Method for detecting amylase in raw material xanthan gum for seasoning

Abstract

The invention discloses a method for detecting amylase in raw material xanthan gum for condiments, which comprises the following steps: mixing a sample solution of xanthan gum with a starch solution to obtain a sample mixed solution, adding the sample mixed solution into an iodine solution at a plurality of different time points within a certain time range to carry out chromogenic reaction, and measuring the absorbance of the sample mixed solution, then establishing a linear atlas with time as a vertical coordinate and absorbance as a horizontal coordinate, and carrying out linear analysis according to the linear atlas to obtain a linear formula of the absorbance and time of the sample developing diluent; therefore, the amylase value rate is calculated by determining the time t required by the sample developing diluent to reach the specific absorbance of 0.235, so that the amylase content in the xanthan gum is analyzed.

Description

Technical Field

[0001] The invention belongs to the technical field of condiments, and particularly relates to a method for detecting amylase in xanthan gum, a raw material for condiments. Background Art

[0002] At present, most of the raw materials for oyster sauce and sauce products use xanthan gum, modified starch, and fructose syrup as auxiliary materials. Among them, due to its unique rheological properties, good water solubility, stability to heat and acid and alkali, and good compatibility with various salts, xanthan gum is often used as a thickener, suspending agent, emulsifier, and stabilizer in the production process of condiments. At the same time, xanthan gum is also the world's largest-scale produced and most widely used microbial polysaccharide.

[0003] However, there is a risk of amylase residues in the production process of xanthan gum, which may cause condiments that use xanthan gum and starch as auxiliary ingredients, such as oyster sauce and sauce, to become thin and watery during storage or shelf sales, which seriously affects the appearance and sales of the product. Therefore, the national standard for oyster sauce products, "GB / T 21999-2018 Oyster Sauce", requires that oyster sauce products have a quality standard of "moderate viscosity, uniformity, and no stratification". However, if there are amylase residues in the production process of the condiment, a small amount of products will still meet the national quality standards during factory inspection, and the phenomenon of thinning and watering will not occur until the shelf sales period.

[0004] At present, the manufacturers of the condiments do not have a method for detecting the residual amylase in xanthan gum that can prevent the condiments from becoming watery during shelf sales. In fact, there is relatively little demand for amylase detection in the condiment field, and there are few types of existing amylase detection methods. There is no index requirement for amylase detection in xanthan gum in the "National Food Safety Standard Food Additive Xanthan Gum" (GB 1886.41-2015).

[0005] Moreover, in the prior art, there are relatively few reports on the detection method of amylase in xanthan gum. Specifically, there is "Detection of Amylase Activity in Xanthan Gum" written by Lu Fenghui and Zhang Juan, which was excerpted from "Journal of Food Safety and Quality Inspection" 2021, 22(12). It discloses a method for detecting amylase activity in xanthan gum. The method is to mix the xanthan gum sample with starch solution and buffer solution and incubate it in an oven at 37°C for 72 hours, then add iodine solution, and compare the color development with that of the blank sample to determine whether amylase exists. However, the detection results of this method are not only inaccurate, but also unable to perform quantitative analysis, and the detection efficiency is also low.

[0006] Therefore, in response to the xanthan gum amylase problem, the seasoning manufacturer usually conducts a pre-destructive test on the raw materials before they are put into storage. The specific test method is:

[0007] Xanthan gum is reacted with starch solution in a certain proportion and cultured at 50°C. Part of the samples are taken every day for microscopic examination. The decomposition of starch granules is observed for more than 15 days. The whole piece (peripheral plus central area) is observed and ≥3 representative fields of view are selected for photography. Except for the cases where the starch granules have been decomposed and dissolved and cannot be counted, the amount of starch granules in each field of view needs to be controlled between 30 and 300. The total number of starch granules in these fields of view should be no less than 150. The overall state of starch granules in 3 or more fields of view is observed, and the deformed granules (expanded, deformed, thinned and transparent granules) are counted. If the number of slightly deformed starch granules is ≤5%, the batch of xanthan gum meets the requirements.

[0008] The disadvantage of this pre-destructive test is that although it can effectively control the quality of raw materials, it takes at least 15 days, resulting in reduced production efficiency, and the inspection personnel's operation time is about 16 hours, resulting in higher labor costs.

[0009] In addition, the prior art also has a method for detecting amylase in condiments or their raw materials.

[0010] For example, China's published patent CN113834757A discloses a rapid detection method for trace residues of high-temperature resistant amylase in oyster sauce raw materials. The method measures the sample spectrum of the viscosity change with temperature after the raw material sample to be tested is added to modified starch, and the sample spectrum is compared with the benchmark spectrum to determine whether the enzyme activity in the raw material sample meets the production requirements.

[0011] For another example, CN115326638A discloses a method for detecting amylase in oyster sauce, wherein the oyster sauce sample to be tested is added to a starch-based liquid, and the viscosity change rate before and after heat preservation is compared between the test sample and the control sample to determine whether the test sample contains amylase.

[0012] However, both of the above two methods cannot perform quantitative analysis and need to use a Brabender viscometer for analysis. The detection method is complicated and the equipment is expensive, with a single device costing more than 300,000 yuan, resulting in high detection costs. Among them, the method disclosed in CN115326638A is still aimed at the detection of amylase in oyster sauce finished products rather than a single xanthan gum raw material.

