase, putting the mixture into an oil bath reactor for enzymolysis reaction at 50 ℃ for 3h, cooling to room temperature, adjusting the pH value of the solution subjected to the enzymolysis of the medium temperature alpha-amylase to 3.5, then adding 0.1g of diastase, putting the mixture into a shaking table for reaction at 50 ℃ for 4h, standing for 2h, centrifuging (4500rpm, 5min), and taking out precipitate to obtain the feed with the precipitate removed and pulverized;
s3, in vitro digestion stage:
a. simulating gastric digestion stage: respectively weighing 3g of two equal-mass feed samples without starch, adding one of the feed samples into 29mL of acetic acid-sodium acetate buffer solution, adding 1mL of enzyme solution to be detected to serve as a sample group to be detected, adding 30mL of acetic acid-sodium acetate buffer solution into the other feed sample to serve as a blank sample group, putting the sample group to be detected and the blank sample group into a water bath shaker at 40 ℃, rotating at 200rpm and swinging amplitude of 2.5mm, and incubating for 4 hours to obtain an incubation solution;
b. and (3) color development stage: standing hatching fluid prepared from a sample group to be detected and a blank sample group for 30min, respectively taking 0.1mL of supernatant after standing, sequentially adding 0.7mL of acetic acid-sodium acetate buffer solution and 0.4mL of DNS reagent, oscillating for 3-5s, heating in a boiling water bath for 5min, cooling to room temperature by using tap water, respectively adding 1.3mL of deionized water, adjusting to zero by taking a standard blank sample as a control, carrying out color comparison at 540nm, and determining absorbance on the sample group to be detected;
s4, determination of reducing sugar: calculating the content of reducing sugar according to the absorbance measured in S3 and a standard curve in S1, and calculating the increment of the reducing sugar according to a formula;
calculating the formula: w ═ C × V/m-C 0 *V 0 /m 0
In the formula, C is the reducing sugar content of the sample to be detected, mg/mL;
v is the volume of the sample to be detected, mL;
m is the mass of the feed after starch removal and pulverization, g;
C 0 the content of reducing sugar in the blank sample is mg/mL;
V 0 volume of blank sample, mL;
m 0 feed mass for the blank sample, g.
Example 2
The embodiment of the application discloses a method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed, which comprises the following steps:
s1, drawing a standard curve: firstly, drawing a standard curve of glucose light absorption luminosity value and glucose concentration;
s2, feed de-starchy: grinding the feed, sieving with a sieve with an aperture of 500 mu m, taking 10g of the ground feed, adding 50g of water, mixing, then adding 0.02g of medium temperature alpha-amylase, putting the mixture into an oil bath reactor for enzymolysis reaction at 50 ℃ for 3h, cooling to room temperature, adjusting the pH value of the solution subjected to the enzymolysis of the medium temperature alpha-amylase to 3.5, then adding 0.1g of saccharifying enzyme, putting the mixture into a shaking table for reaction at 50 ℃ for 4h, standing for 2.5h, centrifuging (4500rpm, 5min), and taking out precipitate to obtain the de-precipitated and powdered feed; s3, in vitro digestion stage:
a. simulating gastric digestion stage: respectively weighing 3g of two equal-mass feed samples without precipitation powder, adding one of the feed samples into 29mL of acetic acid-sodium acetate buffer solution, adding 1mL of enzyme solution to be detected to serve as a sample group to be detected, adding the other feed sample into 30mL of acetic acid-sodium acetate buffer solution to serve as a blank sample group, putting the sample group to be detected and the blank sample group into a water bath shaker at 40 ℃, rotating at 200rpm, swinging at 2.5mm, and incubating for 4 hours to obtain an incubation solution;
b. and (3) color development stage: standing hatching fluid prepared from a sample group to be detected and a blank sample group for 30min, respectively taking 0.1mL of supernatant after standing, sequentially adding 0.7mL of acetic acid-sodium acetate buffer solution and 0.4mL of DNS reagent, oscillating for 3-5s, heating in a boiling water bath for 5min, cooling to room temperature by using tap water, respectively adding 1.3mL of deionized water, adjusting to zero by taking a standard blank sample as a control, carrying out color comparison at 540nm, and determining absorbance on the sample group to be detected;
s4, determination of reducing sugar: calculating the content of reducing sugar according to the absorbance measured in S3 and a standard curve in S1, and calculating the increment of the reducing sugar according to a formula;
calculating the formula: w ═ C × V/m-C 0 *V 0 /m 0
In the formula, C is the reducing sugar content of the sample to be detected, mg/mL;
v is the volume of the sample to be detected, mL;
m is the mass of the feed after starch removal and pulverization, g;
C 0 the content of reducing sugar in the blank sample is mg/mL;
V 0 volume of blank sample, mL;
m 0 feed mass for the blank sample, g.
