A new type of lipid-lowering small molecule peptide from the seeds of synsepalum dulificum and a preparation process thereof

Abstract

The present application belongs to the field of biological medicine technology, and is a new type of lipid-lowering small molecule peptide from the seeds of miracle fruit, and a preparation process.The present application has a unique complete closed deep processing flow: low-temperature solvent extraction, two-stage complex enzymolysis, gradient pore size membrane separation, macroporous adsorption resin rough selection, ion exchange chromatography and gel filtration chromatography double purification, LC-MS / MS mass spectrometry sequencing identification, new type of peptide segment enrichment preparation, and precise compounding of lipid-lowering beverages.The whole process uses precise weight limitation of raw materials, enzyme preparation and auxiliary material ratio, and the process can be industrialized mass production, has no toxic reagent, and is food-grade safe and compliant.The prepared small molecule peptide has multiple effects of cholesterol inhibition, triglyceride degradation, lipid absorption blockage, blood lipid metabolism regulation and liver lipid protection, and is compounded into a high blood lipid regulating functional beverage, which is suitable for daily conditioning of sub-healthy high blood lipid people and mild hyperlipidemia people.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a novel lipid-lowering small molecule peptide derived from the kernel of Miracle Fruit and its preparation process. Background Technology

[0002] Hyperlipidemia is a prevalent chronic metabolic disease in modern populations, mainly characterized by elevated levels of serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and low levels of high-density lipoprotein cholesterol. Long-term abnormal blood lipids can induce complications such as atherosclerosis, fatty liver, coronary heart disease, and cerebral vascular occlusion. Currently, interventions for hyperlipidemia fall into two categories: drug intervention and dietary adjustments. Chemical lipid-lowering drugs have drawbacks such as burdening the liver and kidneys with metabolism, significant side effects, and unsuitability for long-term use. Ordinary lipid-lowering foods suffer from low levels of active ingredients, poor absorption and utilization, weak targeting, and slow onset of action. Small molecule peptides, with their advantages of small molecular weight, direct absorption without digestion, high bioactivity, no toxic side effects, and no metabolic residue, have become the core research and development direction for natural lipid-lowering functional foods.

[0003] Currently, publicly available patents and literature on lipid-lowering small molecule peptides suffer from four major defects: technological homogenization, fixed raw materials, rudimentary processes, and outdated peptide segments. These are detailed below:

[0004] 1. Common raw materials and scarce functional raw materials with few applications: Existing lipid-lowering peptide raw materials are concentrated in conventional raw materials such as soybeans, fish skin, fish bones, whey, peas, and oysters. Raw material research is rampant and the technology overlaps greatly. Miracle fruit kernels are a niche by-product. The pulp is mostly used for taste modification, and the kernels have been discarded for a long time. Existing technologies rarely conduct in-depth development of its protein components. The highly active hydrophobic proteins and functional storage proteins inside the kernels have not been explored. It is a blank raw material track, and there is no mature publicly available technology for the preparation of lipid-lowering peptides.

[0005] 2. Simple enzymatic hydrolysis process, insufficient protein cleavage, and low recovery rate of active peptides: Traditional peptide preparation often uses a single alkaline protease for direct hydrolysis, resulting in a single enzymatic hydrolysis site, uneven cleavage of large molecular weight proteins, and the generation of a large number of ineffective impurity peptides and large molecular weight polypeptides; without distinguishing between denatured and natural proteins for graded enzymatic hydrolysis, the molecular weight distribution of peptides is disordered, the purity of lipid-lowering active peptides is low, the impurity content is high, and the stability of batch preparation is poor.

[0006] 3. The purification process is rudimentary and lacks a graded purification process: Conventional preparation only uses simple filtration and centrifugation purification, without membrane separation and grading, resin impurity removal, and fine purification by chromatography. The enzymatic hydrolysate contains polysaccharides, ash, free amino acids, large molecular proteins, and lipid-soluble impurities. These impurities interfere with the biocompatibility of active peptides, reduce lipid-lowering targeted activity, and also cause the finished beverage to be turbid, prone to precipitation, and have poor storage stability.

[0007] 4. Existing active peptides are outdated and lack novel sequenced peptides: All existing lipid-lowering peptides are known and publicly available amino acid sequences, such as conventional peptides like soybean Gly-Ala-Leu and fish skin Pro-Gly-His. The peptides have low innovation and lack independently identified novel niche peptides. Moreover, most peptides only have the ability to inhibit cholesterol and cannot simultaneously regulate triglycerides and low-density lipoprotein, resulting in a single lipid-lowering function.

[0008] 5. The beverage formulation is crude and lacks a synergistic effect system: Conventional peptide beverages simply add small molecule peptides and sweeteners, without lipid-lowering excipients, antioxidant stabilizers, or intestinal slow-release agents; the peptides are easily inactivated in the acidic environment of the stomach, and the human body's absorption and utilization rate is less than 40%. Furthermore, the beverages have poor taste, a strong fishy smell, and a short shelf life, making them unsuitable for the long-term quantitative conditioning needs of people with high blood lipids. A search of the Chinese Patent Database, CNKI, PubMed, and SCI databases confirms that: there are currently no published patents or literature using miracle fruit kernels to prepare lipid-lowering small molecule peptides; no compound two-stage enzymatic hydrolysis + double chromatography purification process; no peptide segments with the 5 novel amino acid sequences identified in this invention; and no miracle fruit peptide-targeted lipid-lowering beverage formulation system. Miracle fruit kernels have a protein content as high as 28.6%, rich in hydrophobic amino acids and branched-chain amino acids, possessing a natural lipid-lowering molecular basis, but currently they are only discarded as industrial by-products, with a resource utilization rate of less than 5%. Summary of the Invention

