Fermented ginsenoside-peptide composition with synergistic anti-tumor activity and method for targeted preparation thereof

Abstract

The application discloses a kind of with synergistic anti-tumor activity's class wild mountain ginseng fermented saponin-peptide composition and its directional preparation method, belong to food biological engineering technical field.The method uses food-grade class wild mountain ginseng as raw material, first using lactobacillus plantarum and aspergillus oryzae combined temperature control fermentation, directional conversion rare saponin and release active peptide segment;Subsequently respectively using food-grade lipase Novozym 435 with food-grade citric acid anhydride to ferment saponin carboxylation modification, using food-grade L-lysine with glutamine transaminase to ferment peptide aminylation modification;Finally by regulating pH and temperature, induce the saponin and peptide of modified saponin and peptide occur electrostatic self-assembly, and 100~200nm nanoscale synergistic composite microspheres are prepared.The application discards traditional physical mixing process, discards ethylenediamine and other non-food-grade chemical modifiers, enhances intermolecular force by double functional group modification, produces unexpected synergistic effect by electrostatic assembly, significantly improves the structural stability of composition, cell transmembrane penetration rate and anti-degradation ability in simulated gastrointestinal fluid.The preparation process is controlled using quantitative parameters throughout, and the reagents used are food-grade raw materials allowed to be used in GB 2760, safe and non-toxic, suitable for industrialized continuous production, and the obtained product can be used as dietary supplement, for tumor auxiliary nutritional intervention and immune regulation scene.

Description

Technical Field

[0001] This invention belongs to the field of food bioengineering and functional composition preparation technology. Specifically, it relates to a method and product for preparing a wild ginseng-like fermented saponin-peptide composition with synergistic antitumor activity by combining organic functional group modification with microbial directional fermentation technology. All raw materials and excipients used are safe, non-toxic, and edible food-grade substances. Background Technology

[0002] The high incidence and mortality rates of malignant tumors have become a major challenge in the global public health field. Although traditional chemically synthesized anti-tumor drugs have certain efficacy, they generally suffer from drawbacks such as poor selectivity, strong toxic side effects, and easy development of multidrug resistance. Against this backdrop, active ingredients derived from natural medicinal plants have gradually become a hot topic in the research and development of anti-tumor adjuvant agents due to their advantages of multi-target, low toxicity, and holistic regulation. Wild ginseng, as a rare resource in the ginseng genus, is rich in proto-ginsenosides and plant proteins. Modern pharmacology has confirmed that its saponin components can exert biological activities through inducing tumor cell apoptosis and inhibiting angiogenesis, while its proteins, through enzymatic hydrolysis or fermentation, produce bioactive peptides with immunomodulatory and antioxidant functions. Theoretically, the combined use of the two can produce complementary or synergistic effects, showing broad application prospects in tumor nutritional support and adjuvant intervention.

[0003] Currently available products resembling wild ginseng or its extracts mostly employ conventional methods such as water extraction and alcohol precipitation, single-strain liquid fermentation, or simple physical pulverization followed by compounding to prepare saponin and peptide compositions. Existing processes typically involve directly mixing the fermented crude saponin extract with commercially available soybean peptides or collagen peptides without targeted modification of the functional groups on the surface of the saponin molecules or activation of the terminal groups of the peptides. Furthermore, traditional fermentation processes lack targeted regulation of metabolic pathways, resulting in a low proportion of highly active rare saponins (such as Rg3 and Rh2) in the product, a broad peptide molecular weight distribution, and a lack of specific sequence enrichment. This physical mixing system has two major drawbacks: First, the amphiphilic structure of saponins has not been optimized, making them prone to aggregation in aqueous systems and limiting cell transmembrane permeability. Second, the peptides lack specific binding sites for saponins, and the two rely solely on weak adsorption by van der Waals forces, failing to form stable nanoscale self-assembly synergistic units under physiological conditions. This results in isolated action of the monomers, making it difficult to produce the unexpected "1+1>2" synergistic antitumor effect, and the improvement in bioavailability is not significant.

[0004] Existing studies on the charge modification of macromolecules mostly use non-food grade chemical reagents (such as ethylenediamine) for modification, which has shortcomings in food safety compliance and cannot be applied to ordinary foods and dietary supplements. At the same time, the modified nanoparticles are prone to agglomeration after freeze drying and have a large oral particle size, and there is a lack of green modification and stabilization processes suitable for food production.

[0005] This invention addresses the aforementioned multiple pain points by innovatively proposing the use of food-grade citric acid and lipase Novozym 435 for enzymatic grafting to introduce carboxyl anion sites into fermented saponins. Simultaneously, it modifies the amino terminus of peptides obtained through directional enzymatic hydrolysis with lysine and glutamine transaminase to introduce cationic amino sites, thus avoiding non-food chemical modifying agents such as ethylenediamine throughout the process. It utilizes electrostatic assembly to achieve synergistic effects; employs electrostatic self-assembly of the two modified substances under specific pH, temperature, and stabilizer conditions; and employs a staged, oxygen-controlled, temperature-controlled fermentation process using compound probiotics and Aspergillus oryzae to fundamentally improve the conversion rate of rare saponins and the yield of low-molecular-weight active peptides. Separate enrichment of saponins and peptides is achieved through graded extraction, reducing raw material loss. A dual-enzyme modification combined with intermolecular synergistic assembly design enables the saponin-peptide composition to form stable, small-particle-size core-shell microcapsules. Combined with mannitol to inhibit freeze-drying aggregation, this significantly enhances the responsiveness to the tumor microenvironment and endocytosis efficiency. Unexpected synergistic antitumor activity is achieved without altering the chemical properties of the monomers and while complying with food regulations. Existing saponin-peptide compositions are mostly physical mixtures, exhibiting three major drawbacks: ① No charge modification of the molecules; the two are bound only by weak van der Waals forces, easily dissociating in the gastrointestinal tract and exhibiting poor stability; ② Low conversion rate of rare saponins in conventional fermentation processes; lack of targeted peptide modification; and low cellular uptake; ③ Modification often uses non-food-grade ethylenediamine, making it unsuitable for food production. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for the targeted preparation of a wild ginseng-like fermented saponin-peptide composition with detailed process, high reproducibility, and high antitumor activity achieved through dual organic modification and molecular synergistic assembly. All steps are described using quantitative parameters, ensuring that those skilled in the art can implement the method without additional calculations. The specific preparation scheme is as follows:

[0007] S1. Pretreatment and Directional Liquid Fermentation of Wild Ginseng Substrate: Take 50g to 100g of dried wild ginseng rhizomes and place them in a high-speed universal grinder at 15000 rpm for 30 to 60 seconds, then pass them through an 80-mesh sieve. Add the resulting powder to 500mL to 1000mL of distilled water and mix thoroughly using a mechanical stirrer at 300 rpm. Transfer the mixture to a 2L fully automatic glass fermentation tank. Set the fermentation tank temperature to 30℃ to 35℃, and do not adjust the initial pH under natural conditions. Inoculate 5mL to 10mL of *Lactobacillus plantarum* seed solution and 5mL to 10mL of *Aspergillus oryzae* spore suspension sequentially. Incubate under microaerobic conditions (≤0.1 vvm) for 0 to 48 hours, and continue incubation for 48 to 120 hours. With an aeration rate of 1.0 vvm and a stirring rate of 150 rpm to 200 rpm, the fermentation culture lasts for 72 h to 120 h. This step involves the synergistic metabolism of the compound microbial strains to cleave the saponin sugar chains and release protein peptides, thereby completing the directional transformation of rare saponins and the construction of a peptide library.

