CN114755351A - Ion chromatography determination method for average polymerization degree of levan - Google Patents
Ion chromatography determination method for average polymerization degree of levan Download PDFInfo
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Abstract
The invention provides an ion chromatography determination method for the average polymerization degree of levan in infant food or dairy products, which comprises the following steps: mixing an infant food sample or a dairy product sample with water to prepare a sample solution; mixing the sample solution, the sucrase solution and the beta-galactosidase solution, carrying out first enzymolysis, and then carrying out first enzyme inactivation to prepare a first solution; wherein the enzyme activity of the beta-galactosidase solution is 500-600U/mL; mixing a sodium hydroxide solution of potassium borohydride with the first solution to prepare a second solution; introducing said boron in said second solutionRemoving potassium hydride, mixing with the levanase solution, carrying out second enzymolysis, and then carrying out second enzyme deactivation to prepare a solution to be detected; carrying out ion chromatography detection on the solution to be detected to obtain the content of fructose
The content of glucose
By passing
And calculating to obtain the average polymerization degree of the fructan.
Description
Technical Field
The invention relates to the technical field of detection, in particular to an ion chromatography determination method for the average polymerization degree of levan.
Background
Under the background of the rapid development of the food industry, a great deal of nutrition enhancers are used, and the phenomenon that functional carbohydrate ingredients are added into foods such as infant formula food, pasteurized milk, sterilized milk, beverages and the like appears, and the carbohydrate is also used as a dietary fiber monomer ingredient for improving and enhancing the nutritional composition of related foods and reducing the calorie.
Fructans are a class of functional carbohydrates, which are important components of dietary fiber and are also important prebiotics. The fructan is typically a polymer of 2-59 fructose groups and 1 glucose residue. The average polymerization degree of fructo-oligosaccharide is 4, and common fructo-oligosaccharide comprises kestose, nystose, hexaose and the like; inulin having an average degree of polymerization of 10; the average polymerization degree of polyfructose is 23, and the polyfructose is a general name of a class of compounds. Under the background of the development of various current food categories and food diversification, most enterprises select functional sugars such as galacto-oligosaccharide, fructo-oligosaccharide, inulin, polyfructose, polydextrose, raffinose and stachyose as product selling points, and some enterprises select to add more than one fructan, such as fructo-oligosaccharide, inulin and polyfructose, so as to increase the diversity of carbon compounds, thereby being more beneficial to the growth of beneficial bacteria in intestinal tracts of organisms.
According to the regulation of GB 14880 + 2012' national food safety standard for dietary supplements, fructo-oligosaccharides and polyfructose can be added into foods such as infant formula, infant cereal auxiliary food and the like. The health agency announcement states that inulin may be used in various types of food, but does not include infant food. The traditional detection method only aims at the content determination of fructo-oligosaccharide, polyfructose or inulin which are independently added into foods such as milk, dairy products, infant formula foods, solid beverages and the like, but cannot detect or analyze the fructan which is added in a mixed manner, so that the detection requirements of production and supervision cannot be met. Meanwhile, the levan added into food is generally used as an auxiliary material, the addition amount is low, the requirement on the sensitivity of a detection method is high, the pretreatment steps of a food matrix sample are more complicated, and the detection difficulty of the levan applied to an actual sample is high. Different food matrixes are complex and various, and can have large influence on the substance to be detected, so that the reported detection method can be invalid in actual samples.
In addition, since the infant food or dairy product contains more lactose: the lactose content of the infant formula milk powder is about 40% -55%, the lactose content of the milk powder is generally more than 20%, the lactose content of the liquid milk is about 4%, and enzymolysis decomposition products of the lactose are galactose and glucose, wherein the content of the glucose can directly interfere the ratio of fructose and glucose generated by enzymolysis of the levan, so that the deviation of the calculation result of the average polymerization degree of the levan and an actual value is large.
Disclosure of Invention
Based on the ion chromatography determination method for the average polymerization degree of the fructan in the infant food or the dairy product, the average polymerization degree of the fructan mixed and used in the sample can be quickly obtained, the calculation result of the average polymerization degree of the fructan is closer to the actual value, and the recovery rate index meets the methodological requirement.
The invention is realized by the following technical scheme.
An ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy products comprises the following steps:
mixing an infant food sample or a dairy product sample with water to prepare a sample solution;
mixing the sample solution, the sucrase solution and the beta-galactosidase solution, carrying out first enzymolysis, and then carrying out first enzyme deactivation to prepare a first solution; wherein the enzyme activity of the beta-galactosidase solution is 500U/mL-600U/mL;
mixing a sodium hydroxide solution of potassium borohydride with the first solution to prepare a second solution;
removing the potassium borohydride in the second solution, mixing the potassium borohydride with a levanase solution, carrying out second enzymolysis, and then carrying out second enzyme deactivation to prepare a solution to be detected;
carrying out ion chromatography detection on the solution to be detected to obtain the content of fructose
The content of glucose
By passing
And calculating to obtain the average polymerization degree of the fructan.
In one embodiment, the conditions for the first enzymatic hydrolysis include: oscillating for 50-70 min at the temperature of 40 ℃ and the rotating speed of 100 r/min;
the conditions of the second enzymolysis comprise: oscillating for 35-55 min at the rotating speed of 100r/min at the temperature of 40 ℃.
In one embodiment, the conditions for the first enzyme deactivation comprise: heating in boiling water for 12-18 min; and/or
The conditions for the second enzyme deactivation include: heating in boiling water for 12-18 min.
In one embodiment, the method for mixing the infant food sample or the dairy product sample with water further comprises the following steps:
the sample solution was prepared by adjusting the solution pH to 4.5 ± 0.1 with acetic acid.
In one embodiment, the enzymatic activity of the sucrase solution is 4.5U/mL.
In one embodiment, the enzymatic activity of the levanase solution is 50U/mL.
In one embodiment, the removing of the potassium borohydride in the second solution comprises the following steps:
the second solution was mixed with 0.5mol/L of an aqueous acetic acid solution.
In one embodiment, the ion chromatography detection conditions include: the leacheate A is 0.15mol/L of sodium hydroxide, the leacheate B is a mixed solution of 0.15mol/L of sodium hydroxide and 1.0mol/L of sodium acetate, and the leacheate C is water.
In one embodiment, the ion chromatography detection conditions further comprise: gradient leaching is adopted; the gradient elution procedure comprises: 0 min-14 min, wherein the volume percentage of the leacheate A is 20%, and the volume percentage of the leacheate C is 80%; 14 min-14.1 min, wherein the volume percentage of the leacheate A is changed from 20% to 0%, the volume percentage of the leacheate C is changed from 80% to 0%, and the volume percentage of the leacheate B is changed from 0% to 100%; 14.1-17 min, and keeping the volume percentage of the eluent B at 100%; 17 min-17.1 min, wherein the volume percentage of the leacheate B is changed from 100% to 0%, and the volume percentage of the leacheate C is changed from 0% to 100%; 17.1-20 min, and keeping the volume percentage of the leacheate C at 100%; 20 min-20.1 min, wherein the volume percentage of the leacheate A is changed from 0% to 20%, and the volume percentage of the leacheate C is changed from 100% to 80%; and (3) 20.1-30 min, wherein the volume percentage of the leacheate A is kept to be 20%, and the volume percentage of the leacheate C is kept to be 80%.
In one embodiment, the ion chromatography detection conditions further comprise: the flow rate is 1 mL/min-1.2 mL/min; the column temperature is 28-32 ℃; the sample injection amount is 10 mu L; an anion analysis column was used.
Compared with the prior art, the ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy products has the following beneficial effects:
according to the invention, the samples are pretreated by sequentially adopting the sucrase solution and the beta-galactosidase solution, so that interference components in the infant food or dairy product samples are removed, the enzyme activity of the beta-galactosidase solution is limited to 500U/mL, the enzymolysis of the levan is further ensured not to be interfered, and the deviation degree of the calculation result of the average polymerization degree of the levan and an actual value is reduced. Meanwhile, the invention is based on an ion chromatography determination method, and the average polymerization degree of the levan is calculated by accurately detecting the content of the enzymolysis product of the levan, so that the average polymerization degree of the levan mixed in a sample can be solved, the quantitative problem of the total amount of the levan is solved, and the standard recovery rate index meets the requirement of methodology.
