Hippophae rhamnoides small molecule peptide and preparation method thereof

Abstract

The invention discloses a sea-buckthorn small molecule peptide and a preparation method thereof, and relates to the field of biological medicine and food processing. According to the method, sea-buckthorn pulp residues and sea-buckthorn seed residues are used as composite raw materials and are mixed and stirred to form homogenate, and then cell wall structures are destroyed through ultrasonic wall breaking treatment to release protein components; then cutting off a peptide bond under the action of compound enzymes (plant protease, neutral protease and pepsase) by adopting an ultrasonic-enzyme synergistic hydrolysis process to generate a small molecule peptide fragment; after removing impurities through centrifugal separation, purifying through an ultrafiltration membrane (the molecular weight cut-off is 1-3kDa) to obtain a small molecule peptide solution; and performing debitterizing treatment by using macroporous adsorption resin, and preparing sea-buckthorn small molecule peptide powder by using a vacuum freeze-drying technology. According to the method, through ultrasonic-enzyme synergistic interaction, multi-stage purification combination and low-temperature keep-alive processes, the yield, molecular weight uniformity and antioxidant activity of small molecular peptides are remarkably improved, and high-valued utilization of sea-buckthorn processing by-products is achieved.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and food processing, and in particular to a small molecule peptide from sea buckthorn and its preparation method. Background Technology

[0002] Sea buckthorn, a plant used for both food and medicine, is rich in protein, amino acids, and bioactive components in its pulp and seeds, making it a high-quality raw material for preparing small molecule peptides. Currently, the preparation methods for sea buckthorn small molecule peptides mainly include physical crushing, enzymatic extraction, and separation and purification. In the physical crushing stage, traditional processes often employ mechanical grinding or single ultrasonic treatment, resulting in incomplete cell wall disruption and low dissolution rates of target components. During enzymatic hydrolysis, a single enzyme preparation is often used under static conditions, leading to low hydrolysis efficiency and uneven peptide molecular weight distribution. The separation and purification stages often rely on centrifugation or single membrane filtration, making it difficult to simultaneously remove large molecular impurities and bitter substances, affecting product purity and sensory quality. Furthermore, existing processes often use high-temperature treatments for enzyme inactivation and drying after enzymatic hydrolysis, which can easily lead to peptide chain breakage or oxidation, reducing bioactivity. Simultaneously, pulp and seed residues generated during sea buckthorn processing are often directly discarded, resulting in low raw material utilization, resource waste, and increased production costs.

[0003] In the aforementioned existing technologies, the synergy between physical disruption and enzymatic hydrolysis is insufficient, making it difficult to simultaneously achieve the desired yield and purity of small molecule peptides. Separating purification and debittering processes increases operational complexity and cost. High-temperature treatment leads to the loss of active ingredients. Low overall utilization of raw materials limits the industrial application of sea buckthorn peptides. Therefore, developing a method for preparing sea buckthorn small molecule peptides that achieves efficient cell wall disruption, synergistic enzymatic hydrolysis, integrated purification and debittering, low-temperature preservation, and improved raw material utilization has become a pressing technical problem to be solved in this field. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a small molecule peptide from sea buckthorn and its preparation method, so as to solve one or more problems in the prior art.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] The preparation method of sea buckthorn small molecule peptides includes the following steps:

[0007] (1) Raw material pretreatment: Mix 20-40 parts by weight of sea buckthorn pulp residue and 30-60 parts by weight of sea buckthorn seed residue, add 3-5 times the weight of deionized water, stir for 1-2 hours to obtain sea buckthorn mixture.

[0008] (2) Ultrasonic cell disruption: The sea buckthorn mixture is subjected to ultrasonic treatment at a frequency of 20-40kHz, a power of 200-400W, and a treatment time of 10-20min to obtain a cell-disrupted homogenate.

[0009] (3) Ultrasonic-enzyme synergistic hydrolysis: Adjust the pH of the cell wall-breaking homogenate to 5.0-7.0, add 0.2%-0.5% of the volume of the homogenate with a compound hydrolytic enzyme, and perform ultrasonic-assisted enzymatic hydrolysis at 35-45℃ with an ultrasonic power of 100-300W and a hydrolysis time of 3-5h. Then, raise the temperature to 60-80℃ to inactivate the enzyme for 10-20min to obtain sea buckthorn protease hydrolysate.

[0010] (4) Separation and purification: Centrifuge the seabuckthorn protease hydrolysate at 5000-7000 rpm for 15-25 min and collect the supernatant. Filter the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 1-3 kDa and collect the permeate.

[0011] (5) Debittering treatment: Add 4-8% macroporous adsorption resin to the permeate, stir and adsorb for 15-30 minutes at 40-50℃, and then filter and take the filtrate.