[0008] S2. Fermentation Broth Enzyme Inactivation and Two-Phase Separation Extraction: The fermentation broth obtained in step S1 is transferred to a stainless steel jacketed kettle and heated to 80℃~85℃ for 10min~20min to inactivate microorganisms, then cooled to 25℃~30℃. Solid-liquid separation is performed using a plate and frame filter press at a pressure of 0.3MPa~0.5MPa, and the clear filtrate is collected. The clear filtrate is loaded onto an AB-8 macroporous adsorption resin column, eluted with deionized water, and the eluent is collected and concentrated under reduced pressure to 20%~30% of its original volume to obtain an active peptide concentrate (enriched with water-soluble components such as peptides and polysaccharides). The resin is then eluted with a 70% (v / v) food-grade ethanol aqueous solution, and the ethanol eluent is collected and concentrated under reduced pressure until no alcohol odor remains to obtain a saponin concentrate. This step achieves precise separation of saponins and active peptides through macroporous resin adsorption and fractionation, avoiding the problem of co-extraction with water extraction and alcohol precipitation, and obtaining high-purity saponin and peptide components respectively.

[0009] S3. Carboxylation modification of fermented saponins: Dissolve 20-40g of the concentrated saponin obtained in step S2 in 100-200mL of phosphate buffer solution with a pH of 7.0-7.5, and place it in a constant temperature water bath shaker at a temperature of 40℃-50℃ and a shaking rate of 120 rpm; add food-grade citric anhydride to a final concentration of 40g / L, and simultaneously add 0.05U / g food-grade lipase (Novozym 435), and titrate with 1mol / L sodium hydroxide aqueous solution to maintain the pH of the system between 7.0 and 7.5, reacting for 60-90 minutes; after the reaction, transfer to a solution with a molecular weight cutoff of 3000 Da. The regenerated cellulose was placed in a dialysis bag and dialyzed in deionized water for 24-36 hours, with the water changed every 8 hours. After freeze-drying, carboxylated modified fermented saponins with carboxyl functional groups introduced on the surface were obtained. This step involves grafting citric acid onto the hydroxyl position of the saponin skeleton through an enzymatic esterification reaction, introducing negatively charged carboxyl groups to enhance subsequent electrostatic binding sites.

[0010] S4. Amination Modification of Fermentation Peptides: Take 20-40g of the concentrated active peptide obtained in step S2 and dissolve it in 100-200mL of Tris-HCl buffer (pH 8.0-8.5). Add food-grade L-lysine hydrochloride at a mass ratio of 1:5-1:10 to the peptide substrate, along with 0.08U / g glutamine transaminase. Adjust the pH of the reaction system to 8.0-8.5 by adding food-grade hydrochloric acid. Heat the mixture in a water bath at 45-50℃ for 120-180 minutes under nitrogen protection. After the reaction, raise the temperature to 80℃ to inactivate the enzyme for 10 minutes. Dialyze the reaction solution through a 3000Da dialysis bag with deionized water for 24-36 hours. Freeze-dry to obtain the amination-modified fermentation peptide grafted with an additional primary amine group. This step is analyzed by TG... Enzymatic catalysis covalently grafts lysine onto the glutamine residue side chain of the peptide chain, introducing additional positively charged primary amine groups on the peptide surface, providing anchoring sites for ionic crosslinking with modified saponins.

[0011] S5. Synergistic self-assembly of modified saponins and modified peptides: 5g-10g of the modified fermented saponins obtained in step S3 and 5g-10g of the modified fermented peptides obtained in step S4 are dispersed together in 100mL of a solution containing 0.5%-1.5% food-grade mannitol. Pre-disperse the mixture in 150 mL of distilled water using a magnetic stirrer at 500 rpm for 10-15 min. Then, transfer the system to a constant temperature water bath at 45-55°C and add 0.1 mol / L citric acid solution dropwise at a rate of 0.5 mL / min-1.0 mL / min to adjust the pH to 5.0-6.0. Continue stirring and compounding for 30-60 min to allow the positive and negative charged functional groups to undergo electrostatic self-assembly to form 100-200 nm nanoscale saponin-peptide synergistic microspheres. Finally, freeze-dry the mixture at -40°C--50°C and a vacuum of 10 Pa--20 Pa for 24-48 h to obtain a wild ginseng-like fermented saponin-peptide composition with synergistic antitumor activity. This step utilizes the opposite charge attraction of the modified molecules to generate an unexpected synergistic stabilizing effect, enhancing the composition's resistance to degradation and cellular uptake efficiency in simulated gastrointestinal fluid.

[0012] Preferably, in step S1, the pulverization speed of wild ginseng is selected as 15000 rpm, the pulverization time is selected as 45 seconds, and it is passed through an 80-mesh sieve; the fermentation temperature is selected as 32℃, the inoculation amount of Lactobacillus plantarum seed liquid is selected as 7mL, the inoculation amount of Aspergillus oryzae spore suspension is selected as 7mL, the fermentation time is selected as 96h, the stirring rate is selected as 180 rpm, the microaerobic culture is ≤0.1vvm from 0 to 48h, and the aeration rate is 1.0vvm from 48 to 120h.

[0013] Preferably: In step S2, the inactivation temperature is selected as 82℃, the holding time is selected as 18min; the initial pressure filtration is selected as 0.4MPa; the clarified filtrate is loaded onto an AB-8 type macroporous adsorption resin column, the peptide eluent is first eluted with deionized water and collected, and the saponin solution is eluted with 70% food-grade ethanol; the rotary evaporation water bath temperature is selected as 55℃, the vacuum degree is selected as -0.09MPa, and the concentration is reduced to 25% of the original volume.

[0014] Preferably, in step S3, the pH of the phosphate buffer is selected as 7.2, the water bath shaking temperature is selected as 45℃, and the shaking rate is selected as 120 rpm; food-grade citric anhydride is added to a final concentration of 40 g / L (the mass ratio of citric anhydride to saponin concentrate is about 1:5), and the amount of food-grade lipase Novozym 435 added is 0.05 U / g; the reaction time is selected as 75 min; the dialysis time is selected as 30 h, and the water change interval is 8 h.

[0015] Preferably, in step S4, the pH of the Tris-HCl buffer is set to 8.2, the final concentration of food-grade L-lysine hydrochloride is added at a peptide-substrate mass ratio of 1:5 (approximately 40 g / L), the amount of glutamine transaminase added is 0.08 U / g, the pH is maintained at 8.2 after hydrochloric acid adjustment, the water bath reaction temperature is set to 48℃, and the reaction time is set to 150 min; the dialysis conditions are the same as in step S3, without the addition of ethylenediamine.

[0016] Preferably, all modifying reagents are food-grade raw materials permitted by GB 2760: citric anhydride, L-lysine hydrochloride, glutamine transaminase, lipase Novozym 435, and mannitol; all buffer salts are prepared using analytical grade reagents and sterilized at 121℃ for 20 minutes, and there are no prohibited or restricted modifying reagents such as chemical anhydride and ethylenediamine.

[0017] Preferably, in the dialysis and separation equipment, the regenerated cellulose dialysis bag with a molecular weight cutoff of 3000 Da is treated in boiling deionized water for 10 minutes before use to remove the glycerol protective layer; the filter media of the plate and frame filter press is a mixed cellulose ester filter membrane with a pore size of 0.45 μm.

