A method for determining the content of amyloid fibril protein

Abstract

The present invention relates to a method for determining the content of amyloid fibril protein, and belongs to the field of food biotechnology. It uses α-amylase to degrade starch in frozen cooked noodles, and improves the accuracy of determining the content of amyloid fibril protein in frozen cooked noodles by combining and optimizing the addition of α-amylase, proteinase K and trypsin. The present invention uses α-amylase to degrade starch in noodles, and by combining and optimizing the addition of α-amylase, proteinase K and trypsin, establishes a method for determining the content of amyloid fibril protein suitable for a noodle product system, which provides technical support for regulating the quality of frozen cooked noodles from a new perspective, and at the same time provides a new strategy for the preparation and application development of amyloid fibril protein.

Description

Technical Field

[0001] The invention relates to a method for determining the content of amyloid fibril protein, and in particular to a method for improving the accuracy of determining the content of amyloid fibril protein by enzymatic hydrolysis with a composite enzyme, and belongs to the field of food biotechnology. Background Art

[0002] Amyloid fibrils are protein aggregates rich in cross-β-folded structures obtained after protein processing such as heating. Due to their high Young's modulus, high tensile strength, high hydrophobicity, excellent stability and interfacial properties, antioxidant and antibacterial activity, they can be used to improve the foaming properties of materials, stabilize foams and emulsions, and improve the texture properties of food by forming a space-filling network. Therefore, the future application of amyloid fibrils in the food industry will be conducive to promoting the development of functional foods and the development of the food industry.

[0003] At present, it is reported that milk (such as β-lactoglobulin and bovine serum albumin), egg (such as lysozyme and ovalbumin), legume (such as soy and pea protein) and cereal (such as rice, corn and wheat protein) proteins can form amyloid fibrils after heating, and amyloid fibrils are also found to be formed in typical food processing processes such as the cooking process of pasta. Current research focuses on the formation mechanism and application of amyloid fibrils, and there are few studies on the determination of amyloid fibril content. The reported determination and extraction methods of amyloid fibrils are based on the characteristics that amyloid fibrils are difficult to be degraded by proteases. Proteins or peptides other than amyloid fibrils are degraded by trypsin or proteinase K, and then the content of amyloid fibrils is analyzed by thioflavin T ThT. This method is only applicable to pure protein or peptide systems. In food systems, non-protein components with a high content ratio and complex substances formed by the interaction between components during heating and other treatments will affect the binding of amyloid fibrils and ThT during content detection, thereby affecting the accuracy of amyloid fibril content determination. Therefore, it is necessary to establish a method suitable for determining the content of amyloid fibrils in food systems.

[0004] Previous studies have found that amyloid fibrils are also formed during the cooking process of noodles, and that the formation of amyloid fibrils is significantly correlated with noodle quality. In order to systematically study the formation of amyloid fibrils and their impact on noodle quality, a method for determining the content of amyloid fibrils in noodle systems should be established. However, no relevant research reports have been seen so far. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned shortcomings and provide a method for determining the content of amyloid fibril protein. Based on the characteristic that the starch content in the dry noodle system accounts for 60%-70%, α-amylase is creatively used to degrade the starch in the noodles, and a method for determining the content of amyloid fibril protein suitable for a cooked noodle product system is established by combining and optimizing the addition of α-amylase, proteinase K and trypsin.

