CN114292347A - Extraction and detection method of yeast beta-glucan in protein powder - Google Patents
Extraction and detection method of yeast beta-glucan in protein powder Download PDFInfo
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Abstract
The invention discloses an extraction and detection method of yeast beta-glucan in protein powder, which comprises the following steps: and adding the pretreatment solution into the sample, uniformly mixing, and carrying out water bath treatment to obtain a treatment solution. The detection method specifically comprises the following steps: after the yeast beta-glucan in the sample is extracted by the extraction method, the yeast beta-glucan is measured by a sulfuric acid phenol method. Compared with the prior art, the invention can extract, separate and purify the yeast beta-glucan from the protein powder, and has simple and accurate extraction method and low detection cost.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to an extraction and detection method of yeast beta-glucan in protein powder.
Background
The yeast beta-glucan is a new national food raw material, is a polysaccharide which exists in yeast cell walls and has the activity of enhancing immunity, and has the effects of improving blood fat, resisting radiation and improving intestinal functions.
Because the addition amount of the yeast beta-glucan in the food is low, the matrix is more and complicated, and the detection method of the yeast beta-glucan raw material is not suitable for detecting the yeast beta-glucan in the food, such as the method of QB/T4572-2021 yeast beta-glucan and United states Pharmacopeia. At present, the detection methods for the yeast beta-glucan in the food comprise QB/T4572-2021 yeast beta-glucan appendix A and food safety national standard food yeast beta-glucan determination-requisition manuscript, the contents of the two methods are consistent, the two methods can be regarded as the same detection method, and the detection method mainly refers to the method of the United states Pharmacopeia. The method for detecting the yeast beta glucan in the yeast beta glucan protein powder by adopting the existing food yeast beta glucan has the following problems and difficulties:
1. the method is applicable to the determination of insoluble yeast beta-glucan in the recombined milk, the flavored fermented milk, the recombined milk powder and the infant formula milk powder, and the yeast beta-glucan protein powder is a protein food and does not belong to the milk food. The dairy food can be separated into the yeast beta-glucan with higher purity by processing according to the method, the residue amount is very small, which is helpful for subsequent alkali treatment and quantitative operation, and the residue amount obtained by processing the yeast beta-glucan protein powder according to the method is very large, which affects the subsequent alkali treatment and quantitative operation, which indicates that the method has insufficient impurity removal capability on the yeast beta-glucan protein powder. Therefore, the yeast beta-glucan protein powder is not suitable for the method.
2. The method has the advantages that the method has no strict constant volume operation, certain loss can be caused in the extraction and separation process of the yeast beta-glucan, and the detection error is amplified through multiple absorption and dilution operations in the enzymolysis process, so that the precision of the method can only be less than or equal to 15%.
3. The operability problem of the method is that different blank samples and yeast beta-glucan reference substances are required to be introduced to correct the detection result according to different varieties each time, the introduction of the blank samples can increase the detection amount, the blank samples with different formulas are required to be reserved, the yeast beta-glucan reference substances have special requirements, water-insoluble yeast beta-glucan is required, and the daily detection and the external detection are difficult to realize; the method has multiple operation steps, high requirement on the operational capability of people, time consumption of 2-3 days in the whole detection process and low detection efficiency; and the enzymolysis process has a plurality of influencing factors, and the detection process is difficult to control.
4. The method has the problems of high cost, expensive enzyme required by the method and high detection cost for a large amount of samples.
Therefore, it is highly desirable to provide a method for extracting and detecting yeast β -glucan, which is simple in extraction method, good in accuracy and low in detection cost.
Disclosure of Invention
In order to solve the technical problems, the invention provides an extraction and detection method of yeast beta-glucan, the extraction, separation and purification of yeast beta-glucan can be carried out from yeast beta-glucan protein powder, and the extraction method is simple and accurate and has low detection cost.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for extracting yeast beta-glucan comprises the following steps: and adding the pretreatment solution into the sample, uniformly mixing, and carrying out water bath treatment to obtain a treatment solution.
Preferably, the mass-to-volume ratio of the sample to the pretreatment solution is 0.2: 9-11; most preferably 0.2: 10.0.
preferably, the pretreatment solution is a buffer and a thermostable a-amylase.
Further preferably, the volume ratio of the buffer to the thermostable a-amylase is 10: 0.04-0.06; most preferably 10: 0.05.
preferably, the buffer is Tris buffer.
In some preferred embodiments, the method for extracting yeast beta-glucan further comprises proteolysis.
