A soybean glyceollins polypeptide, its preparation method and use, and pancreatic lipase inhibitor

Abstract

This application discloses a soybean epidermal polypeptide, its preparation method, uses, and pancreatic lipase inhibitor, belonging to the field of polypeptide extraction technology. The technical solution is as follows: a soybean epidermal polypeptide, obtained by washing, freeze-drying, crushing, enzymatically hydrolyzing, and then freeze-drying soybean epidermal tissue. The enzyme used in the enzymatic hydrolysis process is at least one selected from pancreatic enzymes, complex proteases, and neutral proteases; the complex protease is composed of endopeptidases, exopeptidases, and flavor enzymes. The soybean epidermal polypeptide of this application has good pancreatic lipase inhibitory ability. Due to the large obese population and the rapid growth of the natural inhibitor market, this product has extremely strong commercial prospects and strong market demand. Furthermore, due to its green and environmentally friendly characteristics, it is expected to be applied in multiple fields such as anti-obesity health products, food additives, functional foods, food-medicine homologous active peptides, and lipase inhibitors.

Description

Technical Field

[0001] This application belongs to the field of polypeptide extraction technology, and more specifically, relates to a soybean epidermal polypeptide, its preparation method, uses, and pancreatic lipase inhibitor. Background Technology

[0002] In recent years, the number of obese people has continued to rise. Pancreatic lipase is the most important enzyme for hydrolyzing dietary fat and can promote fat digestion. However, when pancreatic lipase activity is too high, it may cause excessive digestion and absorption of lipids, thereby leading to obesity.

[0003] Pancreatic lipase inhibitors control and treat obesity by inhibiting the digestion and absorption of lipids. Orlistat is a commonly used representative drug, which reduces fat absorption by approximately 30% and promotes excretion, thus achieving weight loss. However, long-term use of orlistat can cause side effects such as diarrhea, bloating, and fatty stools, limiting its application. Therefore, developing naturally derived pancreatic lipase inhibitors with no toxic side effects is of great significance for the prevention and control of obesity.

[0004] Common methods for preparing pancreatic lipase inhibitory peptides include chemical methods (such as alkali dissolution and acid precipitation, and organic solvent extraction) and biological methods (such as enzymatic hydrolysis and fermentation). Chemical methods, however, are subject to harsh operating conditions, high energy consumption, and can easily damage amino acid structures, posing risks of solvent residue and environmental pollution, thus hindering their application in food and health products. In contrast, biological methods are milder and more environmentally friendly, but still suffer from time-consuming processes, low efficiency, and insufficient product purity. Furthermore, the preparation of bioactive peptides from animal tissues, microorganisms, or milk proteins typically requires defatting, and commonly used organic solvent defatting methods also present residue and pollution problems. Therefore, developing a green, energy-efficient, and highly effective method for obtaining high-purity pancreatic lipase inhibitory peptides has become a critical issue that urgently needs to be addressed in this field.

[0005] Bean worms are the larvae of the bean hawk moth (Spodoptera litura), a moth belonging to the family Sphingidae. Their bodies are cylindrical and bright green, hence the common name "bean green worm." This insect primarily feeds on soybean leaves and locust leaves, and is mainly distributed in Shandong and northern Jiangsu provinces. It is a natural, non-toxic, and green food. Bean worms are highly nutritious, containing approximately 65% ​​protein, as well as calcium, phosphorus, iron, and various vitamins. They are also rich in essential amino acids and are believed to have health benefits such as lowering cholesterol, preventing hypertension, and improving gastrointestinal function. Currently, bean worms have become a geographical indication agricultural product of Guanyun County, Lianyungang City, Jiangsu Province. The local bean worm breeding and processing industry has developed rapidly, with an annual output value exceeding 1.1 billion yuan. Currently, the main way to consume bean worms is by focusing on the white fleshy part inside the insect, while the outer green outer layer is usually discarded due to its hard texture and poor taste.