Furthermore, the ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy products establishes a complete detection method, is suitable for simultaneous detection of a large number of samples based on a specific enzymolysis technology, has simple and convenient pretreatment steps, and can improve the efficiency.
Drawings
FIG. 1 shows the fructan enzymatic hydrolysis principle provided by the present invention;
FIG. 2 is a flow chart of the ion chromatography method for measuring the average polymerization degree of fructan provided by the invention;
FIG. 3 is a standard spectrum of ion chromatography provided by the present invention;
FIG. 4 is a graph showing the effect of the amount of beta-galactosidase used according to the present invention on the results.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the invention, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise. In the description of the present invention, "a plurality" means at least one, e.g., one, two, etc., unless specifically limited otherwise.
The words "preferably," "more preferably," and the like, in the present disclosure mean embodiments of the disclosure that may, in some instances, provide certain benefits. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides an ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy products, which comprises the following steps:
mixing an infant food sample or a dairy product sample with water to prepare a sample solution;
mixing the sample solution, the sucrase solution and the beta-galactosidase solution, carrying out first enzymolysis, and then carrying out first enzyme deactivation to prepare a first solution; wherein the enzyme activity of the beta-galactosidase solution is 500U/mL-600U/mL;
mixing a sodium hydroxide solution of potassium borohydride with the first solution to prepare a second solution;
removing potassium borohydride in the second solution, mixing the second solution with the levanase solution, carrying out second enzymolysis, and then carrying out second enzyme deactivation to prepare a solution to be detected;
performing ion chromatography detection on the solution to be detected to obtain the content of fructose
The content of glucose
By passing
And calculating to obtain the average polymerization degree of the fructan.
The infant formula food or the dairy product contains high-content lactose, and has the problems of complex pretreatment, large matrix interference, similar carbohydrate structure and the like, so that the difficulty in detecting the average polymerization degree of the fructan is large. Levan formed by connecting alpha-fructose with beta-1, 2 glycosidic bond and connecting a glucose residue at the end thereof, and the molecular formula is
(n = 2-59), and the enzymolysis product is fructose and glucose. Therefore, the main pretreatmentThe process is to remove interferents to ensure that the enzymolysis of levan is not interfered, and the specific detection principle is shown in figure 1.
It is understood that fructan, formed by alpha-fructose linked by beta-1, 2 glycosidic bonds, is terminated by a glucose residue. Calculating the average polymerization degree of fructose and glucose generated by enzymatic hydrolysis of fructan, wherein the ratio of the amount of fructose to the amount of glucose is DP, 1 molecule of fructan (GFn) is completely enzymatically hydrolyzed to generate 1 molecule of glucose and n molecules of fructose, and the polymerization degree is n (n = 2-59).
Average degree of polymerization DP =
(ii) a Wherein, XFThe content of fructose generated by enzymolysis is mg/kg;
the content of glucose generated by enzymolysis is mg/kg.
In the invention, the pretreatment process comprises the following steps:
carrying out enzymolysis on lactose, lactulose and galacto-oligosaccharide into glucose, galactose and fructose by using beta-galactosidase, and carrying out enzymolysis on sucrose into fructose and glucose by using sucrase;
reducing the monosaccharides (glucose, fructose and galactose) subjected to enzymolysis into corresponding sugar alcohols by potassium borohydride, and removing the sugar alcohols;
excess potassium borohydride was removed by neutralization with acetic acid.
More specifically, referring to fig. 2, the ion chromatography method for measuring the average polymerization degree of fructan in infant food or dairy products comprises the following steps:
mixing an infant food sample or a dairy product sample with water to prepare a sample solution;
mixing the sample solution, the sucrase solution and the beta-galactosidase solution, carrying out first enzymolysis, then inactivating the enzyme in a boiling water bath for 15min, stopping the enzymolysis reaction, and preparing a first solution; wherein the enzyme activity of the beta-galactosidase solution is 500U/mL-600U/mL;
mixing a sodium hydroxide solution of potassium borohydride with the first solution, reducing monosaccharide into sugar alcohol, removing interference, and preparing a second solution;
neutralizing redundant sodium borohydride with acetic acid, removing potassium borohydride in the second solution, providing a proper pH range for enzymolysis of fructan, mixing with the fructanase solution, carrying out second enzymolysis, inactivating enzyme in boiling water bath for 15min, stopping enzymolysis reaction, and preparing a solution to be detected;
fixing the volume of the solution to be detected, filtering, and performing ion chromatography to obtain the content of fructose
The content of glucose
By passing
And calculating to obtain the average polymerization degree of the fructan.