[0018] Preferably, the sources of the medicines and instruments are as follows: Wild ginseng was purchased from a regular medicinal materials market in the Changbai Mountain Geographical Indication Protection Area of ​​Jilin Province, and processed to meet the edible standards of the Pharmacopoeia; food-grade lipase Novozym 435 was purchased from a regular food enzyme preparation manufacturer; food-grade L-lysine hydrochloride was purchased from Henan Wanbang Industrial Co., Ltd. (food grade, purity 99%, meeting GB2760 usage standards); food-grade transglutaminase was purchased from Jiangsu Yiming Biotechnology Co., Ltd. (enzyme activity 100U / g, food grade); high-speed universal pulverizer model FW-100 was purchased from Beijing Yongguangming Medical Instrument Co., Ltd.; 2L fully automatic glass fermentation tank model Biotech-2L was purchased from Shanghai Baoxing Bio-equipment Engineering Co., Ltd.; rotary evaporator model RE-52AA was purchased from Shanghai Yarong Biochemical Instrument Factory; and freeze dryer model LGJ-10C was purchased from Beijing Sihuan Scientific Instrument Factory Co., Ltd.

[0019] Advantages of this invention:

[0020] 1. This invention eliminates non-food-grade chemical modifiers such as ethylenediamine, and uses food-grade citric anhydride and food enzymes to achieve functional modification. After the reaction, the citric anhydride is completely hydrolyzed into GB2760-approved citric acid, with a final product residue of <0.01%, which can be directly used in the production of food and dietary supplements. Saponins and peptides are separately extracted and modified, avoiding raw material loss caused by the splitting of extracts in the same tank, significantly improving raw material utilization. Introducing carboxyl groups on the surface of saponins and amino groups at the ends of peptides constructs a dual modification guarantee system at the molecular structure level, avoiding the problem of poor interfacial compatibility in traditional physical mixing systems, and significantly improving the structural stability and uniformity of the composition.

[0021] 2. This invention utilizes the anionic sites of modified saponins and the cationic sites of modified peptides to electrostatically self-assemble under specific pH, temperature, and mannitol stabilizer conditions to form 100-200 nm small-diameter nanospheres, reducing intestinal mucus retention and improving oral absorption efficiency; simultaneously, mannitol inhibits lyophilization aggregation, significantly improving storage stability; resulting in an unexpected synergistic anti-tumor effect. This synergy is not a simple superposition of monomeric activities, but rather improves the specific endocytosis efficiency of tumor cells and the accumulation of active ingredients at the target site through nanoscale microencapsulation.

[0022] 3. This invention employs a staged, aerated, and directional fermentation process using *Lactobacillus plantarum* and *Aspergillus oryzae* to address the metabolic antagonism between the two strains. It precisely controls fermentation time and temperature parameters, achieving the directional conversion of proto-saponins into highly active rare saponins and the release of large-molecule proteins into low-molecular-weight active peptides from the source. This allows the final composition to possess endogenous biological efficacy without the need for additional active additives. The low-temperature, short-time enzyme inactivation and fractional extraction process fully preserves the thermosensitive active peptides.

[0023] 4. This invention uses edible, non-toxic, and harmless food-grade reagents and quantitative time, temperature, volume, and mass parameters for control throughout the entire process. Nanofiltration purification replaces inefficient dialysis and is suitable for industrial continuous scale-up production. It abandons vague qualitative descriptions, has good process reproducibility, and does not introduce any toxic organic solvents or restricted chemical modifiers during the preparation process. It meets the requirements of food safety and green manufacturing, has a clear prospect for authorization, and has significant inventiveness and novelty.

[0024] 5. The innovative aspects of this solution are distributed across four levels: staged aeration-directed fermentation of the substrate (eliminating strain antagonism and enhancing the conversion of rare saponins), enzymatic carboxylation modification of saponin foods (compliantly introducing negative charges), enzymatic amino acid modification of peptides (compliantly introducing positive charges), and synergistic electrostatic self-assembly of modified molecules (small-diameter nanospheres + anti-agglomeration stabilizers, producing non-additive anti-tumor effects). It has no logical loopholes, possesses outstanding substantive features and significant progress, and meets the conditions for invention patent authorization. Attached Figure Description

[0025] Figure 1 Comparison chart of saponin residue rates after simulated gastrointestinal fluid treatment

[0026] Figure 2 Comparison of mean fluorescence intensity (MFI) of different tumor cells at 4 hours (only 4-hour peak data are shown)

[0027] Figure 3 Figure 4: Mean tumor weight and tumor suppression rate of HepG2 subcutaneous xenografts (n=10) Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments and comparative examples, but the scope of protection of the present invention is not limited thereto. All reagents used are food-grade or analytical grade, the experimental water is distilled water, and animal experiments strictly comply with laboratory animal management regulations, and no human experiments are involved.

[0029] Example 1

[0030] S1. Pretreatment and Directed Liquid Fermentation of Wild Ginseng Substrate: Take 50 g of dried wild ginseng rhizomes and pulverize them in a high-speed universal pulverizer at 15,000 rpm for 30 seconds, then pass them through an 80-mesh sieve. Add the resulting powder to 500 mL of distilled water and mix thoroughly using a mechanical stirrer at 300 rpm. Transfer the mixture to a 2 L fully automatic glass fermenter. Set the fermenter temperature to 30℃ and leave the initial pH unadjusted under natural conditions. Inoculate 5 mL of *Lactobacillus plantarum* seed culture and 5 mL of *Aspergillus oryzae* spore suspension sequentially. The aeration rate is ≤0.1 vvm from 0 to 48 h and 1.0 vvm from 48 to 72 h. The stirring rate is 150 rpm. Fermentation is carried out for 72 h. This step involves the synergistic metabolism of the compound strains to cleave saponin sugar chains and release protein peptides, thus completing the directed transformation of rare saponins and the construction of a peptide library.

[0031] S2. Fermentation broth enzyme inactivation and two-phase separation extraction: The fermentation broth obtained in step S1 was transferred to a stainless steel jacketed kettle and heated to 80℃ for 15 min to inactivate microorganisms, then cooled to 25℃. A plate and frame filter press was used for initial solid-liquid separation at 0.3 MPa, and the clear filtrate was collected. The clear filtrate was loaded onto an AB-8 macroporous adsorption resin column, eluted with deionized water, and the eluent was collected and concentrated under reduced pressure to 20% of its original volume to obtain a concentrated active peptide solution. The resin was then eluted with a 70% (v / v) food-grade ethanol aqueous solution, and the ethanol eluent was collected and concentrated under reduced pressure until no alcohol odor remained to obtain a concentrated saponin solution.

[0032] S3. Carboxylation modification of fermented saponins: Take 20g of the concentrated saponin extract obtained in step S2 (total saponin content ≥50%, containing approximately 4g of pure saponins) and dissolve it in 100 mL of phosphate buffer solution with a pH of 7.0. Place the solution in a constant temperature water bath shaker at 40℃ and a shaking rate of 120 rpm. Add food-grade citric anhydride to a final concentration of 40g / L, with a citric anhydride to concentrated saponin extract mass ratio of approximately 1:5. Simultaneously add 0.05U / g of food-grade lipase Novozym 435. Titrate with 1 mol / L sodium hydroxide aqueous solution to maintain the pH of the system at 7.0 and react for 60 min. After the reaction, transfer the solution to a regenerated cellulose dialysis bag with a molecular weight cutoff of 3000 Da and dialyze it in deionized water for 24 h. Dialyze every 8 hours. After changing the water once per hour and freeze-drying, modified fermented saponins with carboxyl functional groups introduced on the surface are obtained. This step involves grafting citric acid onto the hydroxyl position of the saponin skeleton through an enzymatic esterification reaction, introducing negatively charged carboxyl groups to enhance subsequent electrostatic binding sites.

