Method for extracting and detecting folic acid in protein-starch-containing food by combining enzymolysis and impurity removal

Abstract

The application discloses a kind of enzyme hydrolysis and impurity removal combined extraction detection methods of protein-containing starch food folate, it is characterized in that, comprising the following steps: (1) sample pretreatment: food sample is homogenized and crushed after classification, accurately sample is weighed in sterile centrifuge tube;(2) complex enzymolysis: 20mL pH7.2 0.05mol / L phosphate buffer is added to sample, then food-grade amylase and neutral protease are added, and enzymolysis is carried out in 50-60 ℃ water bath, and every 10min oscillation is carried out once;(3) heat inactivation and anti-oxidation extraction: enzyme is inactivated, after cooling, supplement 0.1% analytically pure ascorbic acid-containing phosphate buffer to 40mL, and 95 ℃ extraction is carried out for 20min, and during oscillation is carried out 3 times;(4) classification impurity removal: after extraction liquid is cooled to ≤30 ℃, centrifugation is carried out for 10min, and supernatant is filtered by sterile water system filter membrane, and filter liquor is collected, and folate extraction liquid is obtained.The step-by-step addition mode of amylase, neutral protease, pectinase and xylanase is used in the application, to avoid the activity inhibition between different enzyme preparations, and each enzyme species plays a targeted role in optimal reaction period.

Description

Technical Field

[0001] This invention relates to the field of extraction and detection methods, and more particularly to a combined enzymatic hydrolysis and impurity removal extraction and detection method for folic acid in protein-starch foods. Background Technology

[0002] The Forms of Folic Acid in Food Matrices and the Challenges of Detection Folic acid (FA) typically exists in three forms in natural or fortified foods: free, protein-bound, and starch-encapsulated. For complex matrices containing both protein and starch (such as cereal protein blends, meal replacement powders, and infant formula), traditional extraction methods face the following bottlenecks: Protein-bound folic acid needs to be hydrolyzed by proteases to be released, but proteases have poor penetration into the starch matrix, resulting in a low extraction rate. Starch gelatinization causes a sharp increase in viscosity, making centrifugation / filtration difficult and easily clogging the filter membrane. Folic acid is extremely sensitive to light, heat, and oxygen. Conventional high-temperature enzyme inactivation steps (100℃, 5min) can cause a loss of 5-15%. Residual proteins and dextrin particles in the extract can interfere with subsequent microbiological detection, causing blank turbidity and poor linearity of the standard curve.

[0003] Kit-based methods (ELISA or enzyme-mediated colorimetric assays) are rapid, but are susceptible to matrix interference and have a narrow linear range. The national standard microbial method (Lactobacillus rhamnosus ATCC7469) has high accuracy, but the culture time is long (40-48h), and different substrates require empirical dilution, otherwise the "hook effect" is likely to occur.