[0006] The technical solution of the present invention is a method for determining the content of amyloid fibril protein, which uses α-amylase to degrade starch in frozen cooked noodles, and improves the accuracy of determining the content of amyloid fibril protein in frozen cooked noodles by combining and optimizing the addition of α-amylase, proteinase K and trypsin; the specific steps are as follows:

[0007] (1) Raw material preparation: The freeze-dried noodle sample is crushed and sieved, and the sieved material is collected;

[0008] (2) Enzyme treatment: Weigh the frozen cooked noodles and add them to a sodium azide solution, then add α-amylase to the system for enzymatic hydrolysis; then boil in a boiling water bath, cool to room temperature, centrifuge, discard the supernatant, and collect the precipitate; then add proteinase K and trypsin for enzymatic hydrolysis; cool to room temperature, centrifuge, and collect the supernatant SF1 and precipitate P1 respectively; disperse P1 in sodium phosphate buffer, shake and centrifuge to obtain SF2;

[0009] (3) ThT fluorescence analysis: Based on the specific binding characteristics of amyloid fibrils AFs and thioflavin T ThT, SF1, SF2 and ThT were mixed in a 96-well plate. The fluorescence value of the sample was measured under the conditions of excitation and emission wavelengths of 440 and 480 nm, respectively, and the content of AFs was calculated.

[0010] Furthermore, in step (1), the product is sieved through a 80-120 mesh screen.

[0011] Furthermore, in step (2), α-amylase is added to the system until the enzyme activity is 6-12 kEU, and proteinase K and trypsin are added to the system until the enzyme activities are 20-30 EU and 16-35 kEU, respectively.

[0012] Furthermore, in step (2), the enzymatic hydrolysis conditions of α-amylase are 30-42° C. and 50-250 rpm for 6-18 hours.

[0013] Furthermore, in step (2), the enzymatic hydrolysis conditions of proteinase K and trypsin are 30-42° C. and 50-250 rpm for 16-30 hours.

[0014] Furthermore, step (2) is specifically as follows:

[0015] a. Weigh 0.4-5 g of frozen cooked noodles containing 50-400 mg of protein, add to 5.0-20.0 mL of sodium azide solution with a mass volume concentration of 0.02%, then add α-amylase for enzymolysis, boil in a boiling water bath for 4-6 minutes, cool to room temperature; centrifuge at 4000-8000 rpm for 5-15 minutes at 4-25°C, discard the supernatant, and collect the precipitate;

[0016] b. Add proteinase K and trypsin for enzymatic hydrolysis; after cooling to room temperature, centrifuge at 4000-8000 rpm for 5-15 min at 4-25°C, and collect the supernatant SF1 and precipitate P1 respectively;

[0017] c. Disperse P1 in 4-12 mL of sodium phosphate buffer, shake at 15-30°C and 50-250 rpm for 8-24 h, then centrifuge at 4-25°C and 4000-8000 rpm for 5-15 min, and collect the supernatant SF2.

[0018] Furthermore, the sodium phosphate buffer in step (2)c is specifically a sodium phosphate buffer with a concentration of 0.05M and a pH of 7.0.

[0019] Furthermore, in step (3), the collected SF1 and SF2 are first mixed to obtain a mixed solution; 190 μL of the mixed solution of SF1 and SF2 is mixed with 10 μL of ThT with a concentration of 200 μM in a 96-well plate.

[0020] Furthermore, the standard curve uses the mass (μg) of the highly amyloid fibrous polypeptide as the abscissa and the sample fluorescence value as the ordinate, and the obtained standard curve is: y=729.61x-99.63, R 2 =0.9998.

[0021] The method is used for detecting the content of amyloid fibrils in frozen cooked noodles.

[0022] Beneficial effects of the present invention: The present invention utilizes α-amylase to degrade starch in noodles, and establishes a method for determining the content of amyloid fibril protein suitable for noodle product systems by combining and optimizing the addition of α-amylase, proteinase K and trypsin, which provides technical support for regulating the quality of frozen cooked noodles from a new perspective, and also provides a new strategy for the preparation and application development of amyloid fibril protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Mass-fluorescence standard curve for highly amyloidogenic fibrillar peptides. DETAILED DESCRIPTION

[0024] The wheat flour in the following examples was purchased from COFCO Flour (Haining) Co., Ltd., special first-class all-purpose flour with a water content of 3.70%; α-amylase was purchased from Beijing Solebow Technology Co., Ltd., proteinase K, thioflavin T and trypsin were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; the multifunctional microplate reader platform Spark 10M was purchased from TECAN Switzerland, Switzerland, and the JHMZ 200 pin-type noodle mixer and JMTD 168 / 140 experimental noodle machine were both purchased from Oriental Fude Technology Development Center.

