A method for preparing a hickory hullin-pisatin complex having starch-digesting enzyme inhibitory activity

Abstract

The application provides a preparation method of hickory hullin acid-pea protein complex with starch digestive enzyme inhibition activity, and belongs to the technical field of food proteins. Specifically, the method comprises the following steps: step 1, hickory hullin acid extraction; step 2, hickory hullin acid separation; step 3, purification of phenolic acid compounds; and step 4, preparation of hickory hullin acid-pea protein complex. The pea protein complex prepared by the method has good alpha-amylase and alpha-glucosidase inhibition activity, can be used for developing a new type of diabetes drug or as a functional food ingredient, and is helpful for controlling blood sugar. Through the application, the hickory hull, a hickory processing byproduct, can be converted into a product with biological activity, the pollution of hickory hull as garbage waste to the environment is reduced, and the effective utilization of resources is promoted.

Description

Technical Field

[0001] This invention relates to a method for preparing a pecan chitosan-pea protein complex with starch digestive enzyme inhibitory activity, belonging to the field of food protein technology. Background Technology

[0002] Pea protein is a plant-based protein extracted from peas, primarily composed of globulins, including pea 7S globulin, 11S globulin, and conopoisin. Pea protein contains a variety of essential amino acids and is a high-quality food protein source. Compared to soy protein or dairy protein products, pea protein generally does not cause allergic reactions, making it suitable for people with allergies.

[0003] Pecans (Carya cathayensis Sarg.) belong to the genus Carya in the family Juglandaceae and are a common and nutritious nut. The shells of pecans are rich in phenolic acids, which can effectively inhibit α-amylase and α-glucosidase. α-amylase and α-glucosidase are two major starch-digesting enzymes in the human body. Ingested starch is first broken down by α-amylase into intermediate products such as maltodextrin, maltotriose, and maltose, and then converted into glucose by α-glucosidase for absorption and utilization by the body. Stable blood sugar levels are crucial for health; long-term hyperglycemia is a major risk factor for type 2 diabetes and its related complications (such as retinopathy, nephropathy, neuropathy, and cardiovascular disease). Due to their inhibitory effect on α-amylase and α-glucosidase, the phenolic acids in pecan shells can slow down the breakdown of starch in food after meals, reducing the rate of postprandial blood sugar rise.

[0004] In the processing of pecans, the shells are usually considered waste, resulting in resource waste and environmental pollution. This invention discovers a rapid extraction and separation process for phenolic acid compounds from pecan shells. These compounds, through modification, impart inhibitory effects on pea protein against α-amylase and α-glucosidase. This not only enhances the utilization value of pea protein and expands its application areas but also effectively utilizes the pecan shell resource, reducing waste generation during pecan production and mitigating environmental pollution. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a pecan chitosan-pea protein complex with anti-amylase activity. The pea protein complex prepared by this method exhibits good inhibitory activity against α-amylase and α-glucosidase, and can be used to develop novel diabetes drugs or as a functional food ingredient, helping to control blood sugar. This invention also transforms pecan chitosan shells, a byproduct of pecan processing, into a bioactive product, reducing the environmental pollution caused by pecan chitosan shells as waste and promoting the efficient utilization of resources.

[0006] The technical solution of this invention:

[0007] A method for preparing a pecan chitosan-pea protein complex with starch digestive enzyme inhibitory activity, the method comprising the following steps:

[0008] Step 1: Extraction of phenolic acids from pecan shells: Pecan shells are pulverized and mixed with water, with the temperature adjusted between 25℃ and 50℃. The mixture is homogenized using a high-pressure homogenizer at 5–30 MPa. The homogenized mixture is then separated into solid and liquid phases by filtration or centrifugation, and the clear liquid is collected.

[0009] Furthermore, the solid-liquid mass ratio of the powdered pecan shells mixed with water is 1:6 to 1:30.

[0010] Step 2, Separation of pecan chitosan: The supernatant from Step 1 was subjected to ultrafiltration sieving using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the permeate was collected.

[0011] Step 3, purification of phenolic acid compounds: The ultrafiltration permeate from Step 2 is added to an HW-40 gel column. First, it is eluted with a low-concentration ethanol solution for 2 column volumes (Bed Volume, BV) to remove impurities, and then eluted with a high-concentration ethanol solution for 2 column volumes. The eluent is collected, concentrated under reduced pressure, and then freeze-dried or spray-dried. The resulting powder is the phenolic acid extract of hickory shell.