[0004] Existing technologies do not provide a "dual-track" strategy for different regulatory scenarios. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the present invention proposes a method for the combined extraction and detection of folic acid from protein starch-containing foods by enzymatic hydrolysis and impurity removal.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a method for enzymatic hydrolysis and impurity removal combined extraction and detection of folic acid in protein-starch foods, comprising the following steps: (1) Sample pretreatment: After homogenizing and pulverizing the food sample, grade it to 0.3-0.5 mm, and accurately weigh 0.1-2.0 g of the sample into a sterile centrifuge tube; (2) Compound enzymatic hydrolysis: Add 20 mL of phosphate buffer with pH 7.2 and a concentration of 0.05 mol / L to the sample, then add 0.05-0.07 g of food-grade amylase and 0.05-0.07 g of neutral protease, and enzymatically hydrolyze in a water bath at 50-55℃ for 30-60 min, shaking once every 10 min; (3) Heat inactivation and anti-oxidation extraction: Inactivate the enzyme in a 95℃ water bath for 10-12 min, cool and add phosphate buffer containing 0.1% analytical grade ascorbic acid to 40 mL, extract at 95-100℃ for 20-25 min, shaking 3 times during the process; (4) Grading and impurity removal: After the extract is cooled to ≤30℃, it is centrifuged at 0-4℃ and 8000-10000r / min for 10-15min. The supernatant is filtered through a 0.22μm sterile aqueous filter membrane, and the filtrate is collected to obtain folic acid extract. Preferably, in the compound enzymatic hydrolysis step, the mass ratio of amylase to neutral protease is 1:1, and the enzymatic hydrolysis temperature fluctuation range is ≤±1℃. Preferably, the testing is completed through the following steps: (1) Sample dilution: Dilute the extract to 0.160-1.280 μg / 100g using the kit method, or to 0.20-0.30 ng / mL using the national standard microbiological method; (2) Preparation of standard curve: The kit method uses serial dilution of the matching standard solution, and the national standard method is prepared according to GB5009.211-2022 to ensure that the correlation coefficient R² of the standard curve is ≥0.99; (3) Microbial detection: The kit method involves incubating at 37℃ in the dark for 44-48h and measuring the turbidity at 620nm; the national standard method uses Lactobacillus rhamnosus ATCC7469, incubating at 36±1℃ for 20-40h in test tubes and at 36±1℃ for 32-48h in microplates, and measuring the absorbance at 540-610nm. (4) Calculation of results: Substitute into the standard curve formula, take the average of two parallel determinations, retain 2 decimal places for the kit method and 3 significant figures for the national standard method. Preferably, the microbial culture process uses a sterile operating table to control contamination, and the experiment needs to be repeated if the blank tube becomes turbid. Preferably, the food includes cereal protein mixtures, starch-based meal replacement powders, protein starch pastries, canned whole grain protein products, infant protein starch complementary foods, and folic acid-fortified foods. Preferably, during the compound enzymatic hydrolysis process, amylase and neutral protease are added first, vortexed and mixed, and after 20 seconds, 0.01-0.02g of pectinase is introduced and mixed. After mixing for 30 seconds, 0.005-0.01g of xylanase is added.

[0007] Preferably, in the fractionation and impurity removal step, the extract is placed in an ice-water bath at 0-4℃ and magnetically stirred at 200 rpm. The temperature is lowered to ≤30℃ within 3 minutes. First, 0.1-0.2% of the extract volume of chitosan hydrochloride is added. The mixture is stirred at a constant speed of 600-800 rpm for 30-40 seconds, and then the speed is reduced to 100 rpm for 2 minutes to form 50-200µm flocs. After stirring evenly, the mixture is allowed to stand at room temperature for 15 minutes. After the flocs have completely settled, the mixture is centrifuged at 4℃ and 8000 rpm for 10 minutes to remove the flocs.

[0008] Preferably, 1 mL / 100 mg citric acid-ethylenediamine carbon dot solution is added to the residue after centrifugation. After mixing, the mixture is microwaved at 90°C for 30 seconds. After microwave treatment, the mixture is immediately placed in an ice-water bath for 30 seconds to lower the system temperature to ≤40°C to prevent thermal quenching of the carbon dot fluorescence. Then, the mixture is separated and the carbon dots are recovered through an elution-neutralization-recycling process. The mass ratio of citric acid to ethylenediamine is 2-3:0.67-1. First, chitosan hydrochloride is used to precipitate large molecular protein-dextrin flocs. Then, citric acid-ethylenediamine carbon dot solution is used to target and capture residual polyphenols, pigments, and small molecular proteins to form a "CDs-impurity" complex with a particle size of <100nm. The two steps are complementary in particle size, which can further reduce turbidity by 30-40% and extend the filter membrane clogging time from 60s to >5min, thus achieving two-stage purification of "coarse-fine".

[0009] Citric acid-ethylenediamine carbon dot solution has strong fluorescence recognition and adsorption properties, which can specifically bind to the bound folic acid that has not been released in the centrifugation residue. Microwave-assisted coordination binding of folic acid and carbon dots is promoted, and secondary recovery of folic acid accounts for 5%-8% of the total extraction, thereby increasing the overall extraction recovery rate from 92%-95% to 97%-99%.

[0010] Preferably, the microbial culture process uses a sterile operating table to control contamination, and the experiment needs to be repeated if the blank tube becomes turbid. Preferably, the food products include cereal protein mixtures, starch-based meal replacement powders, protein starch pastries, canned whole grain protein products, infant protein starch complementary foods, and folic acid-fortified foods. Preferably, food companies should prioritize using the reagent kit method for factory testing, while third-party testing institutions should use the national standard microbiological method for routine testing and regulatory departments should use the method during supervision and sampling. When optimizing food research and development, a combination of the reagent kit method and the national standard method should be used for verification.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The stepwise addition of amylase, neutral protease, pectinase and xylanase is adopted to avoid the activity inhibition between different enzyme preparations. For example, xylanase has a slight antagonistic effect on amylase. Each enzyme plays a targeted role at the optimal reaction time. The folic acid release rate can be increased by 8%-10% compared with simultaneous addition and by 15%-20% compared with the original single dual enzyme system.

