Method for constructing in vivo absorption active peptide library of dudaim and application thereof

Abstract

This invention discloses a method for constructing an in vivo absorption bioactive peptide library of *Eriocaulon buergerianum* and its application, belonging to the field of biotechnology. The method includes: preparing the edible portion of *Eriocaulon buergerianum* as a test substance; orally administering the test substance to a mammalian individual; collecting a blood sample from the individual after ingesting the test substance; separating serum from the blood sample; and performing mass spectrometry identification on the peptides in the serum to obtain a set of bioactive peptide sequences that enter the bloodstream after digestion and absorption of *Eriocaulon buergerianum*. A total of seven bioactive peptides derived from *Eriocaulon buergerianum* were identified, with sequences EHALLAFTLGVK, IGGIGTVPVGR, IGYNPAAVAFVPISGWHGDNMLEPSTK, LPLQDVYK, QLIVGVNK, VETGILKPGTIVVFAPANITTEVK, and YYVTIIDAPGHR. This invention is the first to construct a peptide library reflecting the actual bioactive components absorbed in vivo from *Eriocaulon buergerianum*, providing technical support for research on the nutritional functional mechanisms of *Eriocaulon buergerianum*, product development, and quality control.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and food nutrition, specifically to a method for constructing an in vivo absorption bioactive peptide library of soybean and its application. Background Technology

[0002] The bean hawk moth, or larva of the bean hawk moth (Clanis bilineata), is an insect resource with a long history of consumption in my country. Rich in high-quality protein, with a high protein content and balanced amino acid composition, it is considered a promising edible insect resource for development.

[0003] Currently, research on active peptides from *Eupolyphaga sinensis* mainly focuses on in vitro enzymatic hydrolysis. Existing technologies, such as CN122189132A, disclose a *Eupolyphaga sinensis* epidermal polypeptide and its preparation method. This method involves washing, freeze-drying, crushing, and enzymatically hydrolyzing the *Eupolyphaga sinensis* epidermis to obtain the polypeptide, and verifying its pancreatic lipase inhibitory ability. Another example is CN121931210A, which discloses a production process for *Eupolyphaga sinensis* oligopeptide powder, using ultrasound-assisted enzymatic hydrolysis to prepare oligopeptides from defatted *Eupolyphaga sinensis* protein powder. These existing technologies all simulate the enzymatic hydrolysis process in vitro, obtaining in vitro enzymatic hydrolysis products of *Eupolyphaga sinensis* protein.

[0004] However, the aforementioned existing technologies have significant shortcomings: the bioactive peptides obtained through in vitro enzymatic hydrolysis are only potential active ingredients, and are not equivalent to the truly effective ingredients that can enter the bloodstream and exert physiological functions after actual digestion and absorption by the body. Food / food-derived bioactive ingredients must undergo complex processes such as enzymatic hydrolysis in the digestive tract and penetration of absorption barriers; only the peptide segments that ultimately enter the bloodstream are the material basis for their actual nutritional or functional effects. Currently, there are no clear research reports on the specific bioactive ingredients that are actually absorbed and utilized through the digestive-circulatory system after consumption of soybeans.

[0005] This technological gap has hindered a deeper understanding of the nutritional and functional mechanisms of soybean worms and has also limited the precise development of functional foods based on soybean worm active peptides. Summary of the Invention

[0006] The technical problem this invention aims to solve is that existing research on active peptides from *Solanum tuberosum* (a type of bean pod) is limited to in vitro enzymatic hydrolysis, failing to reflect the actual active components of *Solanum tuberosum* after ingestion and absorption into the bloodstream. This results in an unclear material basis for the nutritional or functional effects of *Solanum tuberosum*. Unlike existing in vitro enzymatic hydrolysis screening techniques, this invention provides a method for constructing an in vivo absorption active peptide library from *Solanum tuberosum*. This method simulates the actual in vivo digestion and absorption process, using peptides that enter the bloodstream after oral ingestion as the objects of the active peptide library construction.

[0007] The first aspect of this invention provides a method for constructing a library of bioactive peptides absorbed in vivo from soybean.

[0008] Preferably, the bean hawk moth is a larva of the bean hawk moth (Clanis bilineata).

[0009] Preferably, the active peptide library is a collection of active peptide segments that have entered the bloodstream after being digested and absorbed by mammals.

[0010] Preferably, the construction method includes the following steps:

[0011] (1) Prepare the edible part of the bean worm as the test substance;

[0012] (2) The test substance was administered orally to a mammalian individual;

[0013] (3) After the mammalian individual ingests the test substance, a blood sample is collected from it;

[0014] (4) Separate serum from the blood sample;

[0015] (5) Identify the peptides in the serum to obtain the set of active peptide sequences that enter the blood circulation after the soybean is digested and absorbed.

