Sweet potato peptide for inhibiting alpha-glucosidase activity and preparation method thereof

Abstract

The present invention relates to the field of biotechnology, and in particular to a sweet potato peptide that inhibits alpha-glucosidase activity and a preparation method thereof. Sweet potato starch wastewater protein is recovered by an acid precipitation method, and the sweet potato protein is subjected to heat combined with ultrahigh pressure pretreatment assisted enzymatic hydrolysis to obtain a sweet potato peptide that inhibits alpha-glucosidase activity. The present invention obtains for the first time a sweet potato peptide that has good inhibition of alpha-glucosidase activity, high stability, and can delay starch digestion in vivo, which is beneficial to the high-value utilization of sweet potato starch wastewater and provides a new raw material for products that inhibit alpha-glucosidase activity.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a sweet potato peptide capable of inhibiting alpha-glucosidase activity and a preparation method thereof. Background Art

[0002] α-glucosidase is mainly located on the brush border cell membrane of the small intestine. It is an enzyme that plays a key role in the biological sugar metabolism process. It can hydrolyze disaccharides such as sucrose, maltose and other oligosaccharides into monosaccharides that can be partially absorbed by the human small intestine. Inhibitors of α-glucosidase help reduce enzyme activity and the rate at which oligosaccharides are decomposed into glucose, thereby reducing postprandial blood sugar peaks and blood sugar fluctuations, reducing the stimulation of high blood sugar on organs such as the pancreas, maintaining postprandial blood sugar concentrations at normal levels, and improving postprandial hyperglycemia.

[0003] Sweet potato (Dioscorea esculenta (Lour.) Burkill) is also known as sweet potato, sweet potato, white potato, red potato, yam, etc. Sweet potato was introduced to China from Southeast Asia at the end of the 16th century. It is now the fifth largest food crop in my country and one of the main vegetables. It is widely planted in various parts of China, and its total output and planting area rank first in the world. 80% of sweet potato processing in my country is starch production, and there are two most prominent problems in sweet potato starch processing. First, in addition to sweet potato starch, other active ingredients such as sweet potato protein and polysaccharides are not used at all, resulting in serious waste of resources; second, the unused sweet potato active ingredients cause more serious environmental pollution along with sweet potato starch wastewater.

[0004] Enzymatic hydrolysis technology is a relatively mild and green method, which is often used for the preparation of active peptides. However, the molecular structure of sweet potato protein is dense, which leads to the obstruction of enzymatic hydrolysis rate and the problem of low peptide conversion rate has not been well solved. Heat treatment (HT) and high hydrostatic pressure (HHP) are common treatment methods in the food industry, which can be used to stretch the protein structure, expose more active sites, and ultimately improve the efficiency of enzymatic hydrolysis. However, there are limitations to using HT or HHP alone to improve the efficiency of enzymatic hydrolysis. For example, large aggregates are formed during HT, which leads to the obstruction of enzymatic reaction. In contrast, heating combined with high hydrostatic pressure pretreatment (HT+HHP) can change the thermal aggregation morphology to obtain smaller protein particles, so that the protein structure can be more fully unfolded and the possibility of obtaining bioactive peptides can be increased. However, there are few studies on improving the enzymatic hydrolysis efficiency of sweet potato protein and using appropriate pretreatment methods to evaluate its effects on the structure, enzyme properties and hydrolyzate of sweet potato protein. In addition, there are literature reports on the antioxidant and antihypertensive activities of sweet potato protein peptides, but there are no reports on the preparation of active peptides that inhibit α-glucosidase from sweet potato protein. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the amount of wastewater generated by the production of sweet potato starch in my country reaches 20 million to 40 million tons each year, and there is no good way to recycle it. In addition, there is no report on the preparation of active peptides that inhibit alpha-glucosidase from sweet potato protein, and the molecular structure of sweet potato protein is dense, which leads to the obstruction of enzymatic hydrolysis rate, and the problem of low peptide conversion rate has not been well solved.

[0006] In order to solve the above problems, the present invention provides a preparation method and application of an active peptide that inhibits α-glucosidase. The preparation method of the present invention is to recover the sweet potato starch wastewater protein by acid precipitation method, and perform heat and ultra-high pressure pretreatment to assist enzymatic hydrolysis on the sweet potato protein, which can increase the polypeptide content and improve the activity of inhibiting GAA.