[0006] With the continuous expansion of bean weevil farming and the increasing number of processing plants, a large amount of bean weevil skin is discarded as a byproduct, resulting not only in the waste of high-quality protein resources but also imposing a certain burden on the environment. Currently, research on the utilization of bean weevil skin resources is severely lacking, and there are no effective high-value processing methods or industrial application technologies. Therefore, how to efficiently develop and utilize the protein resources in bean weevil skin, especially through processing to prepare high-value-added products such as bioactive peptides, can not only alleviate resource waste and environmental pressure but also enhance the value of the industrial chain, which has significant economic and social implications.

[0007] The problem this solution aims to solve is: how to provide a polypeptide with lipase-inhibiting ability extracted from the epidermis of *Eriocaulon buergerianum*. Summary of the Invention

[0008] The purpose of this application is to provide a polypeptide component extracted from the epidermis of *Eriocaulon buergerianum*. Through extensive experiments, the applicant has discovered that the extracted polypeptide component has good pancreatic lipase inhibition ability and has good application prospects in inhibiting obesity. Furthermore, the above-mentioned extract has the advantage of being green and environmentally friendly. This advancement enhances its application scenarios and application value.

[0009] To achieve the above objectives, this solution provides a soybean skin polypeptide, which is obtained by washing, drying, crushing, enzymatically hydrolyzing, and then freeze-drying soybean skin. The enzyme used in the enzymatic hydrolysis process is at least one selected from trypsin, complex protease, and neutral protease.

[0010] The complex protease is composed of endopeptidase, exopeptidase, and flavor enzyme.

[0011] Preferably, the enzyme used in the enzymatic hydrolysis process is pancreatic enzyme.

[0012] Preferably, the molecular weight of the bean lily epidermal polypeptide is less than or equal to 3 kDa.

[0013] In addition, this application also discloses a method for preparing the above-mentioned *Eriocaulon buergerianum* epidermal polypeptide, comprising the following steps:

[0014] Step 1: Clean the skin of the bean weevil, dry it at low temperature, and crush it to obtain powder;

[0015] Step 2: Mix the powder with water, sonicate, enzymatically hydrolyze and inactivate the enzyme to obtain the enzymatic hydrolysis product;

[0016] Step 3: Centrifuge the enzymatic hydrolysis product, collect the supernatant, concentrate and freeze-dry to obtain the skin polypeptide of Sophora flavescens.

[0017] Preferably, step 2 specifically comprises:

[0018] The powder and water were mixed at a material-to-liquid ratio of 1:15-25 (w / w), and then the pH was adjusted to 11.0 and sonicated for at least 30 minutes to obtain a pretreated solution.

[0019] The pH of the pretreatment solution was then adjusted to 7.2–7.8. Enzymes were added to the pretreatment solution and the mixture was stirred in a water bath at 38–60°C for 1.5–3 hours for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated to obtain the enzymatic hydrolysis product.

[0020] Preferably, step 3 specifically comprises:

[0021] The enzymatic hydrolysis product was centrifuged and the supernatant was concentrated to obtain a concentrate. The concentrate was then ultrafiltered, and the fraction with a molecular weight less than or equal to 3 kDa was concentrated under vacuum and then freeze-dried to obtain the skin peptide of Sophora flavescens.

[0022] In addition, this application also discloses the use of the above-mentioned *Eriocaulon buergerianum* epidermal polypeptide in the preparation of pancreatic lipase inhibitors.

[0023] In addition, this application also discloses the use of the above-mentioned *Eriocaulon buergerianum* epidermal polypeptide in the preparation of pancreatic lipase inhibitor drugs and foods.

[0024] In addition, this application also discloses a pancreatic lipase inhibitor containing 0.01 to 100 wt% of the above-described safflower epidermal polypeptide.

[0025] The beneficial effects of this application are:

[0026] This application uses bean peony skin, a waste product from bean peony processing, as a raw material to prepare active peptides. This not only solves the problems of waste of skin resources and environmental burden caused by discarding them, but also increases the added value of the skin and increases the economic income of farmers.