[0016] Preferably, the edible part of the bean caterpillar in step (1) is selected from the skin, flesh, or a combination thereof of the bean caterpillar.

[0017] Preferably, the test substance in step (1) is prepared by crushing and freeze-drying the edible part of the bean caterpillar into a dry powder; or by cooking the edible part of the bean caterpillar into a cooked food.

[0018] More preferably, the pulverization is performed using a food-grade mixer.

[0019] More preferably, the freeze-drying is performed after freezing at -20°C overnight.

[0020] More preferably, the cooking method is selected from frying or steaming.

[0021] Preferably, the mammalian individual in step (2) is selected from mice or humans.

[0022] Preferably, the blood sample in step (3) is collected 2 to 8 hours after oral ingestion.

[0023] More preferably, the blood sample is collected 4 to 6 hours after oral ingestion.

[0024] Preferably, the serum separation conditions in step (4) are: centrifugation at 2-8°C and 2000-4000 rpm for 10-20 minutes.

[0025] Preferably, the identification in step (5) is performed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) for peptide sequence analysis.

[0026] Preferably, step (5) further includes an enzymatic digestion step of the serum before identification.

[0027] Preferably, the sequence identification of the peptide in step (5) is determined by matching with a protein database.

[0028] Preferably, the set of active peptide sequences constructed by the method comprises one or more peptide segments selected from the group consisting of:

[0029] (a) EHALLAFTLGVK (SEQ ID NO: 1);

[0030] (b) IGGIGTVPVGR (SEQ ID NO: 2);

[0031] (c) IGYNPAAVAFVPISGWHGDNMLEPSTK (SEQ ID NO: 3);

[0032] (d) LPLQDVYK (SEQ ID NO: 4);

[0033] (e) QLIVGVNK (SEQ ID NO: 5);

[0034] (f)VETGILKPGTIVVFAPANITTEVK (SEQ ID NO: 6);

[0035] (g) YYVTIIDAPGHR (SEQ ID NO: 7).

[0036] More preferably, the set of active peptide sequences comprises a combination of peptides (a), (b), (e), and (g).

[0037] More preferably, the set of active peptide sequences includes a combination of peptides (a), (b), (c), (d), (e), (f), and (g).

[0038] Preferably, the peptides (a) to (g) are derived from an elongation factor 1-alpha (eEF1A or EF-1α) fragment or an aromatic-L-amino-acid decarboxylase (AADC) fragment.

[0039] More preferably, the source protein of the peptides (a) to (f) is the elongation factor 1-α fragment.

[0040] More preferably, the source protein of the peptide (g) is an aromatic L-amino acid decarboxylase fragment.

[0041] Preferably, the abundance of peptide (e)QLIVGVNK in human serum is higher than that of other peptides.

[0042] A second aspect of the present invention provides a library of bioactive peptides for in vivo absorption from *Dendrobium nobile* constructed according to the method described in the first aspect.

[0043] Preferably, the active peptide library contains one or more peptides selected from the group consisting of:

[0044] (a) EHALLAFTLGVK (SEQ ID NO: 1);

[0045] (b) IGGIGTVPVGR (SEQ ID NO: 2);

[0046] (c) IGYNPAAVAFVPISGWHGDNMLEPSTK (SEQ ID NO: 3);

[0047] (d) LPLQDVYK (SEQ ID NO: 4);

[0048] (e) QLIVGVNK (SEQ ID NO: 5);

[0049] (f)VETGILKPGTIVVFAPANITTEVK (SEQ ID NO: 6);

[0050] (g) YYVTIIDAPGHR (SEQ ID NO: 7).

[0051] More preferably, the active peptide library comprises a combination of peptides (a), (b), (e), and (g).

[0052] More preferably, the active peptide library comprises a combination of peptides (a), (b), (c), (d), (e), (f), and (g).

[0053] The third aspect of the present invention provides the application of the method described in the first aspect or the active peptide library described in the second aspect in screening active ingredients of soybean source with nutritional or functional activity.

[0054] Preferably, the nutritional or functional activity is selected from at least one of the following: amino acid supplementation, antioxidant activity, antibacterial activity, and enzyme inhibitory activity.

[0055] More preferably, the enzyme inhibitory activity is pancreatic lipase inhibitory activity.

[0056] Preferably, the application includes: using peptides in the active peptide library as candidate molecules, and employing an in vitro activity screening model to screen for peptides with target activity.

