Preparation process and application of small-fruit coffee residue-derived polypeptide

Abstract

The invention belongs to the field of personal care, and relates to a preparation process and application of a polypeptide derived from Coffee arabica L. residues. The invention provides a method for predicting and acquiring functional active ingredients of small-fruit coffee grounds by utilizing a bioinformatics technology, and application of the method in the field of skin care or hair care.

Description

Technical Field

[0001] The invention belongs to the field of personal care, and in particular relates to a preparation process and application of a polypeptide derived from Arabica coffee (Coffea arabica L.) residues. Background Art

[0002] In 2023, the coffee planting area in Yunnan Province was 1.2 million mu, and the green bean output reached 146,000 tons. These data show that Yunnan's coffee planting area and output account for more than 98% of the country, 0.82% of the global area and 1.08% of the output. The main varieties of Yunnan coffee are Arabica, the so-called small-grain coffee (also known as small-fruit coffee) (Coffeaarabica L.). In addition to carbohydrates, fats and proteins, every 100 grams of Yunnan small-fruit coffee beans are also rich in a variety of plant active molecules, such as caffeine, chlorogenic acid, etc., and have a variety of biological activities. Studies have found that small-fruit coffee extract can reduce the inflammation caused by gout by effectively inhibiting the release of pro-inflammatory factors IL-1β, IL-6 and TNF-α.

[0003] In the coffee making process, the residue left after the coffee beans are ground and brewed is called coffee grounds. Coffee grounds are mainly composed of the skin of coffee beans, including the outer peel and the inner fruit membrane. Coffee grounds are rich in protein, polysaccharides, soluble sugars and fats, as well as a large amount of organic acids and polyphenols. These ingredients make coffee grounds widely used in biomass energy, cosmetics and agricultural fertilizers. More than 2.2 billion cups of coffee are sold every day in the world, and more than 20,000 tons of coffee grounds are produced every day. In 2023, China's coffee consumption reached 350,000 tons, a year-on-year increase of 167%. According to the calculation that about 650 kilograms of coffee grounds are produced during the production process of each ton of coffee beans, China will produce 806,000 tons of coffee grounds in 2022. The development of the efficacy of proteins and plant active molecules in coffee grounds has important economic value and can significantly increase the added value of the environmental protection utilization of coffee grounds. At present, there are some applications of coffee grounds in agriculture, environmental protection and food, but coffee grounds are still rare in the field of skin care and beauty.

[0004] In addition, during the roasting and brewing process of coffee beans, proteins are basically not dissolved. Therefore, the protein content in coffee grounds is very high. However, the high-abundance proteins in coffee grounds are mainly storage proteins, and there is no way to play a good role in skin care or hair care. Enzymatic hydrolysis, as a gentle, controllable and efficient technical means, has gradually become an important way to obtain polypeptides with specific functions. However, the application of enzymatic hydrolysis technology to Yunnan small-fruit coffee grounds to prepare polypeptides suitable for the field of personal care is still in a relatively early stage.

[0005] At present, the existing related technologies still have many deficiencies and challenges in the optimization of the enzymatic hydrolysis process, the separation and purification of polypeptides, and their actual application effects in personal care, such as low enzymatic hydrolysis efficiency, unstable polypeptide activity, and insignificant application effects. Therefore, developing an efficient enzymatic hydrolysis method for preparing Yunnan Arabica coffee polypeptides and exploring their wide application in the field of personal care have important practical significance and broad market prospects. Summary of the invention

[0006] The present invention uses high-abundance proteins in coffee grounds as raw materials, and uses bioinformatics techniques based on structural biology and computational biology to predict bioactive peptides targeting specific targets, including The virtual enzymatic hydrolysis module was used to construct a peptide library, and high-throughput activity prediction was performed based on molecular docking. The peptide sequence after enzymatic cleavage was verified by in vitro experiments to finally obtain a highly active small-fruit coffee grounds extract.

[0007] In one aspect, the present invention provides a method for preparing polypeptides derived from Arabica coffee (Coffea arabica L.) grounds, wherein the method comprises enzymolyzing Arabica coffee grounds with ficin.

[0008] In some embodiments, the method further comprises using an alkaline protease.

[0009] In some embodiments, the method comprises the steps of:

[0010] (1) taking coffee grounds, adding ficin to perform enzymolysis;

[0011] (2) Obtaining enzymatic hydrolyzate of small-fruit coffee grounds.

[0012] In some embodiments, the method comprises the steps of:

[0013] (1) taking small fruit coffee grounds and adding alkaline protease for enzymatic hydrolysis;

[0014] (2) adding ficin to the alkaline protease hydrolysis product for enzymolysis;

[0015] (3) Obtaining the enzymatic hydrolyzate of small-fruit coffee grounds.

[0016] In some embodiments, the enzymatic hydrolyzate of small-fruit coffee grounds comprises one or more polypeptides selected from any one of the polypeptides having amino acid sequences as shown in SEQ ID NOs: 12-18.

[0017] In some embodiments, the method comprises the steps of:

[0018] (1) taking small fruit coffee grounds, adding purified water and mixing evenly, adjusting the pH to above 8, preferably 9.5-10.5, adding alkaline protease and performing enzymolysis at above 50° C.;

[0019] (2) cooling to room temperature, adjusting the pH to 7-8, heating to above 60° C., preferably 65° C., and adding ficin for enzymatic hydrolysis;

[0020] (3) Obtaining the enzymatic hydrolyzate of small-fruit coffee grounds.

[0021] In a second aspect, the present invention provides a method for selecting a protease for enzymatic hydrolysis of small-fruit coffee grounds, wherein the method comprises using bioinformatics technology to predict bioactive peptides in small-fruit coffee grounds, and selecting a suitable protease based on the prediction results.

[0022] In some embodiments, the suitable protease is ficin.

[0023] In some embodiments, the suitable proteases are alkaline protease and ficin.