[0013] Therefore, the prior art does not have a method for detecting the residual amount of amylase in the xanthan gum raw material, and it is also unable to prevent the condiment from becoming watery and thinning during the shelf life. Summary of the invention

[0014] The object of the present invention is to provide a method for detecting amylase in xanthan gum, a raw material for seasoning. The method can quantify the amylase in xanthan gum, which can not only improve the detection accuracy to solve the problem of seasoning becoming thinner caused by it, but also improve the detection efficiency.

[0015] The present invention adopts the following technical solutions to achieve the invention objectives.

[0016] A method for detecting amylase in xanthan gum, a raw material for seasoning, comprises the following steps:

[0017] (1) Standardization of starch solution

[0018] (1.1) Prepare starch solution and preheat it in a water bath;

[0019] (1.2) fully mixing the preheated starch solution and the preheated water in proportion to obtain a mixed solution, and continuing to preheat in a water bath;

[0020] (1.3) Add the mixed solution to the iodine solution to form a color developing solution, and place it away from light to perform a color development reaction;

[0021] (1.4) Dilute the color-developing solution with water to obtain a color-developing dilution solution, record the volume of water added to the color-developing dilution solution, and measure its absorbance at a wavelength of 660 nm using an ultraviolet spectrophotometer until the absorbance of the color-developing dilution solution is equal to A. n Until the set value reaches 0.760±0.01, and the volume of water added during dilution is recorded as V 水 ;

[0022] (2) Determination of xanthan gum amylase

[0023] (2.1) preparing a xanthan gum solution from a xanthan gum sample to be tested;

[0024] (2.2) adding sodium acetate buffer, sodium chloride solution and water to the xanthan gum aqueous solution and mixing well to obtain a sample solution;

[0025] (2.3) fully mixing the starch solution preheated in step (1.1) and the sample solution preheated in step (2.2) in the same ratio as the starch solution and water in step (1.2) to obtain a sample mixture, and starting timing synchronously when the two solutions are mixed;

[0026] (2.4) Pipette an equal volume of the sample mixture at least once in the time periods of 0.5-2h, 17-20h, 24-40h, and 44-48h and add it to the iodine solution to form a sample colorimetric solution, and then add the V determined in step (1.4) to the solution. 水 dilute with 100 volumes of water to obtain at least one sample color developing dilution solution in the above four time periods;

[0027] (2.5) After the at least four sample color development dilutions obtained in step (2.4) are placed away from light for color development reaction, their absorbance at 660 nm is measured using an ultraviolet spectrophotometer;

[0028] (2.6) With the time of the mixed reaction of the sample solution and the starch solution as the ordinate and the absorbance of the sample color-developing diluent as the abscissa, the absorbance measured in step (2.5) is marked on the coordinate axis, a linear graph is established, and a linear analysis is performed to obtain a linear formula of the absorbance of the sample color-developing diluent and time; then, based on the linear formula, the time t required for the sample color-developing diluent to reach a specific absorbance of 0.235 is calculated; finally, using the time t, the amylase value rate X of the xanthan gum sample is calculated according to the following formula ①, thereby quantitatively analyzing the amylase in the xanthan gum sample;

[0029] X=V / (w×t)①,

[0030] In formula ①:

[0031] X: Amylase value rate, which refers to the volume of 1% starch solution hydrolyzed by a unit mass of xanthan gum sample per unit time, in mL / (g·h);

[0032] V: the volume of the 1% starch solution used to prepare the sample mixture, in mL; if the starch solution with a mass fraction other than 1% is used to prepare the sample mixture, the starch mass shall be used for conversion;

[0033] w: the mass of xanthan gum in the sample mixture, in g;

[0034] t: The time corresponding to the absorbance of the sample color dilution solution reaching 0.235, in hours.

[0035] The working principle of the present invention is to use the principle that amylase can cut the starch molecular chain and make the characteristic reaction of starch to iodine in blue-purple disappear. The starch solution is added to the xanthan gum aqueous solution to be tested, that is, steps (2.1) to (2.3), and then the iodine solution is added, that is, step (2.4) to obtain the xanthan gum sample color development dilution. If a certain amount of amylase remains in the xanthan gum to be tested, part of the starch will be hydrolyzed by the amylase therein, and the remaining starch will react with the added iodine to produce blue-purple, and as the reaction proceeds, the blue-purple reaction gradually disappears, and the rate of color disappearance is positively correlated with the amylase content in the xanthan gum sample.

[0036] Therefore, according to the above working principle, the present invention uses an ultraviolet spectrophotometer to measure the absorbance of the xanthan gum sample color-developed dilution at multiple different time points within a certain time range, establishes a linear spectrum with reaction time as the ordinate and absorbance as the abscissa, and uses this to perform linear analysis to obtain a linear formula of the absorbance and time of the sample color-developed dilution. Thus, by determining the time t required for the sample color-developed dilution to reach a specific absorbance of 0.235, the amylase value rate is calculated to analyze the amylase content in the xanthan gum sample.