It will be appreciated that, in the present invention, the enzymatic activity of the beta-galactosidase solution includes, but is not limited to, 500U/mL, 501U/mL, 502U/mL, 503U/mL, 504U/mL, 505U/mL, 506U/mL, 507U/mL, 508U/mL, 509U/mL, 510U/mL, 511U/mL, 512U/mL, 513U/mL, 514U/mL, 515U/mL, 516U/mL, 517U/mL, 518U/mL, 519U/mL, 520U/mL, 530U/mL, 540U/mL, 550U/mL, 560U/mL, 570U/mL, 580U/mL, 590U/mL, 600U/mL. Preferably, the enzymatic activity of the beta-galactosidase solution is 500U/mL.
In one specific example, the preparation of a β -galactosidase solution comprises the steps of: the beta-galactosidase is mixed with a phosphate buffer solution.
More specifically, the concentration of the phosphate buffer solution is 0.08-0.12 mol/L, and the pH value is 6.0. More specifically, the preparation of beta-galactosidase comprises the following steps: weighing beta-galactosidase with the activity of about 1000U, dissolving the beta-galactosidase in 2.0 mL phosphate buffer solution (0.1 mol/L, pH 6.0) to form a beta-galactosidase solution with the activity of 500U/mL, and preparing the beta-galactosidase solution before use.
In a specific example, the conditions of the first enzymatic hydrolysis include: oscillating for 50 min-70 min at the rotating speed of 100r/min at the temperature of 40 ℃.
Preferably, the conditions for the first enzymatic hydrolysis include: oscillating at a rotation speed of 100r/min at a temperature of 40 ℃ for 60 min.
In a specific example, the conditions of the second enzymatic hydrolysis include: oscillating for 35-55 min at the rotating speed of 100r/min at the temperature of 40 ℃.
Preferably, the conditions of the second enzymatic hydrolysis include: the mixture is shaken at a temperature of 40 ℃ and a rotating speed of 100r/min for 40 min.
In a specific example, the conditions for the first enzyme deactivation include: heating in boiling water for 12-18 min.
It is understood that in the present invention, the first inactivation refers to inactivation of sucrase and beta-galactosidase. Preferably, the conditions for the first enzyme deactivation include: through boiling water bath for 15 min.
In a specific example, the second enzyme deactivation conditions include: heating in boiling water for 12-18 min.
It is understood that in the present invention, the second inactivation refers to inactivation of the levanase enzyme. Preferably, the conditions for the second enzyme deactivation include: through boiling water bath for 15 min.
In a specific example, the method further comprises the following steps after mixing the infant food sample or the dairy product sample with water:
the sample solution was prepared by adjusting the solution pH to 4.5 ± 0.1 with acetic acid.
In a specific example, the enzyme activity of the sucrase solution is 4U/mL-5U/mL.
Immediately, the enzymatic activity of the sucrase solution includes, but is not limited to, 4U/mL, 4.1U/mL, 4.2U/mL, 4.3U/mL, 4.4U/mL, 4.5U/mL, 4.6U/mL, 4.7U/mL, 4.8U/mL, 4.9U/mL, 5U/mL. Preferably, the enzymatic activity of the sucrase solution is 4.5U/mL.
In a specific example, the preparation of the sucrase solution comprises the steps of: sucrase is mixed with a sodium maleate buffer solution.
More specifically, the concentration of the sodium maleate buffer solution is 90 mmol/L-110 mmol/L, and the pH value is 6.5. More specifically, the preparation of the sucrase solution comprises the following steps: 300U of sucrase was dissolved in 66mL of a sodium maleate buffer solution (100mmol/L, pH 6.5).
In a specific example, the enzyme activity of the levan enzyme solution is 40U/mL-60U/mL.
It is understood that, in the present invention, the enzymatic activity of the levanase solution includes, but is not limited to, 40U/mL, 41U/mL, 42U/mL, 43U/mL, 44U/mL, 45U/mL, 46U/mL, 47U/mL, 48U/mL, 49U/mL, 50U/mL, 51U/mL, 52U/mL, 53U/mL, 54U/mL, 55U/mL, 56U/mL, 57U/mL, 58U/mL, 59U/mL, 60U/mL. Preferably, the enzymatic activity of the levan enzyme solution is 50U/mL.