[0012] Furthermore, the volume fraction of the low-concentration ethanol solution is 5-15%, and the volume fraction of the high-concentration ethanol solution is 40-60%.

[0013] Step 4: Preparation of pecan chitosan-pea protein complex: Disperse pea protein in water to a concentration of 10 g / L–85 g / L, and add the pecan chitosan extract from Step 3 to a concentration of 0.1 g / L–2 g / L. After mixing, add laccase (EC.1.10.3) to a concentration of 0.5 U / L–5 U / L, adjust the pH to 7.2–8.0, and continue stirring until homogeneous. Remove free phenolic acid compounds by dialysis. Dry the mixture in the dialysis bag using freeze-drying or spray drying to obtain pecan chitosan-pea protein complex powder.

[0014] Furthermore, after adding laccase, maintain the temperature within the range of 30-40℃ and continue stirring for 0.5-2 hours.

[0015] The beneficial effects of this invention: Pea shells are a byproduct of pecan processing and are generally treated as waste, causing environmental pollution and resource waste. This invention uses pecan shells as raw material and utilizes high-pressure homogenization and membrane separation technology to achieve rapid extraction and separation of phenolic acid compounds, providing a new technical method to improve the utilization value of pecan shells. A pecan shell phenolic acid-pea protein complex is prepared by the catalytic action of laccase (EC.1.10.3). This complex has strong inhibitory activity against α-amylase and α-glucosidase, endowing pea protein with new functions. It can be used as a raw material in the development of hypoglycemic drugs or functional foods, showing good development and application prospects. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0017] Figure 1 High performance liquid chromatogram of pecan phenolic acid extract (Example 1). Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments, and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The method for determining α-amylase inhibitory activity involved in the embodiments of the present invention is as follows:

[0020] Sample group: 100 μL of α-amylase (1.25 U / mL) was mixed with 100 μL of complex sample solution (500 μg / mL) and incubated in a 37°C water bath for 10 minutes. Then, 200 μL of starch (0.5%, w / v) dissolved in phosphate buffer was added to the mixture, and the reaction was continued in a 37°C water bath for 10 minutes. After terminating the enzymatic reaction with 100 μL of DNS colorimetric reagent, the mixture was boiled in a 100°C boiling water bath for 10 minutes and then cooled to room temperature. Finally, 1.5 mL of water was added to dilute the mixture, and the absorbance value of the sample at 540 nm was recorded using a UV spectrophotometer.

[0021] The determination of the control group (using deionized water instead of the complex sample aqueous solution), the sample background group (using PBS buffer instead of α-amylase solution), and the control background group (using deionized water instead of the complex sample aqueous solution and PBS buffer instead of α-amylase solution) was performed in the same manner as described above. The enzyme inhibition rate was calculated according to the following formula (1).

[0022]

[0023] In equation (1): OD S The absorbance value of the sample group; OD SB The absorbance value of the sample background group; OD C The absorbance value is for the control group; OD CB The absorbance value is used as a reference for the background group.

[0024] The method for determining α-glucosidase inhibitory activity involved in the embodiments of the present invention is as follows:

[0025] Sample group: Mix 100 μL of α-glucosidase (0.25 U / mL) with 100 μL of complex sample solution (500 μg / mL) and incubate at 37°C for 10 minutes. Then, add 150 μL of p-NPG reagent (2.5 mM) to the mixture and continue the reaction at 37°C for 30 minutes. Terminate the enzymatic reaction by adding 2 mL of Na₂CO₃ solution (1 M). Record the absorbance of the sample at 405 nm using a UV spectrophotometer.

[0026] The determination of the control group (using deionized water instead of the complex sample aqueous solution), the sample background group (using PBS buffer instead of α-amylase solution), and the control background group (using deionized water instead of the complex sample aqueous solution and PBS buffer instead of α-amylase solution) was carried out in the same manner as described above, and the inhibition rate was calculated according to Formula 1 above.