[0025] The present invention will be further described below in conjunction with the embodiments of the specification.

[0026] The standard curve is drawn as follows:

[0027] 1. Drawing of standard curve: Prepare standard solutions of highly amyloid fibrosis peptides (residues: 103-112, ie, NFNYNNNLQG, from sup35 yeast, Protein Data Bank (PDB) code: 1YJP) with mass fractions of 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 and 550 mg / L, respectively. Mix 190 μL of standard solution and 10 μL of ThT (200 μM) in a 96-well plate, and measure the sample fluorescence value under the conditions of excitation and emission wavelengths of 440 and 480 nm, respectively. The mass of highly amyloid fibrosis peptides (μg) is used as the horizontal axis, and the sample fluorescence value is used as the vertical axis. The obtained standard curve is: y=729.61x-99.63, R 2 =0.9998.

[0028] 2. Take 190 μL of the mixture of SF1 and SF2 and 10 μL of ThT (200 μM) and mix them in a 96-well plate. Measure the fluorescence value of the sample under the conditions of excitation and emission wavelengths of 440 and 480 nm respectively, and calculate the AFs content of the sample according to the standard curve.

[0029] The samples of the standard curve were selected according to Sebastian Maurer-Stroh1, Maja Debulpaep, Nico Kuemmerer, Manuela Lopez de la Paz, Ivo Cristiano Martins, Joke Reumers, Kyle L Morris, Alastair Copland, Louise Serpell, Luis Serrano, Joost WH Schymkowitz, and Frederic Rousseau. Exploring the sequence determinants of amyloid structure using position-specific scoring matrices [J]. Nature Methods, 2010, 7 (3): 237-242.

[0030] The specific standard curve is as follows Figure 1 shown.

[0031] Example 1

[0032] α-amylase was added to the system with an enzyme activity of 12 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 18 h. Proteinase K and trypsin were added to the system with enzyme activities of 30 EU and 35 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 24 h.

[0033] (1) Raw material preparation: The freeze-dried cooked noodle sample was crushed and passed through a 120-mesh sieve to collect the sieve residue.

[0034] (2) Enzyme treatment: Weigh 0.5 g of noodle sample and add it to 18.0 mL of sodium azide solution (0.02%, w / v), add α-amylase until the system enzyme activity is 10 kEU, and perform enzymolysis at 37°C and 200 rpm for 18 h; then boil in boiling water for 5 min, cool to room temperature, centrifuge at 8000 rpm and 25°C for 15 min, discard the supernatant, and collect the precipitate; then add proteinase K and trypsin until the system enzyme activity is 30 EU and 35 kEU, respectively, and perform enzymolysis at 37°C and 200 rpm for 24 h; cool to room temperature, centrifuge at 8000 rpm and 25°C for 15 min, and collect the supernatant (SF1) and precipitate (P1), respectively; disperse P1 in 5 mL of sodium phosphate buffer, shake at 25°C and 200 rpm for 24 h, and centrifuge at 8000 rpm and 25°C for 15 min to obtain supernatant SF2.

[0035] (3) ThT fluorescence analysis: SF1 and SF2 were mixed, and 190 μL of the mixture was mixed with 10 μL of ThT (200 μM) in a 96-well plate. Under the conditions of excitation and emission wavelengths of 440 and 480 nm, the fluorescence value of the sample was determined to be 70609. According to the standard curve (y = 729.61x-99.63, R 2 =0.9998, y is the sample fluorescence value, x is the microgram number of highly amyloid fibrillary polypeptide) and the content of amyloid fibrillary protein was calculated to be 193.83 μg / g noodles.