[0024] In some embodiments, the method comprises the steps of:

[0025] (1) Using a software program to select different proteases to perform virtual enzymatic digestion on the target protein sequence and construct a virtual peptide library;

[0026] (2) molecular docking the virtual peptide library with different targets;

[0027] (3) The peptide binding energies of the virtual peptide library are compared with the binding energies of the positive drugs for each target to obtain the predicted bioactive peptides.

[0028] In some embodiments, the target protein is 11S storage protein.

[0029] In some embodiments, the 11S storage protein amino acid sequence is as shown in SEQ ID NO:1.

[0030] In some embodiments, the software program is a virtual enzymatic digestion module, such as Peptide Virtual enzymatic hydrolysis module.

[0031] In some embodiments, the protease is an industrial grade protease, which can be purchased commercially.

[0032] In some embodiments, the protease is one or more selected from ficin, papain, pepsin, bromelain, Bacillus subtilis, and trypsin.

[0033] In some embodiments, the targets are soothing and anti-aging targets.

[0034] In some embodiments, the target is one or more selected from kallikrein-7 (KLK7), capsaicin receptor-1 (TRPV1), or matrix metalloproteinase-1 (MMP1).

[0035] In some embodiments, each of the target-positive drugs comprises LM-030 (Gln-lactone [Thr-Leu-Oaa-Tyr-Ile]), ilomastat, or the analgesic SB-366791.

[0036] In some embodiments, the method comprises the steps of:

[0037] (1) Selecting ficin, papain, pepsin, stem bromelain, Bacillus subtilis or trypsin through a virtual enzymatic hydrolysis module to perform virtual enzymatic hydrolysis on the 11S storage protein sequence to construct a virtual peptide library;

[0038] (2) molecular docking the virtual peptide library with KLK7, TRPV1 or MMP1 targets;

[0039] (3) The binding energy of the peptides in the virtual peptide library was compared with that of the positive drugs LM-030, ilomastat, or the analgesic SB-366791 to obtain the predicted bioactive peptides.

[0040] In some embodiments, the target protein structure is preprocessed when constructing the virtual peptide library.

[0041] In some embodiments, the pretreatment comprises: (1) removing the original ligands and other non-bonded heteroatoms; and (2) deleting water molecules in the structure and adding polar hydrogen atoms.

[0042] In some embodiments, the pre-processing comprises: (1) removing the original ligands and other non-bonded heteroatoms using PyMOL; and (2) deleting water molecules in the structure and adding polar hydrogen atoms using AutoDock Vina.

[0043] In some embodiments, the predicted bioactive peptide comprises any one or more polypeptides having amino acid sequences as shown in SEQ ID NOs: 2-11.

[0044] In a third aspect, the present invention provides a polypeptide prepared according to the above method.

[0045] In some embodiments, the polypeptide sequence comprises any one or more polypeptides selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 2-18.

[0046] In a fourth aspect, the present invention provides an enzymatic hydrolyzate of small-fruit coffee grounds prepared according to the above method.

[0047] In some embodiments, the enzymatic hydrolyzate of small-fruit coffee grounds contains any one or more polypeptides with amino acid sequences as shown in SEQ ID NOs: 2-18.

[0048] In a fifth aspect, the present invention provides the use of the above-mentioned polypeptide or the above-mentioned enzymatic hydrolyzate of small-fruit coffee grounds in the in vitro inhibition of soothing and anti-aging targets.

[0049] In some embodiments, the soothing and anti-aging targets include one or more of kallikrein-7 (KLK7), capsaicin receptor-1 (TRPV1), or matrix metalloproteinase-1 (MMP1).

[0050] In some embodiments, the polypeptide having an amino acid sequence comprising any one of the sequences shown in SEQ ID NOs: 2-5 or the enzymatic hydrolyzate of small fruit coffee grounds comprising an amino acid sequence comprising any one of the polypeptides shown in SEQ ID NOs: 2-5 is used in in vitro inhibition of TRPV1.

[0051] In some embodiments, the polypeptide having an amino acid sequence comprising any one of the sequences shown in SEQ ID NOs: 4, 6-7 or the enzymatic hydrolyzate of small fruit coffee grounds comprising an amino acid sequence as shown in any one of the sequences shown in SEQ ID NOs: 4, 6-7 is used for inhibiting MMP-1 in vitro.

[0052] In some embodiments, the polypeptide having an amino acid sequence comprising any one of the sequences shown in SEQ ID NOs: 2-3, 5, 8-11 or the enzymatic hydrolyzate of small fruit coffee grounds comprising an amino acid sequence as shown in any one of SEQ ID NOs: 2-3, 5, 8-11 is used in the in vitro inhibition of KLK7.

[0053] In some embodiments, the polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 or the enzymatic hydrolyzate of small fruit coffee grounds having an amino acid sequence as shown in SEQ ID NO: 2 is used for inhibiting TRPV1 in vitro.

[0054] In some embodiments, the polypeptide having an amino acid sequence as shown in SEQ ID NO: 8 or the enzymatic hydrolyzate of small fruit coffee grounds having an amino acid sequence as shown in SEQ ID NO: 8 is used in the in vitro inhibition of KLK7.

[0055] In some embodiments, the polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 or the enzymatic hydrolyzate of small fruit coffee grounds having an amino acid sequence as shown in SEQ ID NO: 4 is used for inhibiting TRPV1 in vitro.

[0056] In some embodiments, the polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 or the enzymatic hydrolyzate of coffee grounds having an amino acid sequence as shown in SEQ ID NO: 4 is used for inhibiting MMP-1 in vitro.

[0057] In some embodiments, the polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 or the enzymatic hydrolyzate of small fruit coffee grounds having an amino acid sequence as shown in SEQ ID NO: 2 is used in the in vitro inhibition of KLK7.

[0058] In a sixth aspect, the present invention provides the use of the above polypeptide or the above enzymatic hydrolyzate of small fruit coffee grounds for personal care.

[0059] In some embodiments, the application is skin care and / or hair care.

[0060] In some embodiments, the use is inhibiting KLK7, improving ex vivo hair tresses, and / or soothing the scalp.