Example 3
The embodiment of the application discloses a method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed, which comprises the following steps:
s1, drawing a standard curve: firstly, drawing a standard curve of glucose light absorption luminosity value and glucose concentration;
s2, feed de-starchy: grinding the feed, sieving with a sieve with an aperture of 500 mu m, taking 10g of the ground feed, adding 30g of water, mixing, then adding 0.02g of medium temperature alpha-amylase, putting the mixture into an oil bath reactor for enzymolysis reaction at 50 ℃ for 3h, cooling to room temperature, adjusting the pH value of the solution subjected to the enzymolysis of the medium temperature alpha-amylase to 3.5, then adding 0.1g of diastase, putting the mixture into a shaking table for reaction at 50 ℃ for 4h, standing for 3h, centrifuging (4500rpm, 5min), and taking out precipitate to obtain the feed with the precipitate removed and pulverized;
s3, in vitro digestion stage:
a. simulating gastric digestion stage: respectively weighing 3g of two equal-mass feed samples without starch, adding one of the feed samples into 29mL of acetic acid-sodium acetate buffer solution, adding 1mL of enzyme solution to be detected to serve as a sample group to be detected, adding 30mL of acetic acid-sodium acetate buffer solution into the other feed sample to serve as a blank sample group, putting the sample group to be detected and the blank sample group into a water bath shaker at 40 ℃, rotating at 200rpm and swinging amplitude of 2.5mm, and incubating for 4 hours to obtain an incubation solution;
b. and (3) color development stage: standing hatching fluid prepared from a sample group to be detected and a blank sample group for 30min, respectively taking 0.1mL of supernatant after standing, sequentially adding 0.7mL of acetic acid-sodium acetate buffer solution and 0.4mL of DNS reagent, oscillating for 3-5s, heating in a boiling water bath for 5min, cooling to room temperature by using tap water, respectively adding 1.3mL of deionized water, adjusting to zero by taking a standard blank sample as a control, carrying out color comparison at 540nm, and determining absorbance on the sample group to be detected;
s4, determination of reducing sugar: calculating the content of reducing sugar according to the absorbance measured in S3 and a standard curve in S1, and calculating the increment of the reducing sugar according to a formula;
calculating the formula: w ═ C × V/m-C 0 *V 0 /m 0
In the formula, C is the reducing sugar content of the sample to be detected, mg/mL;
v is the volume of the sample to be detected, mL;
m is the mass of the feed after starch removal and pulverization, g;
C 0 the content of reducing sugar in the blank sample is mg/mL;
V 0 volume of blank sample, mL;
m 0 feed mass for the blank sample, g.
Example 4
The difference between the examples of the present application and example 3 is that: in step S2, the alpha-amylase is a high temperature resistant alpha-amylase.
Example 5
The difference between the examples of the present application and example 4 is that: the enzymolysis reaction time is 2 h.
Example 6
The difference between the examples of the present application and example 4 is that: the enzymolysis reaction time is 2.5 h.
Example 7
The difference between the examples of the present application and example 4 is that: the enzymolysis reaction time is 1 h.
Example 8
The difference between the examples of the present application and example 5 is that: before adding the high-temperature resistant alpha-amylase, adjusting the pH value of the mixed solution to 5.0, carrying out enzymolysis reaction for 2 hours at the temperature of 90 ℃, adjusting the pH value of the solution subjected to enzymolysis by the high-temperature resistant alpha-amylase to 5.5, adding the saccharifying enzyme, and carrying out reaction for 4 hours at the temperature of 68 ℃.
Example 9
The difference between the examples of the present application and example 5 is that: before adding the high-temperature resistant alpha-amylase, adjusting the pH value of the mixed solution to 6.0, carrying out enzymolysis reaction for 2 hours at the temperature of 80 ℃, adjusting the pH value of the solution subjected to enzymolysis by the high-temperature resistant alpha-amylase to 4.5, adding the saccharifying enzyme, and carrying out reaction for 4 hours at the temperature of 60 ℃.