[0057] Preferably, the application further includes: comparing bioactive peptide libraries obtained from edible parts of *Eupolyphaga sinensis* from different sources and / or from *Eupolyphaga sinensis* processed in different ways, and evaluating the impact of different sources and / or different processing methods on the absorption and utilization of bioactive peptides.

[0058] More preferably, the edible parts of the bean worm from different sources are the bean worm skin and the bean worm flesh.

[0059] More preferably, the different processing methods are freeze-drying and cooking.

[0060] Fourthly, the present invention provides a quality control method for bean antler products, comprising the following steps:

[0061] The seven peptides identified by the method described in the first aspect were used as quality markers. The peptides are: EHALLAFTLGVK, IGGIGTVPVGR, IGYNPAAVAFVPISGWHGDNMLEPSTK, LPLQDVYK, QLIVGVNK, VETGILKPGTIVVFAPANITTEVK, and YYVTIIDAPGHR.

[0062] Using the seven peptides as target analytes, a multiple reaction monitoring (MRM) mass spectrometry method was established to detect the presence and abundance of the seven biomarker peptides in the tested bean curd product. The results were then compared with standards or standard spectra to evaluate the consistency of the product.

[0063] Preferably, the abundance of QLIVGVNK is used as an evaluation index for the content of the core active ingredient in the product.

[0064] Preferably, the detection of VETGILKPGTIVVFAPANITTEVK is used as a characteristic identifier for identifying bean ant meat source products.

[0065] The present invention has the following advantages over the prior art:

[0066] (i) An active peptide library reflecting the actual in vivo absorption components of *Dendrobium nobile* has been constructed. Existing technologies only reach the level of in vitro enzymatic hydrolysis, and the obtained peptides are only potential active ingredients, making it impossible to confirm whether they can be truly absorbed and utilized by the body. This invention, by tracking peptides that enter the bloodstream after oral ingestion, clarifies for the first time the true material basis for the nutritional or functional effects of *Dendrobium nobile* after digestion and absorption, filling a technological gap in this field.

[0067] (II) Provided clearly defined bioactive peptide sequences. This invention identified seven specific peptide sequences (SEQ ID NO: 1–7) through mass spectrometry, providing precise molecular targets for the functional study and product development of *Dendrobium nobile* bioactive peptides. Specifically: EHALLAFTLGVK, IGGIGTVPVGR, QLIVGVNK, and YYVTIIDAPGHR were detected in all four sample groups (mouse / human, skin / meat), indicating excellent cross-species stable absorption characteristics; QLIVGVNK had the highest abundance in human serum, suggesting optimal bioavailability and making it a priority candidate for functional peptide development; IGYNPAAVAFVPISGWHGDNMLEPSTK was detected only in mouse serum, and VETGILVKPGTIVFAPANITTEVK was only present in the meat protein group, revealing that the absorption of different peptides is species- and tissue-specific.

[0068] (III) This invention provides a quality control method for bean antler products. The seven identified peptides are used as quality markers for bean antler products. A multiple reaction monitoring (MRM) mass spectrometry method can be established, using the detection and abundance ratio of the seven marker peptides as evaluation indicators of product consistency. Specifically, the abundance of QLIVGVNK is used to evaluate the content of the core active ingredient in the product, and the detection of VETGILVKPGTIVVFAPANITTEVK is used as a characteristic identifier for bean antler meat-derived products, providing a scientific means for quality control and authenticity identification of bean antler products.

[0069] (iv) The method is highly versatile and has high application value. This invention's method is not only applicable to the construction of an in vivo bioactive peptide library for *Eriocaulon buergerianum*, but can also be used to compare the effects of different edible parts (skin / flesh) and different processing methods (dry powder / cooked food) on the absorption and utilization of bioactive peptides, providing a basis for the intensive processing and targeted development of *Eriocaulon buergerianum* products. Simultaneously, this method also provides a referable technical paradigm for the study of in vivo bioactive components of other edible insects or food-derived proteins. Attached Figure Description

[0070] Figure 1 Standard curve for determining the concentration of bovine serum albumin (BSA) standard.

[0071] Figure 2 Mass spectrometry results for identifying proteins in the skin and flesh of soybean hawk moth larvae (protein types and abundance distribution).

[0072] Figure 3 This is an SDS-PAGE electrophoresis image of the skin and flesh protein of the soybean hawk moth larvae.

[0073] Figure 4 This is the secondary mass spectrum of the peptide EHALLAFTLGVK.

[0074] Figure 5 This is a secondary mass spectrum of the peptide IGGIGTVPVGR.

[0075] Figure 6 This is the secondary mass spectrum of the peptide IGYNPAAVAFVPISGWHGDNMLEPSTK.

[0076] Figure 7 This is the secondary mass spectrum of the peptide LPLQDVYK.