[0061] In some embodiments, improving the separated hair tresses comprises increasing the shine of the hair tresses or improving the frizziness of the hair tresses.

[0062] In some embodiments, soothing the scalp comprises increasing the moisture content of the scalp stratum corneum or improving the redness of the scalp.

[0063] In a seventh aspect, the present invention provides use of the above polypeptide or the above enzymatic hydrolyzate of small-fruit coffee grounds in the preparation of personal care products.

[0064] In some embodiments, the personal care product comprises a skin care and / or a hair care product.

[0065] In some embodiments, the skin care product comprises a cleansing product, a toner, a lotion, a cream, an essence, an eye cream, a mask, a skin care oil, a serum or a sunscreen.

[0066] In some embodiments, the hair care product comprises shampoo, conditioner, hair care essential oil, hair care spray, elasticizer, hair care nutrient solution or hair mask.

[0067] Advantages of the invention

[0068] (1) Provides a method for predicting highly active polypeptide fragments in the most abundant protein in coffee grounds using bioinformatics techniques of structural biology and computational biology;

[0069] (2) An efficient method for preparing polypeptides from Yunnan Arabica coffee grounds is provided;

[0070] (3) The application of the Arabica coffee hydrolysate in the field of skin care or hair care is provided, and the inhibitory effect on serine protease KLK7, the effect of improving isolated hair bundles and the effect of soothing the scalp are verified. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The gloss difference test results of the control group and the sample group in Example 7 are shown, where "*" indicates a significant difference, 0.01≤P<0.05.

[0072] Figure 2 The results of the hair bundle area change rate test of the control group and the sample group in Example 7 are shown. Among them, "**" indicates a significant difference, 0.001≤P<0.01.

[0073] Figure 3 The figure shows the comparison of the increment of moisture content of the scalp stratum corneum of the control group and the sample group after using the sample in Example 8. Among them, "**" indicates a significant difference, 0.001≤P<0.01; "***" indicates a significant difference, P<0.001.

[0074] Figure 4 A comparison chart showing the reduction in a* value of scalp color between the control group and the sample group in Example 8 after using the sample is shown. DETAILED DESCRIPTION

[0075] For the purpose of clear and concise description, features are described herein as part of some identical or separate embodiments, however, it will be understood that the scope of the present disclosure may include some embodiments having a combination of all or some of the described features. The technical solution of the present invention will be clearly and completely described below. Obviously, based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0076] I. Definitions

[0077] Unless otherwise specified, the following terms used in the specification and claims have the following meanings:

[0078] To the extent that the terms "comprises," "including," and "having," "has," and "with," or variations thereof, are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising."

[0079] As used in this disclosure, the terms "optional", "either", "any", or "any" mean that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. As used in this disclosure, "a" and "an" are used in this disclosure to refer to one or more than one of the grammatical objects.

[0080] As shown in the present disclosure, “and / or” should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0081] As used herein, the terms "polypeptide" or "peptide segment" are used interchangeably and refer to amino acid polymers of any length having at least two amino acids.

[0082] As used herein, the term "ficin (EC 3.4.22.3)" belongs to a proteolytic enzyme mainly found in fig gum. It is a single-chain thiol protease and is considered to be a universal catalyst in the enzymatic preparation of peptides.

[0083] As used herein, the term "alkaline protease" refers to an enzyme that can hydrolyze protein peptide bonds under alkaline conditions, also known as serine protease, which is a complex enzyme. The main source of alkaline protease is microbial extraction, and the most studied and applied ones are mainly Bacillus, with Bacillus subtilis as the most, and there are also a small number of other species, such as Streptomyces.

[0084] In some embodiments, ficin is selected to enzymatically hydrolyze the Arabica coffee grounds.

[0085] In some embodiments, ficin and alkaline protease are selected to be used in combination to enzymatically hydrolyze Arabica coffee grounds.

[0086] As used herein, the terms "binding free energy", "binding energy" and "ΔG" are used interchangeably and refer to the free energy difference between molecules, that is, the free energy difference between molecules before and after binding. ΔG = ligand-receptor complex free energy - (ligand free energy + receptor free energy). ΔG is meaningful only when it is a negative value, indicating that the binding of the ligand and receptor is an energy-reducing reaction (releasing energy). If it is a positive value, it indicates that binding is impossible.

[0087] As used herein, the term "soothing" refers to personal care products that can relieve or reduce skin discomfort after use, making the skin feel comfortable and relaxed. These personal care products usually contain some special ingredients, such as plant extracts, mineral water, vitamins, etc., which have calming, soothing and anti-inflammatory effects. In some embodiments, the soothing refers to soothing the skin or soothing the scalp. In some embodiments, the personal care products include skin care and / or hair care products; in some embodiments, the skin care products include cleansing products, lotions, emulsions, creams, essences, eye creams, facial masks, skin care oils, stock solutions or sunscreens; in some embodiments, the hair care products include shampoo, conditioner, hair care essential oils, hair care sprays, elasticizers, hair care nutrient solutions or hair masks.

[0088] As used herein, the term "KLK7 (Kallikrein-Related Peptidase 7)" is a serine protease that belongs to the human tissue kallikrein family. It is mainly expressed in the stratum corneum of the skin and plays an important role in stratum corneum formation and skin desquamation. It is overexpressed in inflamed skin and is also the main KLK involved in the pathogenesis of AD. KLK7 can degrade intracellular connecting molecules such as corneal desmosomes, desmocollins 1, and desmocollins 1 to cause desquamation and excessive keratinization of the skin. Its abnormal regulation can lead to pathophysiological inflammation of the skin, thereby triggering the occurrence of diseases such as psoriasis, chronic skin diseases, and Netherton syndrome.

[0089] As used in this article, the term "capsaicin receptor-1 (TRPV1)" belongs to the vanilloid subfamily of the transient receptor potential (TRP) ion channel family. It is specifically expressed in sensory neurons that recognize noxious stimuli. It is not only present in oral nerve cells, but also widely present in muscle, intestine, pancreas and other tissue cells. It can be activated by physical and chemical stimuli such as high temperature (>43°C) and low pH (<5.9), as well as endogenous lipid molecules and exogenous ligands. It participates in various physiological and pathological processes such as pain perception, temperature regulation, and inflammatory response. It is a therapeutic target for various preclinical models of chronic pain.