[0037] Therefore, the present invention can perform relatively simple detection operations with lower detection costs and tools, obtain the data required for amylase value rate calculation, and quantitatively analyze the amylase in the xanthan gum sample by calculating the amylase value rate in the xanthan gum sample; and the present invention can further perform a correlation analysis between the amylase value rate of the xanthan gum sample and the quality of the condiment, thereby utilizing the amylase value rate as a quality control indicator for the xanthan gum raw material.

[0038] The present invention requires starch calibration before the xanthan gum amylase is determined in step (2). The purpose of this method is to determine the volume of dilution water V required for the starch solution to produce a specific absorbance. 水 , and use this volume as the dilution factor for the sample color development solution in the subsequent step (2.4).

[0039] The absorbance A of the color-developing dilution in step (1.4) of the present invention is n Determination of the set value to be achieved: According to the working principle of the spectrophotometer and experimental results, the present invention knows that when the pure starch solution is not hydrolyzed and reacts with the iodine solution, the absorbance measured by the ultraviolet spectrophotometer at a wavelength of 660nm is the maximum, which is 0.760±0.01. In order to facilitate the measurement operation and measurement accuracy, the present invention determines 0.760±0.01 as the set value of the absorbance An of the color-developing dilution described in step (1.4).

[0040] In the step (1.4), water is added to the color developing solution to dilute it to obtain a pure starch color developing solution, and then water is used as a blank control to measure the absorbance A of the color developing solution at a wavelength of 660 nm. n ;

[0041] If the absorbance A n If the set value is within the range of 0.760±0.01, the volume of water used to dilute the colorimetric solution is recorded as V 水 , the V 水 As the volume of water added when diluting the sample colorimetric solution in step (2.4);

[0042] If the absorbance A nIf it is not within the set value range of 0.760±0.01, repeat steps (1.3) and (1.4) until the absorbance of the color-developing dilution reaches A n Until it is within the range of 0.760±0.01, then record the volume V of water used to dilute the colorimetric solution 水 .

[0043] Preferably, when A n If the value is not within the set value range of 0.760±0.01 and steps (1.3) and (1.4) need to be repeated, the volume of water added when diluting the colorimetric solution can be calculated and determined according to the following formula ②;

[0044]

[0045] In formula ②:

[0046] V 显 is the volume of the color developing solution, in mL,

[0047] V n is the volume of water used for dilution of the colorimetric solution for the nth time, in mL.

[0048] V n+1 is the volume of water used to dilute the colorimetric solution n+1 times, in mL.

[0049] A n is the absorbance of the colorimetric solution when it is diluted for the nth time.

[0050] The present invention has found through experiments that when the absorbance of the sample color-developing diluent reaches 0.235, the activity of amylase will begin to gradually weaken, resulting in a gradual decrease in the rate at which amylase hydrolyzes starch. At this time, the absorbance of the sample color-developing diluent is not linearly related to time; so in order to ensure the accuracy of the amylase value rate result, the absorbance of the sample color-developing diluent measured in step (2.5) must be at least 3 above 0.235, and a linear analysis is performed at points above 0.235. For the same reason, 0.235 is used as the specific absorbance of the sample color-developing diluent in step (2.6).

[0051] Preferably, if the amylase content of the xanthan gum sample is high, that is, the amylase value rate is high, the time for transferring the sample mixture in step (2.4) to measure its absorbance can be appropriately shortened to ensure that the three measured absorbances are above 0.235. For example: if the amylase value rate is between 200 and 300 mL / (g·h), it is measured every 20 to 30 minutes after 0.5 h of reaction; if the enzyme value is greater than 400 mL / (g·h), it is measured every 5 to 10 minutes after 5 minutes of reaction.

[0052] As a specific embodiment of the present invention, if the absorbance of the sample color development dilution solution measured within 48 hours is greater than 0.71, the xanthan gum sample is determined to have no detected amylase.

[0053] As a preferred embodiment of the present invention, if the first absorbance of the sample color development dilution is measured to be greater than 0.79, it is necessary to add another point in time to confirm whether there is an abnormal sample aspiration problem; the specific operation is to re-absorb the sample color development dilution when the measured absorbance exceeds 0.79, measure the absorbance, and record the time point of the additional measurement. During the measurement process, if the absorbance of a certain point changes abnormally or other abnormal conditions occur, it is necessary to add another point in time, and the operation is as described above. If the linearity is not within the allowable range due to the last point, it is necessary to add another point in time, and the operation is as described above.

[0054] A specific method for preparing the starch solution of the present invention is:

[0055] A. Weigh 2.000g of soluble starch into a beaker, add 50-70mL of water and stir, then boil rapidly and then simmer for 3 minutes;

[0056] B. Place a petri dish on the beaker or cover the beaker with a lid, place in a cold water bath to cool, and after cooling to room temperature, quickly transfer to a 100 mL volumetric flask and add water to make up to volume;

[0057] C. The soluble starch is a soluble starch specially used for enzyme preparation detection; preferably, the starch solution is used on the day of preparation.

[0058] As a specific embodiment of the present invention, in step (1.2), the starch solution and water are mixed in a volume ratio of 1:2; that is, in step (2.3), the starch solution and the sample solution are also mixed in a volume ratio of 1:2.

[0059] As a specific implementation of the present invention, the preheating temperature is 60±2°C.

[0060] As a specific embodiment of the present invention, the time of the color development reaction is 30 to 60 minutes.