[0027] The skin peptide of *Euphorbia hirta* in this application has good pancreatic lipase inhibition ability. Due to the huge obese population and the rapid growth of the natural inhibitor market, this product has a very strong commercial prospect and strong market demand. Moreover, due to its green and environmentally friendly characteristics, it is expected to be used in many fields such as anti-obesity health products, food additives, functional foods, food-medicine homologous active peptides, and fat-inhibiting drugs. Attached Figure Description

[0028] Figure 1 Flowchart for the preparation of pancreatic lipase inhibitory peptides derived from soybean. Detailed Implementation

[0029] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0030] The following is information on the raw materials and suppliers involved in each embodiment:

[0031] The skin of the bean caterpillar was purchased from Xiaobei Fresh Food Department Store in Lianyungang Economic Development Zone.

[0032] Pancreatic enzyme: CAS number 8049-47-6, molecular weight 23.8 kDa, specific activity 1000-2000 U / mg;

[0033] Neutral protease: CAS number 9068-59-1, specific activity 50000 U / mL;

[0034] The complex protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and consists of endopeptidase, exopeptidase and flavor enzyme.

[0035] The bean curd meat was purchased from Xiaobei Fresh Food Department Store in Lianyungang Economic Development Zone;

[0036] Example 1

[0037] refer to Figure 1 Step 1: Wash the skin of the bean caterpillar to remove sand, dirt and other impurities, drain the water and dry it at a low temperature (35±5℃); then crush the dried bean caterpillar skin and filter the crushed material through a 40-mesh sieve to obtain bean caterpillar skin powder.

[0038] Step 2: Mix the bean skin powder obtained in Step 1 with water at a ratio of 1:20 (w / w). Then, adjust the pH to 11.0 with 1 mol / L sodium hydroxide solution and perform ultrasonic pretreatment for 30 min.

[0039] The pH of the ultrasonicated solution was adjusted to 7.6 using a 1 mol / L HCl solution. Then, 5% (by weight) of trypsin was added to the solution, and the mixture was stirred and hydrolyzed in a water bath at 50 °C for 2 h. After hydrolysis, the hydrolysate was heated at 90 °C for 10 min to inactivate the enzyme. The pH of the inactivated hydrolysate was then adjusted to 7.0 using a 1 mol / L HCl solution to obtain the hydrolysate.

[0040] It should be noted that the ultrasonic equipment used in the above-mentioned ultrasonic pretreatment process is a hexagonal multi-frequency flat-panel ultrasonic device; the ultrasonic treatment is dual-frequency synchronous ultrasound, with ultrasonic frequencies of 20 and 28 kHz processed in tandem, ultrasonic treatment for 30 s followed by a 5 s pause, ultrasonic power density of 40 W / L, and ultrasonic temperature of 35±5℃.

[0041] Step 3: Centrifuge the enzymatic hydrolysis product and collect the supernatant. Concentrate the supernatant under vacuum to obtain the concentrated peptide solution of *Eupolyphaga sinensis* skin. Then freeze-dry the peptide solution at -60℃ for 48 h to obtain the peptide solution of *Eupolyphaga sinensis* skin.

[0042] It should be noted that during the centrifugation process in step 3 above, the rotation speed was 4000 rpm, the temperature was 4℃, and the centrifugation time was 10 minutes.

[0043] Example 2

[0044] Step 1: Wash the skin of the bean caterpillar to remove sand, dirt and other impurities, drain the water and dry it at a low temperature (35±5℃); then crush the dried bean caterpillar skin and filter the crushed material through a 40-mesh sieve to obtain bean caterpillar skin powder.

[0045] Step 2: Mix the bean skin powder obtained in Step 1 with water at a ratio of 1:25. Then, adjust the pH to 11 with 0.8 mol / L sodium hydroxide solution and perform ultrasonic pretreatment for 60 min.

[0046] The pH of the ultrasonicated solution was adjusted to 7.2 using 0.8 mol / L HCl solution. Then, 5% (by weight) of trypsin was added to the solution, and the mixture was stirred and hydrolyzed in a water bath at 60 ℃ for 1.5 h. After hydrolysis, the hydrolysate was heated at 90 ℃ for 10 min to inactivate the enzyme. The pH of the inactivated hydrolysate was then adjusted to 7.0 using 1 mol / L HCl solution to obtain the hydrolysate.