[0090] As used herein, the term "matrix metalloproteinase 1 (MMP-1)" is encoded by the MMP1 gene in humans. MMP-1 is produced by fibroblasts, chondrocytes, macrophages, keratinocytes, endothelial cells and osteoblasts, plays an important role in the degradation of collagen fibers in the remodeling of the extracellular matrix, and is involved in a variety of biological processes including rheumatoid arthritis, osteoarthritis, periodontal disease, tumor invasion, angiogenesis, corneal ulcers, tissue remodeling, inflammatory bowel disease, atherosclerosis and aneurysm, while also cleaving a variety of substrates such as casein, gelatin, proteoglycans, endogenous factors, tumor necrosis factor and cartilage junction proteins.

[0091] II. Examples

[0092] The present invention is described in detail below through specific examples. It should be understood that the following examples are only for explanation and illustration and are not intended to limit the scope of the present invention in any form.

[0093] In the following embodiments, biochemical reagents not specifically described are all conventional reagents in the art, and can be prepared according to conventional methods in the art or commercially available, and the specifications are laboratory pure grade.

[0094] Example 1: Highly Abundant Protein Sequences in Arabica Coffee Grounds

[0095] According to a large number of literature reports, 11S storage protein is the most abundant protein in Arabica coffee beans, accounting for more than 50%. The specific sequence of this protein (identifier: Q9ZNY2, name: 11S Storage protein, source: Coffea arabica (Arabian coffee)) retrieved from the UniProt database is as follows:

[0096] SEQ ID NO: 1:

[0097]

[0098] Example 2: Construction of virtual peptide library

[0099] Peptide In the virtual enzymatic hydrolysis module, commercially available industrial proteases, including ficin, papain, pepsin, stem bromelain, Bacillus subtilis, and trypsin, were selected to perform virtual enzymatic hydrolysis on the protein sequence in Example 1 to construct a peptide library. The number of peptides obtained after the six proteases hydrolyzed the protein is shown in Table 1. Except for trypsin, the peptides after hydrolysis by other industrial enzymes were highly expressed, all of which were more than 100 peptides.

[0100] Table 1. Number of peptides produced after virtual enzymatic digestion of high-abundance proteins in coffee grounds by different proteases

[0101]

[0102]

[0103] Example 3: Target selection and molecular docking

[0104] The present invention aims at the soothing and anti-aging effects of the coffee grounds extract, and selects the soothing and anti-aging targets: kallikrein-7 (KLK7, PDB ID: 5Y9L), capsaicin receptor-1 (TRPV1, PDB ID: 8GF9), matrix metalloproteinase-1 (MMP1, PDB ID: 966C). The virtual peptide library obtained by the above 6 enzymatic hydrolysis schemes was molecularly docked with each target. The three-dimensional structure model of the peptide segment in the peptide library was batch constructed by AutoDock Tools (1.5.6, CCSB, CA, USA), and the target protein crystal structure was downloaded from the Protein Data Bank (RCSB-PDB) (http: / / www.rcsb.org / pdb / home / home.do). First, PyMOL (2.6.0, Inc., NY, USA) was used to pre-process the target protein structure to remove the original ligand and other non-bonded heteroatoms. The processed structure was further manipulated using AutoDock Vina (1.2.5, CCSB, CA, USA) to delete water molecules in the structure and add polar hydrogen atoms. The processed peptide and target files were imported into AutoDock Vina for molecular docking. The binding energy was compared with that of each target positive drug (KLK7: LM-030 (Gln-lactone [Thr-Leu-Oaa-Tyr-Ile]), IC50 = 6nM; MMP-1: ilomastat, IC50 = 1.5nM; TRPV1: analgesic SB-366791, IC50 = 5.7nM) to obtain the number of highly effective active peptides. The results are shown in Tables 2-4. Among them, LM-030 was used as a positive control for molecular docking with KLK7, so the peptides with a binding energy ΔG less than -7.0kcal / mol were all positive results. The results showed that each enzymatic hydrolysis method More than 10 active peptides were obtained for all of them; ilomastat was used as the positive control for molecular docking with MMP-1, so the peptides with binding energy ΔG less than -9.0 kcal / mol were all positive results. The results showed that each enzyme cleavage method had peptides that were superior to the positive control, and only trypsin had the least equivalent peptides, while the others were all more than 10; the analgesic SB-366791 was used as the positive control for molecular docking with TRPV1, so the peptides with binding energy ΔG less than -9.1 kcal / mol were all positive results, including 0 for pepsin, 5 for subtilisin, 4 for papain, 3 for ficin, and 1 for the others.

[0105] Table 2. Molecular docking results of theoretical peptide libraries formed by different enzymatic hydrolysis schemes of coffee grounds and KLK7

[0106]

[0107] Note: The docking results use LM-030 as a positive control, so peptides with a binding energy ΔG less than -7.0 kcal / mol are positive results.

[0108] Table 3. Molecular docking results of theoretical peptide libraries formed by different enzymatic hydrolysis schemes and MMP-1

[0109]

[0110] Note: The docking results used Ilomasstat as a positive control, so peptides with a binding energy ΔG less than -9.0 kcal / mol were positive results.

[0111] Table 4 Molecular docking results of theoretical peptide libraries formed by different enzymatic hydrolysis schemes and TRPV1

[0112]

[0113] Note: The docking results use the analgesic SB-366791 as a positive control, so all binding energies ΔG less than -9.1 kcal / mol are positive results.