Example 10
The difference between the examples of the present application and example 5 is that: before adding the high-temperature resistant alpha-amylase, adjusting the pH value of the mixed solution to 5.5, carrying out enzymolysis reaction for 2 hours at the temperature of 85 ℃, adjusting the pH value of the solution subjected to enzymolysis by the high-temperature resistant alpha-amylase to 5.0, adding the saccharifying enzyme, and carrying out reaction for 4 hours at the temperature of 65 ℃.
Example 11
The difference between the examples of the present application and example 10 is that: in step S2, high-voltage pulse electric field treatment (12kV/cm, 80 mL/min) is performed after the high-temperature resistant alpha-amylase is added -1 ) Performing enzymolysis for 0.5h, adding diastase, and performing high-voltage pulsed electric field treatment (13kV/cm, 70 mL/min) -1 ) The reaction was further carried out for 3 hours with shaking.
Example 12
The embodiment of the present application differs from embodiment 11 in that: carrying out enzymolysis reaction for 1.5h, and carrying out shaking reaction for 3.5 h.
Example 13
The difference between the examples of the present application and example 11 is that: carrying out enzymolysis reaction for 0.5h, and carrying out oscillation reaction for 2 h.
Example 14
The difference between the examples of the present application and example 11 is that: carrying out enzymolysis reaction for 1h, and carrying out shaking reaction for 3 h.
Example 15
The difference between the examples of the present application and example 14 is that: in step S2, the amount of the high temperature resistant alpha-amylase added was 0.01g, and the amount of the saccharifying enzyme added was 0.05 g.
Example 16
The difference between the examples of the present application and example 15 is that: in step S2, after the precipitate was centrifuged, distilled water was added to the precipitate for centrifugation again, and the volume of the added distilled water was 5 times the volume of the precipitate, and the centrifugation was repeated 3 times with distilled water.
Example 17
The difference between the examples of the present application and example 16 is that: in step S2, the de-precipitated and powdered feed is dried at 65 ℃ for 4h, cooled to room temperature and then refrigerated at 4 ℃.
Example 18
The difference between the examples of the present application and example 17 is that: step S3 the in vitro digestion phase includes the following steps:
a. simulating gastric digestion stage: respectively weighing 2g of two equal-mass feed samples without precipitation powder, adding one of the two equal-mass feed samples into 25mL of acetic acid-sodium acetate buffer solution, adding 2mL of enzyme solution to be detected to serve as a sample group to be detected, adding 27mL of acetic acid-sodium acetate buffer solution into the other feed sample group to serve as a blank sample group, putting the sample group to be detected and the blank sample group into a 45-DEG C water bath shaker, rotating at 200rpm, swinging at 2.5mm, and incubating for 4.5h to obtain an incubation solution;
b. imitating the digestion stage of the small intestine: respectively and slowly adding 0.1mL of 5mol/L sodium hydroxide solution into the hatching solution prepared by the sample group to be detected and the blank sample group, adjusting the pH of the hatching solution to 7.0 by using the sodium hydroxide solution, respectively adding 1mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and digesting for 7 hours in a 40 ℃ water bath shaking table (rotating speed 200rpm, swing amplitude 2.5mm) to obtain a digestive juice;
c. and (3) color development stage: standing digestive juice prepared from a sample group to be detected and a blank sample group for 25min, respectively taking 0.15mL of supernatant after standing, sequentially adding 0.5mL of acetic acid-sodium acetate buffer solution and 0.3mL of DNS reagent, oscillating for 3-5s, heating in a boiling water bath for 3min, cooling to room temperature by using tap water, respectively adding 1.0mL of deionized water, adjusting to zero by taking a standard blank sample as a control, carrying out color comparison at 540nm, and determining the absorbance of the sample group to be detected.