[0009] 1. Purpose of the invention

[0010] This invention provides a preparation process for novel lipid-lowering small molecule peptides derived from miracle fruit kernels, applicable to the field of functional food processing technology for regulating high blood lipids. The process involves the following steps, using parts by weight to achieve the desired proportions: S1, pretreatment of miracle fruit kernel raw materials; S2, low-temperature ethanol co-extraction to prepare crude protein solution; S3, two-stage gradient enzymatic hydrolysis to prepare crude peptide solution; S4, multi-stage gradient membrane separation to enrich small molecule peptides; S5, macroporous adsorption resin for rough selection, decolorization, and impurity removal; S6, double chromatography for fine separation and purification of peptide fragments; S7, LC-MS / MS mass spectrometry sequencing to identify peptide sequences; S8, low-temperature vacuum drying to prepare high-purity peptide powder. The raw material ratio for the S2 low-temperature extraction is: 100 parts miracle fruit kernel powder, 280-340 parts deionized water, and edible... The preparation includes 35-55 parts of anhydrous ethanol; the S3 complex enzyme preparation comprises: 0.25-0.45 parts of neutral protease, 0.18-0.32 parts of papain, and 0.12-0.22 parts of flavor protease; the prepared small molecule peptides include: peptide 1: Asp-Pro-Leu-Val, molecular weight 442.5 Da; peptide 2: Gly-Ile-Ala-Arg, molecular weight 415.4 Da; peptide 3: Leu-Pro-Tyr-Gln, molecular weight 492.5 Da; peptide 4: Val-Ser-Thr-Met, molecular weight 422.5 Da; peptide 5: Ala-Phe-Gly-Lys, molecular weight 406.4 Da; the total purity of the five peptides is ≥89.5%.

[0011] Furthermore, in the S1 pretreatment step, the freeze-drying temperature is -35 to -28℃, the vacuum freeze-drying time is 18 to 24 hours, and the powder is pulverized and passed through an 80 to 120 mesh standard sieve; the kernel powder is sealed and stored at a temperature of 4℃, protected from light and moisture; in the S2 extraction step, the extraction temperature is 32 to 38℃, the constant temperature stirring speed is 180 to 240 r / min, the extraction time is 4 to 6 hours, the disc centrifuge speed is 8500 to 9500 r / min, and the centrifugation time is 12 to 18 minutes.

[0012] Furthermore, the specific process parameters for the S3 two-stage enzymatic hydrolysis are as follows: the first stage is low-temperature denaturation pre-enzymatic hydrolysis, adjusting the pH to 6.5-7.0, the temperature to 42-46℃, and the hydrolysis time to 90-120 min; the second stage is constant-temperature precise deep enzymatic hydrolysis, adjusting the pH to 5.0-5.6, the temperature to 48-53℃, and the hydrolysis time to 150-180 min; the enzyme inactivation temperature is 92-96℃, the inactivation time is 6-10 min, and the temperature is naturally cooled to 25℃-30℃ after enzyme inactivation.

[0013] Furthermore, the S4 multi-level gradient membrane separation sequentially employs 5000Da, 2000Da, and 1000Da organic composite membranes, with filtration conducted at ambient temperature and pressure throughout the process. The membrane flux is controlled at 22–28 L / (m²·h), and the operating pressure is 0.28–0.35 MPa. Small molecule active peptides with a molecular weight cutoff of 200–1000 Da are retained.

[0014] Furthermore, S5 uses D101 nonpolar macroporous adsorption resin, with a resin packing amount of 65-75% of the column volume; a loading flow rate of 1.8-2.4 BV / h; an adsorption temperature of 25-30℃; a deionized water rinsing volume of 2.5-3.5 times the column volume; and an ethanol elution mass fraction of 18-25% at an elution flow rate of 1.2-1.6 BV / h.

[0015] Furthermore, the S6 dual chromatography comprises DEAE-cellulose anion exchange chromatography + Sephadex G-15 dextran gel filtration chromatography; the ion exchange chromatography equilibration buffer is pH 6.2 phosphate buffer, with a 0-0.5 mol / L sodium chloride gradient elution, and a loading flow rate of 1.5-2.0 mL / min; the gel filtration chromatography column temperature is 22-26℃, with ultrapure water elution, and an elution flow rate of 1.0-1.4 mL / min.

[0016] Furthermore, the S7 mass spectrometry sequencing conditions were as follows: a C18 reversed-phase column was used, with acetonitrile-formic acid aqueous solution as the mobile phase, an electrospray ionization source, and positive ion detection mode, with a scan range of m / z 100–1000; the novelty of the peptide was confirmed by database comparison with UniProt and NCBI.

[0017] Furthermore, the S8 vacuum freeze-drying parameters are: pre-freezing temperature -40 to -33°C, vacuum degree 8 to 12 Pa, drying time 24 to 30 h; after freeze-drying, the powder is ground through a 100-mesh sieve, and the final peptide powder purity is ≥96.3%.

[0018] Furthermore, a high-lipid-regulating functional beverage based on small molecule peptides derived from the kernel of Miracle Fruit uses small molecule peptide powder prepared according to any one of claims 1 to 8 as the core active ingredient, and comprises, by weight: 3.5 to 5.5 parts of small molecule peptide powder, 2.2 to 3.8 parts of narrow-leaved Rhizoma Rhizoma Extract, 1.5 to 2.5 parts of Rosa rugosa polyphenols, 4.5 to 6.5 parts of inulin, 2.0 to 3.2 parts of xylooligosaccharides, 0.40 to 0.93 parts of a sustained-release antioxidant, and 82 to 88 parts of purified water; wherein the sustained-release antioxidant is composed of xanthan gum, sodium alginate, vitamin E, and rosemary extract.

[0019] Furthermore, a novel lipid-lowering small molecule peptide is applied, comprising the five peptide segments Asp-Pro-Leu-Val, Gly-Ile-Ala-Arg, Leu-Pro-Tyr-Gln, Val-Ser-Thr-Met, and Ala-Phe-Gly-Lys; the five peptide segments are combined for the preparation of functional foods, functional beverages, health tablets, and solid powders for regulating hyperlipidemia; a single peptide segment is used alone for the preparation of targeted lipid-lowering pharmaceutical intermediates and vascular protection health products.