[0114] According to the molecular docking results, the activity of peptides that are better than the positive control was predicted. The peptide sequences with high inhibitory activity of TRPV1, MMP1 and KLK7 were analyzed by Peptide Lego software. The analysis results are shown in Tables 5-7. Among them, the peptide APHW has the strongest inhibitory activity against TRPV1; the peptide EYV has the strongest inhibitory activity against MMP-1; the peptide NNPEF has the strongest inhibitory activity against KLK7; the peptides PDR and APHW have dual-target inhibitory activity. Among them, the peptides APHW, NNPEF and PDR are all from the fig protease enzymatic peptide library, so fig protease was determined as the actual enzymatic tool enzyme.

[0115] Table 5. Peptides with high inhibitory activity against TRPV1

[0116]

[0117]

[0118] Table 6. Peptides with high inhibitory activity against MMP-1

[0119]

[0120] Table 7. Peptides with high inhibitory activity against KLK7

[0121]

[0122] Example 4: Enzymatic hydrolysis of coffee grounds using alkaline protease and fig protease

[0123] Alkaline protease is a composite protease. Due to its excellent hydrolysis ability and wide substrate specificity, it can efficiently decompose proteins in a high pH environment and is widely used in industrial fields such as detergents, food processing, leather processing, textile refining and pharmaceutical production. The activity prediction results show that the virtual peptide library generated by fig protease has high inhibitory activity. In order to further improve the functionality of the hydrolyzate, this study finally determined that alkaline protease and fig protease were combined as the final process scheme. The specific process is as follows: Take 100 grams of small fruit coffee grounds, add four times of pure water and mix evenly, add 4 mol / L sodium hydroxide to adjust the pH to between 9.5-10.5, add 0.04% alkaline protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and enzymolyze at 55℃ for 2h (the preparation process of alkaline protease: add 10 times the amount of water to dissolve, dissolve at 55℃ for 15min). Lower to room temperature, adjust pH to 7-8 with 10% lemon water, raise temperature to 65°C, add 0.04% fig proteinase (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) for enzymolysis for 2h, and inactivate at 90-95°C for 15min. Filter with 1μL filter membrane and diatomaceous earth as filter aid. Concentrate to 3 times the amount of feed, cool, add 10% citric acid to adjust the final pH to 7, and obtain the enzymolysis solution of small fruit coffee grounds.

[0124] Example 5: Prediction of highly active peptides in enzymatic hydrolysis products

[0125] Electrostatic field orbital trap mass spectrometry (HRMS) was used to detect the peptide components in the coffee grounds extract before and after enzymatic hydrolysis. The reagents used are shown in Table 8.

[0126] Table 8. Reagents used in HRMS testing

[0127] Reagents purity brand Isopropyl alcohol (IPA) HPLC grade Merck Acetonitrile (ACN) HPLC grade Merck Formic Acid (FA) LC-MS Grade Aladdin Ultrapure water UP Water Made by NeoLab Pure water purifier

[0128] The experimental instruments for peptide sequence determination are as follows:

[0129] (1)Thermo Vanquish LC-Thermo Orbitrap Exploris 120HRMS

[0130] (2) NeoLab Pure Water Purifier

[0131] For the samples of the enzymatic hydrolyzate of small-fruit coffee grounds and the extract of small-fruit coffee grounds (without enzymatic hydrolysis), the supernatant was taken after centrifugation and diluted 5 times with UP water before injection.

[0132] Mobile phase (A1): H2O + 0.1% FA; Mobile phase (B1): ACN + 0.1% FA; Injection volume: 2 μL; Seal flush: 10% IPA; Needle flush: 10% IPA + 1% FA; Flow rate: 0.4 mL / min; Sample chamber temperature: 4°C; Column temperature: 60°C.

[0133] HRMS conditions: ion source: H-ESI; positive mode voltage: 3800 V; ion transfer tube temperature: 275°C; vaporization temperature: 200°C; sheath gas (arbitrary unit): 35; auxiliary gas (arbitrary unit): 8; purge gas (arbitrary unit): 0; scan mode: full scan; full scan orbital trap resolution: 60000; scan range (m / z): 500-3000; RF lens: 130%; micro scan times: 5; data type: profile; scan mode: data-dependent secondary mass spectrometry scan (ddMS2); ddMS2 orbital trap resolution: 15000; high energy collision dissociation collision energy (%): 30.

[0134] The high-abundance peptide fragments after enzymatic cleavage identified by HRMS were compared with the peptide fragments predicted by the above molecular docking model. The results are shown in Table 9. The predicted peptide fragments were detected in the enzymatic cleavage products. Due to the short actual enzymatic cleavage time, the sequence length of the enzymatic cleavage product is usually slightly longer than the peptide predicted by the model. Table 10 lists 7 peptides with high abundance changes after enzymatic cleavage compared to before enzymatic cleavage. These high-abundance peptide sequences were molecularly docked with the targets. The results showed that their binding energy with the targets KLK7, TRPV1 and MMP1 was close to the positive control (the positive control for KLK7 was LM-030, with a binding energy of -7.0 kcal / mol; the positive control for TRPV-1 was the analgesic SB-366791, with a binding energy of -9.1 kcal / mol; the positive control for MMP1 was ilomastat, with a binding energy of -9.0 kcal / mol).

[0135] Table 9. Changes in peptides in coffee grounds before and after treatment with alkaline protease-ficin

[0136]

[0137]

[0138] Table 10. Docking results of actual enzymatically high-abundance peptides and targets

[0139]

[0140] Example 6: Activity verification of enzymatic hydrolysis products

[0141] The samples were divided into three groups for comparison: inhibitor group A1, enzyme control group B1, and sample blank group B0. The inhibitor group A1 was added with the corresponding enzyme to be tested and enzymatic hydrolysate of small-fruit coffee grounds, the enzyme control group B1 was only added with the enzyme to be tested, and the sample blank group B0 was not added with anything.

[0142] 1. MMP-1 inhibitory activity

[0143] Prepare the detection buffer: 50mM Tris, HEPES, 150mM NaCl, 10mM CaCl2, 0.05% Brij-35; dilute the enzyme to 2mM enzyme solution with the detection buffer, add an equal volume of collagenase latent enzyme activator (APMA) to activate the enzyme; complete the reaction in sequence as shown in Table 11 below.