[0047] It should be noted that the ultrasonic equipment used in the above-mentioned ultrasonic pretreatment process is a hexagonal multi-frequency flat-panel ultrasonic device; the ultrasonic treatment is dual-frequency synchronous ultrasound, with ultrasonic frequencies of 20 and 28 kHz processed in tandem, ultrasonic treatment for 30 s followed by a 5 s pause, ultrasonic power density of 40 W / L, and ultrasonic temperature of 35±5℃.

[0048] Step 3: Centrifuge the enzymatic hydrolysis product and collect the supernatant. Concentrate the supernatant under vacuum to obtain the concentrated peptide solution of *Eupolyphaga sinensis* skin. Then freeze-dry the peptide solution at -60℃ for 48 h to obtain the peptide solution of *Eupolyphaga sinensis* skin.

[0049] It should be noted that during the centrifugation process in step 3 above, the rotation speed was 4000 rpm, the temperature was 4℃, and the centrifugation time was 10 minutes.

[0050] Example 3

[0051] Step 1: Wash the skin of the bean caterpillar to remove sand, dirt and other impurities, drain the water and dry it at a low temperature (35±5℃); then crush the dried bean caterpillar skin and filter the crushed material through a 40-mesh sieve to obtain bean caterpillar skin powder.

[0052] Step 2: Mix the bean skin powder obtained in Step 1 with water at a ratio of 1:15. Then adjust the pH to 11 with 1 mol / L sodium hydroxide solution and perform ultrasonic pretreatment for 45 min.

[0053] The pH of the ultrasonicated solution was adjusted to 7.8 using 1.2 mol / L HCl. Then, 5% trypsin was added to the solution, and the mixture was stirred and hydrolyzed in a water bath at 40 ℃ for 3 h. After hydrolysis, the hydrolysate was heated at 90 ℃ for 10 min to inactivate the enzyme. Then, the pH of the inactivated hydrolysate was adjusted to 7.0 using 1 mol / L HCl solution to obtain the hydrolysate.

[0054] It should be noted that the ultrasonic equipment used in the above-mentioned ultrasonic pretreatment process is a hexagonal multi-frequency flat-panel ultrasonic device; the ultrasonic treatment is dual-frequency synchronous ultrasound, with ultrasonic frequencies of 20 and 28 kHz processed in tandem, ultrasonic treatment for 30 s followed by a 5 s pause, ultrasonic power density of 40 W / L, and ultrasonic temperature of 35±5℃.

[0055] Step 3: Centrifuge the enzymatic hydrolysis product and collect the supernatant. Concentrate the supernatant under vacuum to obtain the concentrated peptide solution of *Eupolyphaga sinensis* skin. Then freeze-dry the peptide solution at -60℃ for 48 h to obtain the peptide solution of *Eupolyphaga sinensis* skin.

[0056] It should be noted that during the centrifugation process in step 3 above, the rotation speed was 4000 rpm, the temperature was 4℃, and the centrifugation time was 10 minutes.

[0057] Example 4

[0058] It is basically the same as Example 1, except that neutral protease is used instead of pancreatic enzyme.

[0059] Example 5

[0060] It is basically the same as Example 1, except that a complex protease is used instead of pancreatic enzyme.

[0061] Comparative Example 1

[0062] It is basically the same as Example 1, except that the skin of the bean worm is replaced with the flesh of the bean worm.

[0063] Comparative Example 2

[0064] The process is basically the same as in Example 1, except that the defatted flesh of the bean plant is used to replace the skin of the bean plant. The defatting process of the bean plant is as follows: freeze-dried powder of bean plant is mixed with petroleum ether organic solvent at a temperature of 75±5℃ at a solid-liquid ratio of 1:30 (w / v), then stirred at 200 rpm for 0.5 h, allowed to stand for 30 min, the precipitate is collected, an equal volume of petroleum ether solvent is added again and stirred for 0.5 h, the above operation is repeated twice, and dried at room temperature for 24 h to obtain defatted bean plant powder.

[0065] Comparative Example 3

[0066] It is basically the same as Example 4, except that the skin of the bean worm is replaced with the flesh of the bean worm.

[0067] Comparative Example 4

[0068] This is basically the same as Example 4, except that the defatted bean flesh is used to replace the bean skin, and the defatting method of the bean flesh is the same as that of Comparative Example 2.