Specifically, the proteolysis comprises the following steps:
(1) centrifuging the treated solution to obtain a precipitate, adding a Tris buffer solution, neutral protease and alkaline protease into the precipitate, uniformly mixing, heating in a water bath, and cooling to obtain an enzymolysis solution;
(2) centrifuging the enzymolysis solution obtained in the step (1) to obtain a precipitate, adding water and an aluminum chloride solution into the precipitate, uniformly mixing, adding ammonia water, uniformly mixing, and centrifuging to obtain a precipitate;
(3) and (3) dissolving the precipitate obtained in the step (2) by using a phenol sulfuric acid solution to obtain an extract to be extracted.
Preferably, the mass concentration of the aluminum chloride solution in the step (2) is 4.5-5.5%; most preferably 5%.
Preferably, the volume ratio of the aluminum chloride solution to the ammonia water in the step (2) is 4-6: 1; most preferably 5: 1.
the invention also provides a detection method of the yeast beta-glucan, which comprises the following steps: after the yeast beta-glucan in the sample is extracted by the extraction method, the yeast beta-glucan is measured by a sulfuric acid phenol method.
The invention has the beneficial effects that:
according to the characteristic that yeast beta-glucan is water-insoluble, a sample is hydrolyzed by amylase, centrifuged to remove centrifugate, and water-soluble impurities such as starch, dextrin, water-soluble sugar, water-soluble protein and the like are removed; hydrolyzing with protease to remove insoluble protein, centrifuging to remove centrifugate to obtain residue as yeast beta-dextran, and adsorbing the residue with precipitant to prevent loss during washing; and finally, dissolving the residue by using a phenol-sulfuric acid solution and detecting. Compared with the existing detection method, the method has the following advantages:
(1) the impurity removal effect is good, and the method can finally obtain powder with a loose structure, which is consistent with the character of the yeast beta glucan raw material; the specificity is high, and other impurities are confirmed to have no obvious interference on detection through a negative sample test; the detection result is accurate and can reach 97% of the actual content.
(2) The method has high precision, the loss of the yeast beta-glucan is very little, the volumetric flask is adopted to perform constant volume, the constant volume is accurate, and the detection precision can be less than or equal to 5 percent, which is superior to the conventional method and less than or equal to 15 percent.
(3) The method has good operability, only aims at sample detection, does not need to additionally add a method for correcting a detection result, does not relate to complex enzymolysis and dilution operations, and has stable enzyme property and no unstable factor; the time consumption is short, and the detection can be finished in 1 day.
(4) The method has low cost, does not need to purchase expensive enzyme preparations, and has very low detection cost because other reagents are conventional reagents in laboratories.
(5) The sensitivity is high, the range of the yeast beta-glucan detected by the conventional detection method at present is 1.5mg to 12.5mg, while the range of the yeast beta-glucan detected by the method is 0.25mg to 5mg, and the sensitivity of the method is higher.
Drawings
FIG. 1 is a standard operating curve for the detection method of example 1.
Detailed Description
The present invention will be further explained with reference to specific examples in order to make the technical means, the technical features, the technical objectives and the effects of the present invention easier to understand, but the following examples are only preferred embodiments of the present invention, and not all embodiments of the present invention. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention. In the following examples, unless otherwise specified, all the procedures used were conventional, all the equipment used were conventional, and all the starting materials used were commercially available.
Except for special instructions, all the reagents are analytically pure, and the water is tertiary water, wherein the glucose reference substance: alatin, lot number: h1921084, the purity is 99.5%; 5% phenol solution: taking 5g of phenol, adding water to dissolve the phenol, and fixing the volume to 100mL, wherein the phenol can be stored for one month at 4 ℃; phenol sulfuric acid solution: taking 70mL of concentrated sulfuric acid, carefully adding the concentrated sulfuric acid into 30mL of 5% phenol solution, and shaking up (paying attention to adding water to 100mL after cooling); tris buffer: weighing 6.1g of tris (hydroxymethyl) aminomethane, dissolving with 800mL of water, adjusting the pH to 8.2 with 6mol/L hydrochloric acid, and adding water to dilute to 1000 mL; 5% aluminum chloride solution: 9.05g of aluminum chloride hexahydrate is taken, and water is added to dissolve the aluminum chloride hexahydrate to 100 mL; neutral protease: CAS: 9068-59-1, the activity is more than or equal to 80 ten thousand U/g; alkaline protease: CAS: 9014-01-1, the activity is more than or equal to 25 ten thousand U/g; thermostable a-amylase: CAS, 9000-85-5; the test samples were: yeast beta-glucan protein powder.