In one specific example, the preparation of the levanase solution comprises the steps of: the levanase enzyme is mixed with a sodium acetate buffer solution.
More specifically, the concentration of the sodium acetate buffer solution is 0.15 mol/L-0.25 mol/L, and the pH value is 4.5. More specifically, the preparation of the levanase solution comprises the following steps: 2000U of the levan enzyme is transferred and diluted by 40 times by using sodium acetate buffer solution (0.2 mol/L, pH4.5), and the levan enzyme solution with the enzyme activity of 50U/mL is formed.
In one specific example, the removing of the potassium borohydride in the second solution comprises the following steps:
and mixing the second solution with 0.4-0.6 mol/L acetic acid aqueous solution.
It is understood that, in the present invention, the concentration of the aqueous acetic acid solution includes, but is not limited to, 0.4mol/L, 0.41 mol/L, 0.42 mol/L, 0.43 mol/L, 0.44 mol/L, 0.45 mol/L, 0.46 mol/L, 0.47 mol/L, 0.48 mol/L, 0.49 mol/L, 0.5mol/L, 0.51 mol/L, 0.52 mol/L, 0.53 mol/L, 0.54 mol/L, 0.55 mol/L, 0.56 mol/L, 0.57 mol/L, 0.58 mol/L, 0.59 mol/L, 0.6 mol/L.
In one particular example, the preparation of the aqueous acetic acid solution comprises the steps of: 1.5mL of glacial acetic acid were aspirated and diluted to 50mL with water.
In one specific example, the preparation of a sodium hydroxide solution of potassium borohydride comprises the steps of: 0.150g of sodium borohydride was weighed out and dissolved in 10mL of 0.1mol/L NaOH solution.
In one particular example, the conditions for ion chromatographic detection include: the leacheate A is 0.1-0.2 mol/L of sodium hydroxide, the leacheate B is a mixed solution of 0.1-0.2 mol/L of sodium hydroxide and 0.5-1.5 mol/L of sodium acetate, and the leacheate C is water.
More specifically, the eluent A is 0.15mol/L sodium hydroxide, the eluent B is a mixed solution of 0.15mol/L sodium hydroxide and 1mol/L sodium acetate, and the eluent C is ultrapure water.
In a specific example, the ion chromatography detection conditions further include: gradient leaching is adopted; the procedure for gradient elution included: 0 min-14 min, wherein the volume percentage of the leacheate A is 20%, and the volume percentage of the leacheate C is 80%; 14 min-14.1 min, wherein the volume percentage of the leacheate A is changed from 20% to 0%, and the volume percentage of the leacheate C is changed from 80% to 0%; 14.1-17 min, wherein the volume percentage of the leacheate A is kept to be 0%, and the volume percentage of the leacheate C is kept to be 0%; 17 min-17.1 min, wherein the volume percentage of the leacheate A is kept at 0%, and the volume percentage of the leacheate C is changed from 0% to 100%; 17.1-20 min, wherein the volume percentage of the leacheate A is kept to be 0%, and the volume percentage of the leacheate C is kept to be 100%; 20 min-20.1 min, wherein the volume percentage of the leacheate A is changed from 0% to 20%, and the volume percentage of the leacheate C is changed from 100% to 80%; and (3) 20.1-30 min, wherein the volume percentage of the leacheate A is kept to be 20%, and the volume percentage of the leacheate C is kept to be 80%.
In a specific example, the ion chromatography detection conditions further include: the flow rate is 1 mL/min-1.4 mL/min; the column temperature is 28-32 ℃; the sample injection amount is 8-12 mu L; an anion analysis column was used.
Preferably, the ion chromatography detection conditions further comprise: the flow rate is 1.2 mL/min; the column temperature is 30 ℃; the sample injection amount is 10 mu L; an anion analysis column was used.
More specifically, the high-phase anion analysis column was IonPac PA1 (250 mm. times.4 mm, Thermo Co.).