[0077] Figure 8 This is the secondary mass spectrum of the peptide QLIVGVNK.

[0078] Figure 9 This is the secondary mass spectrum of the peptide VETGILKPGTIVVFAPANITTEVK.

[0079] Figure 10 This is the secondary mass spectrum of the peptide YYVTIIDAPGHR. Detailed Implementation

[0080] The technical solution of the present invention will be described in more detail with reference to specific embodiments and accompanying drawings. In implementing the present invention, except for the experimental steps, conditions, and methods described in detail below, all other techniques and common knowledge in the art are employed, and no additional special limitations are imposed.

[0081] It should be noted that the embodiments described herein are only some typical examples of the present invention, and not all implementation methods. The following description of exemplary embodiments is for illustrative purposes only and does not constitute any limitation on the present invention or its application scenarios.

[0082] In the demonstration and discussion of the various embodiments, the specific numerical values ​​and parameters involved are for illustrative purposes only and are not intended to be limiting. Therefore, other embodiments based on the concept of this invention can have their relevant numerical values ​​adjusted according to actual needs.

[0083] In addition, unless otherwise specified, the experimental materials used in the examples are all commercially available biochemical reagents, and the experimental operations also follow the standard procedures in this field.

[0084] Experimental materials

[0085] The main experimental materials used in the embodiments of this invention and their sources are as follows:

[0086] Bean hawk moth (Clanis bilineata) larvae were purchased from a bean hawk moth breeding base in Guanyun County, Jiangsu Province; C57BL / 6j mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; BCA protein concentration assay kits and sequencing-grade trypsin were purchased from Sigma-Aldrich; liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was performed using a Thermo Scientific™ Orbitrap™ Astral™ high-resolution mass spectrometer; the protein database used was the UniProt database (…). https: / / www.uniprot.org ).

[0087] All other reagents not specified in their origin were of analytical grade and purchased from conventional biochemical reagent suppliers.

[0088] Example 1: Protein Component Analysis of Bean Peel and Meat

[0089] 1.1 Separation of Bean Peel and Flesh and Preparation of Dry Powder

[0090] Take 200 healthy larvae of the bean hawk moth (Clanis bilineata), and use dissecting tools to separate the skin and flesh of the larvae and collect them separately.

[0091] The separated bean curd skin and bean curd flesh were placed separately in a food-grade mixer and thoroughly pulverized into a homogeneous paste. The pulverized sample was then frozen at -20°C overnight, and then transferred to a freeze dryer for freeze drying to obtain bean curd skin powder and bean curd flesh powder, which were then sealed and stored at -80°C for later use.

[0092] 1.2 Determination of protein concentration in bean curd skin and meat using BCA method

[0093] Weigh 5 mg each of the dried powder of soybean bark and dried soybean flesh prepared in step 1.1, dilute with an appropriate amount of PBS buffer, and measure the absorbance at 562 nm according to the instructions of the BCA protein concentration assay kit.

[0094] A standard curve was prepared using bovine serum albumin (BSA) standards. Figure 1 The protein concentrations of bean curd skin and meat were calculated based on the standard curve.

[0095] Results: The protein concentration of the fleshy part of the bean peony root was 4.00 μg / μL, while that of the skin was 3.22 μg / μL. The results indicate that, under the same conditions, the protein content of the fleshy part of the bean peony root was higher than that of the skin.

[0096] 1.3 Mass spectrometric identification of bean curd skin and meat protein

[0097] Take appropriate amounts of the dried powdered soybean bark and dried soybean flesh prepared in step 1.1, and after enzymatic hydrolysis, analyze them using a ThermoScientific™ Orbitrap™ Astral™ high-resolution mass spectrometer with liquid chromatography-tandem mass spectrometry (LC-MS / MS). Identify the protein species using the soybean hawk moth (Clanis bilineata) protein database in the UniProt database as a reference.

[0098] Result: As Figure 2 As shown, three main proteins were identified in both the skin and flesh of the bean peony: aromatic-L-amino-acid decarboxylase (UniProt: B9U744), elongation factor 1-alpha (UniProt: B9U7F4), and carbamoyl-phosphate synthase (UniProt: B9U6T3). The abundance of carbamoyl-phosphate synthase and aromatic-L-amino-acid decarboxylase in the flesh of the bean peony was higher than that in the skin, while the abundance of elongation factor 1-alpha in the skin was significantly higher than that in the flesh.

[0099] 1.4 SDS-PAGE electrophoresis to detect protein composition

[0100] Take appropriate amounts of the dried powder of safflower bark and dried safflower flesh prepared in step 1.1, add them to the loading buffer, and then perform SDS-PAGE electrophoresis in MOPs electrophoresis buffer. After electrophoresis, stain with Coomassie brilliant blue, and observe the distribution of protein bands after destaining.