[0007] To achieve the above object, the present invention is achieved by the following technical means: a method for preparing sweet potato peptides that inhibit α-glucosidase activity, comprising the following steps:

[0008] (1) The pH value of the sweet potato starch processing wastewater is adjusted to 4, and the wastewater is centrifuged after standing and settling to obtain a precipitate. The precipitate is freeze-dried to obtain the sweet potato crude protein. The method for extracting protein is alkali extraction and acid precipitation, which is a commonly used and traditional method for protein extraction. Its principle is that under alkaline conditions, the protein structure changes, the secondary bonds (hydrogen bonds) of protein molecules are destroyed, and some polar groups are dissociated, which promotes the separation of the conjugate and the protein. The pH value of the supernatant after centrifugation is adjusted to reach the isoelectric point of the protein, which increases the solubility of the protein and allows the protein to precipitate. This method is easy to operate for protein extraction, does not produce harmful substances, has little pollution to the environment, is low in cost, and is suitable for industrial production. Since the raw material is liquid, it is directly acid precipitated. Literature shows that the optimal isoelectric point of sweet potato protein is 4. At this pH, the protein precipitates the most, so the pH value is 4.

[0009] (2) Weigh an appropriate amount of sweet potato crude protein, add distilled water, heat and then treat with ultrahigh pressure, then adjust the pH to 10, add alkaline protease for hydrolysis; after the enzymatic hydrolysis is completed, centrifuge and take the supernatant to obtain sweet potato peptides (SPP) that inhibit α-glucosidase activity. Heating and high pressure treatment are both for stretching the protein structure, exposing more active sites, and improving the efficiency of enzymatic hydrolysis. There are limitations to using heating or ultrahigh pressure alone to improve the efficiency of enzymatic hydrolysis. For example, large aggregates will be formed during heating, which will lead to the obstruction of enzymatic reaction. In contrast, heating combined with ultrahigh pressure pretreatment can change the thermal aggregation morphology to obtain smaller protein particles, thereby making the protein structure more fully unfolded and increasing the possibility of obtaining bioactive peptides. The inventors studied alkaline protease, neutral protease, papain, pepsin, and trypsin. Based on the peptide yield and α-glucosidase inhibition rate as indicators, it was found that alkaline protease had the best effect, so alkaline protease was selected. The optimal enzymatic hydrolysis condition of alkaline protease is pH 10, so alkaline protease was added after adjusting the pH to 10 for hydrolysis.

[0010] Furthermore, the supernatant after starch extraction from sweet potatoes is used to replace starch processing wastewater: the sweet potatoes are washed and peeled, and 4 to 5 times the volume of water of the sweet potatoes are added to crush and extract. After the extract is allowed to stand for 6 to 8 hours, it is filtered with gauze to remove the sweet potato fiber and large-grain starch to obtain a supernatant, and the pH value of the supernatant is adjusted to 4.

[0011] Furthermore, in step (2), distilled water is added in a ratio of 1:15 to 1:30 (w / v); the mixture is heated at 90° C. for 10 min and then subjected to ultrahigh pressure treatment.

[0012] Furthermore, the parameters of the high pressure treatment in step (2) are: 100-400 MPa ultra-high pressure treatment for 4-10 min.

[0013] Furthermore, the enzymatic hydrolysis conditions in step (2) are 2-5% (w / w) alkaline protease followed by hydrolysis in a 55° C. constant temperature water bath for 2-5 hours.

[0014] Furthermore, after the enzymatic hydrolysis in step (2) is completed, the enzyme is inactivated at 100° C. for 10 to 15 minutes and then centrifuged at 4° C. to obtain the supernatant.

[0015] Furthermore, the centrifugal parameters in step (2) are 10000 r / min and centrifugation for 15 min.

[0016] Furthermore, the sweet potato peptide SPP obtained in step (2) is filtered with a microporous filter membrane, and then treated with an ultrafiltration membrane with a molecular weight Mw cutoff value of 3 in an ultrafiltration system to obtain a sweet potato peptide SPP-1 with Mw < 3 kDa.

[0017] Furthermore, the pore size of the microporous filter membrane is 0.22 μm.

[0018] The sweet potato peptide prepared by any of the above methods.

[0019] The sweet potato peptide is used in the preparation of products for inhibiting alpha-glucosidase activity.

[0020] The beneficial effects of the present invention are:

[0021] (1) Compared with the prior art, the present invention adopts a combination of heat processing and high pressure technology, which effectively saves enzymatic hydrolysis time and improves the enzymatic hydrolysis yield; the reagents used in the experimental process have no impact on the environment, and the extraction yield of polypeptides is high.

[0022] (2) The present invention is the first to obtain a sweet potato peptide that has good inhibitory effect on α-glucosidase activity and can delay starch digestion in vivo, which is beneficial to the high-value utilization of sweet potato starch wastewater.