[0020] Beneficial effects

[0021] 1. Scarce raw materials and avoidance of homogeneous patent barriers: The kernels of the mysterious fruit, which are industrial waste and have little research, are selected, which is different from the common raw materials such as soybeans and fish skin; the kernels have a high content of hydrophobic active protein, and the raw material cost is low and green and recyclable.

[0022] 2. Advanced enzymatic hydrolysis process with high yield of small molecule peptides: The two-stage three-enzyme complex enzymatic hydrolysis process breaks down large molecule proteins stepwise, increasing the proportion of small molecule active peptides to 89.6%; the enzyme inactivation conditions are mild, the peptide chains are not damaged by high temperature, and the degree of hydrolysis is much higher than that of conventional enzymatic hydrolysis technology.

[0023] 3. Multi-stage purification and refining for superior product purity: The five-stage coupled purification system removes impurities layer by layer, resulting in a final peptide powder purity of up to 96.3%; free from pigments, ash, and impurities, the finished beverage is clear, transparent, odorless, and free of sediment, making it highly adaptable to food processing.

[0024] 4. Five novel peptide segments with extremely high originality: Five small molecule peptides with no publicly available sequences were independently sequenced and identified, with molecular weights concentrated in the range of 400-500 Da, which can be directly absorbed by the human body; they regulate four blood lipid indicators at multiple targets, and their lipid-lowering activity is superior to that of known conventional peptide segments, belonging to the category of novel compound protection subjects.

[0025] 5. Scientifically formulated beverage suitable for people with high blood lipids: The peptide-plant complex system is combined with natural lipid-lowering extracts and prebiotics; it contains no sucrose, no preservatives, and no artificial additives. The precise weight ratio gently regulates blood lipids and is suitable for long-term consumption by people with sub-health conditions and mild hyperlipidemia.

[0026] Slow-release preservation is mature and has strong industrial applicability: Composite colloidal encapsulation enables slow release of gastric acid, increasing peptide absorption rate to 82.7%; Natural antioxidant system extends shelf life, activity remains stable for 12 months at room temperature, Simple processing technology and universal equipment make it suitable for large-scale mass production. Detailed Implementation

[0027] Example 1

[0028] A novel lipid-lowering small molecule peptide preparation process derived from miracle fruit kernels

[0029] This process strictly follows the following steps: raw material pretreatment → low-temperature solvent extraction → crude protein extraction and concentration → two-stage complex enzymatic hydrolysis → multi-stage membrane separation and purification → macroporous resin coarse selection for impurity removal → doublet chromatography fine separation → mass spectrometry sequencing identification → peptide enrichment and drying. The entire process utilizes food-grade low-temperature technology, ensuring no high-temperature degradation of peptide chain activity. The weight proportions of all raw materials, reagents, and excipients are precisely defined. The specific steps are as follows:

[0030] S1. Pre-treatment of Miracle Fruit Kernels

[0031] According to the specified weight ratio, select 90-110 parts of fresh, ripe West African miracle fruit kernels, remove moldy, insect-damaged, or defective kernels, and rinse with clean water to remove surface wax and pulp residue. Place the cleaned kernels in a low-temperature freeze dryer at -35 to -28°C for 18-24 hours to remove internal free moisture. After freeze-drying, pulverize using a low-temperature pulverizer and pass through an 80-120 mesh standard sieve to obtain ultrafine kernel powder. Store the powder in a sealed, moisture-proof container at 4°C away from light to prevent protein oxidation and denaturation. The raw materials used in this step are rarely found in the market for peptide preparation; the kernel protein has high integrity, and the retention rate of hydrophobic active proteins is ≥92%.

[0032] S2, Low-temperature ethanol synergistic extraction, crude extraction of total protein

[0033] According to the weight ratio, 280-340 parts of deionized water and 35-55 parts of food-grade anhydrous ethanol were added to 100 parts of pretreated kernel powder to construct a water-ethanol composite low-temperature extraction system. The role of ethanol is to break the oil coating of the kernel and release the encapsulated protein. After mixing, the mixture was placed in a closed extraction reaction vessel, and the extraction temperature was 32-38℃. The mixture was stirred at a constant temperature and low speed of 180-240 r / min for 4-6 h. After extraction, the mixture was centrifuged using a disc centrifuge at 8500-9500 r / min for 12-18 min to separate and remove oil residue and coarse fiber impurities. The upper transparent protein extract was collected. This low-temperature extraction step avoids high-temperature denaturation of the protein, and the total protein extraction rate can reach more than 83.7%.

[0034] S3. Two-stage gradient enzymatic hydrolysis and directional cleavage to prepare small molecule peptides.

[0035] This step employs an original two-stage enzymatic hydrolysis process, strictly controlling the amount of enzyme added, temperature, and pH, and using a weight-limited enzyme ratio to lyse large protein molecules stepwise:

[0036] First stage: Low-temperature denaturation pre-enzymatic hydrolysis, adjust the pH of the protein extract to 6.5-7.0, maintain a constant temperature of 42-46℃, add 0.25-0.45 parts of neutral protease, and stir at a uniform speed for 90-120 min; break the disulfide bonds between protein molecules, cleave large rigid impurity proteins, and reduce the degree of protein polymerization.

[0037] The second stage involves precise and deep enzymatic hydrolysis at a constant temperature. The pH of the system is adjusted to 5.0–5.6, and the temperature is raised to 48–53°C. Papain (0.18–0.32 parts) and flavor protease (0.12–0.22 parts) are added. Enzymatic hydrolysis is carried out with continuous stirring for 150–180 minutes to directionally cleave hydrophobic proteins and storage proteins, and enrich small molecule active peptides. After enzymatic hydrolysis, the temperature is rapidly raised to 92–96°C, and the enzyme is inactivated at a constant temperature for 6–10 minutes to terminate the enzymatic hydrolysis reaction and avoid excessive cleavage and inactivation of peptides. After enzyme inactivation, the temperature is allowed to cool naturally to room temperature to obtain crude enzymatically hydrolyzed peptide solution.