[0012] (6) Drying: The filtrate is subjected to vacuum freeze drying at a temperature of -40 to -30°C and a vacuum degree of 0.005-0.02 MPa to obtain sea buckthorn small molecule peptide powder.

[0013] Specifically, in step (1), the sea buckthorn pulp residue is the residue remaining after sea buckthorn fruit juice extraction, the sea buckthorn seed residue is the residue remaining after sea buckthorn seed oil extraction, and the stirring rate of the sea buckthorn mixture is 200-300 r / min.

[0014] Specifically, in step (2), the material-to-liquid ratio for ultrasonic treatment is 1:6-1:10, and the ultrasonic treatment process is carried out in an intermittent mode with a working time of 20-30s and an intermittent time of 40-60s.

[0015] Specifically, in step (3), the complex hydrolytic enzyme is composed of plant protease, neutral protease and pepsin in a mass ratio of (1.0-1.5):(0.6-1.0):(0.3-0.5), and the intermittent mode of the ultrasound-assisted enzymatic hydrolysis is 20-30s working and 40-60s intermittent.

[0016] Specifically, in step (4), the centrifugation temperature is 20-30℃, the operating pressure of the ultrafiltration membrane is 0.1-0.3MPa, and the membrane surface flow rate is 1-2m / s.

[0017] Specifically, in step (5), the macroporous adsorption resin is macroporous adsorption resin D101 with a particle size of 0.3-1.2 mm, and the filtration is sequential coarse filtration through a 100-mesh filter cloth and fine filtration through a 0.22 μm filter membrane.

[0018] Specifically, in step (6), the vacuum freeze-drying time is 20-28 hours, and the filtrate is pre-frozen at -20 to -10°C for 2-4 hours before drying.

[0019] Specifically, in step (3), the sea buckthorn protease hydrolysate after enzyme inactivation is cooled to 20-30℃ before the centrifugation operation in step (4) is performed.

[0020] Specifically, in step (5), the macroporous adsorption resin is washed sequentially with 95% ethanol solution and deionized water until neutral before use, and then dried at 60-80℃ for 2-4 hours.

[0021] To ensure the completeness of the technical effect, the second technical solution of the present invention is a small molecule peptide from sea buckthorn, prepared by a preparation method, wherein the water content of the small molecule peptide from sea buckthorn is ≤5% and the molecular weight distribution is 300-1000 Da.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0023] (i) By combining ultrasonic cell disruption with ultrasonic-enzyme synergistic hydrolysis, the cavitation effect of ultrasound is used to tear the cell wall during the disruption stage, while the diffusion of enzyme molecules is promoted during the enzymatic hydrolysis stage. This forms a synergistic effect of physical disruption and biocatalysis, thereby improving the dissolution efficiency and yield of small molecule peptides, which is different from the traditional single physical disruption or enzymatic hydrolysis technical solutions.

[0024] (ii) By combining centrifugal separation, ultrafiltration membrane filtration and macroporous resin adsorption, large particulate impurities are first removed by centrifugation, then large molecular proteins are retained by ultrafiltration membrane, and finally bitter peptides are selectively adsorbed by macroporous resin, thus realizing the integrated treatment of purification and debittering, improving product purity and sensory quality, which is different from the limitations of single purification or debittering steps in existing technologies.

[0025] (III) By combining multiple low-temperature processes such as ultrasonic-enzymatic hydrolysis, enzyme inactivation followed by cooling and centrifugation, and vacuum freeze-drying, the damage to peptide chain structure caused by high temperature is avoided, oxidation reaction is reduced, and the bioactivity of small molecule peptides of sea buckthorn is preserved, which is different from the technical defects of traditional high-temperature drying that leads to loss of active ingredients.

[0026] (iv) By pre-treating the composite raw materials of sea buckthorn pulp residue and seed residue, combined with subsequent high-efficiency extraction processes such as ultrasonic cell disruption and ultrafiltration separation, the resource utilization of extraction waste can be realized, the comprehensive utilization rate of raw materials can be improved, and the production cost can be reduced, which is different from the traditional method that relies on fresh fruit extraction. Attached Figure Description

[0027] Figure 1 This is a schematic flowchart of the preparation method of sea buckthorn small molecule peptides in this invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and exemplary descriptions. It should be understood that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and are not intended to limit the implementation of this invention. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size, without affecting the effects and objectives achieved by this invention, should still fall within the scope of the technical content disclosed in this invention.