[0036] Example 2

[0037] Enzyme treatment: α-amylase was added to the system with an enzyme activity of 14 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 18 h; proteinase K and trypsin were added to the system with enzyme activities of 30 EU and 35 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 24 h.

[0038] The sample preparation method is the same as in Example 1.

[0039] Except that the amount of α-amylase added was 14 kEU, the remaining enzymatic hydrolysis steps and conditions were the same as those in Example 1.

[0040] The fluorescence analysis method was the same as in Example 1. The fluorescence value of the sample was determined to be 70312 a.u., and the content of amyloid fibril protein was calculated to be 193.01 μg / g noodles.

[0041] Example 3

[0042] Enzyme treatment: α-amylase was added to the system with an enzyme activity of 12 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 22 h; proteinase K and trypsin were added to the system with enzyme activities of 30 EU and 35 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 24 h.

[0043] The sample preparation method is the same as in Example 1.

[0044] Except that the enzymatic hydrolysis time of α-amylase was 22 h, the other enzymatic hydrolysis steps and conditions were the same as those in Example 1.

[0045] The fluorescence analysis method was the same as in Example 1. The fluorescence value of the sample was determined to be 70466 a.u., and the content of amyloid fibril protein was calculated to be 193.43 μg / g noodles.

[0046] The results of Examples 1, 2 and 3 show that the content of amyloid fibril protein does not change significantly when the amount of α-amylase added and the hydrolysis time are further increased.

[0047] Example 4

[0048] Enzyme treatment: α-amylase was added to the system with an enzyme activity of 12 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 18 h; proteinase K and trypsin were added to the system with enzyme activities of 35 EU and 40 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 30 h.

[0049] The sample preparation method is the same as in Example 1.

[0050] The remaining enzymatic steps and conditions were the same as those in Example 1, except that the amounts of proteinase K and trypsin added were such that the system enzyme activities were 35EU and 40kEU respectively and the enzymatic hydrolysis time was 30h.

[0051] The fluorescence analysis method was the same as in Example 1. The fluorescence value of the sample was determined to be 70367 a.u., and the content of amyloid fibril protein was calculated to be 193.16 μg / g noodles.

[0052] The results of Examples 1 and 4 show that the content of amyloid fibril protein does not change significantly when the amount of proteinase K and trypsin added and the hydrolysis time are further increased.

[0053] Example 5

[0054] Enzyme treatment: α-amylase was added to the system with an enzyme activity of 14 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 22 h; proteinase K and trypsin were added to the system with enzyme activities of 35 EU and 40 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 30 h.

[0055] The sample preparation method is the same as in Example 1.

[0056] The remaining enzymatic steps and conditions were the same as in Example 1, except that the amount of α-amylase added was such that the system enzyme activity reached 14 kEU and the enzymatic hydrolysis time was 22 h, and the amounts of proteinase K and trypsin added were such that the system enzyme activities reached 35 EU and 40 kEU, respectively, and the enzymatic hydrolysis time was 30 h.

[0057] The fluorescence analysis method was the same as in Example 1. The fluorescence value of the sample was determined to be 70686 a.u., and the content of amyloid fibril protein was calculated to be 194.04 μg / g noodles.

[0058] The results of Examples 1 and 5 show that the content of amyloid fibrils does not change significantly when the amount of α-amylase, proteinase K and trypsin added and the hydrolysis time are further increased. Based on Examples 1-5, it can be seen that the content of amyloid fibrils in the noodle samples is between 193.01 and 194.03 μg / g noodles, with an average value of 193.49 μg / g noodles.

[0059] Example 6

[0060] Enzyme treatment: α-amylase was added to the system with an activity of 8 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 18 h; proteinase K and trypsin were added to the system with enzyme activities of 30 EU and 35 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 24 h.

[0061] The sample preparation method is the same as in Example 1.