[0101] Results: Electrophoresis image ( Figure 3 The results showed that both the skin and flesh of the bean curd are rich in protein, with the Elongation factor 1-alpha protein band at a molecular weight of approximately 39.7 kDa being the most prominent, indicating that this protein is the main protein component of the bean curd.

[0102] Example 2: Construction of a library of bioactive peptides for in vivo absorption by *Dendrobium nobile* in a mouse model.

[0103] 2.1 Administration of drugs to animals via gavage

[0104] Twelve SPF-grade C57BL / 6j mice, weighing 18-22g, were acclimatized for one week and then fasted but allowed to drink water for 12 hours.

[0105] Weigh 10 mg of each of the dried bean peony bark powder and dried bean peony flesh powder prepared in step 1.1, dissolve them in an appropriate amount of purified water, and administer them to mice by gavage using a gavage syringe. Six mice were administered the dried bean peony bark powder and six mice were administered the dried bean peony flesh powder, and were labeled as the skin protein group and the flesh protein group, respectively.

[0106] 2.2 Serum Collection

[0107] Four hours after oral administration, mice were anesthetized with sodium pentobarbital. After complete anesthesia, the eyeballs were removed and blood was collected in blood collection tubes containing an anticoagulant.

[0108] The collected blood samples were immediately centrifuged at 4°C and 3000 rpm for 15 minutes, and the supernatant serum was collected to obtain mouse absorbents of peony bark protein (serum) and peony flesh protein (serum). The serum was aliquoted and stored at -80°C for later use.

[0109] 2.3 Determination of serum protein concentration by BCA method

[0110] Take an appropriate amount of mouse serum separated in step 2.2 and determine the serum protein concentration using the BCA method, following the same method as in Example 1.2.

[0111] Results: The protein concentration in the serum of mice containing *Salvia miltiorrhiza* protein was 13.14 μg / μL, while the protein concentration in the serum of mice containing *Salvia miltiorrhiza* bark protein was 10.22 μg / μL. The serum protein concentration in the *Salvia miltiorrhiza* protein group was higher than that in the *Salvia miltiorrhiza* bark protein group, suggesting that a higher amount of *Salvia miltiorrhiza* protein enters the bloodstream after digestion and absorption.

[0112] 2.4 Serum proteolytic activity

[0113] Take the mouse serum stored in step 2.2, thaw it, and then perform enzymatic hydrolysis according to the following steps:

[0114] (1) Take an appropriate amount of serum sample and add urea solution for denaturation treatment;

[0115] (2) Add dithiothreitol (DTT) for reduction treatment and incubate at 56°C for 30 minutes;

[0116] (3) Add iodoacetamide (IAA) for alkylation treatment and incubate at room temperature in the dark for 30 minutes;

[0117] (4) Add sequencing-grade trypsin and hydrolyze overnight at 37°C;

[0118] (5) After the enzymatic hydrolysis is completed, formic acid is added to terminate the reaction;

[0119] (6) The enzymatically hydrolyzed peptides were desalted using a C18 solid-phase extraction column, and the eluent was freeze-dried and stored for later use.

[0120] 2.5 Identification by LC-MS / MS mass spectrometry

[0121] The peptide samples obtained from enzymatic hydrolysis in step 2.4 were reconstituted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a Thermo Scientific™ Orbitrap™ Astral™ high-resolution mass spectrometer.

[0122] Chromatographic conditions: A C18 reversed-phase column was used, with mobile phase A being 0.1% formic acid aqueous solution and mobile phase B being 0.1% formic acid acetonitrile solution, using gradient elution.

[0123] Mass spectrometry conditions: Data-dependent acquisition mode was used, MS1 scan range 350-1500 m / z, resolution 120,000, and MS / MS was performed in high-energy collisional dissociation (HCD) fragmentation mode.

[0124] 2.6 Peptide sequence identification and database matching

[0125] The MS / MS spectral data obtained in step 2.5 were analyzed using proteomics software, with the Clanis bilineata protein database in the UniProt database as a reference, to perform peptide sequence matching and identification. The false positive rate (FDR) of the identification results was controlled to be below 1%.

[0126] Results: Six bioactive peptides derived from *Dendrobium nobile* were identified from mouse serum samples, namely:

[0127] (1) EHALLAFTLGVK (SEQ ID NO:1), derived from eEF1A, with a molecular weight of 1298 Da, a desminoid abundance of 143662, and a vesinoid abundance of 160875.