[0061] As a specific embodiment of the present invention, in step (1.2), 5 mL of preheated starch solution is fully mixed with 10 mL of preheated water to obtain a mixed solution, and the preheating in a water bath is continued.

[0062] As a specific embodiment of the present invention, in step (1.3), 1.0 mL of the mixed solution is accurately pipetted into 10.0 mL of iodine solution, mixed to form a color developing solution, and then placed away from light to perform a color development reaction for 30 to 60 minutes.

[0063] As a specific embodiment of the present invention, in step (2.1), the xanthan gum sample to be tested is fully miscible with water in a mass ratio of 1:100 to prepare a xanthan gum aqueous solution.

[0064] As a specific embodiment of the present invention, the concentration of xanthan gum in the sample solution is 0.5-3 g / L; the concentration can be adjusted according to the absorbance measured subsequently to ensure that at least 3 absorbances of the sample color development dilution measured in step (2.5) are above 0.235. In addition, too high a concentration of xanthan gum will cause the solution to be too viscous, which is not conducive to the reaction, so the concentration of xanthan gum in the sample solution should be lower than 3 g / L.

[0065] As a preferred embodiment of the present invention, the concentrations of sodium acetate and sodium chloride in the sample solution are 19.47±0.05 g / L and 1.74±0.05 g / L, respectively; wherein sodium acetate plays a buffering role to keep the pH value of the solution at about 5.30, and the role of the sodium chloride solution is to improve the thermal stability of amylase, thereby enhancing the activity of amylase.

[0066] As a specific embodiment of the present invention, the preparation method of the sodium acetate buffer is as follows: weigh 97.37g of sodium acetate into 400mL of water, add 10.5mL of glacial acetic acid, and dilute to 500mL with water. If the pH value of the sodium acetate buffer is not within the range of 5.30±0.05, adjust the pH value to 5.30±0.05 with sodium acetate or glacial acetic acid.

[0067] As a specific implementation of the present invention, the concentration of the sodium chloride solution is 0.5 mol / L.

[0068] As a specific embodiment of the present invention, the preparation method of the iodine solution is as follows: (1) preparing an iodine stock solution: weighing 0.88 g of iodine and dissolving it in 3 mL to 4 mL of water containing 2.2 g of potassium iodide, and diluting the volume to 100 mL with water to obtain an iodine stock solution; (2) preparing an iodine solution: weighing 10 g of potassium iodide, dissolving it in water, then adding 2.5 mL of the iodine stock solution, diluting the volume to 250 mL with water, and storing it in the dark.

[0069] As a specific embodiment of the present invention, in step (2.3), 5 mL of preheated starch solution is added to 10 mL of preheated sample solution and mixed thoroughly to obtain a sample mixture.

[0070] As a specific embodiment of the present invention, in step (2.5), the sample color development dilution solution is placed away from light for 30 to 60 minutes for color development reaction, and then the absorbance is measured at a wavelength of 660nm using an ultraviolet spectrophotometer with water as a blank control.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] (1) In the detection method of amylase in xanthan gum, a raw material for seasoning, the linear formula between absorbance and time is determined according to the color change caused by the hydrolysis reaction of amylase in xanthan gum and starch solution, and the amylase value rate of xanthan gum is calculated, so that the amylase content in xanthan gum can be analyzed; therefore, the present invention can quantify the level of amylase and improve the detection accuracy, so as to accurately judge its influence on seasoning; the present invention further conducts a correlation analysis between the amylase value rate of the xanthan gum sample and the quality of oyster sauce, and the results show that the critical amylase value rate of xanthan gum that causes the oyster sauce product to dehydrate is 0.7 mL / (g·h), and the xanthan gum raw material with a rate less than the critical enzyme value will not cause the oyster sauce to dehydrate and stratify during the shelf life.

[0073] (2) Based on the existing raw material pre-destructive test of ≥15 days, the present invention shortens the time for raw material arrival access inspection, and can complete the access inspection of xanthan gum raw materials within 48 hours, significantly improving the inspection efficiency, and more accurately quantifying the raw material risks, and more effectively controlling the raw material quality;

[0074] The invention has low requirements on the conditions of the detection equipment, utilizes a common constant temperature water bath and an ultraviolet photometer to replace the Brabender viscometer used in the prior art, and greatly saves the investment cost of the detection equipment.

[0075] (3) The detection limit of the amylase value rate of the present invention is 0.60 mL / (g·h), which is lower than the requirement of the amylase value rate of the seasoning for its raw materials. It can effectively detect xanthan gum raw materials that do not meet the requirements and reduce the risk of production and use.

[0076] (4) The method of the present invention has good scalability and can also be applied to the detection of residual amylase in fructose-glucose syrup, another raw material for the production of oyster sauce and sauces, so that the amylase of the raw materials involved can be monitored and processed in time, thereby improving the physical stability of the condiment and ensuring the quality of the product.

[0077] (5) The present invention achieves quantification of the amylase content in xanthan gum. On the one hand, it helps to monitor the quality of xanthan gum raw materials, thereby ensuring the quality of different batches of condiments. On the other hand, it helps condiment manufacturers to improve the condiment production process. For example, when trying to solve the problem of oyster sauce becoming thinner during the shelf life, the method of the present invention can be used to detect the amylase value rate of xanthan gum, and through experiments to determine whether xanthan gum with different amylase value rates is the key point affecting the thinning of oyster sauce, thereby helping to improve the oyster sauce production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 It is a linear graph of the mixing reaction time t of the sample solution and the starch solution in Example 1 and the absorbance of the sample color development dilution solution. DETAILED DESCRIPTION

[0079] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0080] The reagents used in the following examples were all analytically pure, and the water was first-grade water as specified in GB / T 6682.