[0069] Comparative Example 5

[0070] It is basically the same as Example 5, except that the skin of the bean worm is replaced with the flesh of the bean worm.

[0071] Comparative Example 6

[0072] This is basically the same as Example 5, except that the defatted bean flesh is used to replace the bean skin, and the defatting method of the bean flesh is the same as that of Comparative Example 2.

[0073] Performance testing

[0074] The yields of the *Euphorbia hirta* epidermal peptides prepared in each embodiment and comparative example were statistically analyzed, and the pancreatic lipase inhibition rate of the *Euphorbia hirta* epidermal peptides prepared in each embodiment and comparative example was tested.

[0075] The specific method for testing the pancreatic lipase inhibition rate is as follows: Pancreatic lipase was dissolved in Tris-HCl buffer (50 mmol / L, pH = 8.0) to prepare a 1 mg / mL solution. The solution was centrifuged at 6400 × g for 10 min at 4 °C, and the supernatant was collected. The sample (10 µL), buffer (70 µL), and enzyme solution (110 µL) dissolved in Tris-HCl buffer were added to a 96-well plate and incubated at 37 °C for 10 min. Then, 10 µL of substrate pNPB (20 mmol / L) was added, and incubation continued for 30 min. The p-nitrophenol content of the product was then detected at 405 nm using a spectrophotometer. The pancreatic lipase activity without inhibitor was defined as 100%. The inhibitory effect of the *Euphorbia lactea* epidermal peptide on pancreatic lipase was expressed as the half-inhibitory concentration (IC50). 50 The inhibition rate of the peptide is calculated according to the following formula (1):

[0076] Formula 1

[0077] In Equation 1:

[0078] A s The sample group contains the sample, pancreatic lipase, and pNPB.

[0079] A b For the sample blank group, Tris-HCl buffer was used instead of pancreatic lipase solution;

[0080] A t As a control group, Tris-HCl buffer was used instead of the sample solution;

[0081] A c As a control group, Tris-HCl buffer was used instead of the sample and pancreatic lipase solution.

[0082] The test results are shown in Table 1-2:

[0083] Table 1. Pancreatic lipase inhibition rate (%) of peptides prepared by enzymatic hydrolysis of different parts of *Eupolyphaga sinensis* for 2 hours.

[0084]

[0085] Table 2. Yield (%) of polypeptides prepared by enzymatic hydrolysis of different parts of *Eriocaulon buergerianum* using different enzymes for 2 hours.

[0086]

[0087] Results analysis:

[0088] As can be seen from Table 1-2, when neutral protease is used to enzymatically hydrolyze the skin of the soybean, the yield of the final polypeptide is the highest. When enzymatically hydrolyzing the flesh of the soybean, trypsin has a greater advantage in terms of yield. However, when enzymatically hydrolyzing defatted flesh of the soybean, the difference among the three is relatively small.

[0089] Further observation of Table 1 shows that when using pancreatic enzymes and complex proteases for enzymatic hydrolysis, regardless of which part of the bean antler is used for polypeptide preparation, the polypeptides obtained using the epidermis have a significant advantage in lipase inhibition ability.

[0090] When using neutral protease for enzymatic hydrolysis, the polypeptides obtained from defatted soybean flesh have better pancreatic lipase inhibition ability, but the polypeptides obtained from soybean skin still retain a certain pancreatic lipase inhibition ability.

[0091] In summary, although neutral protease yields a high polypeptide yield when using soybean bark as raw material for enzymatic hydrolysis, the polypeptides obtained have significantly lower pancreatic lipase inhibition ability than pancreatic enzymes and complex proteases.

[0092] When pancreatic enzymes are enzymatically hydrolyzed using peony bark as raw material, polypeptide products with extremely excellent pancreatic lipase inhibitory ability are obtained.

[0093] Part Two

[0094] Furthermore, further experiments were conducted on the group with the highest lipase inhibition rate. The specific experimental method was as follows:

[0095] Take a portion of the concentrated peptide solution from the skin of *Eupolyphaga sinensis* from step 3 of Example 1, and then perform ultrafiltration using 3 kDa and 10 kDa filter membranes to obtain low molecular weight (0-3 kDa peptide components), medium molecular weight (3-10 kDa peptide components without 3 kDa), and high molecular weight (peptide components greater than 10 kDa) peptide components, respectively. Then, freeze-dry the peptide concentrate at -60°C for 48 h to obtain the *Eupolyphaga sinensis* skin peptide.