Example 1 extraction and detection method of Yeast beta-glucan
Preparation of standard curve of S1:
103.60mg of glucose reference substance is weighed in a 1000mL volumetric flask, dissolved by a proper amount of water and subjected to constant volume to prepare a 0.10mg/mL glucose reference solution. 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mL of the solution is respectively sucked into a 25mL colorimetric tube, water is added to 1mL (standard series of 0 mug, 10.3 mug, 20.6 mug, 41.2 mug, 61.8 mug, 82.5 mug and 103.1 mug are obtained), 1mL of 5% phenol solution is added, shaking is carried out, 1mL of phenol-sulfuric acid solution is added, shaking is carried out, 5mL of concentrated sulfuric acid is added, shaking is carried out, a boiling water bath is carried out for 15min, and cold water is cooled. A blank tube was used for zeroing, and the absorbance at 490nm was measured with the absorbance as the ordinate and the glucose mass (. mu.g) as the abscissa to prepare a standard curve.
S2, preparation and determination of a test solution:
s2.1 weighing 0.2g (containing 2-3mg of yeast beta-glucan) of the uniformly mixed sample into a dry and clean 15mL pointed-bottom centrifuge tube, adding 10mL Tris buffer solution and 0.05mL thermostable alpha-amylase, uniformly mixing, carrying out water bath in a water bath kettle at 95 ℃ for 1 hour, uniformly mixing for 2-3 times in the midway, taking out, and cooling to room temperature.
S2.2 the sample solution is centrifuged at 8000r/min for 5min, about 8mL of supernatant is carefully aspirated with a pipette (the supernatant cannot be removed by pouring, which would otherwise cause loss of yeast β -glucan), water is added to 10mL, and the above procedure is repeated 3 times to obtain a precipitate containing about 2mL of water. Adding Tris buffer solution into the precipitate to 10mL, respectively adding 0.1mL neutral protease and alkaline protease, mixing, placing in a water bath at 50 deg.C for 2 hr, mixing 5-6 times, taking out, and cooling to room temperature.
S2.3 centrifuge the sample solution at 8000r/min for 5min, carefully pipette about 8mL of supernatant (which cannot be removed by pouring, which would otherwise cause loss of yeast β -glucan), add water to 10mL, and repeat the above procedure 1 time to obtain a pellet containing about 2mL of water. Adding water to the precipitate to 10mL, adding 0.5mL 5% aluminum chloride solution, mixing, adding 0.1mL ammonia, mixing, centrifuging at 8000r/min for 5min, and carefully removing the supernatant (to avoid pouring out the precipitate). The pellet was added to 10mL of water, centrifuged at 8000r/min for 5min and the supernatant carefully discarded (to avoid decanting the pellet).
S2.4 transferring the precipitate to a 50mL volumetric flask by using a phenol-sulfuric acid solution in a fractional manner, fixing the volume to a scale by using the phenol-sulfuric acid solution, standing at room temperature for 30min, shaking up for 2-3 times in the midway, and fully dissolving the precipitate to obtain a test solution.
S2.5 in a 25mL colorimetric tube, adding 1mL of water and 1mL of 5% phenol solution, shaking up, adding 1mL of test solution, shaking up, adding 5mL of concentrated sulfuric acid, shaking up, placing in a boiling water bath for 15min, and cooling with cold water. The absorbance was measured at 490nm, and the amount of glucose (. mu.g) in the test solution was read on the standard curve to calculate the amount of yeast β -glucan in the test.
S3, calculation of Yeast beta-glucan content (in terms of glucose)
In the formula: content of yeast β -glucan (calculated as glucose) in the X-test sample,%;
c-amount of glucose in the test solution, μ g;
v-the dilution volume of the test solution, mL;
v1-test volume of test solution, mL;
m-sample size of the sample, g.
First, methodology validation
1. Specificity test (negative sample test)
1.1 test methods
Taking a yeast beta-glucan protein powder negative sample without yeast beta-glucan, treating the yeast beta-glucan protein powder negative sample according to the method, and determining the absorbance value of the negative sample solution at 490 nm.
1.2 test data, as shown in Table 1.
TABLE 1
| Serial number | Absorbance value |
| 1 | 0.0024 |
| 2 | 0.0015 |
| 3 | 0.0045 |
1.3 conclusion of the test
As can be seen from the above table, the solution of the negative sample has substantially no absorption at 490nm, and the result is substantially free from interference, indicating that the specificity of the method of the present invention is good.
2 Linear Range confirmation
2.1 test data, as shown in Table 2.
TABLE 2
2.2 Standard working Curve
The concentration is plotted on the abscissa and the absorbance is plotted on the ordinate, and a standard working curve is plotted as shown in FIG. 1 below.