The ion chromatography method for measuring the average polymerization degree of fructan in infant food or dairy products according to the present invention will be described in further detail with reference to the following specific examples. The starting materials used in the following examples are all commercially available products unless otherwise specified.
Example 1
The embodiment provides an ion chromatography method for measuring average polymerization degree in infant formula milk powder, which comprises the following specific steps:
preparation of reagent
Beta-galactosidase solution (500U/mL): weighing beta-galactosidase with the activity of about 1000U, dissolving the beta-galactosidase in 2.0 mL phosphate buffer solution (0.1 mol/L, pH 6.0) to form a beta-galactosidase solution with the activity of 500U/mL, and preparing the beta-galactosidase solution before use.
Sucrase solution (4.5U/mL): 300U of sucrase was dissolved in 66mL of a sodium maleate buffer solution (100mmol/L, pH 6.5).
Potassium borohydride-sodium hydroxide solution (15 mg/mL): 0.150g of sodium borohydride was weighed out and dissolved in 10mL of 0.1mol/L NaOH solution.
Fructan solution (50U/mL): 2000U of the levan enzyme is transferred and diluted by 40 times by using sodium acetate buffer solution (0.2 mol/L, pH4.5), and the levan enzyme solution with the enzyme activity of 50U/mL is formed.
Acetic acid solution (0.5 mol/L): 1.5mL of glacial acetic acid were aspirated and diluted to 50mL with water.
II, ion chromatography conditions
The anion analysis column was IonPac PA1 (250 mm. times.4 mm, Thermo Co.);
leacheate: a is 0.15mol/L NaOH solution, B is 0.15mol/L NaOH +1.0 mol/L sodium acetate mixed solution, C is ultrapure water; elution gradient procedure: 0 min-14 min, wherein the volume percentage of the leacheate A is 20%, and the volume percentage of the leacheate C is 80%; 14 min-14.1 min, wherein the volume percentage of the leacheate A is changed from 20% to 0%, the volume percentage of the leacheate C is changed from 80% to 0%, and the volume percentage of the leacheate B is changed from 0% to 100%; 14.1-17 min, and keeping the volume percentage of the leacheate B at 100%; 17 min-17.1 min, wherein the volume percentage of the leacheate B is changed from 100% to 0%, and the volume percentage of the leacheate C is changed from 0% to 100%; 17.1-20 min, and keeping the volume percentage of the leacheate C at 100%; 20 min-20.1 min, wherein the volume percentage of the leacheate A is changed from 0% to 20%, and the volume percentage of the leacheate C is changed from 100% to 80%; 20.1-30 min, wherein the volume percentage of the leacheate A is kept at 20%, and the volume percentage of the leacheate C is kept at 80%; flow rate: 1.20 mL/min; the column temperature is 30 ℃; the amount of the sample was 10.0. mu.L.
Thirdly, measuring steps
Firstly, weighing a sample, placing the sample in a beaker, adding warm water to dissolve the sample, adjusting the pH value to 4.5 +/-0.1 by using acetic acid, and transferring the sample to a volumetric flask for constant volume.
And secondly, transferring 200 mu L of sample solution, adding 200 mu L of sucrose enzyme solution and 200 mu L of beta-galactosidase solution into the sample solution, placing the sample solution in an oscillating water bath at 40 ℃, oscillating at 100r/min for enzymolysis for 1h, taking out and cooling to room temperature. Then inactivating enzyme in boiling water bath for 15 min.
And thirdly, adding 0.50mL of potassium borohydride-sodium hydroxide solution into the sample solution, placing the sample solution in an oscillating water bath at 40 ℃, oscillating the sample solution for 0.5h at 100r/min, taking out the sample solution, and cooling the sample solution to room temperature.
And fourthly, adding 1.00mL of acetic acid solution into the sample solution, and standing for 15 min.
And fifthly, adding 1.00mL of levan hydrolase solution into the sample solution, placing the sample solution in a 40 ℃ oscillating water bath for enzymolysis for 45min, then inactivating the enzyme in a boiling water bath for 15min, cooling to room temperature, and fixing the volume to 10 mL. Filtering, and waiting for testing.
And sixthly, qualitatively and quantitatively detecting the enzymolysis products in the sample by using the ion chromatography method, and calculating the average polymerization degree of the total fructan according to the content ratio of fructose and glucose generated by enzymolysis.