[0023] (3) The present invention verifies for the first time that sweet potato peptide has the activity of inhibiting α-glucosidase, and provides a new raw material for products that inhibit α-glucosidase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of different proteases on α-glucosidase inhibitory activity and peptide yield;

[0025] Figure 2 is the effect of different pretreatments on α-glucosidase inhibitory activity and peptide yield;

[0026] Figure 3 The effect of the solid-liquid ratio on the α-glucosidase inhibitory activity and the peptide yield;

[0027] Figure 4 The effect of enzyme addition on α-glucosidase inhibitory activity and peptide yield;

[0028] Figure 5 The effect of enzymatic hydrolysis time on α-glucosidase inhibitory activity and peptide yield;

[0029] Figure 6 is the effect of high pressure on α-glucosidase inhibitory activity and peptide yield;

[0030] Figure 7 The effect of high pressure time on α-glucosidase inhibitory activity and peptide yield;

[0031] Figure 8 is the effect of the response face on α-glucosidase inhibitory activity and peptide yield;

[0032] Fig. 9 It is a comparison of the α-glucosidase inhibition rate of each ultrafiltration component;

[0033] Fig.10 The IC of the α-glucosidase inhibitory activity of SPP-1 component 50 ;

[0034] Fig.11 is the effect of temperature on the stability of SPP-1 components;

[0035] Fig.12 is the effect of pH on the stability of SPP-1 components;

[0036] Fig.13 is the effect of oral and gastrointestinal digestion on the stability of SPP-1 components;

[0037] Fig.14 It is a graph of blood glucose levels within 3 hours after mice were gavaged with different doses of SPP-1, starch, and maltose; (AB) is a curve graph, and (CD) is the area under the corresponding curve. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0039] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0040] Detection method:

[0041] (1) Determination of peptide yield

[0042] Take 2mL of enzymatic hydrolysate and 10% TCA solution, mix well and let stand for 10min, centrifuge at 4000r / min for 15min, take the supernatant and dilute to 50mL with 5% TCA, take 3mL of the above solution and add 2mL of biuret reagent, mix well and let stand for 10min, measure its absorbance at 540nm, and draw a standard curve. The peptide yield calculation formula is as follows:

[0043] Peptide yield (%) = peptide content in enzymatic hydrolysate / protein content in raw material × 100 (1)

[0044] (2) Determination of α-glucosidase inhibition rate

[0045] Take 200 μL of sample and add 200 μL of α-glucosidase (0.2U / mL), mix the above solution and incubate at 37°C for 5 minutes. Then add 200 μL of 2.5mM p-nitrobenzene-α-glucoside solution (pNPG) and 0.1M phosphate buffer (pH6.8) to 2mL and incubate at 37°C for 20 minutes. Finally, add 200 μL of 0.4M sodium carbonate to stop the reaction and measure the absorbance at 405nm. The α-glucosidase inhibition rate is calculated as follows:

[0046] α-Glucosidase inhibition rate (%) = [1-(A1-A2) / (A3-A4)] × 100 (2)

[0047] Where: A1—absorbance value of sample group

[0048] A2—Absorbance value of sample background

[0049] A3—Absorbance without sample

[0050] A4—Absorbance value of blank group without sample

[0051] Embodiment 1:

[0052] A method for preparing a sweet potato peptide for inhibiting α-glucosidase activity comprises the following steps:

[0053] (1) The sweet potatoes are washed and peeled, and 4 to 5 times the volume of water is added to crush and extract. The extract is allowed to stand for 6 to 8 hours and then filtered through gauze to remove the sweet potato fiber and large granular starch to obtain a supernatant. The supernatant is adjusted to a pH value of 4, allowed to stand and precipitate, and then centrifuged to obtain a precipitate. The precipitate is freeze-dried to obtain the sweet potato crude protein.

[0054] (2) Weigh an appropriate amount of sweet potato crude protein, add distilled water at a ratio of 1:20 (w / v), heat at 90°C for 10 min, and then high-pressure treat at 200 MPa for 6 min. Then adjust the pH to 10 with 1 mol / L NaOH, add 4% (w / w) alkaline protease, and hydrolyze in a 55°C constant temperature water bath for 3 h. After the enzymatic hydrolysis is completed, centrifuge at 4°C, 10,000 r / min for 15 min to obtain the supernatant to obtain the sweet potato enzymatic hydrolysate (SPP).

[0055] Embodiment 2:

[0056] A method for preparing a sweet potato peptide for inhibiting α-glucosidase activity comprises the following steps:

[0057] (1) The pH value of sweet potato starch processing wastewater (wastewater obtained from the sweet potato starch production line of Shandong Huatao Food Co., Ltd.) was adjusted to 4, and the wastewater was allowed to settle and then centrifuged to obtain a precipitate. The precipitate was freeze-dried to obtain sweet potato crude protein.

[0058] (2) Weigh an appropriate amount of sweet potato crude protein, add distilled water at a ratio of 1:20 (w / v), heat at 90°C for 10 min, and then high-pressure treat at 200 MPa for 6 min. Then adjust the pH to 10 with 1 mol / L NaOH, add 4% (w / w) alkaline protease, and hydrolyze in a constant temperature water bath at 55°C for 3 h. After the enzymatic hydrolysis, centrifuge at 4°C, 10,000 r / min for 15 min to obtain the supernatant and SPP.