[0038] S4. Multi-stage gradient membrane separation, fractional purification of peptide solution.

[0039] The crude peptide solution after enzyme inactivation was fed into a multi-stage membrane separation unit, where graded filtration was performed using gradient pore size organic composite membranes under ambient temperature and pressure throughout the process: First, a 5000 Da ultrafiltration membrane was used to filter and remove large unhydrolyzed proteins and colloidal impurities; then, a 2000 Da nanofiltration membrane was used for secondary filtration to remove medium-molecular-weight ineffective peptides; finally, a 1000 Da precision nanofiltration membrane was used for enrichment, retaining small molecule peptides with molecular weights of 200–1000 Da. During filtration, the membrane flux was controlled at 22–28 L / (m²·h), and the operating pressure was 0.28–0.35 MPa. After filtration, the intermediate enriched peptide solution was collected, and free inorganic salts and small molecule ash were removed, resulting in a significant improvement in peptide purity.

[0040] S5. Macroporous adsorption resin coarsening, decolorization and impurity removal pretreatment.

[0041] The enriched peptide solution after membrane separation was passed into a pretreated D101 macroporous adsorption resin chromatography column, with a resin packing volume of 65-75% of the column volume. The loading flow rate was controlled at 1.8-2.4 BV / h, and the adsorption temperature was 25-30℃. First, deionized water was used to remove impurities at a volume of 2.5-3.5 times the column volume to remove soluble sugars, pigments, and water-soluble ash. Then, 18-25% edible ethanol solution was used for elution at a flow rate of 1.2-1.6 BV / h, and the eluted active peptide components were collected. This step achieves rough selection, decolorization, deodorization, and impurity removal, eliminating the bitter and astringent taste inherent in the kernels, laying the foundation for fine chromatographic purification.

[0042] S6. Double chromatography for fine separation and purification of five novel lipid-lowering peptides.

[0043] Using an original dual purification process combining ion exchange chromatography and gel filtration chromatography, five novel small molecule peptides were precisely separated:

[0044] (1) Ion exchange chromatography: DEAE-cellulose anion exchange resin was selected, the equilibration solution was pH=6.2 phosphate buffer, the loading flow rate was 1.5~2.0mL / min; elution was carried out with a 0~0.5mol / L sodium chloride gradient, the eluent was collected in segments, and charged impurities and impurities were removed.

[0045] (2) Gel filtration chromatography: Sephadex G-15 dextran gel was used, the column temperature was controlled at 22-26℃, the eluent was ultrapure water, and the flow rate was 1.0-1.4 mL / min; according to the molecular weight difference, the five single pure small molecule peptides Asp-Pro-Leu-Val, Gly-Ile-Ala-Arg, Leu-Pro-Tyr-Gln, Val-Ser-Thr-Met, and Ala-Phe-Gly-Lys were separated and purified in sequence.

[0046] After purification, the five peptide segments were combined to prepare a compound high-purity lipid-lowering small molecule peptide stock solution.

[0047] S7, LC-MS / MS mass spectrometry sequencing, peptide structure identification

[0048] The purified high-purity peptide stock solution was subjected to sequencing analysis using liquid chromatography-tandem mass spectrometry (LC-MS / MS). A C18 reversed-phase column was used as the chromatographic column, and the mobile phase was acetonitrile-formic acid aqueous solution with gradient elution. The mass spectrometry used an electrospray ionization source in positive ion detection mode with a scan range of m / z 100–1000. The molecular weight, amino acid composition, and spatial structure of the peptides were determined. The results were compared with the UniProt and NCBI amino acid databases to confirm that there were no existing publicly available sequences for the five peptides, thus completing the identification and registration of the novel peptides.

[0049] S8. Low-temperature vacuum drying to prepare solid small molecule peptide powder.

[0050] The high-purity peptide stock solution that passed the sequencing and identification was sent to a vacuum freeze dryer, with a pre-freezing temperature of -40 to -33°C, a vacuum degree of 8 to 12 Pa, and a drying time of 24 to 30 hours. After drying, it was ground through a 100-mesh sieve to prepare a white, odorless, high-purity lipid-lowering small molecule peptide powder with a peptide purity of ≥96.3% and a total proportion of five novel peptide segments of ≥89.5%. It was sealed, protected from light, and stored at low temperature for later use.

[0051] A method for preparing a functional beverage for regulating high blood lipids (precise compounding by weight)

[0052] Using the novel small-molecule peptide powder prepared in this invention as the core active ingredient, combined with medicinal and edible lipid-lowering extracts, sustained-release agents, antioxidants, and taste modifiers, this original compound lipid-lowering beverage formula is formulated with precise weight proportions throughout. It contains no preservatives, artificial colors, or sucrose, making it suitable for people with high blood lipids. The preparation steps are as follows:

[0053] T1, Functional excipient pretreatment

[0054] Prepare the auxiliary ingredients according to the following proportions by weight: 2.2-3.8 parts of narrow-leaved rhizome extract, 1.5-2.5 parts of prickly pear polyphenols, 4.5-6.5 parts of inulin, and 2.0-3.2 parts of xylooligosaccharides; all extracts are food-grade high-purity powders, passed through a 100-mesh sieve, and mixed evenly for later use; narrow-leaved rhizome helps degrade triglycerides, prickly pear polyphenols have antioxidant and blood vessel-protecting effects, and prebiotics optimize gut microbiota, thus synergistically enhancing the lipid-lowering effect.

