Black truffle heptapeptide for inhibiting starch digestion and application of black truffle heptapeptide

Abstract

The invention relates to the technical field of development of active peptides, in particular to a novel small-molecule heptapeptide with a starch digestion inhibiting effect and application thereof. According to the method, perigord truffle is taken as a raw material, and the perigord truffle polypeptide is obtained through protein extraction, protein enzymolysis, dialysis and freeze-drying. Furthermore, a bioinformatics tool is used for predicting the activity, potential toxicity, solubility and physicochemical properties of the peptide fragment, wheat starch is selected as a test material, the digestion characteristic of the polypeptide-starch compound is detected, and the peptide fragment is determined according to the starch hydrolysis rate and the ratio of rapidly digested starch to slowly digested starch to resistant starch. The heptapeptide AADELFR with the effect of inhibiting or delaying starch digestion is obtained and is applied to preparation of food or health food beneficial to maintaining the healthy level of blood sugar.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide development technology, specifically to a novel small-molecule heptapeptide with starch-inhibiting activity and its applications. Background Technology

[0002] Starch, as the most important source of carbohydrates in the human diet, directly affects postprandial blood glucose levels through its digestion rate and extent, and is closely related to the occurrence and development of metabolic syndromes such as obesity and diabetes. Based on the digestion speed and extent, starch can be divided into three categories: rapidly digestible starch (RDS), which is enzymatically digested within 20 minutes of ingestion and causes a rapid rise in blood glucose; slowly digestible starch (SDS), which is gradually digested over 20-120 minutes, maintaining a stable and gradual release of blood glucose; and resistant starch (RS), which is undigested in the small intestine but may undergo bacterial fermentation in the large intestine. Therefore, increasing the proportion of RS and decreasing the proportion of RDS in starch digestion is one of the core strategies for delaying glucose absorption and stabilizing postprandial blood glucose.

[0003] Bioactive peptides have become a hot topic in the research and development of functional foods and drugs due to their good safety, easy absorption, and diverse physiological functions. Some studies have shown that certain bioactive peptides, such as oat peptides, whey protein hydrolysates, and rice protein hydrolysates, can significantly improve starch structure, thereby inhibiting starch digestion.

[0004] Black truffle ( Tuber Sinense Black truffle (also known as black truffle) possesses various physiological activities, including antioxidant, hypoglycemic, anti-inflammatory, immunomodulatory, and lipid-lowering effects. Black truffle protein is a plant-derived protein rich in essential amino acids, but due to its complex molecular structure and large molecular weight, its bioavailability is low when directly ingested, making it difficult to fully realize its nutritional and functional properties.

[0005] This study will provide a plant protein peptide isolated from black truffles and conduct an in vitro starch digestion experiment with wheat starch. By detecting the digestive characteristics of the peptide-starch complex, the study will analyze the starch hydrolysis rate, the proportion of rapidly digestible starch, slowly digestible starch, and resistant starch after the addition of the peptide-starch complex, and evaluate the effect of the peptide fragment on inhibiting starch digestion. Summary of the Invention

[0006] In order to solve the above technical problems, the present application takes black truffle as raw material, and obtains black truffle polypeptide after protein extraction, protein enzymolysis, dialysis and freeze-drying. Further, bioinformatics such as peptide segment activity, potential toxicity, solubility and physicochemical property prediction tools are used, and wheat starch is selected as test material, and the effect of polypeptide on inhibition of starch digestion is evaluated by detecting the digestion characteristics of polypeptide-starch complex, starch hydrolysis rate, rapidly digestible starch, slowly digestible starch and resistant starch ratio. Thus, a new heptapeptide with the characteristics of inhibiting starch digestion is screened.

[0007] The technical route adopted by the present application is as follows: One of the technical solutions provided by the present application is a black truffle heptapeptide, the heptapeptide is AADELFR, the sequence is Ala-Ala-Asp-Glu-Leu-Phe-Arg, and the molecular weight is 820.91 Da. Among them, Ala represents the corresponding residue of an amino acid with the English name Alanine and the Chinese name Alanine; Asp represents the corresponding residue of an amino acid with the English name Aspartic acid and the Chinese name Aspartic acid; Glu represents the corresponding residue of an amino acid with the English name Glutamic acid and the Chinese name Glutamic acid; Leu represents the corresponding residue of an amino acid with the English name Leucine and the Chinese name Leucine; Phe represents the corresponding residue of an amino acid with the English name Phenylalanim and the Chinese name Phenylalanim; Arg represents the corresponding residue of an amino acid with the English name Arginine and the Chinese name Arginine.