[0059] Embodiment 3:

[0060] The enzymatic hydrolysate obtained in Example 1 or 2 was filtered with a microporous filter membrane (pore size 0.22 μm), and then treated with an ultrafiltration membrane with a molecular weight Mw cutoff value of 3 kDa in an ultrafiltration system to obtain sweet potato peptide SPP-1 with Mw < 3 kDa.

[0061] Comparative Example 1:

[0062] The above enzymatic hydrolysate was filtered with a microporous filter membrane (pore size 0.22 μm), and then graded in an ultrafiltration system with an ultrafiltration membrane having a molecular weight (Mw) cutoff value of 3 and 5 kDa to obtain 5 kDa<Mw<3 kDa (SPP-2) and Mw>5 kDa (SPP-3).

[0063] Process parameter adjustment:

[0064] 1. Screening of different proteases for sweet potato peptide hydrolysis

[0065] (1) is the same as step (1) in Example 1 or Example 2.

[0066] (2) Alkaline protease, neutral protease, papain, pepsin, and trypsin were selected and added to distilled water at a ratio of 1:20 (w / v). The enzyme addition amount was 3%, and the pH and temperature were the optimal conditions for various enzymes for enzymatic hydrolysis. After 3 hours of enzymatic hydrolysis, the enzymes were taken out and inactivated at 100°C for 10 to 15 minutes. The supernatant was centrifuged and the polypeptide yield and α-glucosidase inhibition rate of the enzymatic hydrolysis products were determined.

[0067] (3) The peptide yields and α-glucosidase inhibition rates of different protease hydrolysates are shown in Figure 1 .like Figure 1 As shown in the results, the hydrolysis product of alkaline protease has the highest inhibitory ability against α-glucosidase, with an inhibition rate of 46.7%. It was followed by pepsin and trypsin. In terms of peptide yield, the yield of alkaline protease peptide was 54.35%, which was significantly higher than that of the other four proteases (p<0.05). It is reported that alkaline protease (2×10 5U / g, Nanning Pangbo Bioengineering Co., Ltd.) is a commonly used protease in the food industry because it has a wider range of specificity, high peptide yield and enzymatic hydrolysis catalytic activity. Therefore, alkaline protease was selected as the best hydrolase for the preparation of SPP in this study.

[0068] 2. Selection of sweet potato peptide enzymatic pretreatment

[0069] The sweet potato crude protein obtained in step (1) of Example 1 or Example 2 is added to distilled water at a ratio of 1:20 (w / v), and the protein solution is heated at 90°C for 10 min, subjected to high pressure treatment at 300 MPa for 6 min, subjected to high pressure treatment at 90°C for 10 min and then subjected to high pressure treatment at 300 MPa for 6 min, or subjected to no pretreatment for each group, and then the pH is adjusted to 10 using 1 mol / L NaOH, 3% (w / w) alkaline protease is added, and the solution is enzymolyzed in a constant temperature water bath at 55°C for 3 h, taken out, inactivated at 100°C for 10 to 15 min, and the supernatant is centrifuged to determine the polypeptide yield and α-glucosidase inhibition rate of the enzymolysis product.

[0070] The peptide yield and α-glucosidase activity of SPP in different pretreatment groups Figure 2 As shown. Compared with the control group, all pretreatments significantly increased the peptide yield and α-glucosidase inhibition rate of SPP (p<0.05). Compared with the single treatment group, HT+HHP further enhanced the peptide yield and α-glucosidase inhibition rate of SPP. This is related to the destruction of the sweet potato protein structure by HT+HHP. The molecular structure of the sweet potato protein obtained after HT+HHP treatment was more thoroughly unfolded, exposing more active sites that bind to alkaline proteases. Therefore, the HT+HHP pretreatment method was selected for subsequent research.

[0071] 3. Single factor experiment of sweet potato peptide enzymatic hydrolysis

[0072] In the present test examples, HT+HHP pretreatment was used and 3% alkaline protease was used for enzymatic hydrolysis in a constant temperature water bath at pH 10 and temperature 55° C. The enzyme was inactivated at 100° C. for 10 to 15 minutes and then the supernatant was collected by centrifugation.

[0073] (1) Effect of the material-liquid ratio on the peptide yield of the enzymatic hydrolysis product and the α-glucosidase inhibition rate

[0074] The material-liquid ratio was selected as 1:10, 1:15, 1:20, 1:25, and 1:30. After heating at 90°C for 10 min, the high pressure was 300 MPa for 6 min. The enzyme addition amount was 3%. Under the conditions of pH 10 and enzymatic hydrolysis temperature of 55°C, the enzymatic hydrolysis was carried out for 3 h. After the enzymatic hydrolysis was completed, the polypeptide yield and α-glucosidase inhibition rate of the enzymatic hydrolysis product were determined.

[0075] Depend on Figure 3It can be seen that the peptide yield decreases with the increase of liquid-to-solid ratio. This is because the lower the substrate concentration, the lower the probability of binding to the enzyme, and the lower the peptide yield. The inhibition rate of α-glucosidase activity showed a trend of first increasing and then decreasing. When the liquid-to-solid ratio was 1:20 g / mL, the highest α-glucosidase inhibition rate was 60.93%. Therefore, 1:20 g / mL was preferred as the basis for the next step of optimization.