Example 19
The examples of the present application are different from example 17 in that: step S3 the in vitro digestion phase includes the following steps:
a. simulating gastric digestion stage: respectively weighing 5g of two equal-mass feed samples without precipitation powder, adding one of the two equal-mass feed samples into 35mL of acetic acid-sodium acetate buffer solution, adding 3mL of enzyme solution to be detected to serve as a sample group to be detected, adding the other equal-mass feed samples into 38mL of acetic acid-sodium acetate buffer solution to serve as a blank sample group, putting the sample group to be detected and the blank sample group into a 45-DEG C water bath shaker at the rotating speed of 200rpm and the swing amplitude of 2.5mm, and incubating for 4 hours to obtain an incubation solution;
b. simulating the small intestine digestion stage: respectively and slowly adding 0.3mL of 5mol/L sodium hydroxide solution into the hatching solution prepared by the sample group to be detected and the blank sample group, adjusting the pH of the hatching solution to 8.0 by using the sodium hydroxide solution, respectively adding 3mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and digesting for 9 hours in a 45 ℃ water bath shaking table (rotating speed 200rpm, swing amplitude 2.5mm) to obtain a digestive juice;
c. and (3) color development stage: standing digestive juice prepared from a sample group to be detected and a blank sample group for 25min, respectively taking 0.2mL of supernatant after standing, sequentially adding 0.8mL of acetic acid-sodium acetate buffer solution and 0.7mL of DNS reagent, oscillating for 3-5s, heating in a boiling water bath for 5min, cooling to room temperature by using tap water, respectively adding 2.0mL of deionized water, adjusting to zero by taking a standard blank sample as a control, carrying out color comparison at 540nm, and determining the absorbance of the sample group to be detected.
Example 20
The difference between the examples of the present application and example 17 is that: step S3 the in vitro digestion phase includes the following steps:
a. simulating gastric digestion stage: respectively weighing 3g of two equal-mass feed samples without precipitation powder, adding one of the feed samples into 29mL of acetic acid-sodium acetate buffer solution, adding 1mL of enzyme solution to be detected to serve as a sample group to be detected, adding the other feed sample into 30mL of acetic acid-sodium acetate buffer solution to serve as a blank sample group, putting the sample group to be detected and the blank sample group into a water bath shaker at 40 ℃, rotating at 200rpm, swinging at 2.5mm, and incubating for 4 hours to obtain an incubation solution;
b. imitating the digestion stage of the small intestine: and (3) slowly adding 0.2mL of 5mol/L sodium hydroxide solution into the hatching solution prepared by the sample group to be detected and the blank sample group respectively, adjusting the pH of the hatching solution to 7.5 by using the sodium hydroxide solution, then adding 2mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution respectively, and putting the hatching solution into a water bath shaking table (rotating speed is 200rpm, swing amplitude is 2.5mm) at 40 ℃ for digestion for 8 hours to obtain a digestive juice.
c. And (3) color development stage: standing digestive juice prepared from a sample group to be detected and a blank sample group for 30min, respectively taking 0.1mL of supernatant after standing, sequentially adding 0.7mL of acetic acid-sodium acetate buffer solution and 0.4mL of DNS reagent, oscillating for 3-5s, heating in a boiling water bath for 5min, cooling to room temperature by using tap water, respectively adding 1.3mL of deionized water, adjusting to zero by taking a standard blank sample as a control, carrying out color comparison at 540nm, and determining the absorbance of the sample group to be detected.
Example 21
The difference between the examples of the present application and example 20 is that: in the stage of gastric digestion, adding acetic acid-sodium acetate buffer solution, and preheating at 40 deg.C for 20 min.
Example 22
The difference between the examples of the present application and example 20 is that: a color development stage, in which after standing, the supernatant after standing is removed and centrifuged (rotation speed: 5000rpm) for 10 minutes, and then 0.1mL of the supernatant after centrifugation is removed.
Comparative example
Comparative example 1
This comparative example differs from example 1 in that: in step S2, medium temperature alpha-amylase and saccharifying enzyme are not added.
Performance test
1. The method for rapidly detecting the titer of the non-starch polysaccharide enzyme for the feed in the above examples 1-3, 17-22 and comparative example 1 is adopted by respectively adopting broiler chicken materials (purchased from Shandong Hading agriculture and animal husbandry Co., Ltd.) and corn raw materials, wherein NSP enzyme and NSP enzyme + amylase are respectively adopted as enzyme solutions to be detected, and the increment of reducing sugar is respectively measured;
the corn starch residue was measured using corn as a raw material by the method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed in examples 1 to 17 and comparative example 1, and the test results were recorded as shown in table 1:
TABLE 1 results table
2. The total starch content in corn was measured as M1 in reference to GB/T5009.9-2008 "determination of starch in food", and the starch content was measured as M2 in the de-starchy feeds prepared in examples 1-17 and comparative example 1, respectively, and the starch residue was calculated according to the formula P [ (M1-M2)/M1] × 100%, and the experimental results were recorded as in table 1.