[0027] The liquid chromatography analysis method involved in the embodiments of this invention:

[0028] The lyophilized sample powder was dissolved in water and then filtered through a 0.22 μm syringe filter. The filtered sample solution was then injected, and HPLC was performed using LP-C. 18 The column (250 mm × 4.6 mm, 5 μm) was maintained at 25 °C. The flow rate was set to 1 mL / min, and the detection wavelength was 280 nm. The mobile phase consisted of water / formic acid solution (99:1, v / v, as phase A) and acetonitrile (as phase B). The column was first equilibrated with 5% phase B. After injection, gradient elution was performed according to the following program: from 0 to 20 minutes, phase B was increased from 5% to 35%.

[0029] Example 1

[0030] (1) Extraction of phenolic acids from pecan shells: Pecan shells were pulverized and mixed with water at a solid-liquid mass ratio of 1:6. The mixture was heated to 50°C. The mixture was homogenized using a high-pressure homogenizer at 5 MPa. The homogenized mixture was then separated into solid and liquid phases by filtration, and the clear liquid was collected.

[0031] (2) Separation of pecan chitosan: The supernatant from step (1) was subjected to ultrafiltration sieving using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the permeate was collected.

[0032] (3) Purification of phenolic acid compounds: The ultrafiltration permeate from step (2) was added to an HW-40 gel column. First, 2BV was eluted with 5% ethanol solution to remove impurities, and then 2BV was eluted with 40% ethanol solution. The eluent was collected, concentrated under reduced pressure, and then freeze-dried. The resulting powder was the phenolic acid extract of pecan shell.

[0033] (4) Preparation of pecan chitosan-pea protein complex: Pea protein was dispersed in water to a concentration of 10 g / L, and the pecan chitosan extract from step (3) was added to a concentration of 0.1 g / L. After mixing, laccase (EC.1.10.3) was added to a concentration of 0.5 U / L, the pH was adjusted to 7.2, the temperature was maintained at 40℃, and the mixture was stirred continuously for 2 h. The mixture was then placed in a dialysis bag (MWCO 10000 Da) and dialyzed in water to remove free phenolic acid compounds. After dialysis, the mixture in the dialysis bag was freeze-dried to obtain pecan chitosan-pea protein complex powder. Its α-amylase and α-glucosidase inhibitory activities are shown in Table 1.

[0034] Example 2

[0035] The difference between Example 2 and Example 1 is that the solid-liquid mass ratio in step (1) is 1:30, the extraction temperature is room temperature, and the mixture is extracted by homogenization at 30 MPa using a high-pressure homogenizer. Other conditions are the same as in Example 1. The inhibitory activities of α-amylase and α-glucosidase are shown in Table 1.

[0036] Example 3

[0037] The difference between Example 3 and Example 1 lies in the preparation of the pecan chitosan-pea protein complex in step (4). The pea protein concentration was 85 g / L, the pecan chitosan extract concentration was 2 g / L, the pH was adjusted to 8.0, the temperature was maintained at 30°C, and stirring was continued for 0.5 h. After dialysis, the mixture in the dialysis bag was dried by spray drying to obtain pecan chitosan-pea protein complex powder. Other conditions remained the same as in Example 1. The α-amylase and α-glucosidase inhibitory activities are shown in Table 1.

[0038] Example 4

[0039] The difference between Example 4 and Example 2 is that in step (3), after the ultrafiltration permeate is added to the HW-40 gel column, 2BV is first eluted with 15% ethanol solution to remove impurities, and then 2BV is eluted with 60% ethanol solution. Other conditions are the same as in Example 2. The inhibitory activities of α-amylase and α-glucosidase are shown in Table 1.

[0040] Example 5

[0041] The difference between Example 5 and Example 1 is as follows: the solid-liquid mass ratio in step (1) is 1:20; the extraction temperature is 35℃; and the mixture is extracted by homogenization using a high-pressure homogenizer at 20MPa. In step (3), after the ultrafiltration permeate is added to an HW-40 gel column, 2BV is first eluted with 10% ethanol solution to remove impurities, and then 2BV is eluted with 50% ethanol solution. In step (4), the pea protein concentration is 45g / L, the pecan chitosan extract concentration is 1g / L, after mixing, laccase is added to 3U / L, the pH is adjusted to 7.6, the temperature is maintained at 35℃, and stirring is continued for 1.5h. Other operating steps and conditions are consistent with those in Example 1.