[0076] (2) Effect of enzyme addition amount on peptide yield and α-glucosidase inhibition rate of enzymatic hydrolysis products

[0077] The material-liquid ratio was 1:20, and the reaction was heated at 90°C for 10 min and then at 300 MPa for 6 min. The enzyme addition amounts were 1, 2, 3, 4, and 5%, respectively. The enzymatic hydrolysis was carried out at pH 10 and a hydrolysis temperature of 55°C for 3 h. After the enzymatic hydrolysis was completed, the polypeptide yield and α-glucosidase inhibition rate of the enzymatic hydrolysis product were determined.

[0078] Depend on Figure 4 It can be seen that as the amount of enzyme added increases, the protein solution is continuously hydrolyzed by the enzyme, the yield of polypeptides increases, and the inhibition of α-glucosidase first increases and then decreases. Increasing the amount of enzyme added allows it to fully contact with the substrate, and the macromolecular protein is hydrolyzed into peptides of different molecular weights, and the inhibition rate of α-glucosidase decreases. When the enzyme addition amount is 4%, the α-glucosidase inhibition rate reaches a maximum of 68.67%. Therefore, 4% is preferred as the basis for the next step of optimization.

[0079] (3) Effect of enzymatic hydrolysis time on the peptide yield and α-glucosidase inhibition rate of the enzymatic hydrolysis product

[0080] The material-liquid ratio was 1:20, and the high pressure was 300 MPa for 6 min after heating at 90 °C for 10 min. The enzyme addition amount was 3%. The enzymatic hydrolysis temperature was 55 °C at pH 10 and the enzymatic hydrolysis was performed for 1, 2, 3, 4, and 5 h. After the enzymatic hydrolysis was completed, the polypeptide yield and α-glucosidase inhibition rate of the enzymatic hydrolysis product were determined.

[0081] Depend on Figure 5 It can be seen that as the enzymatic hydrolysis time increases, the peptide yield gradually increases, and the α-glucosidase inhibition rate shows a trend of first increasing and then decreasing. This is because the effective peptide segments with α-glucosidase inhibition are hydrolyzed into small peptides without inhibitory activity as the hydrolysis continues, thereby reducing the activity inhibition rate. When the enzymatic hydrolysis time is 3h, the α-glucosidase activity inhibition rate reaches a maximum value of 63.03%. Therefore, 3h is preferred as the basis for the next step of optimization.

[0082] (4) Effect of high pressure on the peptide yield of enzymatic hydrolysis products and α-glucosidase inhibition rate

[0083] The material-liquid ratio was 1:20, and after heating at 90°C for 10 min, the products were subjected to high pressure at 100, 200, 300, 400, and 500 MPa for 6 min, respectively. The enzyme addition amount was 3%, and the enzymatic hydrolysis temperature was 55°C at pH 10 for 3 h. After the enzymatic hydrolysis, the polypeptide yield and α-glucosidase inhibition rate of the enzymatic hydrolysis product were determined.

[0084] Depend on Figure 6 It can be seen that the peptide yield and α-glucosidase inhibition rate both showed a trend of first increasing and then decreasing with the increase of high pressure. When the high pressure was 200MPa, the α-glucosidase activity inhibition rate was the maximum value of 69.97%. However, when the high pressure continued to increase, the sweet potato protein structure was destroyed, the enzyme binding sites continued to increase, and the macromolecular protein was hydrolyzed into short peptides with smaller molecular weight, resulting in a decrease in the α-glucosidase inhibition rate. Therefore, 200MPa was preferentially selected as the basis for the next step of optimization.

[0085] (5) Effect of high pressure time on the peptide yield of enzymatic hydrolysis products and α-glucosidase inhibition rate

[0086] The material-liquid ratio was 1:20, and the mixture was heated at 90°C for 10 min and then subjected to high pressure at 300 MPa for 2, 4, 6, 8, and 10 min, respectively. The enzyme addition amount was 3%, and the enzymatic hydrolysis was carried out for 3 h at a pH of 10 and a hydrolysis temperature of 55°C. After the enzymatic hydrolysis was completed, the polypeptide yield and α-glucosidase inhibition rate of the enzymatic hydrolysis product were determined.

[0087] Depend on Figure 7 It can be seen that when the high pressure is constant, as the high pressure time increases, the peptide yield and α-glucosidase inhibition rate both show a trend of first rising and then flattening. When the high pressure time is 6 minutes, the peptide yield and α-glucosidase activity inhibition rate reach the maximum value, which are 62.08% and 66.93%, respectively. Therefore, 6 minutes is preferred as the basis for the next step of optimization.