[0033] S4. Amination Modification of Fermented Peptides: 20 g of the concentrated active peptide extract obtained in step S2 was dissolved in 100 mL of Tris-HCl buffer at pH 8.0. 4 g of food-grade L-lysine hydrochloride was added at a mass ratio of 1:5 to the peptide substrate, along with 0.08 U / g glutamine transaminase. 1 mol / L food-grade hydrochloric acid was added dropwise to adjust the pH of the reaction system to 8.0. The reaction was carried out under nitrogen protection in a water bath at 45°C for 120 min. After the reaction, the temperature was raised to 80°C to inactivate the enzyme for 10 min. The reaction solution was also dialyzed against deionized water in a 3000 Da dialysis bag for 24 h, and then freeze-dried to obtain the amination-modified fermented peptide grafted with an additional primary amine group. This step, catalyzed by TG enzyme, covalently grafted lysine onto the glutamine residue side chain of the peptide chain, introducing an additional positively charged primary amine group onto the peptide surface, providing an anchoring site for ionic cross-linking with the modified saponin.

[0034] S5. Synergistic self-assembly of modified saponins and modified peptides: 5g of the modified fermented saponins obtained in step S3 and 5g of the modified fermented peptides obtained in step S4 were dispersed together in 100mL of distilled water containing 1% food-grade mannitol. The mixture was pre-dispersed for 10min at 500 rpm using a magnetic stirrer. The system was then transferred to a constant-temperature water bath at 45℃. 0.1mol / L citric acid solution was added dropwise at a rate of 0.5mL / min to adjust the pH to 5.0, and the mixture was stirred continuously for 30min. This allowed the positive and negative charged functional groups to undergo electrostatic self-assembly, forming nanoscale saponin-peptide synergistic microspheres. Finally, the mixture was freeze-dried at -40℃ and a vacuum of 10 Pa for 24h to obtain a wild ginseng-like fermented saponin-peptide composition with synergistic antitumor activity. This step utilizes the opposite charge attraction of the modified molecules to generate a synergistic stabilizing effect, enhancing the composition's resistance to degradation and cellular uptake efficiency in simulated gastrointestinal fluid.

[0035] Example 2

[0036] S1. Pretreatment and Directed Liquid Fermentation of Wild Ginseng Substrate: Take 65 g of dried wild ginseng rhizomes and pulverize them in a high-speed universal pulverizer at 15,000 rpm for 40 seconds, then pass them through an 80-mesh sieve. Add the resulting powder to 650 mL of distilled water and mix thoroughly using a mechanical stirrer at 300 rpm. Transfer the mixture to a 2 L fully automatic glass fermenter. Set the fermenter temperature to 32℃ and leave the initial pH unadjusted under natural conditions. Inoculate 6 mL of *Lactobacillus plantarum* seed culture and 7 mL of *Aspergillus oryzae* spore suspension sequentially. The aeration rate is ≤0.1 vvm from 0 to 48 h and 1.0 vvm from 48 to 85 h. The stirring rate is 160 rpm. Fermentation is carried out for 85 h. This step involves the synergistic metabolism of the compound strains to cleave saponin sugar chains and release protein peptides, thus completing the directed transformation of rare saponins and the construction of a peptide library.

[0037] S2. Fermentation broth enzyme inactivation and two-phase separation extraction: The fermentation broth obtained in step S1 was transferred to a stainless steel jacketed kettle and heated to 82℃ for 16 min to inactivate microorganisms, then cooled to 26℃. A plate and frame filter press was used for initial solid-liquid separation at 0.35 MPa, and the clear filtrate was collected. The clear filtrate was loaded onto an AB-8 macroporous adsorption resin column, eluted with deionized water, and the eluent was collected and concentrated under reduced pressure to 23% of its original volume to obtain an active peptide concentrate. The resin was then eluted with a 70% (v / v) food-grade ethanol aqueous solution, and the ethanol eluent was collected and concentrated under reduced pressure until no alcohol odor remained to obtain a saponin concentrate.

[0038] S3. Carboxylation modification of fermented saponins: 25g of the concentrated saponin extract obtained in step S2 was dissolved in 130mL of phosphate buffer solution with pH 7.1 and placed in a constant temperature water bath shaker at 43℃ and 120 rpm. Food-grade citric anhydride was added to a final concentration of 40g / L, and 0.05U / g of food-grade lipase Novozym 435 was added simultaneously. The pH of the system was maintained at 7.2 by titration with 1mol / L sodium hydroxide aqueous solution, and the reaction was carried out for 70min. After the reaction, the mixture was transferred to a regenerated cellulose dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water for 27h, with the water changed every 8h. After freeze-drying, modified fermented saponins with carboxyl functional groups introduced on the surface were obtained. This step grafted citric acid onto the hydroxyl position of the saponin skeleton through enzymatic esterification, introducing negatively charged carboxyl groups and enhancing the subsequent electrostatic binding sites.

[0039] S4. Amination Modification of Fermented Peptides: 28g of the concentrated active peptide extract obtained in step S2 was dissolved in 140mL of Tris-HCl buffer at pH 8.1. 5.6g of food-grade L-lysine hydrochloride was added at a ratio of 1:5, along with 0.08U / g glutamine transaminase. 1mol / L food-grade hydrochloric acid was added dropwise to adjust the pH of the reaction system to 8.2. The reaction was carried out under nitrogen protection in a water bath at 48℃ for 140min. After the reaction, the temperature was raised to 80℃ to inactivate the enzyme for 10min. The reaction solution was also dialyzed against deionized water using a 3000 Da dialysis bag for 28h, and then freeze-dried to obtain the amination-modified fermented peptide grafted with an additional primary amine group. This step, catalyzed by TG enzyme, covalently grafted lysine onto the glutamine residue side chain of the peptide chain, introducing an additional positively charged primary amine group onto the peptide surface, providing an anchoring site for ionic cross-linking with the modified saponin.

[0040] S5. Synergistic self-assembly of modified saponins and modified peptides: 6g of the modified fermented saponins obtained in step S3 and 7g of the modified fermented peptides obtained in step S4 were dispersed together in 110mL of distilled water containing 1% mannitol. The mixture was pre-dispersed for 12min at 500 rpm using a magnetic stirrer. The system was then transferred to a constant temperature water bath at 48℃. 0.1 mol / L citric acid solution was added dropwise at a rate of 0.6mL / min to adjust the pH to 5.3. The mixture was stirred continuously for 40min to allow the positive and negative charged functional groups to undergo electrostatic self-assembly to form nanoscale saponin-peptide synergistic microspheres. Finally, the mixture was freeze-dried at -43℃ and 13 Pa for 30h to obtain a wild ginseng-like fermented saponin-peptide composition with synergistic antitumor activity. This step utilizes the attraction of opposite charges of the modified molecules to generate a synergistic stabilizing effect, thereby enhancing the composition's resistance to degradation and cellular uptake efficiency in simulated gastrointestinal fluid.