[0008] Further, the heptapeptide AADELFR can be obtained by further separating the black truffle polypeptide obtained by taking black truffle as raw material, protein extraction, protein enzymolysis, dialysis and freeze-drying, or can be obtained by chemical synthesis, preferably chemical solid-phase synthesis method.

[0009] The second technical solution provided by the present application is a composition containing the heptapeptide AADELFR of the first technical solution, the composition takes the heptapeptide AADELFR as active ingredient, and can also contain other active ingredients, and can also contain food science, pharmaceutically acceptable adjuvant.

[0010] The third technical solution provided by the present application is the application of the heptapeptide AADELFR of the first technical solution or the composition of the second technical solution, especially in the application of inhibiting or delaying starch digestion, more particularly in the application of increasing the content of resistant starch in starch digestion; the application further comprises any of the following aspects: (1) Its application in the preparation of foods or health foods that inhibit or delay starch digestion; (2) Its application in the preparation of foods or health foods that help maintain healthy blood sugar levels; Furthermore, the food or health food mentioned includes noodles, bread and other pasta products, mooncakes, pudding and other baked goods, etc. Furthermore, the specific method used in the above application is as follows: the heptapeptide AADELFR described in technical solution one or the composition described in technical solution two is mixed with starch and then gelatinized; Furthermore, the method can be any of the following: (1) The heptapeptide AADELFR described in technical solution one or the composition described in technical solution two is premixed with starch and gelatinized. The resulting product can be directly used for subsequent product processing, such as the processing of bread, mooncakes and other foods. (2) The heptapeptide AADELFR described in technical solution one or the composition described in technical solution two is mixed with starch and then directly made into finished products according to the processing requirements of different categories (such as noodles). The gelatinization process is completed in the subsequent heat processing stage for consumers. Furthermore, the amount of the heptapeptide AADELFR or the composition added is 8% (w / w) or more of the starch mass; Furthermore, the gelatinization conditions are: in an environment with sufficient moisture content, the gelatinization temperature of starch is reached or above and maintained for 0.5-4 hours; Furthermore, the moisture content is not less than 30% (w / w), where moisture content refers to the percentage of water by mass of all materials in the entire gelatinization system; Furthermore, the starch includes, but is not limited to: corn starch, potato starch, wheat starch, sweet potato starch, tapioca starch, mung bean starch, pea starch, rice starch, etc. For example, the gelatinization temperature of corn starch is 62-72℃, that of potato starch is 58-68℃, and that of wheat starch is 45-85℃.

[0011] The fourth technical solution provided by the present invention is the application of the heptapeptide AADELFR described in technical solution one in the preparation of starch digestion inhibitor; further, the inhibitor contains heptapeptide AADELFR.

[0012] Compared with the prior art, the present invention has the following advantages: This invention yields the novel heptapeptide AADELFR for the first time. Bioinformatics methods were used to determine the novelty of the peptide, predict its bioactivity and toxicity, and simultaneously investigate the effect of the synthesized peptide on the proportions of rapidly digestible starch, slowly digestible starch, and resistant starch during starch digestion. Specifically, after adding 8% AADELFR heptapeptide, the contents of both rapidly and slowly digestible starch in wheat starch significantly decreased, while the content of resistant starch significantly increased. Furthermore, the starch hydrolysis rate and hydrolysis percentage both significantly decreased. This indicates that the heptapeptide AADELFR possesses the function of inhibiting or delaying starch digestion and can be applied to the preparation of foods / health foods that help maintain healthy blood sugar levels. Attached Figure Description

[0013] The accompanying drawings are helpful for further understanding of the present invention. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0014] Figure 1 The result of ToxinPred for the Ala-Ala-Asp-Glu-Leu-Phe-Arg peptide; Figure 2 This is a purity identification diagram for the Ala-Ala-Asp-Glu-Leu-Phe-Arg peptide. Figure 3 MS molecular weight identification diagram of the Ala-Ala-Asp-Glu-Leu-Phe-Arg peptide; Figure 4 The effect of Ala-Ala-Asp-Glu-Leu-Phe-Arg peptide on the hydrolysis rate of wheat starch; Figure 5 The effect of Ala-Ala-Asp-Glu-Leu-Phe-Arg peptide on wheat starch digestion; * indicates a significant difference compared to the group with 0 heptapeptide addition. P <0.05, ** indicates a highly significant difference compared to the group with 0 heptapeptide addition. P <0.01, **** indicates a highly significant difference compared to the group with 0 heptapeptide addition. P <0.0001. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description of the patent is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the patent and are not intended to limit the scope of the invention.