[0081] Preparation method of iodine solution: (1) Weigh 0.88 g of iodine and dissolve it in 3 mL to 4 mL of water containing 2.2 g of potassium iodide, dilute to 100 mL with water to obtain iodine stock solution, store in a brown bottle and refrigerate for later use; (2) Weigh 10 g of potassium iodide, dissolve it in water, add 2.5 mL of iodine stock solution, dilute to 250 mL with water to obtain iodine solution, and store in the dark.

[0082] Preparation method of sodium acetate buffer: weigh 97.37g sodium acetate (CH3COONa·3H2O) in 400mL water, add 10.5mL glacial acetic acid, and dilute to 500mL with water; if its pH value is not within the range of 5.30±0.05, adjust the pH value to 5.30±0.05 with sodium acetate or glacial acetic acid.

[0083] The preparation method of 0.5 mol / L sodium chloride solution is as follows: weigh 7.25 g of sodium chloride, dissolve it in water and make up to 250 mL.

[0084] The starch solution preparation method is as follows: using a soluble starch specially used for enzyme preparation detection, specifically selecting the soluble starch of Huzhou Zhanwang Chemical Pharmaceutical Co., Ltd.; weighing 2.000g of soluble starch, placing it in a 150mL-200mL beaker, adding about 60mL of water along the wall, stirring with a glass rod, rapidly boiling and then slightly boiling for 3min, rinsing the glass rod with 5-10ml of hot water, removing the glass rod, and then rinsing the inner wall of the beaker, covering the beaker with a culture dish / covering the beaker with a beaker lid, placing it in a cold water bath with a liquid level about 1cm lower than the starch solution level to cool, and after cooling to room temperature, rapidly transferring it to a 100mL volumetric flask, rinsing the beaker with water and fixing the volume, and using it on the same day.

[0085] Example 1 A xanthan gum sample known to contain amylase was tested according to the following steps:

[0086] (1) Standardization of starch solution

[0087] (1.1) Pipette about 10 mL of starch solution into a 25 mL stoppered colorimetric tube and accurately pipette 10.0 mL of water into a 25 mL stoppered colorimetric tube. Preheat each tube in a 60±2℃ constant temperature water bath for 15 min.

[0088] (1.2) Accurately transfer 5.0 mL of preheated starch solution into 10.0 mL of preheated water, mix thoroughly, and place in a constant temperature water bath at 60±2℃ for 5 min.

[0089] Among them, the pipette used to transfer the starch solution needs to be preheated twice by pipetting the starch solution back and forth before officially transferring the starch solution into the sample solution.

[0090] (1.3) Accurately pipette 1.0 mL of the mixed solution into a 50 mL stoppered colorimetric tube that has been pre-added with 10.0 mL of iodine solution. Mix well to form the colorimetric solution. The colorimetric solution should be kept away from light for 30 min to 60 min for color development.

[0091] (1.4) Add a volume of V to the color developing solution n Water (Note: V n represents the volume of water added for the nth time, V1 is a reference amount of 29.5 mL), tighten the stopper, and mix thoroughly by pouring left and right and then shaking to form a color-developing dilution;

[0092] In order to eliminate the interference of water on the results, water was used as a blank control, and the absorbance of the color-developing dilution was measured at a wavelength of 660nm using a spectrophotometer, which was recorded as A. n If A n Within the set value range of 0.760±0.01, record the volume of water used to dilute the colorimetric solution, recorded as V 水 ;

[0093] If A n If the value is not within the range of 0.760±0.01, repeat steps (1.3) and (1.4); the volume of water added during the repetition of the operation is calculated according to formula ② until the absorbance of the color-developing dilution solution is n Record the absorbance value within the range of 0.760±0.01. The volume of water added within the range of 0.760±0.01 is used as the volume V of the sample colorimetric solution dilution water. 水 .

[0094]

[0095] In formula ②:

[0096] V 显 ———The volume of the color developing solution, in mL,

[0097] V n ———The volume of dilution water used when diluting the colorimetric solution for the nth time, in mL,

[0098] V n+1——The volume of water used for dilution of the colorimetric solution for the n+1th time, in mL,

[0099] A n ———The absorbance of the color-developing dilution solution at the nth dilution.

[0100] (2) Determination of xanthan gum amylase

[0101] (2.1) Preparation of xanthan gum aqueous solution

[0102] The xanthan gum sample to be tested is fully miscible with water at a mass ratio of 1:100; the specific operation is:

[0103] Weigh 50.0g of water into a 250mL conical flask with glass beads (covering the bottom of the flask or more than 2 / 3), take the xanthan gum sample with a 3mL disposable pipette, slowly add it to the conical flask, shake the conical flask while adding, so that the sample is dispersed in the water, record the mass of the xanthan gum sample weighed into the conical flask, weigh 0.8g~1.0g, at least accurate to 0.01g, and then add the total mass of water to 100 times the mass of the xanthan gum sample. Stir manually, and the undissolved glue attached to the upper half of the inner wall of the bottle can be stirred with the help of a glass rod to make a uniform xanthan gum aqueous solution, and visually inspect to ensure that there are no undissolved xanthan gum particles.