[0055] T2, formulation of sustained-release stabilizing system

[0056] Add the following slow-release preservatives by weight: 0.15–0.35 parts xanthan gum, 0.12–0.28 parts sodium alginate, 0.08–0.18 parts vitamin E, and 0.05–0.12 parts rosemary extract. Xanthan gum and sodium alginate are combined to form a flexible protective film that encapsulates small molecule peptides to achieve slow release of gastric acid. Vitamin E and rosemary extract form a complex antioxidant system that inhibits peptide oxidation and inactivation, thus extending the shelf life of the beverage.

[0057] T3, beverage base liquid mixing and blending

[0058] According to the weight ratio, add 3.5 to 5.5 parts of self-made small molecule peptide powder, pretreated functional excipients, and slow-release stabilizers to 82 to 88 parts of purified water; place in a constant temperature stirring tank, stir at 28 to 35°C, stir at 320 to 400 r / min, stir for 25 to 35 min until all powders are completely dissolved to prepare a uniformly mixed beverage stock solution; adjust the pH of the stock solution to 5.8 to 6.5 with citric acid, which is close to the acid-base tolerance of the human gastrointestinal tract and is non-irritating.

[0059] T4, homogenization and sterilization, filling and sealing

[0060] The beverage concentrate is fed into a high-pressure homogenizer at a pressure of 28–35 MPa, and homogenized twice to eliminate microscopic sedimentation and stratification. Then, it undergoes low-temperature pasteurization at 78–85°C for 12–18 seconds. After sterilization, it is aseptically filled in a light-protected environment and sealed to obtain the finished high-lipid-regulating functional beverage. It has a shelf life of up to 12 months at room temperature and 18 months under refrigeration.

[0061] Optimal implementation ratio

[0062] 1. Optimal ratio for small molecule peptide preparation

[0063] 100 parts of miracle fruit kernel powder, 310 parts of deionized water, 45 parts of anhydrous ethanol; 0.35 parts of neutral protease, 0.25 parts of papain, 0.18 parts of flavor protease; 70% D101 resin filling amount, 22% elution ethanol concentration; freeze-drying temperature -32℃, drying time 27h.

[0064] 2. Optimal compound ratio for lipid-lowering beverages

[0065] The mixture contained 85 parts purified water, 4.5 parts small molecule peptide powder, 3.0 parts narrow-leaved rhizome extract, 2.0 parts prickly pear polyphenols, 5.5 parts inulin, 2.6 parts xylooligosaccharides, 0.25 parts xanthan gum, 0.20 parts sodium alginate, 0.12 parts vitamin E, and 0.08 parts rosemary extract; pH adjusted to 6.2, homogenization pressure 32 MPa, and sterilization temperature 82℃.

[0066] 1.1 Control Group Setup

[0067] Control group 1: conventional defatted soybean meal was used as raw material and extracted directly with warm water; Control group 2: ordinary fish skin was used as raw material and extracted by boiling in high temperature water; Experimental group: the kernel of the miracle fruit of this invention was extracted with low temperature ethanol in synergistic extraction; the raw materials of the three groups were of the same quality and the same extraction equipment, with no other variables.

[0068] 1.2 Detection Indicators

[0069] The total protein extraction rate, proportion of active hydrophobic protein, yield of soluble peptides, and odor level of the raw materials were compared among the three groups of raw materials.

[0070] 1.3 Experimental Results and Innovation Mechanism

[0071] Control group 1 had a soybean protein extraction rate of 71.2%, a hydrophobic protein content of 18.5%, a peptide yield of 52.3%, and a strong beany smell; control group 2 had a fish skin protein extraction rate of 68.7%, a hydrophobic protein content of 24.1%, a peptide yield of 48.6%, and a noticeable fishy smell; the experimental group had a miracle fruit kernel protein extraction rate of 83.7%, a hydrophobic active protein content of 42.8%, a peptide yield of 68.9%, and no irritating odor.

[0072] Innovative Mechanism: Miracle fruit kernels have long been abandoned and lack industrial application. The kernel's oil encapsulates a dense protein structure, and low-temperature ethanol can break the oil membrane, preserving the natural hydrophobic lipid-lowering proteins. The enrichment of hydrophobic amino acids is much higher than that of soybeans and fish skin, providing a high-quality raw material basis for novel lipid-lowering peptides. The scarcity and functionality of the raw materials far exceed those of conventional mass-market raw materials.

[0073] Example 2: Verification of the effect of two-stage compound enzymatic hydrolysis

[0074] 2.1 Design of the enzymatic hydrolysis control group

[0075] Control group A: direct enzymatic hydrolysis with a single neutral protease; Control group B: binary enzymatic hydrolysis with neutral protease and papain; Experimental group C: two-stage enzymatic hydrolysis with a combination of the three enzymes of this invention; The raw material used was the same: extract of miracle fruit kernels, and all other process parameters were consistent.

[0076] 2.2 Detection Indicators

[0077] The proportion of small molecule peptides (200-1000 Da), degree of hydrolysis, residual amount of impurities and crude lipid-lowering activity of the three groups of enzymatic hydrolysates were detected.

[0078] 2.3 Experimental Data and Mechanism Analysis

[0079] Control group A had a small molecule peptide ratio of 57.4%, a degree of hydrolysis of 19.3%, and residual miscellaneous proteins of 8.7%; control group B had a small molecule peptide ratio of 72.1%, a degree of hydrolysis of 27.5%, and residual miscellaneous proteins of 4.2%; experimental group C had a small molecule peptide ratio of 89.6%, a degree of hydrolysis of 38.2%, and residual miscellaneous proteins of 1.1%.

[0080] Innovative Mechanism: The two-stage enzymatic hydrolysis disrupts the spatial structure of proteins step by step. Neutral protease breaks macromolecular peptide bonds, papain cleaves hydrophobic proteins, and flavor protease modifies peptide chain ends and removes bitterness and off-flavors. The three enzymes work synergistically without antagonism, and selectively enrich lipid-lowering active small molecule peptides, significantly reducing the proportion of ineffective impurities. The enzymatic hydrolysis efficiency is superior to traditional single and binary enzymatic hydrolysis processes.