[0012] 2. First, chitosan hydrochloride is used to precipitate large molecular protein-dextrin flocs, and then citric acid-ethylenediamine carbon dot solution is used to target and capture residual polyphenols, pigments, and small molecular proteins, achieving a two-stage purification process of "coarse-fine".

[0013] 3. First, the starch-protein cross-linking structure is broken by amylase-protease, then pectinase is used to degrade intercellular pectin, and finally xylanase hydrolyzes hemicellulose, peeling away the three-dimensional network of "starch-protein-dietary fiber" layer by layer, so that the bound folic acid encapsulated in it can be fully released, solving the pain point of traditional enzymatic hydrolysis of "surface degradation and internal encapsulation".

[0014] 4. By utilizing the strong fluorescence recognition and adsorption properties of the citric acid-ethylenediamine carbon dot solution, excess components in the filter residue are filtered out, while folic acid remains in the clear liquid and can be directly used for subsequent detection. Detailed Implementation

[0015] The invention will now be described in detail with reference to the embodiments.

[0016] Example 1

[0017] A method for the combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods, comprising the following steps: (1) Sample pretreatment: After homogenizing and pulverizing the food sample, grade it to 0.3 mm and accurately weigh 0.1 g of the sample into a sterile centrifuge tube; (2) Compound enzymatic hydrolysis: Add 20 mL of phosphate buffer with pH 7.2 and a concentration of 0.05 mol / L to the sample, then add 0.05 g of food-grade amylase and 0.05 g of neutral protease, vortex to mix, and after 20 s, introduce 0.01 g of pectinase and mix. After mixing for 30 s, add 0.005 g of xylanase and hydrolyze in a 50 ℃ water bath for 30 min. The temperature fluctuation range of the enzymatic hydrolysis is ≤ ±1 ℃, and shake once every 10 min. (3) Heat inactivation and anti-oxidation extraction: Inactivate the enzyme in a 95℃ water bath for 10 min, cool and add phosphate buffer containing 0.1% analytical grade ascorbic acid to 40 mL, extract at 95℃ for 20 min, shaking 3 times during the process; (4) Grading and impurity removal: Place the extract in a 0℃ ice-water bath and stir with a magnetic stirrer at 200 rpm. Cool down to ≤30℃ within 3 min. First, add 0.1% of the extract volume of chitosan hydrochloride and stir at a constant speed of 600 rpm for 30 s. Then, reduce the speed to 100 rpm and continue for 2 min to form 50µm flocs. After stirring evenly, let stand at room temperature for 15 min. After the flocs have completely settled, centrifuge at 0℃ and 80000 r / min for 10 min. Filter the supernatant through a 0.22μm sterile aqueous filter membrane and collect the filtrate to obtain the folic acid extract. The testing is then completed through the following steps: (1) Sample dilution: The extract was diluted to 0.160 μg / 100g using the kit method and to 0.20 ng / mL using the national standard microbiological method; (2) Preparation of standard curve: The kit method uses serial dilution of the matching standard solution, and the national standard method is prepared according to GB5009.211-2022 to ensure that the correlation coefficient R² of the standard curve is ≥0.99; (3) Microbial detection: The kit method involves incubating at 37℃ in the dark for 44-48h and measuring the turbidity at 620nm; the national standard method uses Lactobacillus rhamnosus ATCC7469, incubating at 36±1℃ for 20h in test tubes and at 36±1℃ for 32h in microplates, and measuring the absorbance at 540nm. (4) Calculation of results: Substitute into the standard curve formula, take the average of two parallel determinations, retain 2 decimal places for the kit method and 3 significant figures for the national standard method. The microbial culture process uses a sterile operating table to control contamination; if the blank tube becomes turbid, the experiment must be repeated. Food products include cereal protein mixes, starch-based meal replacement powders, protein starch pastries, canned whole grain protein products, infant protein starch complementary foods, and folic acid-fortified foods.