As can be seen by combining example 1 and comparative example 1 with Table 1, if the non-starch polysaccharide enzyme is incorporated with amylase, the increase of reducing sugar measured by using broiler feed or corn feed as a substrate is greatly influenced, so that the evaluation of the titer of the non-starch polysaccharide enzyme for feed is greatly deviated; after the medium-temperature alpha-amylase and the saccharifying enzyme are adopted for carrying out the starch removal treatment on the feed, the interference caused by the amylase doped in the non-starch polysaccharide enzyme can be better shielded, the measurement result of the reducing sugar increment is relatively close, and the titer evaluation accuracy of the non-starch polysaccharide enzyme is improved.
It can be seen from the combination of examples 1-3 and table 1 that, in the process of feed de-starchzation, different ratios of feed to water have different effects on the residual amount of starch in the corn material subjected to de-starchzation treatment, and when amylase is mixed into non-starch polysaccharidase to perform the reducing sugar increase measurement on the same corn feed subjected to de-starchzation treatment, the reducing sugar increase produced by the non-starch polysaccharidase is close to the result of the non-starch polysaccharidase, so that the evaluation of the titer of the non-starch polysaccharidase in the feed is facilitated.
Based on the analysis results of comparative example 1 and examples 1 to 3 described above,the gradual decrease in residual starch content is advantageous for shielding the interference of the amylase incorporated in the non-amylolytic amylase, and when the residual starch content tends to be stable, the measurement results of the reducing sugar increment tend to be consistent even if the amylase is incorporated in the non-amylolytic amylase, and therefore, further optimization (i.e., based on the reaction conditions of the amylase and the saccharifying enzyme in example 3) is performedExamples 4-17) were set to reduce the residual amount of starch in the corn material.
It can be seen from the combination of examples 4-7 and table 1 that the method for rapidly detecting the titer of the non-starch polysaccharide enzyme for feed has the advantages of lower starch residue and higher enzymolysis efficiency by adopting the high-temperature resistant alpha-amylase compared with the medium-temperature alpha-amylase, and can shorten the total time of the method for rapidly detecting the titer of the non-starch polysaccharide enzyme for feed, and is more convenient and faster.
In combination with examples 7-10 and table 1, it can be seen that appropriate pH and reaction temperature are beneficial to improving the enzymolysis efficiency of the enzyme preparation, so that the starch in the feed is sufficiently hydrolyzed, and the residual amount of starch in the corn feed is reduced.
It can be seen from the combination of examples 10-14 and table 1 that the high-voltage pulse electric field treatment is performed on the high-temperature resistant alpha-amylase and the glucoamylase respectively, so that the enzymatic activities of the high-temperature resistant alpha-amylase and the glucoamylase can be further improved, the time for hydrolyzing starch in the feed can be shortened, the purpose of rapid detection can be realized, and the treatment efficiency of the raw material per unit time can be improved, so that the residual amount of starch in the corn raw material can be minimized within a proper time.
As can be seen by combining examples 14-15 and Table 1, the weight ratio of suitable feed to thermostable alpha-amylase and saccharifying enzyme enables a reduction in cost while ensuring starch efficiency in the hydrolyzed feed.
As can be seen by combining examples 15-16 with Table 1, drying the de-starched corn feed had less effect on the residual starch content, but drying was beneficial for long term storage of the de-starched feed, thereby reducing errors in the subsequent determination of the non-starch polysaccharide enzyme titer of the feed.
As can be seen by combining examples 16-17 with Table 1, the dry storage of the destarched feeds facilitates easy determination in further assays.
Based on the analysis results of the above examples 4 to 17,the proper enzyme preparation treatment condition on the raw materials is beneficial to minimizing the residual amount of starch, and the interference of amylase on the evaluation of the titer of the non-starch polysaccharide enzyme of the feed can be reduced. Thus, based on the reaction conditions in the in vitro digestion stage of example 17Further optimization (i.e. setting examples 18-22) is carried out, the environment in animals is fully simulated, and the effect of NSP enzyme action is reflected, so that the increment of reducing sugar produced by the NSP enzyme is maximized. The consistency of the evaluation result and the application effect of the animals is improved.