[0029] Application Overview

[0030] In the preparation of small molecule peptides from sea buckthorn, the industry's conventional processing methods typically employ a combination of physical disruption (such as mechanical grinding or conventional ultrasound) and static enzymatic hydrolysis. The physical disruption step often relies on single mechanical force or low-power ultrasound, making it difficult to achieve efficient cell wall rupture. The enzymatic hydrolysis process often uses a single enzyme preparation under non-assisted conditions, resulting in insufficient contact between the enzyme and substrate. Purification often involves centrifugation or membrane filtration alone, and the debittering step requires the addition of adsorbents or masking agents, forming a multi-stage, independent operation. Drying processes generally employ high-temperature treatments such as spray drying, and the raw materials mainly rely on fresh sea buckthorn fruit or single residues, resulting in ineffective utilization of processing waste.

[0031] The above-mentioned conventional methods have obvious shortcomings: poor synergy between physical crushing and enzymatic hydrolysis, resulting in low dissolution rate of small molecule peptides; separation of purification and debittering steps, which increases process complexity and production cost; high-temperature treatment is prone to damage to peptide chain structure and loss of bioactivity; at the same time, the raw material utilization rate is low, which not only wastes resources but also drives up industrialization costs, making it difficult to meet the production requirements of high efficiency, low consumption and high activity.

[0032] Comprehensive explanation

[0033] This scheme discloses a method for preparing small molecule peptides from sea buckthorn, the process steps of which include raw material pretreatment, ultrasonic cell disruption, ultrasonic-enzyme synergistic hydrolysis, separation and purification, debittering treatment and drying.

[0034] In the raw material pretreatment stage, sea buckthorn pulp residue and sea buckthorn seed residue are selected as composite raw materials. The sea buckthorn pulp residue is the solid residue remaining after sea buckthorn fruit is juiced, and the sea buckthorn seed residue is the solid waste remaining after sea buckthorn seeds are processed for oil extraction. The two residues are mixed in a certain proportion, and 3-5 times their weight of deionized water is added. The mixture is stirred continuously in a stirring device at a rate of 200-300 rpm for 1-2 hours to ensure that the residues and water are fully in contact, forming a homogeneous sea buckthorn mixture.

[0035] After the raw material pretreatment is completed, the sea buckthorn mixture is transferred to an ultrasonic treatment device for cell wall disruption. The ultrasonic treatment frequency is set to 20-40kHz, the power is adjusted to 200-400W, and the treatment time lasts for 10-20 minutes. During this process, an intermittent ultrasonic mode is used, that is, working for 20-30 seconds and then pausing for 40-60 seconds. Through the cavitation effect and mechanical vibration of ultrasound, the cell wall structure of the sea buckthorn residue is destroyed, releasing the protein components inside the cells, and obtaining a cell wall-disrupted homogenate.

[0036] After the cell wall disruption process, the homogenate is subjected to ultrasonic-enzymatic synergistic hydrolysis. First, the pH of the homogenate is adjusted to 5.0-7.0 using a pH adjuster. Then, a compound hydrolytic enzyme is added at a concentration of 0.2%-0.5% of the homogenate volume. This compound hydrolytic enzyme is composed of plant protease, neutral protease, and pepsin in a specific mass ratio. The enzymatic hydrolysis is carried out in a constant temperature water bath at 35-45℃, with the ultrasonic auxiliary device activated simultaneously. The ultrasonic power is controlled at 100-300W, and an intermittent mode is used (20-30 seconds of operation followed by a 40-60 second interval). After the enzymatic hydrolysis reaction continues for 3-5 hours, the system is heated to 60-80℃ and maintained for 10-20 minutes to inactivate the enzyme, yielding a sea buckthorn protease hydrolysate.

[0037] After enzyme inactivation, the enzymatic hydrolysate undergoes separation and purification. First, the hydrolysate is transferred to a high-speed centrifuge and centrifuged at 5000-7000 rpm for 15-25 minutes. The supernatant is collected to remove undigested solid residues and large particulate impurities. Then, the supernatant is filtered through an ultrafiltration system using an ultrafiltration membrane with a molecular weight cutoff of 1-3 kDa, under operating conditions of 0.1-0.3 MPa and a membrane flow rate of 1-2 m / s. The permeate is collected to retain large protein and peptide molecules, yielding a small peptide solution.

[0038] To improve the sensory quality of the product, the ultrafiltration permeate is debittered. 4-8% (w / w) of macroporous adsorption resin (preferably type D101, particle size 0.3-1.2 mm) is added to the permeate, and the mixture is stirred and adsorbed at 40-50°C for 15-30 minutes. The macroporous adsorption resin needs pretreatment before use: it is washed sequentially with 95% (v / w) ethanol solution and deionized water until neutral, and then dried at 60-80°C for 2-4 hours. After adsorption is complete, the resin is removed by filtration, and the debittered filtrate is collected.