[0018] Sample Information The commercially available "high-protein oat meal replacement powder" is labeled with a folic acid fortification of 200µg / 100g. Its ingredients include: 45% oat flour, 30% soy protein isolate, 15% maltodextrin, and 10% mineral / vitamin premix.

[0019] Results: The reagent kit method yielded 198.68 µg / 100g, while the national standard method yielded 199 µg / 100g.

[0020] Example 2

[0021] A method for the combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods, comprising the following steps: (1) Sample pretreatment: After homogenizing and pulverizing the food sample, grade it to 0.4 mm and accurately weigh 0.5 g of the sample into a sterile centrifuge tube; (2) Compound enzymatic hydrolysis: Add 20 mL of phosphate buffer with pH 7.2 and a concentration of 0.05 mol / L to the sample, then add 0.06 g of food-grade amylase and 0.06 g of neutral protease, vortex to mix, introduce 0.01 g of pectinase after 20 s and mix, after mixing for 30 s, add 0.005 g of xylanase, and enzymatically hydrolyze in a 52℃ water bath for 35 min. The enzymatic hydrolysis temperature fluctuation range is ≤ ±1℃, and shake once every 10 min. (3) Heat inactivation and anti-oxidation extraction: Inactivate the enzyme in a 95℃ water bath for 10 min, cool and add phosphate buffer containing 0.1% analytical grade ascorbic acid to 40 mL, extract at 95℃ for 20 min, shaking 3 times during the process; (4) Grading and impurity removal: Place the extract in an ice-water bath at 1℃ and stir with a magnetic stirrer at 200rpm. Cool down to ≤30℃ within 3min. First, add 0.2% of the extract volume of chitosan hydrochloride and stir at a uniform speed of 800rpm magnetic rotor for 30s. Then, reduce the speed to 100rpm and continue for 2min to form 50-200µm flocs. After stirring evenly, let stand at room temperature for 15min. After the flocs have completely settled, centrifuge at 1℃ and 8000r / min for 13min. Filter the supernatant through a 0.22μm sterile aqueous filter membrane and collect the filtrate to obtain folic acid extract. The testing is then completed through the following steps: (1) Sample dilution: The extract was diluted to 0.160 μg / 100g using the kit method and to 0.20 ng / mL using the national standard microbiological method; (2) Preparation of standard curve: The kit method uses serial dilution of the matching standard solution, and the national standard method is prepared according to GB5009.211-2022 to ensure that the correlation coefficient R² of the standard curve is ≥0.99; (3) Microbial detection: The kit method involves incubating at 37℃ in the dark for 44h and measuring the turbidity at 620nm; the national standard method uses Lactobacillus rhamnosus ATCC7469, incubating at 36±1℃ for 20h in test tubes and at 36±1℃ for 32h in microplates, and measuring the absorbance at 540nm. (4) Calculation of results: Substitute into the standard curve formula, take the average of two parallel determinations, retain 2 decimal places for the kit method and 3 significant figures for the national standard method. The microbial culture process uses a sterile operating table to control contamination; if the blank tube becomes turbid, the experiment must be repeated. Food products include cereal protein mixes, starch-based meal replacement powders, protein starch pastries, canned whole grain protein products, infant protein starch complementary foods, and folic acid-fortified foods.

[0022] Sample Information The commercially available "high-protein oat meal replacement powder" is labeled with a folic acid fortification of 200µg / 100g. Its ingredients include: 45% oat flour, 30% soy protein isolate, 15% maltodextrin, and 10% mineral / vitamin premix.

[0023] Results: The reagent kit method yielded 201.61 µg / 100g, while the national standard method yielded 203 µg / 100g.