As can be seen by combining examples 17-20 and Table 1, the simulated gastric digestion stage and the simulated small intestine digestion stage are adopted, so that the digestion environment of the feed in an animal body can be better simulated, and the titer of the non-starch polysaccharidase in the feed can be more objectively evaluated; and the proper simulated environment in each stage is favorable for fully performing enzymolysis on the non-starch polysaccharidase, so that reducing sugar increment results measured by the NSP enzyme and the NSP enzyme plus amylase gradually tend to be consistent, and the action accuracy of the non-starch polysaccharidase in the feed is improved.
As can be seen by combining examples 20-21 and Table 1, the process of preheating the de-starchy feed subjected to the gastric digestion simulation stage in advance simulates the flowing process of the feed in the animal body before entering the stomach of the animal, and the influence of the temperature deviation of the enzyme on the feed before reaction on the measurement result is reduced, so that the result is more stable and accurate, and the titer of the non-starch polysaccharide enzyme is better evaluated.
It can be seen from the combination of examples 21-22 and table 1 that the digestive juice obtained after the gastric-simulated digestion stage and the small intestine-simulated digestion stage is centrifuged again, so that the colorimetric digestive juice keeps better transparency and is not easily affected by impurities, thereby improving the accuracy of absorbance determination and the accuracy of the titer evaluation of the non-starch polysaccharide enzyme in the feed.
Application example
In addition, the method for rapidly detecting the titer of the non-starch polysaccharide enzyme for the feed can be applied to the evaluation of the effect of the concentration gradient of the non-starch polysaccharide enzyme, so as to determine the optimal dosage of the NSP enzyme in the feed.
Test method
The broiler feed and corn after the starch removal treatment are used as action feed, the non-starch polysaccharide enzyme mixture is adopted, 4 treatments are respectively set, and the addition amount of the non-starch polysaccharide enzyme mixture in each treatment is as follows in sequence: treatment 1:100 g non-starch polysaccharidase mixture/ton feed, treatment 2: 200g non-starch polysaccharidase mix/ton feed, treatment 3: 400g non-starch polysaccharidase mix/ton feed, treatment 4: 800g of non-starch polysaccharide enzyme mixture per ton of feed, the increment of reducing sugar was determined according to the method for rapid determination of the titer of non-starch polysaccharide enzyme for feed in this application, and the results of the experiment were recorded as in Table 2.
TABLE 2 results recording sheet
According to the analysis in table 2, the reducing sugar increment in the de-starched broiler feed and the de-starched corn shows linear increase along with the increase of the addition amount of the non-starch polysaccharidase mixture in different feeds; the result is not increased higher after reaching a certain gradient, and the effect achieved when the addition amount of the non-starch polysaccharide enzyme mixture is 400g in each ton of feed is the best. The result is consistent with the action rule of the enzyme on the feed and the influence rule on the animal performance, and shows that the application effect of the enzyme preparation can be objectively evaluated. And determining the gradient effect and optimal dosage of the non-starch polysaccharide enzyme.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed is characterized by comprising the following steps:
s1, drawing a standard curve: firstly, drawing a standard curve of glucose light absorption luminosity value and glucose concentration;
s2, feed de-starchy: mixing the feed with water according to the weight ratio of 1 (2-5), adding alpha-amylase to react for 3 hours, adding diastase to react for 4 hours, standing for 2-3 hours, centrifuging to obtain precipitate, and obtaining the feed with the precipitate removed and pulverized;
s3, in vitro digestion stage: carrying out in-vitro enzymolysis digestion on the feed subjected to precipitation powder removal and an enzyme solution to be detected to obtain a sample to be detected, and determining the absorbance of the sample to be detected and a blank sample;
s4, determination of reducing sugar: calculating the content of reducing sugar according to the absorbance measured in S3 and a standard curve in S1, and calculating the increment of the reducing sugar according to a formula;
calculating the formula: w = C V/m-C 0 *V 0 /m 0
In the formula, C is the reducing sugar content of the sample to be detected, mg/mL;
v is the volume of the sample to be detected, mL;
m is the mass of the feed after starch removal and pulverization, g;
C 0 the content of reducing sugar in the blank sample is mg/mL;
V 0 volume of blank sample, mL;
m 0 feed mass for the blank sample, g.
2. The method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed according to claim 1, wherein: and in the step S2, the alpha-amylase is high-temperature-resistant alpha-amylase, and the reaction is carried out for 1-2h after the alpha-amylase is added.