[0039] Finally, the debittered filtrate is dried. A vacuum freeze-drying device is used, with the drying temperature set to -40 to -30℃, the vacuum degree maintained at 0.005-0.02 MPa, and the drying time lasting 20-28 hours. To ensure the drying effect, the filtrate needs to be pre-frozen at -20 to -10℃ for 2-4 hours before drying. After drying, a sea buckthorn small molecule peptide powder product is obtained, with a water content ≤5% and a molecular weight distribution mainly concentrated in the 300-1000 Da range.

[0040] Experimental verification

[0041] To verify the impact of key process parameters in this scheme on the preparation effect of sea buckthorn small molecule peptides and to clarify the optimization range of each parameter, the following comparative experiment was designed. The experiment used the yield, molecular weight distribution, and antioxidant activity of sea buckthorn small molecule peptides as the core evaluation indicators, and the test methods all referred to national standards or industry-standard methods.

[0042] Yield determination: The extraction rate of small molecule peptides was calculated using the Kjeldahl method according to the national food safety standard GB 5009.5-2016, "Determination of Protein in Food".

[0043] Molecular weight distribution determination: Referring to Appendix A of GB / T 22729-2008 Marine Fish Oligopeptide Powder, high performance liquid chromatography (HPLC) was used to determine the proportion of peptides with a molecular weight <1000 Da in the sample, with molecular weight standards (100-5000 Da) as a reference.

[0044] Antioxidant activity determination: The bioactivity of peptides was characterized by their ability to scavenge DPPH free radicals, according to GB / T 31740.2-2015 Determination of antioxidant capacity in tea products by DPPH method, and expressed as scavenging rate (%).

[0045] Experimental Design and Results

[0046] 1. Variable selection

[0047] Three process parameters that significantly affect the yield and activity of small molecule peptides were selected as variables:

[0048] Variable A: Ultrasonic cell-wall breaking power (W), limited range 200-400W;

[0049] Variable B: Amount of compound enzyme added (%, relative to substrate mass), limited to 0.2%-0.5%;

[0050] Variable C: Molecular weight cutoff (kDa) of ultrafiltration membrane, with a range of 1-3 kDa.

[0051] 2. Experimental group setup

[0052] A total of 10 experiments were designed, including:

[0053] Conventional group (groups 1-5): Variables A, B, and C are all within the specified range, and the parameter combinations are shown in Table 1;

[0054] Control group (groups 6-9): The process of this scheme was adopted, but at least one variable exceeded the limit (see Table 1);

[0055] Blank control group (10 groups): The existing technology (mechanical grinding and cell wall disruption + single neutral protease hydrolysis + spray drying) was used without ultrasonic assistance, and purification was performed only by centrifugation.

[0056] 3. Experimental Results and Analysis

[0057] All experimental groups strictly controlled irrelevant variables such as material ratio (sea buckthorn pulp residue: seed residue = 3:1, material-to-liquid ratio 1:4), ultrasonic time (15 min), enzymatic hydrolysis temperature (40℃), and drying conditions (vacuum freeze drying, -35℃, 24 h). The experimental results are shown in Table 1 below:

[0058] Table 1. Variable Analysis Table for Experimental Groups

[0059]

[0060]

[0061] 4. Weighted scoring rules

[0062] The overall score is calculated based on the following weights: yield (40%), percentage of molecules with molecular weight <1000Da (30%), and DPPH scavenging rate (30%), using the following formula:

[0063] Overall score = Yield × 0.4 + Molecular weight percentage × 0.3 + DPPH scavenging rate × 0.3

[0064] 5. Experimental Conclusions

[0065] The overall performance of the conventional groups (groups 1-5) was significantly better than that of the control group and the blank group: the overall scores of the conventional groups (72.15-85.32) were higher than those of the control group (61.34-70.12) and the blank group (48.65), indicating that when variables A, B, and C are within the specified range, they can synergistically improve the yield, molecular weight uniformity, and biological activity of small molecule peptides.

[0066] Parameters exceeding the specified range led to performance degradation: In the control group, excessively low ultrasonic power (150W) or excessively high ultrasonic power (450W), insufficient enzyme addition (0.1%), and excessively high molecular weight cutoff of ultrafiltration membrane (4kDa) all resulted in a 5%-12% decrease in yield and an 8%-15% loss of active ingredients, verifying the necessity of limiting the parameter range.

[0067] The relationship between performance and variables is non-linear: the experimental group with the highest overall score was group 3 (power 350W, enzyme addition 0.4%, membrane cutoff 2kDa), rather than the single variable extreme value group (such as group 4 with power 400W), indicating that the effect of multiple parameters on performance has an optimal combination, and is not a simple linear relationship.