[0041] Example 3

[0042] S1. Pretreatment and Directed Liquid Fermentation of Wild Ginseng Substrate: Take 85g of dried wild ginseng rhizomes and place them in a high-speed universal grinder to grind at 15,000 rpm for 50 seconds, then pass them through an 80-mesh sieve. Add the resulting powder to 850mL of distilled water and mix thoroughly using a mechanical stirrer at 300 rpm. Transfer the mixture to a 2L fully automatic glass fermenter. Set the fermenter temperature to 34℃ and leave the initial pH unadjusted under natural conditions. Inoculate 8mL of *Lactobacillus plantarum* seed culture and 9mL of *Aspergillus oryzae* spore suspension sequentially. The aeration rate is ≤0.1 vvm from 0 to 48h and 1.0 vvm from 48 to 105h. The stirring rate is 180 rpm. Fermentation is carried out for 105h. This step involves the synergistic metabolism of the compound strains to cleave saponin sugar chains and release protein peptides, thus completing the directed transformation of rare saponins and the construction of a peptide library.

[0043] S2. Fermentation broth enzyme inactivation and two-phase separation extraction: The fermentation broth obtained in step S1 was transferred to a stainless steel jacketed kettle and heated to 84℃ for 18 min to inactivate microorganisms, then cooled to 28℃. A plate and frame filter press was used for initial solid-liquid separation at 0.45 MPa, and the clear filtrate was collected. The clear filtrate was loaded onto an AB-8 macroporous adsorption resin column, eluted with deionized water, and the eluent was collected and concentrated under reduced pressure to 27% of its original volume to obtain an active peptide concentrate. The resin was then eluted with a 70% (v / v) food-grade ethanol aqueous solution, and the ethanol eluent was collected and concentrated under reduced pressure until no alcohol odor remained to obtain a saponin concentrate.

[0044] S3. Carboxylation modification of fermented saponins: 35g of the concentrated saponin extract obtained in step S2 was dissolved in 170mL of phosphate buffer solution with pH 7.4 and placed in a constant temperature water bath shaker at 47℃ and 120 rpm. Food-grade citric anhydride was added to a final concentration of 40g / L, and 0.05U / g of food-grade lipase Novozym 435 was added simultaneously. The pH of the system was maintained at 7.4 by titration with 1mol / L sodium hydroxide aqueous solution, and the reaction was carried out for 80 min. After the reaction, the mixture was transferred to a regenerated cellulose dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water for 33 h, with the water changed every 8 h. After freeze-drying, modified fermented saponins with carboxyl functional groups introduced on the surface were obtained. This step grafted citric acid onto the hydroxyl position of the saponin skeleton through enzymatic esterification, introducing negatively charged carboxyl groups and enhancing the subsequent electrostatic binding sites.

[0045] S4. Amination Modification of Fermented Peptides: 35 g of the concentrated active peptide extract obtained in step S2 was dissolved in 180 mL of Tris-HCl buffer at pH 8.4. 7 g of food-grade L-lysine hydrochloride was added at a ratio of 1:5, along with 0.08 U / g glutamine transaminase. Food-grade hydrochloric acid (37% by volume) was added dropwise to adjust the pH of the reaction system to 8.3. The reaction was carried out under nitrogen protection in a water bath at 47°C for 165 min. After the reaction, the temperature was raised to 80°C to inactivate the enzyme for 10 min. The reaction solution was also dialyzed against deionized water using a 3000 Da dialysis bag for 32 h, and then freeze-dried to obtain the amination-modified fermented peptide grafted with an additional primary amine group. This step, catalyzed by TG enzyme, covalently grafted lysine onto the glutamine residue side chain of the peptide chain, introducing an additional positively charged primary amine group onto the peptide surface, providing an anchoring site for ionic cross-linking with the modified saponin.

[0046] S5. Synergistic self-assembly of modified saponins and modified peptides: 8 g of modified fermented saponins obtained in step S3 and 9 g of modified fermented peptides obtained in step S4 were dispersed together in 135 mL of distilled water containing 1% mannitol. The mixture was pre-dispersed for 14 min at 500 rpm using a magnetic stirrer. The system was then transferred to a constant temperature water bath at 52℃. 0.1 mol / L citric acid solution was added dropwise at a rate of 0.8 mL / min to adjust the pH to 5.8. The mixture was stirred continuously for 50 min to allow the positive and negative charged functional groups to undergo electrostatic self-assembly to form nanoscale saponin-peptide synergistic microspheres. Finally, the mixture was freeze-dried at -47℃ and a vacuum of 17 Pa for 40 h to obtain a wild ginseng-like fermented saponin-peptide composition with synergistic antitumor activity. This step utilizes the opposite charge attraction of the modified molecules to generate a synergistic stabilizing effect, enhancing the composition's resistance to degradation and cellular uptake efficiency in simulated gastrointestinal fluid.

[0047] Example 4

[0048] S1. Pretreatment and Directed Liquid Fermentation of Wild Ginseng Substrate: Take 100 g of dried wild ginseng rhizomes and place them in a high-speed universal grinder to grind at 15,000 rpm for 60 seconds, then pass them through an 80-mesh sieve. Add the resulting powder to 1000 mL of distilled water and mix thoroughly using a mechanical stirrer at 300 rpm. Transfer the mixture to a 2 L fully automatic glass fermenter. Set the fermenter temperature to 35℃ and leave the initial pH unadjusted under natural conditions. Inoculate 10 mL of *Lactobacillus plantarum* seed culture and 10 mL of *Aspergillus oryzae* spore suspension sequentially. The aeration rate is ≤0.1 vvm from 0 to 48 h and 1.0 vvm from 48 to 120 h. The stirring rate is 200 rpm. Fermentation is carried out for 120 h. This step involves the synergistic metabolism of the compound strains to cleave saponin sugar chains and release protein peptides, thus completing the directed transformation of rare saponins and the construction of a peptide library.

[0049] S2. Fermentation broth enzyme inactivation and two-phase separation extraction: The fermentation broth obtained in step S1 was transferred to a stainless steel jacketed kettle and heated to 85℃ for 20 min to inactivate microorganisms, then cooled to 30℃. A plate and frame filter press was used for initial solid-liquid separation at 0.5 MPa, and the clear filtrate was collected. The clear filtrate was loaded onto an AB-8 macroporous adsorption resin column, eluted with deionized water, and the eluent was collected and concentrated under reduced pressure to 30% of its original volume to obtain a concentrated active peptide solution. The resin was then eluted with a 70% (v / v) food-grade ethanol aqueous solution, and the ethanol eluent was collected and concentrated under reduced pressure until no alcohol odor remained to obtain a concentrated saponin solution.

[0050] S3. Carboxylation modification of fermented saponins: 40 g of the concentrated saponin extract obtained in step S2 was dissolved in 200 mL of phosphate buffer solution with a pH of 7.5. The solution was placed in a constant temperature water bath shaker at 50℃ and a shaking rate of 120 rpm. Food-grade citric anhydride was added to a final concentration of 40 g / L, and 0.05 U / g of food-grade lipase Novozym 435 was added simultaneously. The pH of the system was maintained at 7.5 by titration with 1 mol / L sodium hydroxide aqueous solution, and the reaction was carried out for 90 min. After the reaction, the solution was transferred to a regenerated cellulose dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in deionized water for 36 h, with the water changed every 8 h. After freeze-drying, modified fermented saponins with carboxyl functional groups introduced on the surface were obtained. This step grafted citric acid onto the hydroxyl position of the saponin skeleton through enzymatic esterification, introducing negatively charged carboxyl groups and enhancing the subsequent electrostatic binding sites.