3. The method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed according to claim 2, wherein: in step S2, the feed with the weight ratio of 1 (2-5) is mixed with water, the pH of the mixed solution is adjusted to 5.0-6.0, then high temperature resistant alpha-amylase is added, the enzymolysis reaction is carried out for 1-2h at the temperature of 80-90 ℃, after the solution is cooled to room temperature, the pH of the solution after the enzymolysis by the high temperature resistant alpha-amylase is adjusted to 4.5-5.5, then diastase is added, the oscillation reaction is carried out for 4h at the temperature of 60-68 ℃, the solution is kept stand for 2-3h, and the precipitate is centrifugally taken out, so that the feed with the precipitate removed and the powder is obtained.
4. The method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed according to claim 1, wherein: in step S2, after adding alpha-amylase, performing high-voltage pulse electric field treatment and then performing enzymolysis reaction for 0.5-1.5h, and after adding glucoamylase, performing high-voltage pulse electric field treatment and then performing oscillation reaction for 2-3.5 h.
5. The method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed according to claim 1, wherein: in step S2, the mass ratio of the alpha-amylase to the feed is 1:900-1300, and the mass ratio of the saccharifying enzyme to the feed is 1: 150-250.
6. The method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed according to claim 3, wherein: in step S2, after the precipitate is centrifuged, distilled water is added for centrifugation again, and the centrifugation with distilled water is repeated for 3 to 5 times.
7. The method for rapidly detecting the titer of non-starch polysaccharide enzymes for feed according to any one of claims 1 to 6, wherein: in step S2, the starch-removed feed is dried at 60-67 deg.C for 3-5h, cooled to room temperature, and then refrigerated at 0-4 deg.C.
8. The method for rapidly detecting the titer of non-starch polysaccharide enzyme for feed according to claim 1, wherein the method comprises the following steps: step S3 the in vitro digestion phase includes the following steps:
simulating gastric digestion stage: respectively weighing 2-5g of two equal-mass feed samples without precipitation powder, adding one of the two equal-mass feed samples into 25-35mL of acetic acid-sodium acetate buffer solution, adding 1-3mL of enzyme solution to be detected to serve as a sample group to be detected, adding the other equal-mass feed sample into acetic acid-sodium acetate buffer solution to serve as a blank sample group, wherein the volume of the acetic acid-sodium acetate buffer solution in the blank sample group is the sum of the volumes of the acetic acid-sodium acetate buffer solution in the sample group to be detected and the enzyme solution to be detected, and placing the sample group to be detected and the blank sample group into a water bath shaker at 40-45 ℃ for incubation for 4-5 hours to obtain an incubation solution;
simulating the small intestine digestion stage: respectively and slowly adding 0.1-0.3mL of 5mol/L sodium hydroxide solution into the hatching solution prepared from the sample group to be detected and the blank sample group, adjusting the pH of the hatching solution to 7.0-8.0 by using the sodium hydroxide solution, respectively adding 1-3mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and putting the hatching solution into a water bath shaker at the temperature of 40-45 ℃ for digestion for 7-9h to obtain a digestive juice;
and (3) color development stage: standing the digestive juice prepared from the sample group to be detected and the blank sample group for 20-30min, respectively taking 0.1-0.2mL of the supernatant after standing, sequentially adding 0.5-0.8mL of acetic acid-sodium acetate buffer solution and 0.3-0.7mL of the LDNS reagent, heating in a boiling water bath for 3-5min, cooling to room temperature, respectively adding 1.0-2.0mL of deionized water, and carrying out color comparison at 540 nm.
9. The method for rapidly detecting the titer of non-starch polysaccharide enzymes for feed according to claim 8, wherein: in the stage of gastric digestion simulation, adding acetic acid-sodium acetate buffer solution, and preheating at 40-50 deg.C for 15-25 min.
10. The method for rapidly detecting the titer of non-starch polysaccharide enzymes for feed according to claim 8, wherein: and in the color development stage, after standing, centrifuging the supernatant for 6-10min, and then respectively taking 0.1-0.2mL of the centrifuged supernatant.
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| CN117491351B (en) * | 2023-12-27 | 2024-04-05 | 中国农业科学院北京畜牧兽医研究所 | Bionic digestion determination kit and determination method for effective phosphorus of broiler feed |
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