[0024] Example 3

[0025] A method for the combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods, comprising the following steps: (1) Sample pretreatment: After homogenizing and pulverizing the food sample, grade it to 0.5 mm and accurately weigh 2.0 g of sample into a sterile centrifuge tube; (2) Compound enzymatic hydrolysis: Add 20 mL of phosphate buffer with pH 7.2 and a concentration of 0.05 mol / L to the sample, then add 0.07 g of food-grade amylase and 0.07 g of neutral protease, vortex to mix, and after 20 s, introduce 0.02 g of pectinase and mix. After mixing for 30 s, add 0.01 g of xylanase and hydrolyze in a 55 ℃ water bath for 60 min. The temperature fluctuation range of the enzymatic hydrolysis is ≤ ±1 ℃, and shake once every 10 min. (3) Heat inactivation and anti-oxidation extraction: Inactivate the enzyme in a 95℃ water bath for 12 min, cool and add phosphate buffer containing 0.1% analytical grade ascorbic acid to 40 mL, extract at 100℃ for 25 min, shaking 3 times during the process; (4) Grading and impurity removal: Place the extract in a 4℃ ice-water bath and stir with a magnetic stirrer at 200 rpm. Cool down to ≤30℃ within 3 min. First, add 0.2% of the extract volume of chitosan hydrochloride and stir at a uniform speed of 800 rpm for 30-40 s. Then, reduce the speed to 100 rpm and continue for 2 min to form 200µm flocs. After stirring evenly, let stand at room temperature for 15 min. After the flocs have completely settled, centrifuge at 4℃ and 10000 r / min for 15 min. Filter the supernatant through a 0.22μm sterile aqueous filter membrane and collect the filtrate to obtain the folic acid extract. The testing is then completed through the following steps: (1) Sample dilution: The extract was diluted to 0.160 μg / 100g using the kit method and to 0.20 ng / mL using the national standard microbiological method; (2) Preparation of standard curve: The kit method uses serial dilution of the matching standard solution, and the national standard method is prepared according to GB5009.211-2022 to ensure that the correlation coefficient R² of the standard curve is ≥0.99; (3) Microbial detection: The kit method involves incubating at 37℃ in the dark for 44h and measuring the turbidity at 620nm; the national standard method uses Lactobacillus rhamnosus ATCC7469, incubating at 36±1℃ for 20h in test tubes and at 36±1℃ for 32h in microplates, and measuring the absorbance at 540nm. (4) Calculation of results: Substitute into the standard curve formula, take the average of two parallel determinations, retain 2 decimal places for the kit method and 3 significant figures for the national standard method. The microbial culture process uses a sterile operating table to control contamination; if the blank tube becomes turbid, the experiment must be repeated. Food products include cereal protein mixes, starch-based meal replacement powders, protein starch pastries, canned whole grain protein products, infant protein starch complementary foods, and folic acid-fortified foods.

[0026] Sample Information The commercially available "high-protein oat meal replacement powder" is labeled with a folic acid fortification of 200µg / 100g. Its ingredients include: 45% oat flour, 30% soy protein isolate, 15% maltodextrin, and 10% mineral / vitamin premix.

[0027] Results: The reagent kit method yielded 199.68 µg / 100g, while the national standard method yielded 201 µg / 100g.

[0028] Compare with Example 1 Compared with Example 2, Control Example 1 did not contain pectinase and xylanase, but was otherwise the same as Example 2.

[0029] Sample Information The commercially available "high-protein oat meal replacement powder" is labeled with a folic acid fortification of 200µg / 100g. Its ingredients include: 45% oat flour, 30% soy protein isolate, 15% maltodextrin, and 10% mineral / vitamin premix.

[0030] Results: The reagent kit method yielded 169.88 µg / 100g, while the national standard method yielded 174 µg / 100g.

[0031] Compare with Example 2 Compared with Example 2, in the fractionation and impurity removal process, chitosan hydrochloride was not added to the extract, but otherwise it was the same as Example 2.

[0032] Sample Information The commercially available "high-protein oat meal replacement powder" is labeled with a folic acid fortification of 200µg / 100g. Its ingredients include: 45% oat flour, 30% soy protein isolate, 15% maltodextrin, and 10% mineral / vitamin premix.

[0033] Results: The reagent kit method yielded 181.89 µg / 100g, while the national standard method yielded 183 µg / 100g.

[0034] Compare with Example 3 Compared to Example 2, only papain was applied; the rest was the same as in Example 2.

[0035] Sample Information The commercially available "high-protein oat meal replacement powder" is labeled with a folic acid fortification of 200µg / 100g. Its ingredients include: 45% oat flour, 30% soy protein isolate, 15% maltodextrin, and 10% mineral / vitamin premix.

[0036] Results: The reagent kit method yielded 125.83 µg / 100g, while the national standard method yielded 132 µg / 100g.

[0037] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the patent and its protection scope should be determined by the appended claims.