[0062] Except that the amount of α-amylase added was such that the system enzyme activity was 8 kEU, the remaining enzymatic hydrolysis steps and conditions were the same as in Example 1.

[0063] The fluorescence analysis method was the same as in Example 1. The fluorescence value of the sample was determined to be 54686 a.u., and the content of amyloid fibril protein was calculated to be 150.18 μg / g noodles, which was 77.61% of the content of amyloid fibril protein in the noodle sample.

[0064] Example 7

[0065] Enzyme treatment: α-amylase was added to the system with an enzyme activity of 8 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 6 h; proteinase K and trypsin were added to the system with enzyme activities of 30 EU and 35 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 24 h.

[0066] The sample preparation method is the same as in Example 1.

[0067] Except that the amount of α-amylase added was 6 kEU and the enzymatic hydrolysis time was 6 h, the remaining enzymatic hydrolysis steps and conditions were the same as in Example 1.

[0068] The fluorescence analysis method was the same as in Example 1. The fluorescence value of the sample was determined to be 38588 a.u., and the content of amyloid fibril protein was calculated to be 106.05 μg / g noodles, which was 54.81% of the content of amyloid fibril protein in the noodle sample.

[0069] Based on Examples 1, 5-7, reducing the amount of α-amylase added and the hydrolysis time will reduce the accuracy of the analysis of the amyloid fibril content. This may be because the incompletely degraded gelatinized starch interferes with the hydrolysis of protein by proteinase K and trypsin and the binding efficiency of amyloid fibrils with ThT.

[0070] Example 8

[0071] Enzyme treatment: Proteinase K and trypsin were added to the system with enzyme activities of 30EU and 35kEU, respectively. The enzymatic hydrolysis conditions were 37°C and 250rpm for 24h.

[0072] The sample preparation method is the same as in Example 1.

[0073] Enzyme treatment: Weigh 3.0 g of noodle sample, add it to 18.0 mL of sodium azide solution (0.02%, w / v), add proteinase K and trypsin until the enzyme activities of the system are 30 EU and 35 kEU, respectively, and enzymatic hydrolysis conditions are 37 ° C and 200 rpm for 24 hours; after cooling to room temperature, centrifuge at 8000 rpm and 25 ° C for 15 minutes, and collect the supernatant (SF1) and precipitate (P1), respectively; P1 is dispersed in 5 mL of sodium phosphate buffer, shaken at 25 ° C and 200 rpm for 24 hours, and then centrifuged at 8000 rpm and 25 ° C for 15 minutes to obtain SF2.

[0074] The fluorescence analysis method was the same as in Example 1. The fluorescence value of the sample was determined to be 14425 a.u., and the content of amyloid fibril protein was calculated to be 39.81 μg / g noodles, which was 20.58% of the content of amyloid fibril protein in the noodle sample.

[0075] Based on Examples 1 and 5-8, reducing the amount of α-amylase added and the hydrolysis time will reduce the accuracy of the analysis of the amyloid fibril content. This may be because the incompletely degraded gelatinized starch interferes with the hydrolysis of protein by proteinase K and trypsin and the binding efficiency of amyloid fibrils with ThT.

[0076] Example 9

[0077] Enzyme treatment: α-amylase was added to the system with an enzyme activity of 12 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 18 h; proteinase K and trypsin were added to the system with enzyme activities of 20 EU and 16 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 24 h.

[0078] The sample preparation method is the same as in Example 1.

[0079] The remaining enzymatic hydrolysis steps and conditions were the same as those in Example 1, except that the amounts of proteinase K and trypsin added were such that the system enzyme activities were 20EU and 16kEU, respectively.

[0080] The fluorescence analysis method was the same as in Example 1, and the fluorescence value of the sample was measured to be 60238 a.u., and the content of amyloid fibril protein was calculated to be 165.40 μg / g noodles, which was 85.48% of the content of amyloid fibril protein in the noodle sample. This indicates that reducing the amount of proteinase K and trypsin added will also affect the accuracy of the determination of the content of amyloid fibril protein in the noodle sample.

