Silk polyphenol content detection method and detection equipment

Abstract

The invention relates to the field of silk quality detection, in particular to a silk polyphenol content detection method which specifically comprises the following steps: adding a silk sample into deionized water, performing ultrasonic extraction, and then filtering; adding polyethylene glycol into the filtrate, standing to precipitate sericin, and centrifuging to take supernate; enabling the supernate to pass through an acidic cation exchange column to replace and discharge impurity cations, and collecting column effluent; according to the present invention, the column effluent is subjected to evaporation concentration and filtration to obtain the extraction liquid, the extraction liquid is subjected to HPLC detection, and the polyethylene glycol can effectively promote the sericin precipitation so as to reduce the occurrence of the chromatographic column blocking in the subsequent HPLC detection process as much as possible, such that the detection process can be smoothly performed.

Description

Technical Field

[0001] This application relates to the field of silk quality testing, and in particular to a method and testing equipment for detecting the polyphenol content of silk. Background Technology

[0002] To evaluate the quality of silk, it is necessary to test the content of bioactive components such as polyphenols in the silk to determine its quality, which helps in quality control and stability. For example, because fresh cocoon raw silk involves fewer processes and is cheaper than dried cocoon raw silk, many silk reeling companies choose to produce fresh cocoon raw silk. However, it is difficult to distinguish between fresh cocoon raw silk and dried cocoon raw silk.

[0003] Some publicly available literature, such as "Extraction Method of Polyphenolic Trace Components from Fresh Cocoon Silk," mentions that the polyphenol content in fresh cocoon silk differs significantly from that in dried cocoon silk. Therefore, extracting and measuring polyphenolic substances in silk can effectively distinguish between fresh and dried cocoon silk. The specific extraction method involves using pure water as the solvent, with a bath ratio of 1:25 (g / ml) between the silk sample and the solvent. Ultrasonic extraction is performed at 40℃ for 90 minutes. The extract is then filtered, and the filtrate is evaporated and concentrated to dryness. Pure water and the extract are then placed in an ultrasonic device to dissolve the extract, followed by filtration. The filtrate is transferred to a sample bottle, and finally, HPLC analysis is performed to determine the specific components and content of polyphenols in the silk for traceability.

[0004] In HPLC detection, sericin in the extracted sample is prone to denaturation and precipitation when it encounters high concentrations of methanol, which in turn causes blockage of the chromatographic column, affecting the normal separation function of the column and making the detection process difficult to proceed smoothly. Summary of the Invention

[0005] To ensure the smooth progress of the testing process, this application provides a method and equipment for detecting the polyphenol content in silk.

[0006] The method for detecting the polyphenol content in silk provided in this application adopts the following technical solution.

[0007] A method for detecting the polyphenol content in silk includes the following steps.

[0008] S1. Add the silk sample to deionized water and perform ultrasonic extraction, then filter;

[0009] S2. Add polyethylene glycol to the filtrate, let it stand to precipitate the sericin, and then centrifuge to collect the supernatant.

[0010] S3. Pass the supernatant through an acidic cation exchange column to displace and remove impurity cations, and collect the column effluent;

[0011] S4. The column effluent is evaporated and concentrated, then filtered to obtain the extract, and the extract is analyzed by HPLC.

[0012] By adopting the above technical solution, polyethylene glycol can form a complex with sericin and precipitate it. The precipitate of the complex is then removed by filtration, so as to minimize the occurrence of subsequent sericin precipitation clogging the chromatographic column and ensure the smooth progress of the detection work.

[0013] Optionally, sodium citrate is added to the deionized water in S1.

[0014] By adopting the above technical solution, sodium citrate can dissolve the calcium bridge structure in sericin, thereby reducing the tendency of sericin to aggregate and improving the polyphenol extraction rate.

[0015] Optionally, after adding α-cyclodextrin solution to the column effluent of S3 and shaking, S4 can be performed.

[0016] By adopting the above technical solution, the hydrophobic cavity of α-cyclodextrin can encapsulate the hydrophobic residues of sericin to form a water-soluble complex, so that it is not easy to precipitate when encountering organic phase during subsequent HPLC detection.

[0017] Optionally, disodium ethylenediaminetetraacetate is added to the α-cyclodextrin solution.

[0018] By adopting the above technical solution, disodium ethylenediaminetetraacetate can chelate residual metal ions, further blocking the possibility of calcium bridge regeneration, and greatly improving the stability of sericin in the organic phase, thus making it less prone to precipitation.

[0019] Optionally, in step S4, the pH of the column effluent after evaporation and concentration is adjusted to 6.5-7.5.

[0020] By adopting the above technical solution, an oxidation inhibition buffer zone is established to reduce the possibility of polyphenol oxidative degradation in an acidic environment and improve the polyphenol extraction rate.