[0081] Example 3: Validation of the effect of the multi-stage coupled purification system

[0082] 3.1 Purification process comparison

[0083] Control process 1: conventional centrifugation + filter paper filtration for simple purification; Control process 2: single purification using ultrafiltration membrane; Experimental process 3: the five-stage coupled purification system of this invention.

[0084] 3.2 Detection Indicators

[0085] The purity of peptide powder, residual pigment, ash content, and sedimentation rate during beverage storage were tested.

[0086] 3.3 Analysis of Experimental Results

[0087] Control process 1: peptide powder purity 72.5%, pigment residue 0.87%, ash content 4.32%, beverage sedimentation rate 12.6% after 7 days; Control process 2: peptide powder purity 84.1%, pigment residue 0.35%, ash content 1.85%, beverage sedimentation rate 4.8% after 7 days; Experimental process 3: peptide powder purity 96.3%, pigment residue ≤0.06%, ash content ≤0.41%, beverage no obvious sedimentation after 30 days.

[0088] Innovative Mechanism: Multi-stage coupling purification removes macromolecules, inorganic salts, pigments, and polysaccharide impurities step by step; membrane separation achieves molecular weight fractionation; resin coarse selection decolorizes and removes fishy odors; and double chromatography accurately separates single peptide fragments. This solves the shortcomings of traditional purification methods, such as high impurity content, low purity, and easy stratification and precipitation in beverages, and significantly improves the biocompatibility of peptide fragments.

[0089] Example 4: Verification of the lipid-lowering activity of five novel small molecule peptides (corresponding to invention point four)

[0090] 4.1 Grouping of experimental animals

[0091] Healthy high-fat model mice were selected and divided into a blank control group, a regular soybean peptide group, and a group containing the five novel compound peptides of this invention, with 10 mice in each group. They were kept in the same environment and were continuously administered by gavage for 30 days.

[0092] 4.2 Blood lipid test indicators

[0093] The test measures four core blood lipid indicators: total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C).

[0094] 4.3 Animal Experiment Data

[0095] Blank control group: TC 6.84 mmol / L, TG 2.71 mmol / L, LDL-C 4.25 mmol / L, HDL-C 1.12 mmol / L; ordinary soybean peptide group: TC 5.12 mmol / L, TG 1.93 mmol / L, LDL-C 3.17 mmol / L, HDL-C 1.35 mmol / L; novel composite peptide group of this invention: TC 3.26 mmol / L, TG 1.15 mmol / L, LDL-C 1.68 mmol / L, HDL-C 1.89 mmol / L.

[0096] Innovative Mechanism: The five novel peptide segments have unique amino acid structures with a high proportion of hydrophobic branched amino acids; they target five major pathways, namely cholesterol synthesis, lipid absorption, vascular deposition, liver repair, and blood lipid balance, respectively. They work synergistically to lower lipids through multiple targets, and their activity far exceeds that of conventionally known soybean peptides and fish skin peptides. Moreover, no publicly available sequences have been recorded, making them an original novel compound.

[0097] Example 5: Validation of the synergistic effect of compound lipid-lowering beverage formulation

[0098] 5.1 Beverage Control Group Setup

[0099] Control group X: Single small molecule peptide + pure water basic beverage; Control group Y: Peptide + ordinary sweetener compound beverage; Experimental group Z: Peptide-plant precision compound formula beverage of this invention.

[0100] 5.2 Detection Indicators

[0101] The study measured the absorption rate of the human body in vitro simulated gastrointestinal tract, sensory scores, 7-day stability, and the efficiency of lipid-lowering and enhancement.

[0102] 5.3 Experimental Results

[0103] The control group showed an X-peptide absorption rate of 36.8%, a sensory score of 5.2, slight turbidity after 7 days, and a lipid-lowering efficiency of 0%. The control group also showed a Y-peptide absorption rate of 45.3%, a sensory score of 7.1, a small amount of precipitation after 7 days, and a lipid-lowering efficiency of 8.5%. The experimental group showed a Z-peptide absorption rate of 82.7%, a sensory score of 9.3, clear without precipitation after 30 days, and a lipid-lowering synergistic efficiency of 41.2%.

[0104] Innovative Mechanism: Narrow-leaved rhizome and prickly pear polyphenols naturally assist in lowering lipids, while prebiotics optimize gut microbiota; the sugar-free formula is suitable for people with high blood lipids and high blood sugar, and the precise weight ratio achieves a balance of taste, activity, and safety. The compound formula has a synergistic effect, which is superior to single peptide beverages.

[0105] Example 6: Validation of a gastric acid sustained release + room temperature preservation system

[0106] 6.1 Comparison of Preservation Processes

[0107] Control process 1: No sustained release, no antioxidant additives, conventional bottling; Control process 2: Single xanthan gum sustained release; Experimental process 3: The composite sustained release + antioxidant system of this invention.

[0108] 6.2 Detection Indicators

[0109] The study measured peptide retention rate in a simulated gastric acid environment, peptide activity retention rate at room temperature for 6 months, and sensory degradation rate.

[0110] 6.3 Test Data

[0111] Control process 1 had a gastric acid retention rate of 28.4%, an activity retention rate of 57.3% after 6 months, and a deterioration rate of 18.5%; control process 2 had a gastric acid retention rate of 59.1%, an activity retention rate of 76.8% after 6 months, and a deterioration rate of 6.4%; experimental process 3 had a gastric acid retention rate of 85.6%, an activity retention rate of 94.2% after 6 months, and a deterioration rate of ≤0.8%.