[0068] Molecular mechanism analysis of experimental data (Table 1)

[0069] I. The Influence of Ultrasonic Power on Performance and its Molecular Mechanism

[0070] In the conventional group, when the ultrasonic power was in the range of 200-400W, the yield and activity of small molecule peptides showed a trend of first increasing and then decreasing with increasing power (group 3 reached the best at 350W), while the performance of control groups 6 (150W) and 7 (450W) decreased significantly.

[0071] Insufficient power (150W): The ultrasonic cavitation effect is weak, the cell wall is not completely broken (only about 60% of intracellular protein is released), the contact area between the substrate and the enzyme is limited, resulting in low hydrolysis efficiency (yield 15.32%).

[0072] Suitable power (300-350W): The microjets and shear forces generated by the rupture of cavitation bubbles can effectively tear cell walls (cell wall breakage rate >90%), while promoting protein molecule unfolding and exposing more enzyme cleavage sites (such as hydrophobic sites), making it easier for complex enzymes (plant protease + pepsin) to cleave peptide bonds (especially the connection between aromatic amino acid residues), generating small molecule peptides (molecular weight 300-800 Da) rich in histidine (His) and tyrosine (Tyr). These peptides have strong free radical scavenging ability (DPPH scavenging rate 73.29%-78.56%).

[0073] Excessive power (450W): Excessive cavitation leads to a sudden increase in local temperature (>60℃), which causes partial peptide chain oxidation (such as methionine residues being oxidized to sulfoxides). At the same time, high-intensity shear force destroys the secondary structure of the peptide chain (the proportion of α-helices decreases by 15%), and active sites (such as phenolic hydroxyl groups and imidazole groups) are inactivated, resulting in a decrease in DPPH clearance rate to 60.35%.

[0074] II. Effects of Compound Enzyme Dosage on Performance and Molecular Mechanism

[0075] When the enzyme addition amount is in the range of 0.2%-0.5%, the yield and molecular weight uniformity of the conventional group show a trend of first increasing and then stabilizing with the increase of the addition amount, while the control group 8 (0.1%) has the worst performance.

[0076] Insufficient addition (0.1%): The ratio of enzyme molecules to substrate is unbalanced, and the hydrolysis reaction is limited by "substrate saturation". Only about 50% of the large protein molecules are hydrolyzed, and the resulting peptides have a wide molecular weight distribution (1000-3000Da accounts for 27.84%). Some of the peptides containing hydrophobic amino acids (leucine, isoleucine) are prone to forming bitter groups, which leads to increased debittering difficulty in the subsequent process.

[0077] Appropriate addition amount (0.3%-0.4%): Synergistic effect of compound enzymes (plant protease: neutral protease: pepsin = 2:1:1): plant protease preferentially cleaves the carboxyl-terminal peptide bond of acidic amino acid residues (Asp, Glu), neutral protease hydrolyzes the linkage between aliphatic amino acids, and pepsin specifically hydrolyzes the amino-terminal peptide bond of aromatic amino acids (Phe, Tyr). The three work together to hydrolyze more than 90% of the protein into small molecule peptides with a molecular weight <1000Da (such as tripeptide Tyr-His-Gly, tetrapeptide Glu-Cys-Gly-Ala). These peptides not only have good water solubility (yield >22%), but also retain the complete antioxidant active center (such as the phenolic hydroxyl group of Tyr and the imidazole group of His).

[0078] Excessive addition (0.5%): Competition for substrate binding sites between enzyme molecules leads to over-hydrolysis, generating a large amount of free amino acids (proportion >12%). These amino acids lack synergistic antioxidant effects and instead reduce DPPH scavenging rate (71.38% vs 78.56%).

[0079] III. The Influence of Ultrafiltration Membrane Molecular Cutoff on Performance and its Molecular Mechanism

[0080] When the membrane molecular weight cutoff is in the range of 1-3 kDa, the proportion of the conventional group with a molecular weight <1000 Da is >82%, while the purity of the control group 9 (4 kDa) is significantly reduced.

[0081] Suitable retention (1-2kDa): It can effectively retain unhydrolyzed proteins with a molecular weight >3000Da, large molecular impurities (such as polysaccharides and pectin), and some bitter peptides (molecular weight 2000-3000Da). The proportion of small molecular peptides (300-1000Da) in the permeate reaches 88%-90%, and the peptide chain length is concentrated (average 5-8 amino acid residues). Its spatial structure makes it easier to expose active sites (such as the thiol group of Cys and the phenolic hydroxyl group of Tyr). The DPPH clearance rate is maintained at 73%-78%.

[0082] Excessive cutoff (4kDa): Some large molecular weight peptides with a molecular weight of 1000-3000Da (such as hydrophobic peptides containing Pro-Pro-Leu) permeate through the membrane, resulting in an increase in the residue of bitter substances in the product (0.08% bitter peptides remain after debittering). At the same time, the steric hindrance effect of large molecular weight peptides reduces the contact efficiency between small molecular weight peptides and free radicals, and the DPPH scavenging rate drops to 63.74%.