[0021] Optionally, in step S4, acetonitrile is added to the column effluent after evaporation and concentration.

[0022] By adopting the above technical solution, sericin can be pre-adapted to the organic environment and then filtered, reducing the possibility of phase change precipitation clogging the chromatographic column during subsequent HPLC detection.

[0023] Optionally, in S4, the mobile phase A of the HPLC detection uses ammonium acetate and formic acid, and the mobile phase B uses acetonitrile.

[0024] By adopting the above technical solution, a non-denaturing chromatographic environment is constructed to effectively inhibit the precipitation of sericin.

[0025] Optionally, after adding polyethylene glycol in step S2, the solution temperature is controlled at 5-10℃ and allowed to stand.

[0026] By adopting the above technical solution, on the one hand, the binding between polyethylene glycol and sericin is improved, thereby increasing the sericin removal rate; on the other hand, the stability of polyphenols is maintained, and the polyphenol loss rate is reduced.

[0027] Optionally, the filtration in S4 uses a nylon membrane that has been rinsed with an ammonium bicarbonate solution.

[0028] By adopting the above technical solution, the nylon membrane carries a positive charge, which can better adsorb the negatively charged sericin fragments, reduce the sericin residue in the extract, and the washing with ammonium bicarbonate can increase the positive charge density on the nylon membrane.

[0029] A device for detecting the content of silk polyphenols, wherein the content of silk polyphenols is detected according to the above-mentioned method for detecting the content of silk polyphenols.

[0030] By adopting the above technical solution, the sericin is less likely to form a large amount of precipitate during HPLC detection, thereby reducing the possibility of column blockage.

[0031] In summary, this application includes at least the following beneficial effects:

[0032] 1. Polyethylene glycol can form a complex with sericin and precipitate it. The precipitate is then filtered out to minimize the occurrence of subsequent sericin precipitation clogging the chromatographic column.

[0033] 2. Sodium citrate can dissolve the calcium bridge structure in sericin, thereby reducing the tendency of sericin to aggregate and improving the polyphenol extraction rate.

[0034] 3. The hydrophobic cavity of α-cyclodextrin can encapsulate the hydrophobic residues of sericin to form a water-soluble complex, making it less likely to precipitate when it encounters the organic phase during subsequent HPLC detection. Disodium EDTA can chelate residual metal ions, further blocking the possibility of calcium bridge regeneration, greatly improving the stability of sericin in the organic phase and thus making it less likely to precipitate.

[0035] 4. After adding polyethylene glycol, control the solution temperature at 5-10℃. This will improve the binding between polyethylene glycol and sericin, thereby increasing the sericin removal rate, and will also maintain the stability of polyphenols and reduce the polyphenol loss rate.

[0036] 5. Nylon membranes carry a positive charge, which can better adsorb negatively charged sericin fragments, reducing sericin residue in the extract. Furthermore, the washing with ammonium bicarbonate can increase the positive charge density on the nylon membrane. Attached Figure Description

[0037] Figure 1 This is a flowchart of a method for detecting the polyphenol content in silk. Detailed Implementation

[0038] This application discloses a method for detecting the polyphenol content in silk, referring to... Figure 1 Specifically, it includes the following steps.

[0039] S1. Crush the silk sample to 1mm. 3 Then add deionized water containing 2% g / L sodium citrate, maintain at 38-42℃ and perform ultrasonic extraction at 200w for 20min, then filter and retain the filtrate;

[0040] S2. Add a 10% polyethylene glycol solution to the filtrate. The polyethylene glycol can be polyethylene glycol-6000. Then let it stand at 5-10℃ for 30 minutes to precipitate the sericin. Then centrifuge at 4000 rpm for 10 minutes and take the supernatant.

[0041] S3. Pass the supernatant through a strongly acidic cation exchange column filled with sulfonated polystyrene to displace and remove impurity cations. The flow rate of the supernatant is 1 ml / min, and the column bed is pre-activated with 0.1 mol / L ammonium formate solution. At the same time, the column effluent is collected.

[0042] S4. Add a 5% α-cyclodextrin solution to the column effluent. The α-cyclodextrin solution also contains 0.05% disodium ethylenediaminetetraacetate. Shake at 58-62℃ for 15 min. Then evaporate and concentrate the column effluent, and concentrate it under reduced pressure at 40℃ to a suitable volume. Adjust the pH of the concentrated column effluent to 6.5-7.5, add acetonitrile, and then filter through a 0.22μm nylon membrane. The nylon membrane is pre-washed with ammonium bicarbonate solution. Transfer the filtrate to a sample vial to obtain the extract. Perform HPLC analysis on the extract. The mobile phase A for HPLC analysis uses ammonium acetate and formic acid, and the mobile phase B uses acetonitrile.