[0042] Comparative Example 1:

[0043] The difference between Comparative Example 1 and Example 1 is that in step (4), the pea protein solution does not contain pecan chitosan extract, while other conditions remain the same as in Example 1. The inhibitory activities of α-amylase and α-glucosidase are shown in Table 1.

[0044] Comparative Example 2:

[0045] The difference between Comparative Example 2 and Example 2 is that in step (4), laccase is not added after the pea protein is mixed with pecan chitosan; other conditions remain the same as in Example 2. The inhibitory activities of α-amylase and α-glucosidase are shown in Table 1.

[0046] Table 1. Inhibitory activities of α-amylase and α-glucosidase

[0047] α-Amylase inhibition rate (%) α-glucosidase inhibition rate (%) Example 1 83.2 85.1 Example 2 84.0 86.2 Example 3 86.2 91.7 Example 4 81.3 85.6 Example 5 82.6 87.1 Comparative Example 1 1.6 2.8 Comparative Example 2 2.4 3.5

[0048] This invention extracts and separates high-purity phenolic acid compounds from pecan shells, and prepares a phenolic acid-pea protein complex using laccase catalysis. Converting pecan shells into valuable active extracts reduces environmental pollution from agricultural waste, achieves resource recycling, and aligns with the principles of green chemistry and sustainable development. The pecan phenolic acid-pea protein complex prepared by this invention exhibits an inhibition rate of 81.3%–86.2% against α-amylase and 85.1%–91.7% against α-glucosidase, significantly higher than comparative examples 1-2. This invention enhances the utilization value of pea protein and expands its application areas. It endows pea protein with additional biological activity, making it an excellent source for functional foods or nutritional supplements.

[0049] The above description only illustrates the technical solution of the present invention with reference to preferred embodiments. However, those skilled in the art should be able to make changes in specific implementation methods and application scope based on the ideas of the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for preparing a pecan chitosan-pea protein complex with starch digestive enzyme inhibitory activity, characterized in that, The method includes the following steps: Step 1: Extraction of phenolic acid from pecan shells: Pecan shells are processed by a pulverizer and then mixed with water, with the temperature adjusted between 25℃ and 50℃; the mixture is homogenized using a high-pressure homogenizer at 5 to 30 MPa; the homogenized mixture is then separated into solid and liquid by filtration or centrifugation, and the clear liquid is collected. Step 2, Separation of pecan chitosan: The supernatant from Step 1 was subjected to ultrafiltration sieving using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the permeate was collected. Step 3, purification of phenolic acid compounds: The ultrafiltration permeate from step 2 is added to an HW-40 gel column. First, it is eluted with a low-concentration ethanol solution for 2 column volumes to remove impurities, and then eluted with a high-concentration ethanol solution for 2 column volumes. The eluent is collected, concentrated under reduced pressure, and then freeze-dried or spray-dried. The resulting powder is the phenolic acid extract of pecan shell. Step 4: Preparation of pecan chitosan-pea protein complex: Pea protein is dispersed in water at a concentration of 10 g / L to 85 g / L. Pecan chitosan extract from Step 3 is added to a concentration of 0.1 g / L to 2 g / L. After mixing, laccase is added to a concentration of 0.5 U / L to 5 U / L. The pH is adjusted to 7.2 to 8.0, and the mixture is stirred and mixed. Free phenolic acid compounds are removed by dialysis. The mixture in the dialysis bag is dried by freeze-drying or spray drying to obtain pecan chitosan-pea protein complex powder.

2. The method for preparing a pecan chitosan-pea protein complex with starch digestive enzyme inhibitory activity according to claim 1, characterized in that, In step 1, the solid-liquid mass ratio of the powder obtained from crushing the hickory shells to water is 1:6 to 1:

30.

3. The method for preparing a pecan chitosan-pea protein complex with starch digestive enzyme inhibitory activity according to claim 1, characterized in that, In step 3, the volume fraction of the low-concentration ethanol solution is 5-15%, and the volume fraction of the high-concentration ethanol solution is 40-60%.

4. The method for preparing a pecan chitosan-pea protein complex with starch digestive enzyme inhibitory activity according to claim 1, characterized in that, In step 4, after adding laccase, the temperature is maintained within the range of 30-40℃, and stirring is continued for 0.5-2 hours.

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