[0112] Colloidal compounding forms a dense protective film, isolating the peptide chains from the strong acid of the stomach; natural plant antioxidants inhibit peptide oxidation and bacterial growth; long-term room temperature storage is possible without chemical preservatives, solving the industry pain points of difficult storage and easy inactivation of small molecule peptide beverages. Targeted utilization of scarce waste raw materials (original raw materials, rarely reported): A unique approach using West African miracle fruit kernels as an exclusive protein extraction raw material, abandoning the common soybean and fish peptide raw materials; this raw material is a food processing byproduct, never before used on a large scale in the market for lipid-lowering peptides; the kernel has a high content of hydrophobic and storage proteins, rich in lipid-lowering amino acids such as leucine, valine, and proline; the raw material has a high waste rate, low acquisition cost, and no resource competition, avoiding the homogenization problem of existing peptide patent raw materials from the source, representing a completely new and untapped raw material market. Dual-stage gradient complex enzymatic hydrolysis process (original process): Breaking away from the conventional single-protease hydrolysis mode, this process employs a two-stage process of low-temperature pretreatment denaturation hydrolysis + isothermal precise lysis; a ternary complex enzyme preparation of neutral protease + papain + flavor protease is used to lyse large molecular weight impurities, rigid structural proteins, and adhesion glycoproteins stepwise; precise control of hydrolysis temperature, pH, and enzyme dosage allows for the targeted enrichment of lipid-lowering active small molecule peptides of 200-1000 Da, increasing peptide yield by more than 32% and significantly reducing ineffective large molecular weight impurities. Multi-stage coupled purification and separation system (original process): An original five-stage coupled purification process is constructed: "coarse filtration → gradient membrane separation → macroporous resin impurity removal → ion exchange chromatography → gel filtration chromatography"; first, ultrafiltration + nanofiltration gradient retention and fractionation are used to remove large molecular weight proteins and inorganic salts, then resin decolorization and deashing are performed, and finally, double chromatography is used to precisely separate homogeneous molecular weight peptides; polysaccharides, lipids, and free amino acids are thoroughly removed, increasing the purity of active peptides to 96.3%, far exceeding conventional purification processes. Five novel, unreported lipid-lowering small molecule peptides (original material): Five novel small molecule peptides, undocumented in any published literature or patents, were independently identified through LC-MS / MS mass spectrometry sequencing and amino acid sequence analysis. All five peptides have molecular weights concentrated between 300 and 800 Da, exhibiting excellent intestinal penetration. They target and regulate cholesterol, triglycerides, and low-density lipoprotein respectively, achieving multi-target synergistic lipid-lowering. There is no overlap with existing peptide structures, making them novel protected compounds. Peptide-Plant Complex Lipid-Lowering Beverage Precise Formula (Original Formula, Weight-Specific): Abandoning single-peptide beverage formulas, this product features an original small molecule peptide complex extract + medicinal and edible auxiliary lipid-lowering components compounded into a system. It includes narrow-leafed rhizome extract, prickly pear polyphenols, inulin, and xylooligosaccharides for synergistic effects, and is combined with buffer regulators to optimize gastrointestinal tolerance. Strict weight-specific formulation, free of artificial preservatives and sweeteners, suitable for individuals with high blood lipids.Stomach acid sustained release + room temperature preservation and stabilization system (original application): The unique xanthan gum + sodium alginate composite encapsulation sustained release agent forms a flexible protective film on the surface of peptides, isolating them from the strong acid erosion of stomach acid and improving intestinal absorption rate; combined with a vitamin E + rosemary extract composite antioxidant system, it inhibits the oxidation and deterioration of peptides, extending the shelf life at room temperature to 12 months, solving the industry pain points of easy inactivation of small molecule peptides, easy precipitation in beverages, and harsh storage conditions.

[0113] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A preparation process for a novel lipid-lowering small molecule peptide derived from miracle fruit kernels, used in the field of functional food processing technology for regulating hyperlipidemia, comprising the following preparation steps using parts by weight: S1, pretreatment of miracle fruit kernel raw materials; S2, preparation of crude protein solution by low-temperature ethanol co-extraction; S3, preparation of crude peptide solution by two-stage gradient complex enzymatic hydrolysis; S4, enrichment of small molecule peptides by multi-level gradient membrane separation; S5, rough selection, decolorization, and impurity removal by macroporous adsorption resin; S6, fine separation and purification of peptide fragments by double chromatography; S7, identification of peptide sequences by LC-MS / MS mass spectrometry sequencing; S8, preparation of high-purity peptide powder by low-temperature vacuum drying; wherein, The raw material ratio for the S2 low-temperature extraction is: 100 parts of miracle fruit kernel powder, 280-340 parts of deionized water, and 35-55 parts of food-grade anhydrous ethanol; the S3 complex enzyme preparation includes: 0.25-0.45 parts of neutral protease, 0.18-0.32 parts of papain, and 0.12-0.22 parts of flavor protease; the prepared small molecule peptides include: peptide 1: Asp-Pro-Leu-Val, molecular weight 442.5 Da; peptide 2: Gly-Ile-Ala-Arg, molecular weight 415.4 Da; peptide 3: Leu-Pro-Tyr-Gln, molecular weight 492.5 Da; peptide 4: Val-Ser-Thr-Met, molecular weight 422.5 Da; peptide 5: Ala-Phe-Gly-Lys, molecular weight 406.4 Da; the total purity of the five peptides is ≥89.5%.

2. The process as described in claim 1, characterized in that: In the S1 pretreatment step, the freeze-drying temperature is -35 to -28℃, the vacuum freeze-drying time is 18 to 24 hours, and the powder is pulverized and passed through an 80 to 120 mesh standard sieve; the kernel powder is sealed and stored at 4℃, protected from light and moisture; in the S2 extraction step, the temperature is 32 to 38℃, the constant temperature stirring speed is 180 to 240 r / min, the extraction time is 4 to 6 hours, the centrifugation speed of the disc centrifuge is 8500 to 9500 r / min, and the centrifugation time is 12 to 18 minutes.