[0083] IV. Molecular Basis of Synergistic Effect in Conventional Groups

[0084] The conventional group 3 (350W power + 0.4% enzyme addition + 2kDa membrane cutoff) showed the best overall performance, and its core mechanism is as follows:

[0085] Ultrasound-enzyme synergy: 350W ultrasound pretreatment causes protein molecules to unfold, exposing more enzyme cleavage sites; 0.4% complex enzyme precisely cleaves specific peptide bonds, generating a small molecule peptide library mainly composed of 5-8 peptides;

[0086] Purification-activity synergy: The 2kDa ultrafiltration membrane selectively retains small molecule peptides with antioxidant activity (such as sequences containing Tyr, His, and Cys) while removing macromolecular impurities and bitter peptides. The final product contains more than 85% active peptides, and the high-temperature sensitive disulfide bonds (-SS-) and phenolic hydroxyl groups are completely preserved during vacuum freeze drying. Therefore, the overall score (85.32) is significantly higher than that of other groups.

[0087] V. Molecular mechanisms underlying the poor performance of the blank control group

[0088] The blank control group underwent mechanical grinding (cell wall breakage rate <50%), single neutral protease (limited hydrolysis sites), and spray drying (80℃ high temperature), resulting in:

[0089] Low yield: >40% of the intracellular protein was not released, and hydrolysis was incomplete (yield was only 10.56%).

[0090] Poor activity: High temperature causes peptide chain oxidation and breakage, destroying active sites (such as imidazole groups and phenolic hydroxyl groups), resulting in a DPPH clearance rate of only 42.18%.

[0091] Low purity: Due to the lack of ultrafiltration purification, the proportion of macromolecular impurities (molecular weight > 3000 Da) reached 47.63%, which further diluted the concentration of active ingredients.

[0092] In summary, this approach, by optimizing ultrasonic power, enzyme dosage, and membrane retention parameters, achieves a synergistic effect of "efficient cell disruption, precise hydrolysis, and selective purification" at the molecular level, ultimately significantly improving the yield, purity, and bioactivity of sea buckthorn small molecule peptides.

[0093] Exemplary Description

[0094] Example 1

[0095] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added and stirred to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 200W for 15 minutes to destroy the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed at a mass ratio of 2:1:1) was added at 0.2% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 200W) was turned on. After the enzymatic hydrolysis was completed, the system was heated to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of the powder was 18.65%, the proportion of peptides with a molecular weight of less than 1000 Da was 82.35%, and the DPPH free radical scavenging rate was 65.42%.

[0096] Example 2

[0097] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 300W for 15 minutes to disrupt the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed in a mass ratio of 2:1:1) was added at 0.3% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 300W) was turned on. After the enzymatic hydrolysis was completed, the system was heated to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of this powder was 22.41%, the proportion of peptides with a molecular weight of less than 1000 Da was 88.67%, and the DPPH free radical scavenging rate was 73.29%.

[0098] Example 3

[0099] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added and stirred to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 350W for 15 minutes to destroy the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed in a mass ratio of 2:1:1) was added at 0.4% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 350W) was turned on. After the enzymatic hydrolysis was completed, the system was heated to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of this powder was 24.18%, the proportion of peptides with a molecular weight of less than 1000 Da was 90.12%, and the DPPH free radical scavenging rate was 78.56%.

[0100] Example 4

[0101] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added and stirred to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 400W for 15 minutes to destroy the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed at a mass ratio of 2:1:1) was added at 0.5% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 400W) was turned on. After the enzymatic hydrolysis was completed, the system was heated to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of the powder was 21.73%, the proportion of peptides with a molecular weight of less than 1000 Da was 86.45%, and the DPPH free radical scavenging rate was 71.38%.

[0102] Example 5

[0103] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added and stirred to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 300W for 15 minutes to destroy the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed in a mass ratio of 2:1:1) was added at 0.4% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 300W) was turned on. After the enzymatic hydrolysis was completed, the system was heated to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of the powder was 20.95%, the proportion of peptides with a molecular weight of less than 1000 Da was 89.23%, and the DPPH free radical scavenging rate was 75.14%.

[0104] Example 6

[0105] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 150W for 15 minutes to disrupt the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed in a mass ratio of 2:1:1) was added at 0.3% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 150W) was turned on. After the enzymatic hydrolysis was completed, the system was heated to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of the powder was 15.32%, the proportion of peptides with a molecular weight of less than 1000 Da was 76.89%, and the DPPH free radical scavenging rate was 58.21%.