[0043] This application also discloses a silk polyphenol content detection device, which detects the silk polyphenol content according to the above-mentioned silk polyphenol content detection method.

[0044] The following provides a detailed description with reference to several embodiments and comparative examples.

[0045] Example 1:

[0046] A method for detecting the polyphenol content in silk includes the following steps.

[0047] S1. Crush the silk sample to 1mm. 3Weigh 2.0g, add 50ml of deionized water containing 2% g / L sodium citrate, maintain at 40℃ and perform ultrasonic extraction at 200w for 20min, then filter and retain the filtrate;

[0048] S2. Add 5 ml of 10% polyethylene glycol solution to the filtrate. The polyethylene glycol can be polyethylene glycol-6000. Then let it stand at 5°C for 30 min to precipitate the sericin. Then centrifuge at 4000 rpm for 10 min and take the supernatant.

[0049] S3. Pass the supernatant through a strongly acidic cation exchange column filled with sulfonated polystyrene to displace and remove impurity cations. The flow rate of the supernatant is 1 ml / min, and the column bed is pre-activated with 0.1 mol / L ammonium formate solution. At the same time, the column effluent is collected.

[0050] S4. Add 0.5 ml of 5% α-cyclodextrin solution to the column effluent. The α-cyclodextrin solution also contains 0.05% disodium ethylenediaminetetraacetate. Shake at 60°C for 15 min. Then evaporate and concentrate the column effluent, and concentrate it under reduced pressure at 40°C to a suitable volume. Adjust the pH of the concentrated column effluent to 6.5, add 1 ml of acetonitrile, and filter through a 0.22 μm nylon membrane. The nylon membrane is pre-washed with ammonium bicarbonate solution. Transfer the filtrate to a sample vial to obtain the extract. Perform HPLC analysis on the extract. The mobile phase for HPLC analysis consists of ammonium acetate and formic acid in phase A and acetonitrile in phase B.

[0051] The chromatographic column can be a Poroshell 120 EC-C18, with a column temperature of 20℃, an injection volume of 20 μL, a flow rate of 1.2 ml / min, and a detection wavelength of 280 nm, and gradient elution is used. The mobile phase ratios are: 85% phase A and 15% phase B at 0 min; 75% phase A and 25% phase B at 15 min; 55% phase A and 45% phase B at 20 min; and 0% phase A and 100% phase B at 25 min.

[0052] Example 2:

[0053] The difference from Example 1 is that in S2, the temperature at which polyethylene glycol is added to the filtrate and then allowed to stand is 10°C.

[0054] Example 3:

[0055] The difference from Example 1 is that the pH of the concentrated column effluent in S4 is adjusted to 7.5.

[0056] Comparative Example 1:

[0057] The difference from Example 1 is that a method for detecting the content of silk polyphenols specifically includes the following steps.

[0058] S1. Crush the silk sample to 1mm. 3 Weigh 2.0g, add 50ml of deionized water, maintain at 40℃ and perform ultrasonic extraction at 200w for 20min, then filter and retain the filtrate;

[0059] S2. The filtrate is evaporated and concentrated, and then concentrated to dryness under reduced pressure at 40°C to obtain the extract. The extract is then dissolved by sonication in 1 ml of deionized water for 2 min. The solution is then filtered through a 0.22 μm PTFE membrane. The filtrate is transferred to a sample vial to obtain the extract. The extract is then analyzed by HPLC. The mobile phase A for HPLC analysis is methanol and the mobile phase B is water.

[0060] Comparative Example 2:

[0061] The difference from Example 1 is that sodium citrate was not added to the deionized water in S1.

[0062] Comparative Example 2:

[0063] The difference from Example 1 is that no α-cyclodextrin solution was added to the column effluent of S4.

[0064] Comparative Example 3:

[0065] The difference from Example 1 is that disodium ethylenediaminetetraacetate was not added to the α-cyclodextrin solution of S4.

[0066] Comparative Example 4:

[0067] The difference from Example 1 is that acetonitrile was not added to the column effluent after evaporation and concentration in S4.

[0068] Comparative Example 5:

[0069] The difference from Example 1 is that in S4, filtration is performed using a PTFE membrane that has been rinsed with an ammonium bicarbonate solution.

[0070] Comparative Example 6:

[0071] The difference from Example 1 is that in S4, filtration is performed via a nylon membrane that has been rinsed with deionized water.

[0072] Comparative Example 7:

[0073] The difference from Example 1 is that the pH of the column effluent after evaporation and concentration was not adjusted in S4.

[0074] Comparative Example 8:

[0075] The difference from Example 1 is that after adding polyethylene glycol in S2, the solution temperature is controlled at 15°C and allowed to stand.