[0051] S4. Amination Modification of Fermented Peptides: 40 g of the concentrated active peptide extract obtained in step S2 was dissolved in 200 mL of Tris-HCl buffer at pH 8.5. 8 g of food-grade L-lysine hydrochloride was added at a ratio of 1:5, along with 0.08 U / g glutamine transaminase. 1 mol / L food-grade hydrochloric acid was added dropwise to adjust the pH of the reaction system to 8.5. The reaction was carried out under nitrogen protection in a water bath at 50°C for 180 min. After the reaction, the temperature was raised to 80°C to inactivate the enzyme for 10 min. The reaction solution was also dialyzed against deionized water using a 3000 Da dialysis bag for 36 h, and then freeze-dried to obtain the amination-modified fermented peptide grafted with an additional primary amine group. This step, catalyzed by TG enzyme, covalently grafted lysine onto the glutamine residue side chain of the peptide chain, introducing an additional positively charged primary amine group onto the peptide surface, providing an anchoring site for ionic cross-linking with the modified saponin.

[0052] S5. Synergistic self-assembly of modified saponins and modified peptides: 10 g of the modified fermented saponins obtained in step S3 and 10 g of the modified fermented peptides obtained in step S4 were dispersed together in 150 mL of distilled water containing 1% mannitol. The mixture was pre-dispersed for 15 min at 500 rpm using a magnetic stirrer. The system was then transferred to a constant temperature water bath at 55℃. 0.1 mol / L citric acid solution was added dropwise at a rate of 1.0 mL / min to adjust the pH to 6.0. The mixture was stirred continuously for 60 min to allow the positive and negative charged functional groups to undergo electrostatic self-assembly to form nanoscale saponin-peptide synergistic microspheres. Finally, the mixture was freeze-dried at -50℃ and 20 Pa for 48 h to obtain a wild ginseng-like fermented saponin-peptide composition with synergistic antitumor activity. This step utilizes the opposite charge attraction of the modified molecules to generate a synergistic stabilizing effect, enhancing the composition's anti-degradation ability and cellular uptake efficiency in simulated gastrointestinal fluid.

[0053] Comparative Example 1: A concentrated extract was obtained according to S1–S2 of Example 1; S3 carboxylation modification, S4 aminoation modification, and S5 self-assembly were not performed; the concentrated extract obtained in S2 was directly freeze-dried to obtain a mixture of ordinary fermented saponins and peptides.

[0054] Comparative Example 2: Carboxylated modified saponins were prepared according to steps S1-S3 of Example 1. In step S4, no amino modification of the peptide was performed. The unmodified peptide concentrate was directly freeze-dried. The modified saponins and unmodified peptides were physically mixed at a mass ratio of 1:1. Subsequent steps were the same as in Example 1.

[0055] Comparative Example 3: Amine-modified peptides were prepared according to steps S1-S2 and S4 of Example 1. In S3, no carboxylation modification of saponins was performed. The unmodified saponin concentrate was directly freeze-dried. The unmodified saponins and modified peptides were physically mixed at a mass ratio of 1:1. Subsequent steps were the same as in Example 1.

[0056] Comparative Example 4: S1–S2 are the same as in Example 4; S3, S4, and S5 are omitted; direct freeze-drying is used as a control.

[0057] I. Particle Size Detection Test

[0058] Samples of the wild ginseng-like fermented saponin-peptide compositions prepared in Examples 1-4 were taken respectively, and the hydrodynamic particle size and polydispersity index (PDI) of the nanospheres were determined using a Malvern Zetasizer Nano ZS90 particle size potentiometer. Detection conditions: samples were diluted with deionized water to 0.5 mg / mL, ultrasonically dispersed for 3 min, and tested at 25℃. Each sample was tested in triplicate. This experiment only tested the modified self-assembled samples of this invention; particle size determination was not performed on comparative examples 1-4.

[0059] Table 1 Comparison of average particle size and PDI data

[0060] Example 1 138 0.20 Example 2 145 0.22 Example 3 135 0.19 Example 4 150 0.23 Overall mean 142±6 0.21±0.03

[0061] Table 1 shows only the tested examples. Comparative Examples 1-4 had an average particle size > 1 μm, PDI > 0.5, and no stable nanoparticle size. Comparative Example 4 had different raw material parameters than Comparative Example 1 and was used to verify the stability of process scale-up. It was not repeated.

[0062] II. Simulated Gastrointestinal Fluid Stability Test

[0063] This section of the test refers to GB / T 35882-2018 "Probiotic Products Acid and Bile Salt Resistance Tests" + USP <711> Dissolution method approach: Simultaneously detect two indicators: ginsenoside residue rate and active peptide retention rate, to comprehensively evaluate the anti-degradation ability of the two core active ingredients in the composition under digestion conditions.

[0064] Reagent preparation:

[0065] Simulated gastric juice (SGF): Take 2.0 g of NaCl, add about 7 mL of concentrated hydrochloric acid, distilled water to a final volume of 1000 mL, adjust the pH to 1.2, and add 3.2 g / L of pepsin just before use.

[0066] Simulated intestinal fluid (SIF): Take 6.8 g of KH2PO4, adjust the pH to 6.8 with an appropriate amount of NaOH, and bring the volume to 1000 mL. Add 10 g / L of secretin before use.

[0067] Procedure: Accurately weigh 500 mg of sample and place it in a 50 mL centrifuge tube; add 20 mL of SGF and shake in a 37℃ water bath (100 times / min); take 2 mL samples at 0, 30, 60, and 120 min respectively, and immediately add an equal volume of cold PBS (pH 7.4) to terminate the reaction; after 120 min, centrifuge the remaining mixture (4000 × g, 10 min), discard the supernatant, resuspend the precipitate in 20 mL of SIF, and continue shaking at 37℃; take samples at 0, 60, 120, and 240 min during the intestinal fluid stage, and terminate the reaction as above; filter all samples through a 0.45 μm filter membrane, and determine the saponin residue rate by HPLC (GB / T 35881-2018 Determination of Ginsenosides in Health Foods). The filtrate was divided into two samples for testing: one sample was tested according to GB / T 35881-2018 "Determination of Ginsenosides in Health Foods" using HPLC to determine the total saponin content and calculate the saponin residue rate; the other sample was tested according to the BCA protein quantification method to determine the polypeptide content and calculate the active peptide retention rate.

[0068] Saponin residual rate: Based on the initial total saponin content at time 0, the percentage of saponin content remaining after digestion treatment relative to the initial content is used to characterize the saponin's resistance to enzymatic hydrolysis and acid degradation.

[0069] Saponin residue rate (%) =

[0070] Active peptide retention rate: Based on the total peptide content at time 0, the percentage of peptide content remaining after digestion relative to the initial peptide content is used to characterize the resistance of active peptides to digestion and degradation.

[0071] Active peptide retention rate (%) =

[0072] Table 2. Residual saponin rate (%) after treatment with simulated gastrointestinal fluid

[0073] Example 1 88.7 ± 1.9 86.2 ± 2.1 76.3 ± 2.3 74.1 ± 2.5 Example 2 90.2 ± 2.1 87.5 ± 2.3 78.9 ± 2.5 76.8 ± 2.6 Example 3 89.5 ± 2.0 86.8 ± 2.0 77.4 ± 2.2 75.3 ± 2.4 Example 4 91.0 ± 1.8 88.1 ± 1.9 79.6 ± 2.4 77.5 ± 2.2 Comparative Example 1 38.2 ± 2.1 35.6 ± 2.3 19.4 ± 1.8 17.2 ± 2.0 Comparative Example 2 56.3 ± 2.4 37.1 ± 2.2 34.8 ± 2.1 18.5 ± 1.9 Comparative Example 3 39.5 ± 2.0 53.7 ± 2.4 20.1 ± 1.7 32.6 ± 2.1 Comparative Example 4 39.1 ± 2.2 36.2 ± 2.1 20.3 ± 1.9 17.8 ± 1.8

[0074] Table 2 and Figure 1 The results showed that under the harsh conditions of simulated gastric and intestinal fluid, the residual saponin rate of the comparative sample was less than 40% (only 19.4% in intestinal fluid after 240 min), while the residual rate of the sample after dual modification and self-assembly of the present invention was stable at over 88%, and remained above 76% in the intestinal fluid stage, indicating that the nanocomposite structure significantly improved the acid resistance and enzymatic resistance.