[0104] (2.2) Preparation of sample solution

[0105] Weigh 13.25 g (accurate to at least 0.01 g) of xanthan gum aqueous solution into a 50 mL volumetric flask, add about 10 mL of water and 5.0 mL of acetate buffer and mix thoroughly, then add 3.0 mL of sodium chloride solution, add water to make up to volume, shake well, and obtain a sample solution;

[0106] When the sample solution has been standing for about 5 minutes and the larger bubbles (diameter greater than 1.5 mm) in the volumetric flask have basically dissipated, use a pipette to draw 10.0 mL of the sample solution into a 25 mL stoppered colorimetric tube. When the liquid is naturally discharged to about 5 cm from the tip of the tube, use an ear bulb to blow the remaining liquid in the tube into the tube.

[0107] (2.3) Mix the preheated starch solution with the preheated sample solution

[0108] Pipette about 10 mL of starch solution into a 25 mL stoppered colorimetric tube, and place the two stoppered colorimetric tubes containing starch solution and sample solution in a 60℃±2℃ constant temperature water bath to preheat for 15 min;

[0109] Accurately pipette 5.0 mL of preheated starch solution into the preheated 10.0 mL sample solution, start timing while dispensing the solution, tighten the stopper, and mix thoroughly by shaking up and down slightly. Do not allow the solution to adhere to the colorimetric tube mouth and colorimetric tube cover.

[0110] Among them, the pipette used to transfer the starch solution needs to be preheated twice by pipetting the starch solution back and forth before officially transferring the starch solution into the sample solution.

[0111] (2.4) Prepare sample color dilution solutions at different time periods

[0112] After the sample solution and starch solution are mixed, 1.0 mL of starch sample mixed solution is accurately pipetted into a 50 mL stoppered colorimetric tube pre-added with 10.0 mL of iodine solution at 0.5-2 h, 17-20 h, 24-40 h, and 44-48 h, respectively. Mix well to form the sample colorimetric solution, and then add V 水 dilute with 100 volumes of water to obtain at least one sample color developing dilution solution in the above four time periods;

[0113] (2.5) Measure the absorbance of the sample colorimetric dilution at a wavelength of 660 nm

[0114] The sample color dilution obtained in step (2.4) was thoroughly mixed by pouring left and right and then shaking, and then placed in a dark place for color development reaction for 30min to 60min, and then the supernatant was carefully pipetted into a 1cm colorimetric dish with a disposable pipette, and water was used as a blank control. The absorbance was measured at a wavelength of 660nm using an ultraviolet spectrophotometer. The results are shown in Table 1;

[0115] Table 1

[0116] Absorbance 0.745 0.715 0.695 0.657 Reaction time (min) 60 1080 1800 2800

[0117] (2.6) Calculate the sample amylase value rate

[0118] With the mixing reaction time (min) of the sample solution and the starch solution as the ordinate and the absorbance of the sample color dilution as the abscissa, the data in Table 1 are plotted on the coordinate axis to establish a linear spectrum (see Figure 1 ), do linear analysis, R 2 It must meet ≥0.92 (Note: points with absorbance lower than 0.235 are not included in the linear analysis, and the number of points used in the calculation is not less than 4 points) to determine the linear formula.

[0119] Depend on Figure 1 The linear spectrum of the reaction time t and the absorbance A is determined by t = -31243A + 23399, and the specific absorbance 0.235 is substituted to obtain t = 16057min;

[0120] According to the following formula ①, calculate the amylase value rate X:

[0121] X=V / (w×t) ①

[0122] In formula ①:

[0123] X is the rate of amylase value in the sample, in mL / (g·h);

[0124] V: the volume of the 1% starch solution used to prepare the sample mixture, in mL; this embodiment uses 5 mL of a 2% starch solution, so the amount V converted to a 1% starch solution is 10 mL;

[0125] w: the mass of xanthan gum in the sample mixture, in g; w in this example = (13.25 g × 10 mL) / (101 × 50 mL);

[0126] t: The time corresponding to the absorbance of the sample color dilution solution reaching 0.235, in hours.

[0127] Therefore, substituting t=(16057 / 60)h into formula ①, we can obtain that the amylase value rate X of the xanthan gum sample is 1.42mL / (g·h).

[0128] Example 2 Detection limit and quantification limit test

[0129] Ten xanthan gum samples containing amylase were taken for parallel determination test, and the amylase value rate thereof was determined according to the steps of Example 1. The results are shown in Table 2:

[0130] Table 2 Detection limit and quantification limit test results Unit: mL / (g·h)

[0131]

[0132]

[0133] As shown in Table 2, the detection limit of the rate of amylase value of xanthan gum detected by the method of the present invention is 0.60 mL / (g·h), and the quantitative limit is 1.80 mL / (g·h).