[0144] Table 11. MMP-1 inhibitory activity reaction system

[0145]

[0146]

[0147] The MMP-1 inhibition rate was calculated using the following formula:

[0148] Fluorescence slope S = (F A1 -F A0 ) / (T A1 -T A0 )

[0149]

[0150] F--Fluorescence intensity, measured by microplate reader, the unit is relative fluorescence intensity (RFU)

[0151] T--Reaction time

[0152] S--fluorescence slope, that is, the change in fluorescence intensity per unit reaction time

[0153] 2. KLK7 inhibitory activity

[0154] Prepare the detection buffer: 50mM Tris-HCl, 0.01% Tween-20, 150mM NaCl, adjust the pH to 8.0; mix equal volumes of 200μg / mL rhKLK7 and 20μg / mL Thermolysin, incubate at 37°C for 3h to activate the enzyme; complete the reaction in sequence as shown in Table 12. The materials described in the experiment are shown in Table 13.

[0155] Table 12. KLK7 inhibitory activity reaction system

[0156]

[0157] The excitation wavelength was 320 nm and the emission wavelength was 405 nm (top reading) every 5 min for 1 h.

[0158] Table 13. Reagents used in the KLK7 inhibitory activity reaction system

[0159] Reagents brand Concentrated hydrochloric acid HCl Sinopharm Chemical Reagent Co., Ltd. Tris Shanghai MacLean Biochemical Technology Co., Ltd. Tween-20 Sinopharm Chemical Reagent Co., Ltd. Sodium Chloride Sinopharm Chemical Reagent Co., Ltd. KLK7 R&D Thermolsin MedChemExpress

[0160] The KLK7 inhibition rate was calculated using the following formula:

[0161] Fluorescence slope S = (F A1 -F A0 ) / (T A1 -T A0 )

[0162]

[0163] F--Fluorescence intensity, measured by microplate reader, the unit is relative fluorescence intensity (RFU)

[0164] T--Reaction time

[0165] S--fluorescence slope, that is, the change in fluorescence intensity per unit reaction time

[0166] The inhibitory activity of KLK7 and MMP-1 before and after enzymatic hydrolysis is shown in Table 14. The experimental results show that the enzymatic hydrolysis process significantly increased the inhibition rate of KLK7 and MMP-1. Before enzymatic hydrolysis, the inhibition rate of KLK7 was 7.2% ± 1.9%, and after enzymatic hydrolysis, the inhibition rate was significantly increased to 61.1% ± 2.1%. Similarly, the inhibition rate of MMP-1 was 65.8% ± 1.7% before enzymatic hydrolysis, and increased to 81.8% ± 3.4% after enzymatic hydrolysis. This result shows that enzymatic hydrolysis can greatly enhance the inhibitory effect on KLK7 and MMP-1, especially the inhibitory effect on KLK7 is particularly significant.

[0167] Table 14. Comparison of inhibition rates of KLK7 and MMP-1 before and after enzymatic hydrolysis

[0168] Target Inhibition rate before enzymatic hydrolysis Inhibition rate after enzymatic hydrolysis KLK7 7.2％±1.9％ 61.1％±2.1％ MMP-1 65.8％±1.7％ 81.8％±3.4％

[0169] Example 7: Effect of Arabica coffee grounds enzymatic hydrolysate on improving isolated hair bundles

[0170] 1. Test materials and instruments:

[0171] Mildly damaged cylindrical (Asian) hair bundle (3g, 17cmx3g, free 15cm);

[0172] 14% sodium lauryl sulfate (SLS) aqueous solution;

[0173] Flat narrow-tooth comb: The tooth density is moderate (tooth spacing is 0.9-1.1mm), the tooth length is 2.0-3.0cm, and the comb length is not less than 10cm;

[0174] Facial imaging analyzer: Visia;

[0175] Skin gloss test probe: Skin-Glossymeter GL 200;

[0176] Electronic balance: FA2004 (0.0001g).

[0177] 2. Hair bundle pretreatment

[0178] Wet and rinse the hair with clean water, take 1 mL of 14% sodium dodecyl sulfate (SLS) aqueous solution and evenly apply it on the surface of the hair, gently rub until rich foam lasts for 60 seconds, rinse at a water temperature of 37.0±1.0℃ and a water flow rate of 4L / min, wash the hair twice with 14% SLS aqueous solution, comb the hair with a flat narrow-tooth comb for 10 times on each side after rinsing, and hang the hair in a constant temperature and humidity chamber at a temperature of 21±1℃ and a relative humidity of 50±10% RH for 16-24 hours.

[0179] 3. Hair gloss test:

[0180] 1) 10 hair bundles after pretreatment were selected and randomly divided into 2 groups, 5 bundles in the sample group and 5 bundles in the control group;

[0181] 2) Initial value test: Use Skin-Glossymeter GL 200 to measure the gloss parameter value of the hair bundle. Each hair bundle is evenly measured 5 times from top to bottom, and the average value is taken as the initial value (before treatment);

[0182] 3) Hair bundle treatment of the sample group (each hair bundle is treated separately): the hair bundle is treated according to the standard operating procedure for hair bundle treatment in the instructions for use of the test sample, and the shampoo contains enzymatic hydrolyzate of Arabica coffee grounds;

[0183] 4) Treatment of hair bundles in the control group (each hair bundle is treated separately): the hair bundles are treated in the same manner as the hair bundles in the sample group except that the control sample is used instead, and the shampoo does not contain the enzymatic hydrolyzate of Arabica coffee grounds;

[0184] 5) After the treatment, the hair bundle is hung in a constant temperature and humidity chamber at a temperature of 21±1°C and a relative humidity of 50±10% RH for 16-24 hours;

[0185] 6) Test after using the sample: Use Skin-Glossymeter GL 200 to measure the gloss parameter value of the hair bundle, and measure each hair bundle evenly from top to bottom 5 times, and take the average value as the test value after using the sample (after treatment).