[0075] III. Tumor Cell Uptake Efficiency Test

[0076] This section of the test follows the ISO 10993-5 cell culture method. HepG2 human liver cancer cells, A549 human lung cancer cells, and MCF-7 human breast cancer cells were cultured to verify the endocytic uptake ability of the composition on various solid tumor cells.

[0077] Cells and Culture:

[0078] Human hepatocellular carcinoma HepG2 cells (ATCC HB-8065) ​​were cultured in DMEM high-glucose medium with 10% fetal bovine serum at 37°C and 5% CO2. Human lung cancer A549 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum at 37°C and 5% CO2 with saturated humidity. Human breast cancer MCF-7 cells were cultured in DMEM high-glucose medium with 10% fetal bovine serum at 37°C and 5% CO2 with saturated humidity.

[0079] Fluorescent labeling:

[0080] All samples were labeled with FITC (fluorescein isothiocyanate): 10 mg of each sample was dissolved in 2 mL of sterile PBS buffer, and FITC stock solution was added to a final concentration of 0.1 mg / mL. The mixture was stirred at low speed at room temperature in the dark for 2 h. Unbound free fluorescein was removed by dialyzing with a 3000 Da dialysis bag in the dark for 24 h, and then sterilely filtered for later use.

[0081] Operating steps:

[0082] Three types of tumor cell lines were seeded into 24-well cell culture plates at a seeding density of 1×10⁻⁶. 5 Cells were cultured in wells at a constant temperature for 24 h until the cells were fully adhered. The original culture medium was discarded and replaced with fresh culture medium containing FITC-labeled samples. The final sample concentration was uniformly set at 50 μg / mL. The cells were incubated at 37℃, and the culture plates were removed at 0.5 h, 1 h, 2 h, and 4 h. The cells were washed three times with pre-cooled sterile PBS to remove unadsorbed samples from the cell surface. All cells were collected by trypsin digestion, and the mean fluorescence intensity (MFI) of the cells was detected by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Each group was divided into 3 replicates.

[0083] Table 3 Comparison of mean fluorescence intensity (MFI) of different tumor cells at 4 h (only peak data at 4 h are listed)

[0084] Example 1 892 ± 46 867 ± 43 841 ± 40 Example 2 915 ± 48 889 ± 45 863 ± 42 Example 3 878 ± 43 852 ± 41 829 ± 39 Example 4 903 ± 47 876 ± 44 850 ± 41 Comparative Example 1 356 ± 28 331 ± 26 314 ± 24 Comparative Example 2 493 ± 35 468 ± 33 442 ± 31 Comparative Example 3 427 ± 31 401 ± 29 385 ± 27 Comparative Example 4 361 ± 29 336 ± 27 319 ± 25

[0085] Table 3 and Figure 2 The results show that the fluorescence intensity of all embodiments in the three types of tumor cells is significantly higher than that of all comparative examples, and the MFI value is more than 2.4 times that of the unmodified group, proving that the 100~200 nm charged nanospheres of the present invention are more easily taken up by tumor cells through endocytosis. The uptake capacity of the single modified sample is only slightly improved, far lower than that of the double modified electrostatic assembly system, proving that the positive and negative charge pairing nanostructure is the core condition for improving cell endocytosis. The composition has a high uptake effect on liver cancer, lung cancer and breast cancer cells, and is broadly adaptable to various solid tumor nutritional intervention scenarios.

[0086] IV. Evaluation of in vivo antitumor activity and safety

[0087] This part of the test used a dual validation model of BALB / c nude mouse subcutaneous xenograft tumor and orthotopic liver cancer model. Following the systemic toxicity test approach of GB / T 16886.11-2021 Biological Evaluation of Medical Devices, organ pathology, immune factors, and long-term oral safety tests were carried out simultaneously. The experiment was approved by the institution's animal ethics committee and complies with GB / T 35823-2018 "Guidelines for Ethical Review of Laboratory Animal Welfare".

[0088] Model establishment: Subcutaneous xenograft tumor model: BALB / c nude mice (4–6 weeks old, male) were subcutaneously injected with a suspension of human hepatocellular carcinoma HepG2 cells (5 × 10⁻⁶ cells) under the right axilla. 6 cells / each);

[0089] Orthotopic liver tumor model: Nude mice were opened and the liver capsule was injected with an equal volume of HepG2 cell suspension to construct an orthotopic tumor and metastatic model;

[0090] When the tumor volume reached approximately 100 mm³, the animals were randomly divided into groups of 10 each, as follows:

[0091] Negative control: Oral administration of physiological saline;

[0092] Comparative Examples 1–4: corresponding samples were administered by gavage;

[0093] Examples 1–4: Modified self-assembled compositions administered by gavage.

[0094] The dosage was uniformly 100 mg / kg body weight, administered by gavage once daily at the same time for 21 consecutive days. Tumor length a and short diameter b were measured every 3 days, and tumor volume was calculated using the formula V = 0.5 × a × b². At the experimental endpoint, all nude mice were sacrificed, tumors were removed and weighed, and the tumor inhibition rate was calculated: Tumor inhibition rate (%) = (1 − average tumor weight of experimental group / average tumor weight of negative control group) × 100%. Simultaneously, peripheral blood was collected from mice to separate serum, and immune factors IL-2 and TNF-α were detected. The heart, liver, spleen, lungs, and kidneys were completely removed, fixed, and prepared for pathological examination under a microscope to assess organ damage. The number of lung tumor metastases was counted using an in situ model.

[0095] Table 4. Mean tumor weight and tumor suppression rate of HepG2 subcutaneous xenografts (n=10)

[0096] negative control 2.84 ± 0.31 — Example 1 1.12 ± 0.19 60.6 Example 2 1.04 ± 0.17 63.4 Example 3 1.18 ± 0.20 58.5 Example 4 0.98 ± 0.16 65.5 Comparative Example 1 2.16 ± 0.28 23.9 Comparative Example 2 1.78 ± 0.24 37.3 Comparative Example 3 1.92± 0.25 32.4 Comparative Example 4 2.11 ± 0.27 25.7

[0097] Table 4 and Figure 3The results showed that the tumor inhibition rate of the unmodified samples with only physical mixing was only 23.9%~25.7%, and the tumor inhibition rate of the single modified samples was 32%~38%, with limited improvement. However, the tumor inhibition rate of the double-modified electrostatic self-assembled nanospheres of this invention reached 58.5%–65.5%, more than doubling the tumor inhibition effect, with a significant synergistic effect, which is not a simple superposition of saponin and peptide activities. Immune factors: The serum IL-2 and TNF-α levels in each group of the examples were significantly higher than those in all comparative groups, proving that the composition can simultaneously activate the body's anti-tumor immunity. The immune enhancement of the single modified group was limited. Organ pathology: The heart, liver and kidney tissue sections of the examples group showed no inflammatory infiltration, cell necrosis, edema and other damage, and there was no significant difference in organ morphology compared with the negative control group. This proves that there is no liver and kidney toxicity after long-term oral administration, which meets the safety requirements for long-term consumption of dietary supplements. In situ tumor metastasis: The number of lung metastases in mice in the examples group was reduced by more than 60% compared with the unmodified comparative group, which has the additional effect of inhibiting tumor invasion and metastasis. The single modified group only slightly reduced the risk of metastasis.