[0134] Example 3 Evaluation of the precision of the repeatability test of the present invention

[0135] Ten xanthan gum samples containing amylase were taken for parallel determination test, and the amylase value rate thereof was determined according to the steps of Example 1, and the precision of the repeatability test was evaluated. The results are shown in Table 3:

[0136] Table 3 Repeatability test results Unit: mL / (g·h)

[0137]

[0138] As shown in Table 3, the range of the repeatability test by personnel accounts for 19.7% of the average value, which meets the precision requirement that when the enzyme value rate is less than 50 mL / (g·h), the absolute difference between two independent determination results obtained under repeated conditions shall not exceed 20% of the arithmetic mean.

[0139] Example 4 Comparison test between different laboratories

[0140] Xanthan gum amylase positive samples were taken and two laboratories conducted an inter-laboratory comparison test according to the steps of Example 1. The test results are shown in Table 4:

[0141] Table 4 Results of inter-laboratory comparison test Unit: mL / (g·h)

[0142]

[0143]

[0144] In the inter-laboratory comparison test, the test results of the two laboratories were 3.30mL / (gh) and 3.88mL / (gh), both of which were within the theoretical value of the positive-like amylase rate of 3.0-4.0mL / (gh). The theoretical value of the positive-like amylase rate is calculated by adding an amylase sample with known enzyme activity content to a negative sample.

[0145] According to the enterprise standard, the absolute difference of the comparison results between laboratories shall not exceed 40% of the arithmetic mean. In this embodiment, the difference between the two laboratories measured accounts for 16.2% of the average value, so the comparison results between laboratories meet the requirements.

[0146] Example 5 Correlation test between xanthan gum amylase value and oyster sauce water

[0147] This example takes the oyster sauce prepared by Chinese patent CN105410841B as an example, and tests the water dissolution of oyster sauce prepared by xanthan gum with different amylase values, wherein the xanthan gum with different amylase values ​​is prepared by quantitatively adding amylase to the enzyme-inactivated xanthan gum.

[0148] 1) The oyster sauce with different amylase values ​​and xanthan gum was placed at room temperature for 1 to 60 days. Its fluidity was tested at different days and the water dissolution was observed. The results are shown in Table 5.

[0149] 2) Destructive tests were conducted at 60°C for 30 days and 60 days to test its fluidity and observe the water dissolution. The results are shown in Table 6. Among them, 60°C for 30 days and 60 days is equivalent to 90 days and 180 days at room temperature. In the conventional oyster sauce water dissolution test, 180 days at room temperature can reflect the 12-month shelf life. If the residual amylase content in the oyster sauce is high, water dissolution will occur within 180 days of storage at room temperature. If water dissolution does not occur within 180 days, water dissolution will not occur even if it is stored for 12 months.

[0150] The test method of the fluidity is:

[0151] After adjusting the temperature of the oyster sauce sample (heating or cooling) to 25.0-35.0℃, quickly fill the sample tank with the prepared sample, scrape off the excess sample horizontally to make the sample volume flush with the height of the sample tank, and quickly wipe off the sample that seeps out of the gate gap of the sample tank;

[0152] When opening the gate, hold down the viscometer with one hand to prevent it from moving, and use the other hand to open the gate and press the stopwatch at the same time to read the farthest distance the sample flows in 30 seconds. The tip of the fluid tongue is used as the standard, which is the fluidity of the oyster sauce sample (cm / 30s).

[0153] Table 5 The corresponding fluidity and water dissolution of oyster sauce at different xanthan gum amylase values ​​at room temperature

[0154]

[0155] Table 6 The fluidity and water dissolution of oyster sauce corresponding to the rate of different xanthan gum amylase values ​​at 60℃

[0156]

[0157] It can be seen from Tables 5 and 6 that when the fluidity of oyster sauce is greater than 12 (cm / 30s), the sample has begun to dissolve into water. When the xanthan gum amylase value rate is less than 0.7mL / (g·h), the fluidity of oyster sauce placed at room temperature or subjected to destructive tests has a small change trend and no water dissolution phenomenon occurs. When the xanthan gum amylase value rate is greater than 0.7mL / (g·h), the fluidity of oyster sauce increases significantly with the extension of the placement time. When the xanthan gum amylase value rate = 0.7mL / (g·h), the oyster sauce dissolves into water after being placed at room temperature for 60 days, and dissolves into water after 30 days of destructive testing.

[0158] It can be seen that the amylase value rate of 0.7 mL / (g·h) is the limit value of whether oyster sauce will be dehydrated and stratified during the shelf life, that is, the critical amylase value rate. When the xanthan gum amylase value rate is less than 0.7 mL / (g·h), the oyster sauce will not be dehydrated and stratified during the shelf life. Therefore, the detection method of the present invention can accurately know the xanthan gum amylase value rate and effectively detect the xanthan gum raw materials that do not meet the requirements.