[0186] 4. Anti-frizz test:

[0187] 1) Fifteen hair bundles after pretreatment were selected and randomly divided into three groups: a sample group of 5 bundles, a control group of 5 bundles, and a blank group of 5 bundles;

[0188] 2) Initial value test: Use VISIA to take pictures (take 5 pictures for each hair bundle), IPP to analyze the hair bundle area, and take the average value as the initial value (before treatment);

[0189] 3) Hair bundle treatment of the sample group (each hair bundle is treated separately): the hair bundle is treated according to the standard operating procedure for hair bundle treatment in the instructions for use of the test sample, and the shampoo contains enzymatic hydrolyzate of Arabica coffee grounds;

[0190] 4) Treatment of hair bundles in the control group (each hair bundle is treated separately): the hair bundles are treated in the same manner as the hair bundles in the sample group except that the control sample is used instead, and the shampoo does not contain the enzymatic hydrolyzate of Arabica coffee grounds;

[0191] 5) The hair bundles in the blank group were not treated in any way;

[0192] 6) After the treatment, the hair bundles were hung in a constant temperature and humidity chamber at a temperature of 21±1°C and a relative humidity of 50±10% RH for 16-24 hours to balance, and the hair bundles were combed 30 times before testing;

[0193] 7) Test after using the sample: Use VISIA to take pictures (take 5 pictures for each hair bundle), IPP to analyze the hair bundle area, and take the average value as the test value after using the sample (after treatment).

[0194] 5. Test results

[0195] according to Figure 1 The gloss difference test results between the control group and the sample group showed that the glossiness of the hair in the sample group was higher than that in the control group, indicating that Yunnan Arabica coffee residue enzymatic hydrolyzate can effectively improve the glossiness of hair.

[0196] according to Figure 2 The test results of the change rate of hair area between the control group and the sample group showed that compared with before use, the hair area increased by 11.92% after using the test sample, and the hair area increased by 25.07% after using the control sample; compared with the control group, the hair area of ​​the sample group decreased by 13.15%, indicating that the hair in the control group was more frizzy than that in the sample group, and Yunnan small fruit coffee residue enzymatic hydrolyzate can effectively improve the frizziness of hair.

[0197] In summary, Yunnan coffee grounds enzymatic hydrolysate has the effect of improving in vitro hair strands.

[0198] Example 8: Scalp-soothing effect of Arabica coffee grounds enzymatic hydrolysate

[0199] 1. The test materials and instruments used in this embodiment are as follows:

[0200] PhotoMax PRO, a hair growth analysis and management system;

[0201] Hydration test probe Moisture with Pin Probe;

[0202] The formulas of the sample group and the control group are shown in Tables 15 and 16.

[0203] Table 15. Sample set formulation

[0204]

[0205]

[0206] Table 16. Control group formulation

[0207]

[0208] 2. Experimental process:

[0209] On D0: 1) Subject information was registered, informed consent was obtained and signed, and the laboratory technicians screened the subjects according to the inclusion and exclusion criteria; 2) The laboratory technicians shaved the 1.5cm*1.5cm area on the temporal side of the subject's head, and the hair was cut to a residual length of no more than 1mm; 3) The subjects sat quietly in a laboratory with a temperature of 21±1℃ and a humidity of 50±10% RH for 30 minutes; 4) The scalp basal value of the subjects was tested: Photomax-Pro image acquisition, Moistuture with PinProbe test of skin stratum corneum moisture content, and expert evaluation; 5) The subjects filled in the pre-use self-assessment questionnaire and were randomly divided into groups; 6) The subjects in the control group received the test samples, and the subjects in the sample group received the test samples; 7) The laboratory technicians guided the subjects to use the samples according to the sample use requirements and the use site, and provided written test precautions and instructions for use; 9) Leave the laboratory.

[0210] On D14: 1) The test technician shaved the subject's head in an area of ​​1.5cm*1.5cm on the temporal side, and the hair was cut to a residual length of no more than 1mm; 2) The subject sat quietly in a laboratory at a temperature of 21±1℃ and a humidity of 50±10% RH for 30 minutes; 3) The scalp data values ​​of the test subjects after using the samples for 14 days: Photomax-Pro image acquisition, Moisture withPin Probe test of skin stratum corneum moisture content, expert evaluation; 4) The subject filled out a self-assessment questionnaire.

[0211] On D28: 1) The test technician shaved the subject's head in an area of ​​1.5cm*1.5cm on the temporal side, and the hair was cut to a residual length of no more than 1mm; 2) The subject sat quietly in a laboratory at a temperature of 21±1℃ and a humidity of 50±10% RH for 30 minutes; 3) The skin data values ​​of the test subjects after using the samples for 28 days: Photomax-Pro image acquisition, Moisture withPin Probe test of the moisture content of the skin stratum corneum, and expert evaluation; 4) The subject filled out a self-assessment questionnaire, and the samples and usage logs were collected.

[0212] 3. Experimental results:

[0213] The results of the moisture content of the scalp stratum corneum are as follows Figure 3 As shown, after 14 days of use in the control group, the incremental moisture content (au) of the scalp stratum corneum increased by 11.39, while after 14 days of use in the sample group, the incremental moisture content (au) of the scalp stratum corneum increased by 17.22, after 28 days of use in the control group, the incremental moisture content (au) of the scalp stratum corneum increased by 24.38, while after 28 days of use in the sample group, the incremental moisture content (au) of the scalp stratum corneum increased by 33.94, the sample group was significantly improved compared with the control group, indicating that the addition of Yunnan small fruit coffee residue enzymatic hydrolyzate can significantly increase the moisture content of the scalp stratum corneum.

[0214] The scalp color a* value (redness) results are as follows Figure 4 As shown, after 14 days of use in the control group, the a* value of the scalp color decreased by 1.75, while after 14 days of use in the sample group, the a* value of the scalp color decreased by 2.63, after 28 days of use in the control group, the a* value of the scalp color decreased by 2.36, while after 28 days of use in the sample group, the a* value of the scalp color decreased by 3.55. The reduction in the a* value of the scalp color in the sample group was greater than that in the control group, indicating that the addition of Yunnan small fruit coffee grounds enzymatic hydrolyzate can improve the redness of the scalp.