[0016] This invention uses black truffles as raw material, and obtains black truffle polypeptides through protein extraction, enzymatic hydrolysis, and dialysis freeze-drying. Analysis revealed that the black truffle polypeptides exhibit good processing characteristics in wheat starch and have the ability to inhibit starch digestion. Further analysis of the amino acid sequence structure of the black truffle polypeptides screened out potential bioactive peptide fragments.

[0017] This invention discloses for the first time a heptapeptide, AADELFR, isolated from black truffles, with the amino acid sequence: Ala-Ala-Asp-Glu-Leu-Phe-Arg, and a molecular weight of 820.91 Da. Bioinformatics and in vitro experiments have confirmed that AADELFR is a novel polypeptide with an inhibitory effect on starch digestion.

[0018] The heptapeptide AADELFR can be obtained by enzymatic hydrolysis of black truffles or by chemical synthesis, with solid-phase chemical synthesis being preferred.

[0019] The present invention will be further explained and described below with reference to specific embodiments.

[0020] Example 1: Preparation of Black Truffle Peptides Dried black truffles were used as raw materials, pulverized by a grinder and passed through an 80-mesh sieve. Distilled water was added at a material-to-liquid ratio of 1:50 (g / mL), and the pH was adjusted to 11.5 with 1M sodium hydroxide. After standing for 10 min, the mixture was extracted at 50 ℃ for 3 h. The supernatant was collected by centrifugation, cooled to room temperature, and the pH was adjusted to 3.6 with 1M hydrochloric acid. After standing at 4 ℃ for 5 h, the precipitate was collected by centrifugation. The precipitate was washed multiple times with distilled water to remove impurities. Then, an appropriate amount of distilled water was added to reconstitute the precipitate, and vortexing was used to promote dissolution, resulting in a protein solution. The solution was dialyzed for 3 days at 4 ℃ using a 6000-8000 Da dialysis bag. After low-temperature freeze-drying, black truffle protein powder was obtained.

[0021] Black truffle protein powder was prepared into a stock solution with a concentration of 5 mg / mL using distilled water, dissolved by sonication, and the pH was adjusted to 9 with sodium hydroxide. Then, 3% (w / w) of trypsin was added, and the mixture was placed in a magnetic stirrer at 30 ℃ for 4 h for hydrolysis. After the reaction was completed, the enzyme was inactivated by boiling in a water bath for 15 min. After cooling, the supernatant was collected by centrifugation and dialyzed in a 100 Da dialysis bag at 4 ℃ for 3 days. The black truffle polypeptide powder was obtained by low-temperature freeze drying and used for subsequent experiments.

[0022] Example 2: Determination of the relative molecular weight of black truffle polypeptides The relative molecular weight distribution of black truffle peptides was determined by high-performance gel filtration chromatography. An appropriate amount of the sample to be tested (prepared in Example 1) was weighed, dissolved in the mobile phase solution, filtered through a 0.22 μm filter membrane, and then injected into the chromatography system.

[0023] The relative molecular weight distribution of black truffle peptides is detailed in Table 1. They are mainly composed of low molecular weight peptides, with those ≤ 1000 Da being the dominant component (81.32%), followed by those between 1000-2000 Da (11.19%). Peptides with molecular weights of 2000-3000 Da, 3000-5000 Da, and >5000 Da accounted for relatively low proportions, at 3.89%, 2.42%, and 1.19%, respectively. This distribution characteristic suggests that the trypsin hydrolysis products are mainly small molecular weight peptides, which may possess good biological activity.

[0024] Table 1. Relative molecular weight distribution of black truffle polypeptides

[0025] Example 3: Determination of the amino acid sequence structure of black truffle polypeptide The black truffle peptide sample prepared in Example 1 was dissolved in a washing buffer containing 0.1% formic acid and 2% acetonitrile and vortexed. After ultrafiltration centrifugation (12000 g, 10 min) in a 10 kDa centrifuge tube, the filtrate was collected and purified by desalting using a C18 solid-phase extraction column. Procedure: The sample was loaded onto an activated and equilibrated C18 column, and the eluent was discarded; the target peptide was eluted with an elution buffer containing 0.1% formic acid and 2% acetonitrile. The eluent was concentrated by vacuum centrifugation, dried, and stored at -80 °C for later use.