Claims

1. A method for the combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods, characterized in that, Includes the following steps: (1) Sample pretreatment: After homogenizing and pulverizing the food sample, grade it to 0.3-0.5 mm, and accurately weigh 0.1-2.0 g of the sample into a sterile centrifuge tube; (2) Compound enzymatic hydrolysis: Add 20 mL of phosphate buffer with pH 7.2 and a concentration of 0.05 mol / L to the sample, then add 0.05-0.07 g of food-grade amylase and 0.05-0.07 g of neutral protease, and enzymatically hydrolyze in a water bath at 50-55℃ for 30-60 min, shaking once every 10 min; (3) Heat inactivation and anti-oxidation extraction: Inactivate the enzyme in a 95℃ water bath for 10-12 min, cool and add phosphate buffer containing 0.1% analytical grade ascorbic acid to 40 mL, extract at 95-100℃ for 20-25 min, shaking 3 times during the process; (4) Grading and impurity removal: After the extract is cooled to ≤30℃, it is centrifuged at 0-4℃ and 8000-10000r / min for 10-15min. The supernatant is filtered through a 0.22μm sterile aqueous filter membrane, and the filtrate is collected to obtain folic acid extract.

2. The method for combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods as described in claim 1, characterized in that, In the compound enzymatic hydrolysis step, the mass ratio of amylase to neutral protease is 1:1, and the enzymatic hydrolysis temperature fluctuation range is ≤±1℃.

3. The method for combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods as described in claim 2, characterized in that, The testing is then completed through the following steps: (1) Sample dilution: Dilute the extract to 0.160-1.280 μg / 100g using the kit method, or to 0.20-0.30 ng / mL using the national standard microbiological method; (2) Preparation of standard curve: The kit method uses serial dilution of the matching standard solution, and the national standard method is prepared according to GB5009.211-2022 to ensure that the correlation coefficient R² of the standard curve is ≥0.99; (3) Microbial detection: The kit method involves incubating at 37℃ in the dark for 44-48h and measuring the turbidity at 620nm; the national standard method uses Lactobacillus rhamnosus ATCC7469, incubating at 36±1℃ for 20-40h in test tubes and at 36±1℃ for 32-48h in microplates, and measuring the absorbance at 540-610nm. (4) Calculation of results: Substitute into the standard curve formula, take the average of two parallel determinations, retain 2 decimal places for the kit method and 3 significant figures for the national standard method.

4. The method for combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods according to claim 3 or 4, characterized in that, The food products include cereal protein mixtures, starch-based meal replacement powders, protein starch pastries, canned whole grain protein products, infant protein starch complementary foods, and folic acid-fortified foods.

5. The method for combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods according to claim 2, characterized in that, In the complex enzymatic hydrolysis process, first add amylase and neutral protease, vortex and mix well, then introduce 0.01-0.02g of pectinase and mix well after 20s. After mixing for 30s, add 0.005-0.01g of xylanase.

6. The method for combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods according to claim 5, characterized in that, In the fractionation and impurity removal step, the extract is placed in an ice-water bath at 0-4℃ and magnetically stirred at 200 rpm. The temperature is lowered to ≤30℃ within 3 minutes. First, 0.1-0.2% of the extract volume of chitosan hydrochloride is added. The mixture is stirred at a constant speed of 600-800 rpm for 30-40 seconds, then the speed is reduced to 100 rpm and stirred for another 2 minutes to form 50-200µm flocs. After stirring evenly, the mixture is allowed to stand at room temperature for 15 minutes. After the flocs have completely settled, the mixture is centrifuged at 4℃ and 8000 rpm for 10 minutes to remove the flocs.

7. The method for combined enzymatic hydrolysis and impurity removal extraction and detection of folic acid in protein-starch foods according to any one of claims 2-6, characterized in that, Add 1 mL / 100 mg citric acid-ethylenediamine carbon dot solution to the centrifuged residue, mix, and microwave at 90°C for 30 s. After microwave treatment, immediately place in an ice-water bath for 30 s to lower the system temperature to ≤40°C to prevent thermal quenching of carbon dot fluorescence. Then, separate the carbon dots by sequentially performing an elution-neutralization-recycling process. The mass ratio of citric acid to ethylenediamine is 2-3:0.67-1.