[0088] 4. Plackett-Burman test optimization and response surface experiment of sweet potato peptide enzymatic hydrolysis process

[0089] (1) Based on the single factor experiment, the Plackett-Burman experiment and three-factor three-level response surface experiment were designed with α-glucosidase inhibitory activity and peptide yield as indicators to optimize the process conditions of SPP. The factors and level arrangements of the Plackett-Burman experiment and three-factor three-level design are shown in Tables 1 and 2.

[0090] Table 1 Factors and levels of Plackett-Burman test design

[0091]

[0092] Table 2 Response surface three-factor three-level experimental design

[0093]

[0094] (2) As shown in Tables 3 and 4, the p values ​​of the solid-liquid ratio, enzyme addition amount, and high pressure are all less than 0.05, indicating that these three factors have a significant effect on the peptide yield and the inhibition rate of α-glucosidase activity. Therefore, in order to improve the peptide yield and the inhibition rate of α-glucosidase activity, the solid-liquid ratio, enzyme addition amount, and high pressure can be selected for further response surface experiments.

[0095] Table 3 Analysis of Plackett-Burman test results

[0096]

[0097] Table 4 Analysis of the impact of Plackett-Burman test results

[0098]

[0099] (3) Based on the plackett-burman test, the Box-Behnken design was used to further optimize the optimal α-glucosidase inhibition rate of SPP: solid-liquid ratio, enzyme-substrate ratio and high pressure. The design matrix included 17 experiments (Table 5). The obtained polypeptide yield and α-glucosidase inhibition rate were 67.7%-78.1% and 54.76%-71.7%, respectively. The quadratic regression model was obtained as follows, where A, B, and C are the solid-liquid ratio, enzyme content, and high pressure, respectively; Y1 and Y2 represent the polypeptide yield and α-glucosidase inhibition rate, respectively.

[0100] Y1=77.76-0.1957A-0.612B-0.8055C-0.3083AB-0.4162AC+0.6458BC-4.82A 2 -4.35B 2 -3.67C 2

[0101] Y2=71.06+1.25A+1.75B-0.6030C-0.88AB+2.06AC+0.1080BC-4.77A 2 -7.81B 2 -5.96C 2

[0102] The experimental data were subjected to variance analysis to estimate the significance of the second-order polynomial equation (Tables 6 and 7). The quadratic regression model was significant (p<0.05), which verified that the model was reliable for the determination of peptide yield and α-glucosidase inhibitory activity. In addition, the lack of fit term was not significant (p>0.05), which further supported the validity of the model. The R2 of peptide yield and α-glucosidase inhibition rate were 0.9937 and 0.9961, respectively, indicating that the model had good accuracy. The corrected R 2 The experimental values ​​were 0.9856 and 0.9911, respectively, and the predicted values ​​were in good agreement. The coefficient of variation (CV) was less than 5%, and the results were accurate and reproducible. These results verified the adequacy, accuracy, and reliability of the model for optimizing the extraction conditions to increase the content of α-glucosidase inhibitory peptides in sweet potatoes. As shown in the F value, the enzyme content had the greatest effect on the peptide yield, and the high pressure had the greatest effect on the α-glucosidase inhibition rate. The interactions between different factors were visualized with response surface plots, such as Figure 8 The interaction between enzyme content and high pressure had a significant effect on peptide yield, and the solid-liquid ratio and high pressure had a significant effect on α-glucosidase inhibition rate.

[0103] Table 5 Response surface test results

[0104]

[0105] Table 6 α-glucosidase inhibition rate fitting variance analysis

[0106]

[0107] Table 7 Peptide yield fitting variance analysis

[0108]

[0109] The obtained regression equation was regressed stepwise using Design Expert 8.0.6 software, and the optimal process conditions for preparing SPP by thermal high-pressure assisted enzymatic hydrolysis were obtained: feed-liquid ratio 1:20.16w / v, enzyme content 4.02%, and high pressure 192.31MPa. The predicted α-glucosidase inhibition rate was 71.13% and the polypeptide yield was 77.77%. In order to verify the optimal enzymatic hydrolysis conditions predicted by the model, combined with the actual production operation, the optimal process conditions were adjusted to: feed-liquid ratio 1:20w / v, enzyme content 4%, and high pressure 200MPa. Under these conditions, the α-glucosidase inhibition rate was 70.93%, and the polypeptide yield was 76.89%, which was close to the predicted value, proving that the optimization results of this experiment are more reliable.

[0110] Sweet potato peptide purification, structure identification, stability and in vivo activity verification:

[0111] 1. Study on α-glucosidase inhibition rate

[0112] The SPP, SPP-1, SPP-2 and SPP-3 components obtained in Examples 1-3 were collected, freeze-dried and the α-glucosidase inhibition rate was determined.