[0128] (2) IGGIGTVPVGR (SEQ ID NO:2), derived from eEF1A, with a molecular weight of 1025 Da, a desminoid abundance of 1352770, and a lecithinoid abundance of 419853.

[0129] (3) IGYNPAAVAFVPISGWHGDNMLEPSTK (SEQ ID NO:3), derived from eEF1A, with a molecular weight of 2872 Da, was detected only in the skin protein group (abundance 1573.04), and not detected in the meat protein group;

[0130] (4) LPLQDVYK (SEQ ID NO:4), derived from eEF1A, with a molecular weight of 975 Da, a skin protein abundance of 3347330, and a meat protein abundance of 1062380;

[0131] (5) QLIVGVNK (SEQ ID NO:5), derived from eEF1A, with a molecular weight of 870 Da, a desminoid abundance of 551674, and a vesinoid abundance of 337552.

[0132] (6) VETGILKPGTIVVFAPANITTEVK (SEQ ID NO:6), derived from eEF1A, with a molecular weight of 2497 Da, was detected only in the meat protein group (abundance 35107.9), and not detected in the skin protein group;

[0133] (7) YYVTIIDAPGHR (SEQ ID NO:7) is derived from aromatic-L-amino-acid decarboxylase (AADC), with a molecular weight of 1404 Da, a skin protein abundance of 865904, and a meat protein abundance of 518291.

[0134] Example 3: Construction of an in vivo bioactive peptide library for *Dendrobium nobile* (human trial model)

[0135] 3.1 Preparation of cooked bean caterpillar food

[0136] Take 150 soybean hawk moth larvae and separate their skins and flesh. Cook the separated soybean skins and flesh separately:

[0137] Bean curd skin: After washing and draining, deep-fry in oil at 160-180℃ until golden brown, then drain and cool.

[0138] Bean butterflies: After cleaning, add appropriate seasonings and cook by steaming or boiling.

[0139] 3.2 Human Food Trials and Serum Collection

[0140] Six healthy volunteers (half male and half female, aged 25-45, with no history of erysipelas allergy or digestive system diseases) were recruited. Informed consent was obtained before the experiment. Volunteers fasted for 12 hours but were allowed to drink water, and then randomly divided into two groups:

[0141] Peel protein group (3 people): Fried bean curd skin, about 50g per person;

[0142] Meat protein group (3 people): Cooked bean curd meat, about 50g per person.

[0143] Six hours after consumption, venous blood was collected from the median cubital vein of the volunteers and placed in blood collection tubes containing anticoagulants.

[0144] The collected blood samples were immediately centrifuged at 4°C and 3000 rpm for 15 minutes, and the supernatant serum was collected to obtain human absorbent extracts of soybean bark protein (serum) and soybean flesh protein (serum). The serum was aliquoted and stored at -80°C for later use.

[0145] 3.3 Determination of serum protein concentration by BCA method

[0146] The concentration of human serum proteins separated in step 3.2 was determined using the BCA method, following the same method as in Example 1.1.2.

[0147] Results: The protein concentration of human absorbable protein from *Paeonia lactiflora* peel (serum) was 5.67 μg / μL, and the protein concentration of human absorbable protein from *Paeonia lactiflora* meat (serum) was 5.69 μg / μL. The concentrations were essentially equivalent, suggesting that the total protein content entering the bloodstream after digestion and absorption by the human body is similar for both *Paeonia lactiflora* peel and meat.

[0148] 3.4 Serum protease digestion and mass spectrometry identification

[0149] The human serum samples preserved in step 3.2 were subjected to enzymatic digestion and LC-MS / MS analysis in the same manner as in Examples 2.4 and 2.5.

[0150] 3.5 Peptide Sequence Identification

[0151] Database matching and peptide identification were performed using the same method as in Example 2.6.

[0152] Results: Five bioactive peptides derived from *Eriocaulon buergerianum* were identified from human serum samples. Their sequence information, source proteins, molecular weights, and abundance distribution in samples from different tissue sources (skin / flesh) are detailed in Table 1.

[0153] Table 1: Bioactive peptides derived from *Eriocaulon buergerianum* identified in human serum samples

[0154]

[0155] Example 4: Distribution Pattern Analysis of Active Peptides in Samples from Different Sources

[0156] Based on the identification results of Examples 2 and 3, the distribution and abundance of each peptide in the four groups of samples were summarized and analyzed (Table 2).

[0157] Table 2: Distribution and abundance of each active peptide in different samples

[0158]

[0159] Analysis conclusion:

[0160] (1) Common active peptides: EHALLAFTLGVK, IGGIGTVPVGR, QLIVGVNK, and YYVTIIDAPGHR were detected in all four groups of samples, indicating that the above peptides have excellent cross-species absorption stability and are not affected by the ingested object (mouse / human) or the source part of the bean lily (skin / meat). They are the most important active components of bean lily that enter the blood circulation after digestion and absorption.