2.3 conclusion of the Linear test
And (3) linear evaluation: the correlation is 0.99995, and the yeast beta-glucan determined by the method presents good linearity between 10.3082 mu g and 103.0820 and all meet the requirements of GB/T27404-2008 laboratory quality control Specification [ GB/T27404- > 2008 requires that the correlation is more than or equal to 0.99 ].
3 detection limit
3.1 test methods:
the absorbance of the blank solution was measured in parallel at 490nm for 20 times, and the lower limit of the measurement was calculated according to the following formula:
CL＝3Sb/b
in the formula: cL-a determination threshold of the method;
Sb-blank value standard deviation;
b-method the slope of the calibration curve.
3.2 test data, as shown in Table 3.
TABLE 3
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Absorbance of the solution | 0.0009 | 0.0005 | 0.0006 | 0.0011 | 0.0013 | 0.0003 | -0.0001 |
| Serial number | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| Absorbance of the solution | 0.0003 | 0.0010 | 0.0036 | -0.0006 | -0.0002 | 0.0022 | 0.0007 |
| Serial number | 15 | 16 | 17 | 18 | 19 | 20 | Sb |
| Absorbance of the solution | 0.0000 | 0.0015 | 0.0010 | -0.0001 | 0.00081 | 0.0009 | 0.000934 |
3.3 conclusion of the test:
the detection amount of the method is as follows: 3 × 0.000934/0.00783820 ═ 0.357 μ g, calculated according to the actual sample processing procedure, the detection limit of the method is: 0.357 × 50 × 100/1/0.2/1000 ═ 8.9mg/100 g.
4 precision test
4.1 test methods
9 samples were weighed into three groups of three samples, the first group weighed 0.1g, the second group weighed 0.2g, and the third group weighed 0.3g, and the content of yeast β -glucan in the samples was determined by the method described in example 1 above, and the RSD (%) thereof was calculated.
4.2 test data, as shown in Table 4.
TABLE 4
4.3 conclusion of the test
The RSD of the content of the beta-glucan in 9 samples is 1.8 percent, which shows that the method has better precision and meets the requirement of GB/T27404-2008 laboratory quality control Specification (GB/T27404-.
5 durability test (stability)
5.1 test methods:
the sample was treated as in example 1 above, and the absorbance values were measured and calculated after leaving the developed standard solution and sample solution at room temperature for 0 hour, 0.5 hour, 1.0 hour, 1.5 hour, 2 hours, and 3 hours, respectively.
5.2 test data, as shown in Table 5.
TABLE 5
5.3 conclusion of the test
After the developed standard solution and sample solution were left at room temperature for 0 hour, 0.5 hour, 1.0 hour, 1.5 hour, 2 hours, and 3 hours, RSD (%) was 0.3% and 0.2%, respectively, indicating that the developed sample solution had good stability at room temperature for 3 hours.
6 accuracy test (comparison with theoretical content)
6.1 test methods
According to the formula of the product and the feeding amount of the yeast beta-glucan, the theoretical content of the yeast beta-glucan in the product is calculated to be 1.34g/100g, and according to the measurement result of the yeast beta-glucan in the 4-precision test, the deviation between the measurement average value and the theoretical content is calculated by comparing with the theoretical content.
6.2 test data
Accuracy deviation (%) ═ i (measured average-theoretical content)/theoretical content i x 100%
＝|(1.30-1.34)/1.34|×100％＝3.0％
6.3 conclusion of the test
The yeast β -glucan accuracy bias was: 3.0 percent, meets the requirement of GB/T27404 & 2008 laboratory quality control Specification (GB/T27404 & 2008 requires accuracy deviation less than 5 percent).
In order to further highlight the inventive step of the present invention, the technical effects achieved by the present invention will be described in detail with reference to specific comparative examples.
Comparative example 1
This comparative example differs from example 1 in that:
s2.1, weighing 0.2g (containing 2-3mg of yeast beta-glucan) of the uniformly mixed sample, adding 10mL of Tris buffer solution and 0.1mL of thermostable alpha-amylase into a dry and clean 15mL of pointed-bottom centrifuge tube, uniformly mixing, carrying out water bath in a water bath kettle at 95 ℃ for 1 hour, uniformly mixing for 2-3 times in the midway, taking out, and cooling to room temperature; and
s2.3 centrifuge the sample solution at 8000r/min for 5min, carefully pipette about 8mL of supernatant (which cannot be removed by pouring, which would otherwise cause loss of yeast β -glucan), add water to 10mL, and repeat the above procedure 1 time to obtain a pellet containing about 2mL of water. Adding water to the precipitate to 10mL, adding 1mL of 5% aluminum chloride solution, mixing, adding 0.3mL of ammonia water, mixing, centrifuging at 8000r/min for 5min, and carefully removing the supernatant (to avoid pouring out the precipitate). The pellet was added to 10mL of water, centrifuged at 8000r/min for 5min and the supernatant carefully discarded (to avoid decanting the pellet).