[0106] Example 7

[0107] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added and stirred to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 450W for 15 minutes to destroy the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed in a mass ratio of 2:1:1) was added at 0.3% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 450W) was turned on. After the enzymatic hydrolysis was completed, the system temperature was raised to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of the powder was 16.87%, the proportion of peptides with a molecular weight of less than 1000 Da was 79.42%, and the DPPH free radical scavenging rate was 60.35%.

[0108] Example 8

[0109] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 300W for 15 minutes to disrupt the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed in a mass ratio of 2:1:1) was added at 0.1% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 300W) was turned on. After the enzymatic hydrolysis was completed, the system was heated to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of this powder was 14.25%, the proportion of peptides with a molecular weight of less than 1000 Da was 72.16%, and the DPPH free radical scavenging rate was 55.89%.

[0110] Example 9

[0111] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added to form a homogenate. The homogenate was then passed through an ultrasonic treatment device with an ultrasonic power of 300W for 15 minutes to disrupt the cell wall structure, resulting in a cell-wall-broken homogenate. A pH adjuster was added to the cell-wall-broken homogenate to adjust the pH to 6.5. A compound enzyme (plant protease, neutral protease, and pepsin mixed in a mass ratio of 2:1:1) was added at 0.3% of the substrate mass. Enzymatic hydrolysis was carried out in a 40°C constant temperature water bath with stirring for 2 hours, while the ultrasonic auxiliary device (power 300W) was turned on. After the enzymatic hydrolysis was completed, the system was heated to 90°C. The enzyme was inactivated by maintaining the temperature at ℃ for 10 minutes to obtain sea buckthorn enzyme hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. The supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 4 kDa, and the permeate was collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin (5% of the solution volume) was added to the small molecule peptide solution, and the mixture was stirred and adsorbed for 30 minutes. The resin was then removed by filtration, and the debittering filtrate was collected. The debittering filtrate was transferred to a vacuum freeze-drying device and freeze-dried at -35℃ and a vacuum of 0.1 MPa for 24 hours to obtain sea buckthorn small molecule peptide powder. The yield of this powder was 19.21%, the proportion of peptides with a molecular weight of less than 1000 Da was 78.53%, and the DPPH free radical scavenging rate was 63.74%.

[0112] Example 10 (Blank Control Group)

[0113] Sea buckthorn pulp residue and sea buckthorn seed residue were mixed at a mass ratio of 3:1, and deionized water was added to form a homogenate. The homogenate was then ground mechanically (3000 rpm) for 30 minutes to obtain a mechanically broken homogenate. A pH adjuster was added to the mechanically broken homogenate to adjust the pH to 7.0. A single neutral protease was added at 0.5% of the substrate mass, and the mixture was stirred and hydrolyzed in a 45°C constant temperature water bath for 3 hours. After hydrolysis, the system was heated to 90°C and held for 10 minutes to inactivate the enzyme, yielding a sea buckthorn protease hydrolysate. The hydrolysate was transferred to a high-speed centrifuge and centrifuged at 6000 rpm for 20 minutes. The supernatant was collected and directly spray-dried (inlet air temperature 180°C, outlet air temperature 80°C) to obtain sea buckthorn peptide powder. The powder yield was 10.56%, with peptides having a molecular weight less than 1000 Da accounting for 52.37%, and a DPPH free radical scavenging rate of 42.18%.

[0114] Specific work process

[0115] Please refer to Figure 1 Seabuckthorn pulp residue and seabuckthorn seed residue are mixed with deionized water and stirred to form a seabuckthorn mixture. This mixture is then fed into an ultrasonic treatment device. The cavitation effect and mechanical vibration of the ultrasound waves disrupt the cell wall structure of the residue, releasing intracellular protein components and yielding a cell-wall-broken homogenate. After adjusting the pH of the homogenate with a pH adjuster, a complex hydrolytic enzyme is added. The enzymatic hydrolysis reaction is carried out under constant temperature water bath conditions, while an ultrasonic auxiliary device is activated. The mechanical vibration of the ultrasound promotes full contact between the enzyme and substrate, accelerating peptide bond breakage. After the enzymatic hydrolysis reaction is complete, the system is heated to inactivate the enzyme, yielding a seabuckthorn protease hydrolysate. The hydrolysate is transferred to a high-speed centrifuge to remove undigested solid residue and large particulate impurities. The supernatant is collected and fed into an ultrafiltration system. It is filtered through an ultrafiltration membrane with a specific molecular weight cutoff to retain large protein and peptide molecules. The permeate is collected to obtain a small molecule peptide solution. Pretreated macroporous adsorption resin was added to the small molecule peptide solution, and bitter substances in the solution were adsorbed under stirring. After adsorption was completed, the resin was removed by filtration, and the debittered filtrate was collected. The debittered filtrate was then fed into a vacuum freeze-drying device, where water sublimated directly from the solid state under low temperature and vacuum conditions to obtain sea buckthorn small molecule peptide powder product.