[0096] Performance testing

[0097] Test method: The determination of inhibition of pancreatic lipase activity is the same as above, and its IC50 is calculated. 50 value.

[0098] The test results are shown in Table 3:

[0099] Table 3. IC50 values ​​for inhibiting pancreatic lipase activity of polypeptide components of different molecular weights obtained from pancreatic enzyme hydrolysis. 50 Value (mg / mL)

[0100] polypeptide components <![CDATA[IC 50 (mg / mL)]]> > 10 kDa 7.95 3~10 kDa 33.62 ≤3 kDa 0.19

[0101] Note: Different lowercase letters in the same column indicate significant differences (p < 0.05).

[0102] Results Analysis

[0103] As shown in Table 3, further analysis of the IC50 values ​​for the inhibitory activity of peptides with different molecular weights on pancreatic lipase was conducted. 50 Value testing revealed that the IC50 of the components in the 0–3 kDa range was [value missing]. 50 The values ​​are much lower than those of the 3–10 kDa polypeptide components and the >10 kDa polypeptide components, indicating that it has the strongest inhibitory activity against pancreatic lipase.

Claims

1. A polypeptide from the skin of a bean lily, characterized in that, The bean lily epidermal polypeptide is obtained by washing, drying, crushing, enzymatic hydrolysis and freeze-drying the bean lily epidermal skin. The enzyme used in the enzymatic hydrolysis process is selected from at least one of pancreatic enzyme, complex protease and neutral protease. The complex protease is composed of endopeptidase, exopeptidase, and flavor enzyme.

2. The bean lily epidermal polypeptide according to claim 1, characterized in that, The enzyme used in the enzymatic hydrolysis process is pancreatic enzyme.

3. The bean lily epidermal polypeptide according to claim 1, characterized in that, The molecular weight of the bean lily epidermal polypeptide is less than or equal to 3 kDa.

4. A method for preparing the skin polypeptide of *Euonymus alatus* according to claim 1, characterized in that, Includes the following steps: Step 1: Clean the skin of the bean weevil, dry it at low temperature, and crush it to obtain powder; Step 2: Mix the powder with water, sonicate, enzymatically hydrolyze and inactivate the enzyme to obtain the enzymatic hydrolysis product; Step 3: Centrifuge the enzymatic hydrolysis product, collect the supernatant, concentrate and freeze-dry to obtain the skin polypeptide of Sophora flavescens.

5. The method for preparing the skin polypeptide of *Euonymus alatus* according to claim 4, characterized in that, Step 2 specifically involves: The powder and water were mixed at a mass ratio of 1:15 to 25, then the pH was adjusted to 11 and sonicated for at least 30 minutes to obtain a pretreated solution. The pH of the pretreatment solution was then adjusted to 7.2–7.

8. Enzymes were added to the pretreatment solution and the mixture was stirred in a water bath at 38–60°C for 1.5–3 hours for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated to obtain the enzymatic hydrolysis product.

6. The method for preparing the skin polypeptide of *Euonymus alatus* according to claim 4, characterized in that, Step 3 specifically involves: The enzymatic hydrolysis product was centrifuged and the supernatant was concentrated to obtain a concentrate. The concentrate was then ultrafiltered, and the fraction with a molecular weight less than or equal to 3 kDa was concentrated under vacuum and then freeze-dried to obtain the skin peptide of Sophora flavescens.

7. Use of the *Euonymus alatus* epidermal polypeptide as described in any one of claims 1-3 for preparing a pancreatic lipase inhibitor.

8. Use of the soybean epidermal polypeptide as described in any one of claims 1-3 for preparing pancreatic lipase inhibitor drugs and foods.

9. A pancreatic lipase inhibitor, characterized in that, Contains 0.01 to 100 wt% of the skin polypeptide of *Euphorbia hirta* as described in any one of claims 1-3.

Images