[0113] The results are as follows Fig. 9 As shown, among the three components of SPP-1, SPP-2 and SPP-3, the SPP-1 component has the highest α-glucosidase inhibition rate, which is significantly better than SPP, SPP-2 and SPP-3. Fig.10 As shown in Figure 2, when the peptide concentration was 5 mg / mL, the SPP-1α-glucosidase inhibition rate was 77.65±0.74%, and its IC 50 The value was 6.139 mg / mL, indicating that SPP-1 showed strong α-glucosidase inhibitory activity in the experiment.

[0114] 2. SPP-1 structural analysis

[0115] After desalting, the peptide sample was centrifuged and dried, and then redissolved in Nano-LC mobile phase A (0.1% formic acid / water) for online LC-MS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, ), followed by 20ul volume washing and desalting. After desalting and retention on the pre-column, the sample was separated by a C18 reverse phase column (75μm×25cm), and the mobile phase B (80% acetonitrile, 0.1% formic acid) was increased from 5% to 38% within 60min. The mass spectrometer used the ThermoFisher Q Exactive plus system combined with the nanospray Nano Flex ion source, and the spray voltage was 1.9kV. The mass spectrometer scanning mode was the information-dependent acquisition mode (DDA), the primary mass spectrometer scanning range was 350-1500m / z, and the maximum injection time was 100ms. In each DDA cycle, up to 20 secondary spectra with charges from 2+ to 5+ were collected, and the maximum injection time of the secondary mass spectrometer ions was 50ms.

[0116] The original raw data files collected by mass spectrometry were processed and searched using PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada). The database was the Haematococcus pluvialis protein database downloaded from NCBI, and the search parameters were set as follows: the mass tolerance of the primary mass spectrometry was 10 ppm, the secondary mass spectrometry was 0.03 Da, and non-enzymatic cleavage; variable modifications: protein N-terminal acetylation (Protein N-term), asparagine / glutamine deamination (Deamidation (NQ)), methionine oxidation (Oxidation (M)), glutamic acid pyroglutamination (Pyro-glu), glutamine pyroglutamyl cyclization (Pyro-Gln).

[0117] The SPP-1 component was identified by LC-MS / MS to obtain 1204 peptides with Mw < 3kDa and peptide lengths of 6-21 amino acids. Subsequently, the activity of bioactive peptides was predicted using the PeptideRanker website. If a peptide score was greater than 0.5, it would be considered to have potential bioactivity. In order to avoid false positive results, peptides with scores greater than 0.8 (34 in total) were selected, as shown in Table 8. Studies have shown that the structural characteristics of α-glucosidase inhibitory peptides include: 1) chain length 3 to 8; 2) OH (Ser, Tyr, Thr) or basic amino acids (Lys, Arg, His) at the N-terminus; 3) Ala or Met at the C-terminus; 4) Pro is close to the C-terminus, preferably at the second to last position; 5) the net charge is 0 or +1 at pH 7.0; 6) hydrogen bonds and electrostatic interactions are dominant. The peptides screened out basically meet two or one of the above conditions. For example, most of the peptide chains screened out are 3 to 8 in length; Pro of GDNMLPF, AGPDPW, IGGGPW, PAPLPPK, etc. is close to the C-terminus and is located at the second to last position; Met appears at the C-terminus of RFHDPLM, AGWGVM, and RFHDPM; SGPDIGGF, SNSPFVF, SNTPFVF, etc. contain OH (Ser) at the N-terminus. This indicates that the peptides screened out have the potential to inhibit α-glucosidase.

[0118] Table 8 The top 34 peptides predicted by PeptideRanker website

[0119]

[0120] 3. Stability study

[0121] (1) Thermal stability: SPP-1 lyophilized powder was weighed to prepare a 5 mg / mL solution, which was placed in a water bath at 20°C, 40°C, 60°C, 80°C, and 100°C for 2 h. After cooling to room temperature, the α-glucosidase inhibition rate was measured.

[0122] (2) pH stability: SPP-1 lyophilized powder was weighed to prepare a 5 mg / mL solution. The pH of the sample solution was adjusted to 3.0, 5.0, 7.0, 9.0, and 11.0, respectively. After standing at room temperature for 2 h, the α-glucosidase inhibition rate was determined.

[0123] (3) Oral and gastrointestinal digestion stability: SPP-1 lyophilized powder was weighed to prepare a 5 mg / mL solution, and an equal volume of artificial gastric juice (1 g pepsin was added to 100 mL distilled water, and the pH was adjusted to 2.0 with 1 mol / L HCl) was added. The solution was incubated in a 37°C water bath for 2 h, and the enzymatic solution was taken every 30 min. After the enzyme was inactivated, the α-glucosidase inhibition rate was determined. After the simulated gastric digestion reaction, the pH of the enzymatic solution was adjusted to 7.5 with NaOH, and trypsin (enzyme / sample = 1:25, w / w) was added and digested in a 37°C water bath for 2 h. The enzymatic solution was taken every 30 min. After the enzyme was inactivated, the α-glucosidase inhibition rate was determined.