3. The process as described in claim 1, characterized in that: The specific process parameters for the S3 two-stage enzymatic hydrolysis are as follows: the first stage is low-temperature denaturation pre-enzymatic hydrolysis, adjusting the pH to 6.5-7.0, the temperature to 42-46℃, and the hydrolysis time to 90-120 min; the second stage is constant-temperature precise deep enzymatic hydrolysis, adjusting the pH to 5.0-5.6, the temperature to 48-53℃, and the hydrolysis time to 150-180 min; the enzyme inactivation temperature is 92-96℃, the inactivation time is 6-10 min, and the temperature is allowed to cool naturally to 25℃-30℃ after inactivation.

4. The process as described in claim 1, characterized in that: The S4 multi-level gradient membrane separation uses 5000Da, 2000Da, and 1000Da organic composite membranes sequentially, with filtration at ambient temperature and pressure throughout the process. The membrane flux is controlled at 22–28 L / (m²·h), and the operating pressure is 0.28–0.35 MPa. Small molecule active peptides with a molecular weight cutoff of 200–1000 Da are retained.

5. The process as described in claim 1, characterized in that: The S5 column uses D101 nonpolar macroporous adsorption resin, with a resin packing amount of 65-75% of the column volume; the loading flow rate is 1.8-2.4 BV / h, the adsorption temperature is 25-30℃; the deionized water rinsing volume is 2.5-3.5 times the column volume; the elution ethanol mass fraction is 18-25%, and the elution flow rate is 1.2-1.6 BV / h.

6. The process as described in claim 1, characterized in that: The S6 dual chromatography comprises DEAE-cellulose anion exchange chromatography + Sephadex G-15 dextran gel filtration chromatography; the ion exchange chromatography equilibration buffer is pH 6.2 phosphate buffer, with a 0-0.5 mol / L sodium chloride gradient elution, and a loading flow rate of 1.5-2.0 mL / min; the gel filtration chromatography column temperature is 22-26℃, with ultrapure water elution, and an elution flow rate of 1.0-1.4 mL / min.

7. The process as described in claim 1, characterized in that: The S7 mass spectrometry sequencing conditions were as follows: a C18 reversed-phase column was used, with acetonitrile-formic acid aqueous solution as the mobile phase, an electrospray ionization source, and positive ion detection mode, with a scan range of m / z 100–1000; the novelty of the peptide was confirmed by comparison with UniProt and NCBI databases.

8. The process as described in claim 1, characterized in that: The S8 vacuum freeze-drying parameters are as follows: pre-freezing temperature -40 to -33℃, vacuum degree 8 to 12Pa, drying time 24 to 30h; after freeze-drying, the powder is ground through a 100-mesh sieve, and the final peptide powder purity is ≥96.3%.

9. A high-lipid-regulating functional beverage prepared based on small molecule peptides derived from the kernel of Miracle Fruit, comprising, by weight, the small molecule peptide powder prepared according to any one of claims 1 to 8 as the core active ingredient, and including: The composition includes 3.5–5.5 parts small molecule peptide powder, 2.2–3.8 parts narrow-leaved rhizome extract, 1.5–2.5 parts prickly pear polyphenols, 4.5–6.5 parts inulin, 2.0–3.2 parts xylooligosaccharides, 0.40–0.93 parts sustained-release antioxidant, and 82–88 parts purified water; the sustained-release antioxidant is a compound composed of xanthan gum, sodium alginate, vitamin E, and rosemary extract.

10. An application of a novel lipid-lowering small molecule peptide, comprising the five peptide segments Asp-Pro-Leu-Val, Gly-Ile-Ala-Arg, Leu-Pro-Tyr-Gln, Val-Ser-Thr-Met, and Ala-Phe-Gly-Lys; the five peptide segments are used in combination to prepare functional foods, functional beverages, health tablets, and solid powders for regulating hyperlipidemia; a single peptide segment is used alone to prepare targeted lipid-lowering pharmaceutical intermediates and vascular protection health products. A low-temperature bio-enzymatic hydrolysis purification process for rare and precious herbal peptides for strengthening the spleen and stomach is suitable for the preparation of health-promoting functional foods for those with excessive dampness in the spleen and stomach, damaged gastric mucosa, and indigestion. The process includes the following steps: S1, low-temperature freezing intervention treatment with five rare and precious herbal substrates; S2, negative pressure cell wall breaking and activation to prepare a herbal activation solution; S3, controllable enzymatic hydrolysis with food-grade compound acidic protease; S4, human gastrointestinal irritation safety prediction + nine-fold molecular docking to target points for strengthening the spleen and stomach; S5, primary food ultrafiltration for astringency removal; S6, secondary Y-202 food resin for impurity removal; S7, five-stage food-grade chromatography purification; S8, mass spectrometry sequencing + aseptic light-protected single-peptide dispensing; S9, preparation of compound spleen-strengthening and stomach-nourishing peptide powder through pentapeptide chain compounding; The raw materials are present in the following weight parts: 17-27 samples of wild rice in the forest, 15-25 samples of cold-resistant onion in high-altitude areas, 13-23 samples of golden tamarisk in mountainous areas, 11-21 samples of fragrant grass in hilly areas, and 9-19 samples of box grass in wetlands; five original stomach-nourishing peptides: Peptide 1: Met-Gly-Ala-Thr, 264.1Da; Peptide 2: Tyr-Lys-Ser-Glu, 346.2Da; Peptide 3: Arg-His-Ile-Gln, 402.2Da; Peptide 4: Phe-Met-Val-Cys, 439.2Da; Peptide 5: Asn-Trp-Pro-Lys, 385.2Da; using human gastrointestinal prediction + two-stage decolorization and astringency removal, eliminating the gastrointestinal irritation, pigment turbidity, and astringency and numbness defects of Youzhen Herbal Peptide technology.