[0081] Example 10

[0082] Enzyme treatment: α-amylase was added to the system with an enzyme activity of 12 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 18 h; proteinase K and trypsin were added to the system with enzyme activities of 30 EU and 35 kEU respectively, and the enzymatic hydrolysis conditions were 30°C and 250 rpm for 24 h.

[0083] The sample preparation method is the same as in Example 1.

[0084] Except that the enzymatic hydrolysis temperature of proteinase K and trypsin was 30° C., the remaining enzymatic hydrolysis steps and conditions were the same as those in Example 1.

[0085] The fluorescence analysis method was the same as in Example 1, and the fluorescence value of the sample was determined to be 64227 a.u., and the content of amyloid fibril protein was calculated to be 176.33 μg / g noodles, which was 91.13% of the content of amyloid fibril protein in the noodle sample. This indicated that the hydrolysis temperature of proteinase K and trypsin was not appropriate, which would reduce the hydrolysis efficiency of proteinase K and trypsin, and thus reduce the accuracy of the determination of the content of amyloid fibril protein in the noodle sample.

[0086] Embodiment 11

[0087] Enzyme treatment: α-amylase was added to the system with an enzyme activity of 12 kEU, and the enzymatic hydrolysis conditions were 37°C and 200 rpm for 18 h; proteinase K and trypsin were added to the system with enzyme activities of 30 EU and 35 kEU respectively, and the enzymatic hydrolysis conditions were 37°C and 250 rpm for 16 h.

[0088] The sample preparation method is the same as in Example 1.

[0089] Except that the enzymatic hydrolysis time of proteinase K and trypsin was 16 h, the other enzymatic hydrolysis steps and conditions were the same as those in Example 1.

[0090] The fluorescence analysis method was the same as in Example 1, and the fluorescence value of the sample was measured to be 60432 a.u., and the content of amyloid fibril protein was calculated to be 165.93 μg / g noodles, which was 85.75% of the content of amyloid fibril protein in the noodle sample. This indicates that the reduction of the hydrolysis time of proteinase K and trypsin will also reduce the hydrolysis effect of proteinase K and trypsin, thereby reducing the accuracy of the determination of the content of amyloid fibril protein in the noodle sample.

[0091] Example 12

[0092] Enzyme treatment: first hydrolyze with proteinase K and trypsin under optimal conditions, then hydrolyze with α-amylase.

[0093] The sample preparation method is the same as in Example 1.

[0094] Enzyme treatment: Weigh 3.0 g of noodle sample, add to 18.0 mL of sodium azide solution (0.02%, w / v), add proteinase K and trypsin until the system enzyme activities are 30 EU and 35 kEU respectively, and the enzymatic hydrolysis conditions are 37°C and 200 rpm for 24 hours; then boil in boiling water for 5 minutes, cool to room temperature, centrifuge at 8000 rpm and 25°C for 15 minutes, discard the supernatant and collect the precipitate; then add α-amylase until the system enzyme activity is 10 kEU, and enzymatic hydrolysis is carried out at 37°C and 200 rpm for 18 hours; after cooling to room temperature, centrifuge at 8000 rpm and 25°C for 15 minutes, and collect the supernatant (SF1) and precipitate (P1) respectively; P1 is dispersed in sodium phosphate buffer, shaken at 25°C and 200 rpm for 24 hours, and then centrifuged at 8000 rpm and 25°C for 15 minutes to obtain SF2.

[0095] The fluorescence analysis method was the same as in Example 1, and the fluorescence value of the sample was determined to be 55132 a.u., and the content of amyloid fibril protein was calculated to be 151.40 μg / g noodles, which was 78.25% of the content of amyloid fibril protein in the noodle sample. This indicates that hydrolyzing protein first and then hydrolyzing starch will reduce the accuracy of determining the content of amyloid fibril protein in the noodle sample.