[0026] The dried peptides were reconstituted in 0.1% formic acid aqueous solution. Separation was performed using an UltiMate 3000 RSLCnano system. Mobile phase: Solution A (0.1% formic acid aqueous solution), Solution B (0.1% formic acid acetonitrile solution). Procedure: Samples were enriched on a trapping column (RP-C18, 100 μm × 20 mm) at 3 μL / min (100% Solution A); the enriched peptides were transferred to an analytical column (PepMap RSLC C18, 2 μm, 75 μm × 25 cm) and eluted at a gradient of 300 mL / min. A 30-min blank gradient elution was performed between every two injections to eliminate cross-contamination.

[0027] After chromatographic separation, the peptides were analyzed using a Q-Exactive Plus mass spectrometer (positive ion mode). MS1 parameters: scan range 300–1500, resolution 70000, AGC target 3×10⁻⁶. 6 maxIT 100 ms. DDA settings: After full scan, select the top 20 precursor ions with the highest abundance for MS2 (HCD): NCE 28%, resolution 17500, AGC target 1×10⁻⁶. 5 maxIT 50ms. Dynamic exclusion time 25s.

[0028] The sequences of black truffle peptides were identified using LC-MS / MS, and a total ion chromatogram was generated through database matching. A total of 1816 peptides with a -10LgP greater than 25 were obtained after screening, with molecular weights ranging from 640 to 4450 Da and lengths between 6 and 41 amino acids. Regarding amino acid composition, all 20 common amino acids were detected, with glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), leucine (Leu), and valine (Val) being the main components. Glu and Asp appeared significantly more frequently than other amino acids. Given the strong hydrophilicity of Glu and Asp, it is speculated that this black truffle peptide component may exhibit good solubility.

[0029] Example 4: Screening of Potential Bioactive Peptides from Black Truffles 1. Bioactivity prediction The bioactivity of peptides derived from the amino acid sequence of black truffle polypeptides was predicted using the online website Peptide Ranker, and peptides with an activity score greater than 0.55 were screened out.

[0030] 2. Prediction of potential toxicity, solubility, and physicochemical properties. Peptides with an activity score greater than 0.55 were used to predict their physicochemical properties, such as toxicity, solubility, hydrophilicity, amphiphilicity, and hydrophilic pathways, in the ToxinPred database. Peptides with good solubility and no toxicity were then screened for further analysis.

[0031] According to Peptide Ranker screening, 87 black truffle peptides with an activity greater than 0.55 were identified. Further analysis based on toxicity, solubility, and physicochemical properties revealed one potentially bioactive peptide with good solubility and non-toxicity, as shown in Table 2. Figure 1 As shown, and by comparing it with bioactive peptides with known functions in the BIOPEP-UWM database, it was found to be a new bioactive peptide.

[0032] Table 2. Virtual screening of potentially bioactive black truffle peptides (one of them)

[0033] Example 5: Solid-phase synthesis of peptides A synthetic company was commissioned to synthesize the peptide according to the Ala-Ala-Asp-Glu-Leu-Phe-Arg formula.

[0034] Using a high-molecular-weight resin, following the characteristic amino acid sequence Ala-Ala-Asp-Glu-Leu-Phe-Arg, the carboxyl group of Ala is first covalently linked to a resin. Then, the amino and carboxyl groups of Ala undergo a dehydration reaction. After this treatment, Asp is added, and the amino and carboxyl groups of Ala react sequentially from right to left. After adding the last Arg amino acid, the resin is cleaved to obtain the target peptide. After purification, the final product is obtained, requiring a purity of over 95%. Purity is determined by HPLC analysis and molecular weight determination by MS (e.g., [missing information]). Figure 2 , 3 (As shown).

[0035] Example 6: Effect of synthesized heptapeptide on starch digestibility Weigh 0.3 g of wheat starch with 8% heptapeptide AADELFR added (0.3 g of pure wheat starch was used as a blank control), add 5 mL of distilled water, and dissolve it completely under stirring. Then, gelatinize it in a 95 ℃ water bath for 30 min with continuous stirring. Cool the composite solution to room temperature.

[0036] 10 mL of sodium acetate buffer (0.2 mol / L, pH 5.5) and 3 glass beads were added to the composite solution. The mixed slurry was equilibrated in a 37 ℃ constant temperature shaking water bath (120 r / min) for 10 min. Then, 2.5 mL of a mixed enzyme solution of porcine pancreatic α-amylase (250 U / mL) and amyloglucosidase (260 U / mL) was added, and the digestion reaction was carried out at 37 ℃. At 0, 20, 40, 60, 90, and 120 min, 1 mL of the digest was taken and boiled in water for 10 min to inactivate the enzymes. After cooling, the sample was centrifuged (10000 r, 5 min), and the supernatant was collected. The glucose content (Gt) in the sample was determined using a glucose detection reagent.