[0076] Comparative Example 9:

[0077] The difference from Example 1 is that after adding polyethylene glycol in S2, the solution temperature is controlled at 0°C and allowed to stand.

[0078] In each example and comparative example, the column pressure rise rate and polyphenol extraction rate were statistically analyzed during HPLC detection. The initial column pressure was 50 bar, and the warning value was 400 bar. A higher column pressure rise rate indicated a higher degree of column clogging. The polyphenol extraction rate was calculated using gallic acid as a standard, and was obtained by comparing the measured content with the theoretical content. Specific experimental results are shown in the table below.

[0079]

[0080] As can be seen from Examples 1 to 3 and Comparative Example 1, the detection method of this application can effectively reduce column clogging compared with the traditional detection method of directly extracting silkworm silk by ultrasound and then filtering.

[0081] As can be seen from Examples 1 to 3 and Comparative Example 2, the addition of sodium citrate can effectively prevent sericin polymerization, reduce the possibility of calcium ions chelating the phenolic hydroxyl groups of polyphenols, and help improve the polyphenol extraction rate.

[0082] As can be seen from Examples 1 to 3 and Comparative Examples 3 and 4, the hydrophobic cavity of α-cyclodextrin can encapsulate the hydrophobic residues of sericin to form a water-soluble complex, making it less likely to precipitate when encountering the organic phase during subsequent HPLC detection. Disodium ethylenediaminetetraacetate can chelate residual metal ions, further blocking the possibility of calcium bridge regeneration, greatly improving the stability of sericin in the organic phase and thus making it less likely to precipitate.

[0083] As can be seen from Examples 1 to 3 and Comparative Example 5, nylon membranes can more effectively remove sericin fragments from solutions.

[0084] As can be seen from Examples 1 to 3 and Comparative Example 6, washing the nylon membrane with ammonium bicarbonate can effectively improve the electrostatic adsorption capacity of the nylon membrane, thereby further enhancing its ability to remove sericin.

[0085] Based on Examples 1 to 3 and Comparative Example 7, it can be seen that adjusting the pH of the column effluent to near neutral can reduce the degradation rate of polyphenols in an acidic environment, thereby effectively improving the polyphenol extraction rate.

[0086] Based on Examples 1 to 3 and Comparative Examples 8 and 9, it is evident that when the static temperature of the solution after adding polyethylene glycol exceeds 10°C, the binding between polyethylene glycol and sericin becomes relatively poor, resulting in a higher residual sericin content in the solution. Conversely, when the static temperature of the solution after adding polyethylene glycol is below 5°C, ice crystal growth in the solution generates shear force, causing sericin strips to break and increasing sericin fragments. This leads to an increase in the total sericin content in the final extract, and polyphenols bind to these fragments, thus affecting the polyphenol extraction rate.

[0087] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for detecting the polyphenol content in silk, characterized in that: Specifically, the following steps are included: S1. Add the silk sample to deionized water and perform ultrasonic extraction, then filter; S2. Add polyethylene glycol to the filtrate, let it stand to precipitate the sericin, and then centrifuge to collect the supernatant. S3. Pass the supernatant through an acidic cation exchange column to displace and remove impurity cations, and collect the column effluent; S4. The column effluent is evaporated and concentrated, then filtered to obtain the extract, and the extract is analyzed by HPLC.

2. The method for detecting the polyphenol content of silk according to claim 1, characterized in that: Sodium citrate is added to the deionized water in S1.

3. The method for detecting the polyphenol content of silk according to claim 1, characterized in that: After adding α-cyclodextrin solution to the column effluent of S3 and shaking, S4 is performed.

4. The method for detecting the polyphenol content of silk according to claim 3, characterized in that: Disodium ethylenediaminetetraacetate was added to the α-cyclodextrin solution.

5. The method for detecting the polyphenol content of silk according to claim 1, characterized in that: In step S4, the pH of the column effluent after evaporation and concentration is adjusted to 6.5-7.

5.

6. The method for detecting the polyphenol content of silk according to claim 1, characterized in that: In step S4, acetonitrile is added to the column effluent after evaporation and concentration.

7. The method for detecting the polyphenol content of silk according to claim 1, characterized in that: In S4, the mobile phase A of the HPLC detection uses ammonium acetate and formic acid, and the mobile phase B uses acetonitrile.

8. The method for detecting the polyphenol content of silk according to claim 1, characterized in that: After adding polyethylene glycol to S2, the solution temperature is controlled at 5-10℃ and allowed to stand.

9. The method for detecting the polyphenol content of silk according to claim 1, characterized in that: The filtration in S4 uses a nylon membrane that has been rinsed with ammonium bicarbonate solution.

10. A silk polyphenol content detection device, wherein the silk polyphenol content is detected according to any one of claims 1-9.

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