[0096] It can be seen from the above examples that the amount of α-amylase added and the enzymolysis time have the greatest impact on the accuracy of determining the content of amyloid fibril protein, which also illustrates the necessity of the results of this patent. In addition, the amount of proteinase K and trypsin added, the enzymolysis temperature and enzymolysis time, and the hydrolysis order of protease and amylase will also affect the accuracy of the determination of the content of amyloid fibril protein.

Claims

1. A method for detecting the content of amyloid fibril protein in frozen cooked noodles, characterized in that Here are the steps: (1) Raw material preparation: The freeze-dried noodle sample was crushed and sieved, and the sieved material was collected; (2) Enzyme treatment: Weigh the frozen cooked noodles and add them to the sodium azide solution, then add α-amylase to the system for enzymatic hydrolysis; then boil in water, cool to room temperature and centrifuge, discard the supernatant and collect the precipitate; then add proteinase K and trypsin for enzymatic hydrolysis; after cooling to room temperature, centrifuge and collect the supernatant SF1 and precipitate P1 respectively; disperse P1 in sodium phosphate buffer and shake and centrifuge to obtain SF2; add α-amylase to the system with an enzymatic activity of 6-12 kEU, add proteinase K and trypsin to the system with an enzymatic activity of 20-30 EU and 16-35 kEU respectively; (3) ThT fluorescence analysis: Based on the specific binding characteristics of amyloid fibrils AFs and thioflavin T ThT, SF1, SF2 and ThT were mixed in a 96-well plate. The fluorescence value of the sample was measured under the conditions of excitation and emission wavelengths of 440 and 480 nm, and the content of AFs was calculated.

2. The method for determining the content of amyloid fibril protein as claimed in claim 1, characterized in that: In step (1), the product is passed through a 80-120 mesh sieve.

3. The method for determining the content of amyloid fibril protein as claimed in claim 1, characterized in that: In step (2), the α-amylase enzymatic hydrolysis conditions are 30-42°C and 50-250 rpm for 6-18 hours.

4. The method for determining the content of amyloid fibril protein as claimed in claim 1, characterized in that: In step (2), the enzymatic hydrolysis conditions of proteinase K and trypsin are 30-42°C and 50-250 rpm for 16-30 hours.

5. The method for determining the content of amyloid fibril protein as claimed in claim 1, characterized in that Step (2) is as follows: a. Weigh 0.4-5 g of frozen cooked noodles containing 50-400 mg of protein, add them to 5.0-20.0 mL of 0.02% sodium azide solution by mass volume, then add α-amylase for enzymolysis, boil in a boiling water bath for 4-6 minutes, cool to room temperature; centrifuge at 4000-8000 rpm for 5-15 minutes at 4-25°C, discard the supernatant, and collect the precipitate; b. Add proteinase K and trypsin for enzymatic hydrolysis; after cooling to room temperature, centrifuge at 4000-8000 rpm for 5-15 min at 4-25°C, and collect the supernatant SF1 and precipitate P1 respectively; c. Disperse P1 in 4-12 mL of sodium phosphate buffer, shake at 15-30°C and 50-250 rpm for 8-24 h, then centrifuge at 4-25°C and 4000-8000 rpm for 5-15 min, and collect the supernatant SF2.

6. The method for determining the content of amyloid fibril protein as claimed in claim 5, characterized in that: The sodium phosphate buffer in step (2) c is specifically a sodium phosphate buffer with a concentration of 0.05 M and a pH of 7.

0.

7. The method for determining the content of amyloid fibril protein as claimed in claim 1, characterized in that: In step (3), the collected SF1 and SF2 are first mixed to obtain a mixed solution; 190 μL of the mixed solution of SF1 and SF2 is mixed with 10 μL of ThT with a concentration of 200 μM in a 96-well plate.

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