[0161] (2) The peptide with the highest abundance: The abundance of QLIVGVNK in human serum (skin group 4,934,610; meat group 355,903) was significantly higher than that in mouse serum (skin group 551,674; meat group 337,552), and much higher than other peptides, suggesting that QLIVGVNK has the highest bioavailability in the human body and can be the first choice candidate molecule for the development of functional peptides from soybean.

[0162] (3) Tissue-specific peptides: VETGILKPGTIVVFAPANITTEVK is only found in the serum of mice in the bean ant meat protein group, indicating that this peptide is a bean ant meat specific absorption active peptide, and its production depends on the specific amino acid sequence and digestive enzyme cleavage site of the meat protein.

[0163] (4) Species-specific peptides: IGYNPAAVAFVPISGWHGDNMLEPSTK was detected only in mouse serum and not in human serum, suggesting that this peptide may have higher enzymatic hydrolysis efficiency in the mouse digestive system or that the human intestine has lower permeability to this peptide, indicating species differences.

[0164] Example 5: Confirmation of Mass Spectrometry Identification Results of Active Peptides

[0165] To further confirm the accuracy of the peptide sequences identified in Examples 2 and 3, the secondary mass spectra of each peptide were analyzed in detail.

[0166] Secondary mass spectra of each peptide segment are as follows Figures 4 to 10 As shown, all spectra display the complete b / y ion series, with mass deviations between the parent ion and fragment ions less than 10 ppm, sequence coverage of 90%–100%, and matching scores greater than 95%, indicating reliable identification results.

[0167] Taking QLIVGVNK as an example, its secondary mass spectrum ( Figure 6The complete b / y ion series is displayed: b1(Q) m / z 130.1, b2(QL) m / z 243.1, b3(QLI) m / z 356.2, b4(QLIV) m / z 455.3, b5(QLIVG) m / z 512.3, b6(QLIVGV) m / z 611.4, b7(QLIVGVN) m / z 725.4, and the corresponding y ion series. The full sequence coverage reaches 100%, the matching score is >99%, and the identification results are reliable.

[0168] The secondary mass spectra of the other peptides also showed complete b / y ion series, high sequence coverage, and matching scores of >95%, verifying the accuracy of sequence identification of each peptide.

[0169] Example 6: Application of the Active Peptide Library for In Vivo Absorption by Soybean Blossom

[0170] 6.1 Application in screening highly bioavailable bioactive peptides

[0171] Based on the cross-species abundance analysis results of each peptide in Example 4, peptides that simultaneously meet the following conditions are identified as highly bioavailable active peptides:

[0172] (1) It was detected in all four groups of samples (good stability across species and tissues);

[0173] (2) It ranks among the top two in abundance in human serum (high utilization efficiency in the human body).

[0174] According to the data in Table 2, the peptides that meet the above conditions are QLIVGVNK and YYVTIIDAPGHR. Among them, QLIVGVNK has the highest abundance in human serum (4,934,610 in skin group and 355,903 in meat group), and is therefore identified as the optimal candidate.

[0175] 6.2 Application in evaluating the nutritional value of different edible parts

[0176] A comparative analysis of bioactive peptide libraries derived from soybean bark and soybean flesh revealed that:

[0177] Shared peptides (present in both skin and flesh): EHALLAFTLGVK, IGGIGTVPVGR, QLIVGVNK, YYVTIIDAPGHR, indicating that both the skin and flesh of the bean peony have nutritional value in providing core active peptides.

[0178] Meat-specific peptides: VETGILKPGTIVVFAPANITTEVK are only found in meat-derived samples, suggesting that bean antler meat has unique active ingredients that cannot be replaced by the skin.

[0179] These results indicate that although bean peony bark is a processing byproduct, it still contains core active peptide components and has development and utilization value; while bean peony flesh, due to its unique peptide segments, is irreplaceable in the development of specific functional products. The above analysis provides a scientific basis for the graded utilization of bean peony resources and the development of differentiated products.

[0180] 6.3 Application in the quality control of bean curd products

[0181] The seven peptides identified in Examples 2 and 3 were used as quality markers for bean antler-related products, and the specific application method is as follows:

[0182] (1) Using the seven peptides as target analytes, a multiple reaction monitoring (MRM) mass spectrometry detection method was established;

[0183] (2) Test the bean products of different batches, different raw material sources and different processing technology, and use the detection status and abundance ratio of 7 marker peptides as the evaluation index of product consistency.