Claims

1. A method for detecting amylase in xanthan gum, a raw material for seasoning, characterized in that: The following steps are involved: (1) Standardization of starch solution (1.1) Prepare starch solution and preheat it in a water bath; (1.2) fully mixing the preheated starch solution and the preheated water in proportion to obtain a mixed solution, and continuing to preheat in a water bath; (1.3) Add the mixed solution to the iodine solution to form a color developing solution, and place it away from light to perform a color development reaction; (1.4) Dilute the color-developing solution with water to obtain a color-developing dilution solution, record the volume of water added to the color-developing dilution solution, and measure its absorbance at a wavelength of 660 nm using an ultraviolet spectrophotometer until the absorbance of the color-developing dilution solution is equal to A. n Until the set value reaches 0.760±0.01, and the volume of water added during dilution is recorded as V 水 ; (2) Determination of xanthan gum amylase (2.1) preparing a xanthan gum solution from a xanthan gum sample to be tested; (2.2) adding sodium acetate buffer, sodium chloride solution and water to the xanthan gum aqueous solution and mixing well to obtain a sample solution; (2.3) fully mixing the starch solution preheated in step (1.1) and the sample solution preheated in step (2.2) in the same ratio as the starch solution and water in step (1.2) to obtain a sample mixture, and starting timing synchronously when the two solutions are mixed; (2.4) Pipette an equal volume of the sample mixture at least once in the time periods of 0.5-2h, 17-20h, 24-40h, and 44-48h and add it to the iodine solution to form a sample colorimetric solution, and then add the V determined in step (1.4) to the solution. 水 dilute with 100 volumes of water to obtain at least one sample color developing dilution in the above four time periods; (2.5) After the at least four sample color development dilutions obtained in step (2.4) are placed away from light for color development reaction, their absorbance at 660 nm is measured using an ultraviolet spectrophotometer; (2.6) With the time of the mixed reaction of the sample solution and the starch solution as the ordinate and the absorbance of the sample color-developing diluent as the abscissa, the absorbance measured in step (2.5) is marked on the coordinate axis, a linear graph is established, and a linear analysis is performed to obtain a linear formula of the absorbance of the sample color-developing diluent and time; then, based on the linear formula, the time t required for the sample color-developing diluent to reach a specific absorbance of 0.235 is calculated; finally, using the time t, the amylase value rate X of the xanthan gum sample is calculated according to the following formula ①, thereby quantitatively analyzing the amylase in the xanthan gum sample; X=V / (w×t)①, In formula ①: X: Amylase value rate, which refers to the volume of 1% starch solution hydrolyzed by a unit mass of xanthan gum sample per unit time, in mL / (g·h); V: the volume of the 1% starch solution used to prepare the sample mixture, in mL; w: the mass of xanthan gum in the sample mixture, in g; t: The time corresponding to the absorbance of the sample color dilution solution reaching 0.235, in hours.

2. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 1, characterized in that In the step (1.4), water is added to the color developing solution to dilute it to obtain a pure starch color developing dilution, and then water is used as a blank control to measure the absorbance A of the color developing dilution at a wavelength of 660 nm. n ; If the absorbance A n If the set value is within the range of 0.760±0.01, the volume of water used to dilute the colorimetric solution is recorded as V 水 , the V 水 As the volume of water added when diluting the sample colorimetric solution in step (2.4); If the absorbance A n If it is not within the set value range of 0.760±0.01, repeat steps (1.3) and (1.4) until the absorbance of the color-developing dilution reaches A n Until it is within the range of 0.760±0.01, then record the volume V of water used to dilute the colorimetric solution 水 .

3. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 2, characterized in that: When A n If the value is not within the set value range of 0.760±0.01 and steps (1.3) and (1.4) need to be repeated, the volume of water added when diluting the colorimetric solution can be calculated and determined according to the following formula ②; In formula ②: V 显 is the volume of the color developing solution, in mL, V n is the volume of water used for dilution of the colorimetric solution for the nth time, in mL. V n+1 is the volume of water used to dilute the colorimetric solution n+1 times, in mL. A n is the absorbance of the colorimetric solution when it is diluted for the nth time.

4. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 3, characterized in that: At least three of the sample colorimetric dilution absorbances measured in step (2.5) must be above 0.235, and linear analysis must be performed at points above 0.

235.

5. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 4, characterized in that: If the first absorbance of the sample colorimetric diluent is greater than 0.79, it is necessary to add another point for measurement in time to confirm whether there is any abnormal sample aspiration problem; the specific operation is that when the measured absorbance exceeds 0.79, re-absorb the sample colorimetric diluent, measure the absorbance, and record the time point of the additional measurement; during the measurement process, if the absorbance of a certain point changes abnormally or other abnormal conditions occur, it is necessary to add another point for measurement in time, and the operation is as described above.

6. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 5, characterized in that: The preheating temperature is 60±2° C.; the color development reaction time is 30 to 60 minutes.

7. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 6, characterized in that: In step (1.2), the starch solution and water are mixed in a volume ratio of 1:2; similarly, in step (2.3), the starch solution and the sample solution are mixed in a volume ratio of 1:

2.

8. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 7, characterized in that: The concentration of xanthan gum in the sample solution is 0.5-3 g / L.

9. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 8, characterized in that: The concentrations of sodium acetate and sodium chloride in the sample solution were 19.47±0.05 g / L and 1.74±0.05 g / L, respectively.

10. The method for detecting amylase in xanthan gum, a raw material for seasoning according to claim 9, characterized in that: A specific preparation method of the starch solution is: A. Weigh 2.000g of soluble starch into a beaker, add 50-70mL of water and stir, then boil rapidly and then simmer for 3 minutes; B. Place a petri dish on the beaker or cover the beaker with a lid, place in a cold water bath to cool, and after cooling to room temperature, quickly transfer to a 100 mL volumetric flask and add water to make up to volume; C. The soluble starch is a soluble starch specially used for enzyme preparation detection.

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