[0215] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention are all covered within the scope of the present invention.

Claims

1. A method for preparing a polypeptide derived from Arabica coffee (Coffea arabica L.) residues, wherein: The method comprises enzymatically hydrolyzing Arabica coffee grounds using ficin.

2. The method according to claim 1, wherein: The method further comprises using alkaline protease.

3. The method according to claim 1, wherein: The method comprises the following steps: (1) taking coffee grounds, and adding ficin to perform enzymolysis; (2) Obtaining enzymatic hydrolyzate of small-fruit coffee grounds.

4. The method according to claim 2, wherein: The method comprises the following steps: (1) taking small fruit coffee grounds and adding alkaline protease for enzymatic hydrolysis; (2) adding ficin to the alkaline protease hydrolysis product for enzymolysis; (3) Obtaining the enzymatic hydrolyzate of small-fruit coffee grounds.

5. The method according to claim 3 or 4, wherein: The enzymatic hydrolyzate of small-fruit coffee grounds contains any one or more polypeptides selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 12-18.

6. The method according to any one of claims 4, wherein: The method comprises the following steps: (1) taking small fruit coffee grounds, adding purified water and mixing evenly, adjusting the pH to above 8, preferably 9.5-10.5, adding alkaline protease and performing enzymolysis at above 50° C.; (2) cooling to room temperature, adjusting the pH to 7-8, heating to above 60° C., preferably 65° C., and adding ficin for enzymatic hydrolysis; (3) Obtaining the enzymatic hydrolyzate of small-fruit coffee grounds.

7. A method for selecting a protease for enzymatic hydrolysis of small-fruit coffee grounds, wherein: The method comprises using bioinformatics technology to predict bioactive peptides in Arabica coffee grounds, and selecting a suitable protease according to the prediction results.

8. The method according to claim 7, wherein: The method comprises the following steps: (1) Using a software program to select different proteases to perform virtual enzymatic digestion on the target protein sequence and construct a virtual peptide library; (2) molecular docking the virtual peptide library with different targets; (3) Compare the binding energy of the peptides in the virtual peptide library with the binding energy of each target positive drug to obtain the predicted bioactive peptides; Preferably, the target protein is 11S storage protein; Preferably, the 11S storage protein amino acid sequence is shown in SEQ ID NO: 1; Preferably, the software program is a virtual enzymolysis module; Preferably, the protease is an industrial grade protease; Preferably, the protease is one or more selected from ficin, papain, pepsin, stem bromelain, Bacillus subtilis, and trypsin; Preferably, the targets are soothing and anti-aging targets; Preferably, the target is one or more selected from kallikrein-7 (KLK7), capsaicin receptor-1 (TRPV1) or matrix metalloproteinase-1 (MMP1); Preferably, each target-positive drug comprises LM-030 (Gln-lactone [Thr-Leu-Oaa-Tyr-Ile]), ilomastat or the analgesic SB-366791.

9. The method according to claim 8, wherein: The method comprises the following steps: (1) Selecting ficin, papain, pepsin, stem bromelain, Bacillus subtilis or trypsin through a virtual enzymatic hydrolysis module to perform virtual enzymatic hydrolysis on the 11S storage protein sequence to construct a virtual peptide library; (2) molecular docking the virtual peptide library with KLK7, TRPV1 or MMP1 targets; (3) The binding energy of the peptides in the virtual peptide library was compared with that of the positive drugs LM-030, ilomastat, or the analgesic SB-366791 to obtain the predicted bioactive peptides.

10. The method according to claim 8 or 9, wherein: Preprocessing the target protein structure when constructing the virtual peptide library; Preferably, the pretreatment comprises: (1) removing the original ligands and other non-bonded heteroatoms; and (2) deleting water molecules in the structure and adding polar hydrogen atoms.

11. The method according to any one of claims 7 to 10, wherein: The predicted bioactive peptides include any one or more polypeptides with amino acid sequences as shown in SEQ ID NOs: 2-11.

12. A polypeptide prepared according to the method according to any one of claims 1 to 6, preferably, the polypeptide comprises any one or more polypeptides selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 2 to 18.

13. The enzymatic hydrolyzate of small-fruit coffee grounds prepared according to the method according to any one of claims 1 to 6.

14. The enzymatic hydrolyzate of small-fruit coffee grounds according to claim 13, wherein The enzymatic hydrolyzate of small-fruit coffee grounds contains any one or more polypeptides with amino acid sequences as shown in SEQ ID NOs: 2-18.

15. Use of the polypeptide according to claim 12 or the enzymatic hydrolyzate of coffee grounds according to claim 13 or 14 in in vitro inhibition of soothing and anti-aging targets; Preferably, the soothing and anti-aging targets include one or more of kallikrein-7 (KLK7), capsaicin receptor-1 (TRPV1) or matrix metalloproteinase-1 (MMP1).

16. Use of the polypeptide according to claim 12 or the enzymatic hydrolyzate of coffee grounds according to claim 13 or 14 for personal care; Preferably, the application is skin care and / or hair care; Preferably, the application is to inhibit KLK7, improve isolated hair bundles and / or soothe the scalp; More preferably, the improving the separated hair bundle comprises enhancing the glossiness of the hair bundle or improving the frizziness of the hair bundle; More preferably, soothing the scalp comprises increasing the moisture content of the scalp stratum corneum or improving the redness of the scalp.

17. Use of the polypeptide according to claim 12 or the enzymatic hydrolyzate of small-fruit coffee grounds according to claim 13 or 14 in the preparation of personal care products; Preferably, the personal care product comprises a skin care and / or hair care product; Preferably, the skin care product comprises a cleansing product, a toner, a lotion, a cream, an essence, an eye cream, a mask, a skin care oil, a stock solution or a sunscreen; Preferably, the hair care product comprises shampoo, conditioner, hair care essential oil, hair care spray, elasticizer, hair care nutrient solution or hair mask.

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