Comparative example 2
This comparative example differs from example 1 in that: step S2.1 is omitted, 0.2g of a weighed and uniformly mixed sample is directly put into a dry and clean 15mL pointed-bottom centrifuge tube, a Tris buffer solution is added to 10mL, then 0.1mL neutral protease and alkaline protease are respectively added, the mixture is uniformly mixed, the mixture is subjected to water bath in a water bath kettle at 50 ℃ for 2 hours, the mixture is uniformly mixed for 5-6 times in the midway, and the mixture is taken out and cooled to room temperature.
7 investigation of Performance
7.1 the content of yeast β -glucan in the sample was measured according to the methods of "4 precision test" and "6 accuracy test" in the methods of comparative examples 1 and 2, and the RSD (%) thereof was calculated, and the accuracy deviation was calculated as shown in tables 6 and 7.
TABLE 6
TABLE 7
Comparative example 1
Accuracy deviation (%) ═ i (measured average-theoretical content)/theoretical content i x 100%
＝|(3.22-1.34)/1.34|×100％＝140.3％。
Comparative example 2
Accuracy deviation (%) ═ i (measured average-theoretical content)/theoretical content i x 100%
＝|(3.79-1.34)/1.34|×100％＝182.8％。
And (4) test conclusion: the accuracy deviation of the yeast beta-glucan is 140.3 percent in the comparative example 1 and 182.8 percent in the comparative example 2, which does not meet the requirement of GB/T27404-.
The present invention is not limited to the above-described preferred embodiments, but rather, the present invention is to be construed broadly and cover all modifications, equivalents, and improvements falling within the spirit and scope of the present invention.
Claims (10)
1. The extraction method of the yeast beta-glucan is characterized by comprising the following steps of: and adding the pretreatment solution into the sample, uniformly mixing, and carrying out water bath treatment to obtain a treatment solution.
2. The extraction method according to claim 1, wherein the mass-to-volume ratio of the sample to the pretreatment solution is 0.2: 9-11.
3. The extraction process according to claim 2, wherein the pretreatment solution is a buffer and a thermostable α -amylase.
4. The extraction process according to claim 3, wherein the volume ratio of the buffer to the thermostable a-amylase is 10: 0.04-0.06.
5. The method of claim 3, wherein the buffer is Tris buffer.
6. The extraction method according to claim 3, further comprising subjecting the treatment solution to proteolysis.
7. The extraction method according to claim 6, characterized in that said proteolytic digestion comprises the following steps:
(1) centrifuging the treated solution to obtain a precipitate, adding a Tris buffer solution, neutral protease and alkaline protease into the precipitate, uniformly mixing, heating in a water bath, and cooling to obtain an enzymolysis solution;
(2) centrifuging the enzymolysis solution obtained in the step (1) to obtain a precipitate, adding water and an aluminum chloride solution into the precipitate, uniformly mixing, adding ammonia water, uniformly mixing, and centrifuging to obtain a precipitate;
(3) and (3) dissolving the precipitate obtained in the step (2) by using a phenol sulfuric acid solution to obtain an extract to be extracted.
8. The extraction method according to claim 7, wherein the mass concentration of the aluminum chloride solution in the step (2) is 4.5-5.5%.
9. The extraction method according to claim 7, wherein the volume ratio of the aluminum chloride solution to the ammonia water in the step (2) is 4-6: 1.
10. a method for detecting yeast beta-glucan is characterized by comprising the following steps: extracting yeast β -glucan in the sample by the extraction method according to any one of claims 1 to 9, and then measuring the yeast β -glucan by a phenol sulfate method.
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| CN107976503A (en) * | 2017-11-24 | 2018-05-01 | 北京康比特体育科技股份有限公司 | A kind of detection method for adding the yeast dextran in albumen powder |
| CN113075141A (en) * | 2021-03-30 | 2021-07-06 | 汤臣倍健股份有限公司 | Extraction method and content determination method of yeast beta-glucan |
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Patent Citations (6)
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| CN102351943A (en) * | 2011-09-28 | 2012-02-15 | 南京同凯兆业生物技术有限责任公司 | Method for co-producing glucan and mannoprotein by using industrial waste yeast |
| CN102818779A (en) * | 2012-08-07 | 2012-12-12 | 黄河三角洲京博化工研究院有限公司 | Method for detecting content of water-insoluble glucan in yeast glucan |
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