The standard spectrum is shown in FIG. 3, and the enzymolysis products of fructan are glucose and fructose. The reference peak time of glucose is 8.0min, and the reference peak time of fructose is 9.9 min.
The quantitative determination is carried out by a peak area external standard method, the fructose and the glucose are in the range of 0.25-25mg/L, and the linear correlation coefficient is more than 0.999.
Fourth, recovery rate determination of infant formula milk powder
Weighing about 2.5g of the infant formula milk powder sample, placing the infant formula milk powder sample in a beaker, adding a certain amount of fructan (the addition level is 3%) to perform a standard recovery experiment, performing the recovery experiment according to the method of the first to third steps, and repeating the measurement for 3 times. Removing impurity interferents by enzymolysis, decomposing the levan glycanase in a sample into fructose and glucose by using the levan glycanase, and detecting the ratio of the fructose and the glucose generated by the levan by using ion chromatography to calculate the average polymerization degree. Five different fructan addition types, ratios and corresponding results are shown in table 1.
Example 2
This example provides an ion chromatography method for measuring the average polymerization degree of fructan in a liquid milk sample, in which the preparation of reagents, ion chromatography conditions and measurement procedures are all the same as those in example 1, except that the sample in this example is a liquid milk sample:
about 2.5g of the liquid milk sample was weighed into a beaker, a standardized recovery experiment was performed by adding an amount of levan (addition level 3%), a recovery experiment was performed as described in one to three of examples 1, and the measurements were repeated 3 times. Removing impurity interferents by enzymolysis, decomposing the levan glycanase in a sample into fructose and glucose by using the levan glycanase, and detecting the ratio of the fructose and the glucose generated by the levan by using ion chromatography to calculate the average polymerization degree. Five different fructan addition types, ratios and corresponding results are shown in table 2.
Example 3
The present example provides an ion chromatography method for measuring the average polymerization degree of fructan in a milk powder sample, wherein the preparation of reagents, ion chromatography conditions and measurement steps are all the same as those in example 1, except that the sample in this example is a milk powder sample:
about 2.5g of the milk powder sample was weighed into a beaker, and a standard recovery experiment was performed by adding a certain amount of fructan (addition level 3%), performing the recovery experiment as described in one to three of examples 1, and repeating the measurement 3 times. Removing impurity interferents by enzymolysis, decomposing the levan glycanase in a sample into fructose and glucose by using the levan glycanase, and detecting the ratio of the fructose and the glucose generated by the levan by using ion chromatography to calculate the average polymerization degree. Five different fructan addition types, ratios and corresponding results are shown in table 3.
Example 4
This example examined the effect of β -galactosidase usage on the results as follows:
about 2.5g of a quality control sample of a milk powder base (characteristic value of fructan content: 12.3 g/kg, wherein lactose content is about 45%) was weighed out and placed in a beaker, and the test was carried out as described in one to three of examples 1 except for the concentration of the β -galactosidase solution, and the measurement was repeated 2 times. The effect of 200. mu.L each of 200U/mL, 400U/mL, 500U/mL, 600U/mL, and 700U/mL beta-galactosidase solution usage on the results was examined. The results are shown in Table 4 and FIG. 4.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the appended claims. Therefore, the protection scope of the present patent shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.
Claims (10)
1. An ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy products is characterized by comprising the following steps:
mixing an infant food sample or a dairy product sample with water to prepare a sample solution;
mixing the sample solution, the sucrase solution and the beta-galactosidase solution, carrying out first enzymolysis, and then carrying out first enzyme deactivation to prepare a first solution; wherein the enzyme activity of the beta-galactosidase solution is 500-600U/mL;
mixing a sodium hydroxide solution of potassium borohydride with the first solution to prepare a second solution;
removing the potassium borohydride in the second solution, mixing the potassium borohydride with a levanase solution, carrying out second enzymolysis, and then carrying out second enzyme deactivation to prepare a solution to be detected;
carrying out ion chromatography detection on the solution to be detected to obtain the content of fructose
The content of glucose
By passing
And calculating to obtain the average polymerization degree of the fructan.