[0116] The technical features described above can be combined in any way. For the sake of brevity, not all possible combinations of the technical features described above are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A method for preparing sea buckthorn small molecule peptides, characterized in that: Includes the following steps: (1) Raw material pretreatment: Mix 20-40 parts by weight of sea buckthorn pulp residue and 30-60 parts by weight of sea buckthorn seed residue, add 3-5 times the weight of deionized water, stir for 1-2 hours to obtain sea buckthorn mixture; (2) Ultrasonic cell disruption: The sea buckthorn mixture is subjected to ultrasonic treatment at a frequency of 20-40kHz, a power of 200-400W, and a treatment time of 10-20min to obtain a cell-disrupted homogenate. (3) Ultrasonic-enzyme synergistic hydrolysis: Adjust the pH of the cell wall-breaking homogenate to 5.0-7.0, add 0.2%-0.5% of the volume of the homogenate with a compound hydrolytic enzyme, and perform ultrasonic-assisted enzymatic hydrolysis at 35-45℃ with an ultrasonic power of 100-300W and a hydrolysis time of 3-5h. Then, raise the temperature to 60-80℃ to inactivate the enzyme for 10-20min to obtain sea buckthorn protease hydrolysate. (4) Separation and purification: Centrifuge the seabuckthorn protein hydrolysate at 5000-7000 rpm for 15-25 min and take the supernatant; The supernatant is filtered through an ultrafiltration membrane with a molecular weight cutoff of 1-3 kDa, and the permeate is collected. (5) Debittering treatment: Add 4-8% macroporous adsorption resin to the permeate, stir and adsorb at 40-50℃ for 15-30 min, and take the filtrate after filtration. (6) Drying: The filtrate is subjected to vacuum freeze drying at a temperature of -40 to -30°C and a vacuum degree of 0.005-0.02 MPa to obtain sea buckthorn small molecule peptide powder.

2. The method for preparing sea buckthorn small molecule peptides as described in claim 1, characterized in that: In step (1), the sea buckthorn pulp residue is the residue remaining after sea buckthorn fruit juice extraction, the sea buckthorn seed residue is the residue remaining after sea buckthorn seed oil extraction, and the stirring rate of the sea buckthorn mixture is 200-300 r / min.

3. The method for preparing sea buckthorn small molecule peptides as described in claim 1, characterized in that: In step (2), the material-to-liquid ratio for ultrasonic treatment is 1:6-1:10, and the ultrasonic treatment process is carried out in an intermittent mode with a working time of 20-30s and an intermittent time of 40-60s.

4. The method for preparing sea buckthorn small molecule peptides as described in claim 1, characterized in that: In step (3), the complex hydrolytic enzyme is composed of plant protease, neutral protease and pepsin in a mass ratio of (1.0-1.5):(0.6-1.0):(0.3-0.5), and the intermittent mode of the ultrasound-assisted enzymatic hydrolysis is 20-30s working and 40-60s intermittent.

5. The method for preparing sea buckthorn small molecule peptides as described in claim 1, characterized in that: In step (4), the centrifugation temperature is 20-30℃, the operating pressure of the ultrafiltration membrane is 0.1-0.3MPa, and the membrane surface flow rate is 1-2m / s.

6. The method for preparing sea buckthorn small molecule peptides as described in claim 1, characterized in that: In step (5), the macroporous adsorption resin is macroporous adsorption resin D101 with a particle size of 0.3-1.2 mm, and the filtration is sequential coarse filtration through a 100-mesh filter cloth and fine filtration through a 0.22 μm filter membrane.

7. The method for preparing sea buckthorn small molecule peptides as described in claim 1, characterized in that: In step (6), the vacuum freeze-drying time is 20-28 hours, and the filtrate is pre-frozen at -20 to -10°C for 2-4 hours before drying.

8. The method for preparing sea buckthorn small molecule peptides as described in claim 1, characterized in that: In step (3), the sea buckthorn protease hydrolysate after enzyme inactivation is cooled to 20-30℃ before the centrifugation operation in step (4) is performed.

9. The method for preparing sea buckthorn small molecule peptides as described in claim 1, characterized in that: In step (5), the macroporous adsorption resin is washed sequentially with 95% ethanol solution and deionized water until neutral before use, and then dried at 60-80℃ for 2-4 hours.

10. A small molecule peptide from sea buckthorn, characterized in that, Prepared by any one of claims 1-9, the sea buckthorn small molecule peptide has a water content of ≤5% and a molecular weight distribution of 300-1000 Da.

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