[0037] Hydrolysis rate (%) = (1) RDS(%) = (2) SDS(%) = (3) RS(%) = 1 – RDS(%) – SDS(%) Where G0, G20, and G120 represent the glucose content (mg) generated by enzymatic hydrolysis of the mixture at 0, 20, and 120 min, respectively; TS represents the total starch mass (mg) in the mixture; and RDS, SDS, and RS represent rapidly digestible starch, slowly digestible starch, and resistant starch, respectively.

[0038] like Figure 4As shown, after adding 8% AADELFR heptapeptide from black truffle, the hydrolysis rate of gelatinized wheat starch in the time range of 0-120 min was lower than that of pure wheat starch without peptides. In particular, at 120 min, the hydrolysis rate of starch after adding 8% heptapeptide was 47.95%, while the hydrolysis rate of the control group was 54.93%, indicating that the heptapeptide has an inhibitory effect on starch hydrolysis.

[0039] Depend on Figure 5 The results showed that the addition of 8% black truffle AADELFR heptapeptide significantly reduced the content of both rapidly digestible starch (RDS) and slowly digestible starch (SDS) in gelatinized wheat starch. P <0.05), and the decrease in SDS was more significant; at the same time, the content of resistant starch (RS) increased extremely significantly ( P <0.0001). The above data indicate that the heptapeptide AADELFR can effectively increase the proportion of RS in wheat starch and reduce the proportion of RDS and SDS, thus possessing the potential function of delaying the rise in postprandial blood glucose and improving the body's blood glucose homeostasis.

[0040] In summary, the AADELFR heptapeptide synthesized in this invention was found to have no relevant records in the NCBI database, indicating that this sequence is a novel heptapeptide that has not been reported before. Bioactivity prediction using PeptideRanker yielded a score of 0.627, suggesting good bioactivity potential. ToxinPred toxicity prediction analysis showed that this peptide has no toxic risk and good solubility, making it suitable for use as a food additive. The amino acid sequence of this heptapeptide is Ala-Ala-Asp-Glu-Leu-Phe-Arg (abbreviated AADELFR), with hydrophobic amino acids accounting for 42.86%. The efficacy evaluation results in inhibiting starch digestion showed that the synthetic heptapeptide AADELFR significantly reduced the content of rapidly digestible starch (RDS) and slowly digestible starch (SDS) in gelatinized wheat starch, and significantly increased the content of resistant starch (RS), while reducing the starch hydrolysis rate and hydrolysis percentage, indicating that this synthetic polypeptide has the effect of regulating starch digestion behavior.

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A heptapeptide, characterized in that, The heptapeptide is AADELFR, with the amino acid sequence: Ala-Ala-Asp-Glu-Leu-Phe-Arg.

2. A composition comprising the heptapeptide AADELFR of claim 1.

3. The use of the heptapeptide AADELFR of claim 1 or the composition of claim 2, characterized in that, It is used in non-disease treatment purposes to inhibit or delay starch digestion.

4. The application as described in claim 3, characterized in that, It is an application of increasing the content of resistant starch in starch digestion.

5. The application as described in claim 3, characterized in that, The application also includes any of the following aspects: (1) Its application in the preparation of foods or health foods that inhibit or delay starch digestion; (2) It is used in the preparation of food or health food that helps maintain healthy blood sugar levels.

6. The application as described in claim 3, characterized in that, The specific method used in the application is as follows: the heptapeptide AADELFR or the composition is mixed with starch and then gelatinized.

7. The application as described in claim 6, characterized in that, The method is any one of the following: (1) The heptapeptide AADELFR or the composition is premixed with starch and gelatinized, and the resulting product can be directly used for subsequent product processing; (2) The heptapeptide AADELFR or the composition is mixed with starch and then directly made into finished products according to the processing requirements of different product categories. The gelatinization process is completed in the subsequent heat processing stage for consumers.

8. The application as described in claim 6, characterized in that, The amount of the heptapeptide AADELFR or the composition added is 8% (w / w) or more of the starch mass.

9. The use of the heptapeptide AADELFR according to claim 1 in the preparation of starch digestion inhibitors.

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