[0184] (3) Specifically, the abundance of QLIVGVNK is used as an evaluation index for the content of the core functional components of the product, and the detection of VETGILKPGTIVVFAPANITTEVK is used as a characteristic identifier of meat source products.

[0185] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above. Devices, reagents, and operating procedures not described in detail herein should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for constructing a library of bioactive peptides for in vivo absorption by *Dendrobium nobile*, characterized in that, Includes the following steps: The edible part of the bean caterpillar was prepared as the test substance; The test substance was administered orally to mammalian individuals. Blood samples were collected from the mammalian individual after it ingested the test substance. Serum was separated from the blood sample; The peptides in the serum were identified to obtain a set of active peptide sequences that enter the bloodstream after the soybean is digested and absorbed. Preferably, the bean caterpillar is the larva of the bean hawk moth (Clanis bilineata), and the edible part of the bean caterpillar is selected from the skin, flesh, or a combination thereof. The test substance is prepared by crushing and freeze-drying the edible part of the bean caterpillar into a dry powder, or by cooking the edible part of the bean caterpillar into a cooked food. More preferably, the cooking method is selected from frying or steaming.

2. The method according to claim 1, characterized in that, The mammalian individuals are selected from mice or humans.

3. The method according to claim 1, characterized in that, The blood sample is collected 2 to 8 hours after oral ingestion, preferably 4 to 6 hours.

4. The method according to claim 1, characterized in that, The serum was separated under the following conditions: centrifuged at 2-8°C and 2000-4000 rpm for 10-20 minutes.

5. The method according to claim 1, characterized in that, The identification process employs liquid chromatography-tandem mass spectrometry (LC-MS / MS) for peptide sequence analysis, and the serum is further subjected to enzymatic digestion before identification.

6. The method according to claim 1, characterized in that, The sequence of the peptide was determined by matching with a protein database, preferably the UniProt database.

7. The method according to any one of claims 1 to 6, characterized in that, The set of active peptide sequences includes one or more peptide segments selected from the following group: (a) EHALLAFTLGVK; (b) IGGIGTVPVGR; (c)IGYNPAAVAFVPISGWHGDNMLEPSTK; (d) LPLQDVYK; (e)QLIVGVNK; (f)VETGILKPGTIVVFAPANITTEVK; (g)YYVTIIDAPGHR; Preferably, the set of active peptide sequences comprises a combination of peptides (a), (b), (e), and (g), or a combination of peptides (a), (b), (c), (d), (e), (f), and (g); More preferably, the peptides (a) to (g) are derived from an elongation factor 1-alpha fragment or an aromatic-L-amino-acid decarboxylase fragment, wherein the peptides (a) to (f) are derived from an elongation factor 1-alpha fragment and the peptide (g) is derived from an aromatic-L-amino-acid decarboxylase fragment. More preferably, the abundance of the peptide (e)QLIVGVNK in human serum is higher than that of other peptides.

8. A library of bioactive peptides for in vivo absorption from *Solanum nigrum* constructed using the method described in any one of claims 1 to 7, characterized in that... It contains one or more peptides selected from the following groups: (a) EHALLAFTLGVK; (b) IGGIGTVPVGR; (c)IGYNPAAVAFVPISGWHGDNMLEPSTK; (d) LPLQDVYK; (e)QLIVGVNK; (f)VETGILKPGTIVVFAPANITTEVK; (g)YYVTIIDAPGHR; Preferably, the active peptide library comprises a combination of peptides (a), (b), (e), and (g), or a combination of peptides (a), (b), (c), (d), (e), (f), and (g).

9. The application of the method according to any one of claims 1 to 7 or the active peptide library of *Dendrobium nobile* for in vivo absorption according to claim 8 in screening active ingredients derived from *Dendrobium nobile* that have nutritional or functional activity; Preferably, the nutritional or functional activity is selected from at least one of amino acid supplementation, antioxidant activity, antibacterial activity, and enzyme inhibitory activity, and the enzyme inhibitory activity is preferably pancreatic lipase inhibitory activity.

10. A quality control method for a bean-shaped product, characterized in that, Includes the following steps: The seven peptides described in claim 7 are used as quality markers; A multiple reaction monitoring (MRM) mass spectrometry method was established using the seven peptides as target analytes. The detection status and abundance ratio of the seven biomarker peptides mentioned above were determined in the tested soybean product. The test results are compared with the standard or standard spectrum to evaluate the consistency of the product; Preferably, the abundance of QLIVGVNK is used as an evaluation index for the content of the core active ingredient in the product; More preferably, the detection of VETGILKPGTIVVFAPANITTEVK is used as a characteristic marker for identifying bean ant meat source products.

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