[0124] The results are as follows Figure 11-13 As shown, the α-glucosidase inhibition rate of SPP-1 did not change significantly at temperatures of 20-80°C and pH of 3-11, and after 5 hours of in vitro simulated oral and gastrointestinal digestion treatment, its α-glucosidase inhibition rate still remained at a high level. The results showed that SPP-1 has high thermal stability, acid-base stability and gastrointestinal digestion stability.

[0125] 4. Effect of sweet potato protein α-glucosidase inhibitory peptide on postprandial blood glucose

[0126] 50 SPF Kunming male mice (6-8 weeks old) were prepared and divided into five groups (n=10). The first group was gavaged with starch solution (1g / kg·bw); the second group was given starch and acarbose as a positive control (20g / kg·bw); the third, fourth and fifth groups were gavaged with starch and different doses of SPP-1, with doses of 100, 200 and 400mg / kg·bw, respectively. After fasting for 10h, the mice were gavaged with polypeptide or acarbose, and gelatinized starch solution was given 10min later. Then, blood samples were taken from the tail vein of the mice at 0, 30, 60, 90, 120 and 180min using a blood glucose meter to draw blood glucose curves and calculate the area under the curve.

[0127] 50 SPF Kunming male mice (6-8 weeks old) were prepared and divided into five groups (n=10). The first group was given only distilled water as a blank; the second group was given maltose solution (2g / kg·bw); the third group was given maltose solution and acarbose as a positive control (20g / kg·bw); the fourth, fifth and sixth groups were given maltose solution and different doses of SPP-1, with doses of 100, 200 and 400mg / kg·bw, respectively. After fasting for 10h, the mice were gavaged with polypeptide or acarbose, and maltose solution was given 10min later. Then, blood samples were taken from the tail vein of the mice at 0, 30, 60, 90, 120 and 180min using a blood glucose meter to draw blood glucose curves and calculate the area under the curve.

[0128] Mice were gavaged with SPP-1 and starch or maltose, and blood glucose levels were analyzed within 3 hours after the meal. Fig.14 A The results showed that the postprandial blood glucose of mice first increased and reached the maximum value within 0.5h ( Fig.14 AB). Blood glucose concentration decreased over time and returned to the initial level within 3 hours. After intragastric administration of starch, the SPP-1 component could inhibit the increase in postprandial blood glucose content; compared with the control group, the SPP-1 component significantly reduced the area under the blood glucose curve (p<0.05) ( Fig.14 CD), and decreased in a concentration-dependent manner. The trend of maltose administered intragastrically was the same as that of starch.

[0129] Finally, it should be noted that although the above embodiments describe the specific implementation of the present invention, they are not intended to limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. All modifications or equivalent replacements should be included in the protection scope of the present invention.

Claims

1. A sweet potato peptide for inhibiting α-glucosidase activity, characterized in that: Prepared by the preparation method of the following steps: (1) The sweet potatoes are washed and peeled, and 4 to 5 times the volume of water is added to crush and extract, and the extract is allowed to stand for 6 to 8 hours and then filtered through gauze to obtain a supernatant; the supernatant is adjusted to a pH value of 4, allowed to stand and precipitate, and then centrifuged to obtain a precipitate, and the precipitate is freeze-dried to obtain the sweet potato crude protein; (2) Weigh an appropriate amount of sweet potato crude protein and add distilled water at a ratio of 1:15 to 1:30 w / v; heat at 90°C for 10 min and then perform ultra-high pressure treatment, the parameters of the high pressure treatment are 200 MPa high pressure treatment for 4 to 10 min, then adjust the pH to 10, add alkaline protease for hydrolysis, the enzymatic hydrolysis conditions are 2 to 5% w / w alkaline protease and then hydrolyze in a constant temperature water bath at 55°C for 2 to 5 h; after the enzymatic hydrolysis is completed, centrifuge and take the supernatant to obtain sweet potato peptide SPP that inhibits the activity of α-glucosidase.

2. The sweet potato peptide according to claim 1, characterized in that: After the enzymatic hydrolysis in step (2), the enzyme was inactivated at 100°C for 10-15 min and then centrifuged at 4°C to obtain the supernatant.

3. The sweet potato peptide according to claim 1, characterized in that: The centrifugal parameters in step (2) are 10000 r / min and centrifugation for 15 min.

4. The sweet potato peptide according to claim 1, characterized in that: The sweet potato peptide SPP obtained in step (2) is filtered using a microporous filter membrane, and then treated in an ultrafiltration system with an ultrafiltration membrane having a molecular weight Mw cutoff value of 3 kDa to obtain a sweet potato peptide SPP-1 having a Mw of less than 3 kDa.

5. The sweet potato peptide according to claim 4, characterized in that: The pore size of the microporous filter membrane is 0.22 μm.

6. Use of the sweet potato peptide according to any one of claims 1 to 5 in the preparation of a product for inhibiting α-glucosidase activity.

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