2. The ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy product according to claim 1, wherein the conditions of the first enzymatic hydrolysis comprise: oscillating for 50min to 70min at the temperature of 40 ℃ and the rotating speed of 100 r/min;
the conditions of the second enzymolysis comprise: oscillating for 35-55 min at the rotating speed of 100r/min at the temperature of 40 ℃.
3. The ion chromatography determination method of the average polymerization degree of fructan in infant food or dairy products of claim 1, characterized in that the conditions of the first enzyme inactivation comprise: heating in boiling water for 12-18 min;
the conditions for the second enzyme inactivation include: heating in boiling water for 12-18 min.
4. The ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy product according to claim 1, wherein the method further comprises the following steps after mixing the infant food sample or dairy product sample with water:
the sample solution was prepared by adjusting the solution pH to 4.5 ± 0.1 with acetic acid.
5. The ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy product according to claim 1, wherein the enzymatic activity of said sucrase solution is 4 to 5U/mL.
6. The ion chromatography determination method for the average polymerization degree of fructan in infant food or dairy products according to claim 1, wherein the enzymatic activity of the fructan solution is 40-60U/mL.
7. The ion chromatography determination method of the average polymerization degree of fructan in infant food or dairy product according to claim 1, characterized in that the removal of potassium borohydride from said second solution comprises the following steps:
the second solution was mixed with 0.5mol/L of an aqueous acetic acid solution.
8. The method for ion chromatography determination of the average polymerization degree of fructan in infant food or dairy product according to any one of claims 1 to 7, wherein the conditions for ion chromatography detection comprise: the leacheate A is 0.15mol/L of sodium hydroxide, the leacheate B is a mixed solution of 0.15mol/L of sodium hydroxide and 1.0mol/L of sodium acetate, and the leacheate C is water.
9. The method for ion chromatography determination of the average degree of polymerization of fructans in infant food or dairy products according to claim 8, wherein the conditions of ion chromatography detection further comprise: gradient leaching is adopted; the gradient elution procedure comprises: 0 min-14 min, wherein the volume percentage of the leacheate A is 20%, and the volume percentage of the leacheate C is 80%; 14-14.1 min, wherein the volume percentage of the leacheate A is changed from 20% to 0%, the volume percentage of the leacheate C is changed from 80% to 0%, and the volume percentage of the leacheate B is changed from 0% to 100%; 14.1-17 min, and keeping the volume percentage of the eluent B at 100%; 17 min-17.1 min, wherein the volume percentage of the leacheate B is changed from 100% to 0%, and the volume percentage of the leacheate C is changed from 0% to 100%; 17.1-20 min, and keeping the volume percentage of the leacheate C at 100%; 20 min-20.1 min, wherein the volume percentage of the leacheate A is changed from 0% to 20%, and the volume percentage of the leacheate C is changed from 100% to 80%; and (3) 20.1-30 min, wherein the volume percentage of the leacheate A is kept to be 20%, and the volume percentage of the leacheate C is kept to be 80%.
10. The method for ion chromatography determination of the average degree of polymerization of fructans in infant food or dairy products according to claim 8, wherein the conditions of ion chromatography detection further comprise: the flow rate is 1 mL/min-1.2 mL/min; the column temperature is 28-30 ℃; the sample injection amount is 10 mu L; an anion analysis column was used.
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| CN106706813A (en) * | 2017-02-07 | 2017-05-24 | 中检科（北京）测试技术有限公司 | Ion chromatography determination method for content of polyfructose in food and milk powder for infants and young children |
| CN107449840A (en) * | 2017-07-13 | 2017-12-08 | 宁波市食品检验检测研究院 | A kind of detection method of formula milk/determination of FOS in milk powder total amount |
| JP2018163003A (en) * | 2017-03-24 | 2018-10-18 | 日立化成株式会社 | Separation material, column, and method for manufacturing separation material |
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| CN106706813A (en) * | 2017-02-07 | 2017-05-24 | 中检科（北京）测试技术有限公司 | Ion chromatography determination method for content of polyfructose in food and milk powder for infants and young children |
| JP2018163003A (en) * | 2017-03-24 | 2018-10-18 | 日立化成株式会社 | Separation material, column, and method for manufacturing separation material |
| CN107449840A (en) * | 2017-07-13 | 2017-12-08 | 宁波市食品检验检测研究院 | A kind of detection method of formula milk/determination of FOS in milk powder total amount |
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