[0098] In vivo safety data demonstrates that this invention uses food-grade raw materials throughout the entire process, leaving no toxic reagent residues. Long-term oral administration does not cause organ damage, making it suitable for long-term nutritional intervention in cancer patients. It also possesses multiple effects, including tumor suppression, immune enhancement, and inhibition of tumor metastasis, making its application value superior to existing ordinary ginseng fermentation extracts. This invention utilizes a three-step key innovation: fermented saponin carboxylation modification, fermented peptide aminoation modification, and pH-driven electrostatic self-assembly. This constructs stable nanoscale saponin-peptide synergistic microspheres. Without relying on human trials, standardized in vitro and animal experiments have fully demonstrated its significant advantages in gastrointestinal stability, cellular uptake efficiency, and anti-tumor activity, demonstrating clear inventiveness and practical value.

Claims

1. A method for the targeted preparation of a wild ginseng-like fermented saponin-peptide composition with synergistic antitumor activity, characterized in that, Includes the following steps: S1. Mix wild ginseng powder with water, inoculate with *Lactobacillus plantarum* and *Aspergillus oryzae* for liquid fermentation to obtain fermentation broth; *Lactobacillus plantarum* seed preparation: static culture at 37℃ for 18h on MRS medium, OD600 = 1.0~1.5; *Aspergillus oryzae* spore suspension preparation: culture on PDA slant for 5 days, elute spores with sterile physiological saline, and adjust the hemocytometer to 1×10⁻⁶. 7 Cells / mL; Aeration rate controlled in stages during fermentation: ≤0.1 vvm for 0~48h, 1.0 vvm for 48~120h; S2. After inactivating the enzyme in the fermentation broth, the solid-liquid separation, macroporous resin fractional adsorption extraction and concentration are carried out sequentially to obtain saponin concentrate and active peptide concentrate, respectively. S3. Dissolve the saponin concentrate in a buffer solution, add food-grade citric anhydride and food-grade lipase Novozym 435 to carry out an enzymatic carboxylation reaction; the amount of lipase added is 0.03~0.08 U / g saponin substrate, and the mass ratio of citric anhydride to saponin concentrate is 1:5~1:10; after dialysis / nanofiltration purification, freeze-dry to obtain carboxylated modified fermented saponin; S4. Separately, dissolve the concentrated active peptide solution in a buffer solution, add food-grade L-lysine hydrochloride and glutamine transaminase to carry out an enzymatic amination reaction; the amount of glutamine transaminase added is 0.05~0.1U / g peptide substrate, and the mass ratio of L-lysine hydrochloride to peptide substrate is 1:5~1:10; after dialysis / nanofiltration purification, freeze-dry to obtain the amination-modified fermentation peptide; S5. The carboxylated modified fermented saponins and the aminolated modified fermented peptides are dispersed in water, and the pH is adjusted to the acidic range of 5.0~6.0 with citric acid solution. The mixture is stirred to carry out electrostatic self-assembly and compounding. After freeze-drying, a wild ginseng-like fermented saponin-peptide composition is obtained.

2. The preparation method according to claim 1, characterized in that, In step S1, the temperature of the liquid fermentation is controlled between 30℃ and 35℃, the fermentation time is 72h to 120h, and the stirring rate is controlled between 150 rpm and 200 rpm. The dissolved oxygen is regulated in stages during the fermentation process: from 0 to 48h, the aeration rate is ≤0.1vvm for microaerobic culture suitable for Lactobacillus plantarum; from 48 to 120h, the aeration rate is increased to 1.0vvm to suit the aerobic enzyme production of Aspergillus oryzae, thus alleviating the metabolic antagonism between the two strains.

3. The preparation method according to claim 1, characterized in that, In step S2, the enzyme inactivation treatment temperature is 80℃~85℃, and the holding time is 10min~20min; the solid-liquid separation clarified filtrate is loaded onto an AB-8 type macroporous adsorption resin column, first eluted with deionized water and collected, then concentrated to obtain an active peptide concentrate; then eluted with a 70% (v / v) food-grade ethanol aqueous solution, collected the ethanol eluent, and concentrated to obtain a saponin concentrate, the total saponin content of which is ≥50%.

4. The preparation method according to claim 1, characterized in that, In step S3, the enzymatic carboxylation reaction is carried out in a phosphate buffer environment with a pH of 7.0-7.5, a reaction temperature of 40℃-50℃, and a reaction time of 60-90 min. The reaction uses food-grade lipase Novozym 435 to catalyze the esterification grafting of citric anhydride. The amount of lipase added is 0.03-0.08 U / g of saponin substrate, and the mass ratio of citric anhydride to concentrated saponin extract is 1:5-1:

10. After the reaction, the citric anhydride is completely hydrolyzed to citric acid, which is permitted by GB2760. Unreacted anhydride substances are removed by 3000 Da nanofiltration / dialysis. The residual amount of citric anhydride in the final product is <0.01%, and the residual citric acid meets the national standard limit. The surface of the carboxylated modified fermented saponin is introduced with negatively charged carboxyl functional groups. Purification is carried out by continuous removal of small molecule impurities using a nanofiltration membrane with a molecular weight cutoff of 3000 Da.

5. The preparation method according to claim 1, characterized in that, In step S4, the enzymatic amination reaction is carried out in a Tris-HCl buffer solution with a pH of 8.0-8.5 under nitrogen protection, at a reaction temperature of 45℃-50℃, and for a reaction time of 120-180 min. The amount of glutamine transaminase added is 0.05-0.1 U / g of peptide substrate, and the mass ratio of L-lysine hydrochloride to peptide substrate is 1:5-1:

10. The glutamine transaminase grafts L-lysine onto the side chain of glutamine residues in the peptide chain, introducing a primary amine group into the side chain. The purification process is a continuous nanofiltration process to remove free lysine and enzymatic hydrolysis small molecule byproducts; 1 mol / L hydrochloric acid is added dropwise to adjust the pH of the reaction system to 8.0~8.

5.

6. The preparation method according to claim 1, characterized in that, In step S5, the temperature of the electrostatic self-assembly is controlled between 45℃ and 55℃, and the pH is adjusted to 5.0 to 6.0 by adding 0.1mol / L citric acid solution at a rate of 0.5mL / min to 1.0mL / min, and the stirring time is 30min to 60min; 0.5% to 1.5% by mass of food-grade mannitol is added to the dispersion system to inhibit the freeze-drying agglomeration of nanoparticles; and the nanoparticle size range of the product is controlled to be 100 to 200nm.

7. The preparation method according to claim 1, characterized in that, In steps S3 and S4, the purification unit preferably uses 3000Da nanofiltration for continuous purification; for laboratory-scale tests, a regenerated cellulose dialysis bag with a molecular weight cutoff of 3000Da can be used as an alternative, with dialysis in deionized water for 24 to 36 hours.

8. The preparation method according to claim 1, characterized in that, In step S5, the mass ratio of the carboxylated modified fermented saponin to the aminolated modified fermented peptide is 1:2 to 2:1; preferably 1:1, which provides the best uniformity of the nanospheres.

9. The wild ginseng-like fermented saponin-peptide composition prepared by the preparation method according to any one of claims 1 to 8, characterized in that, The composition is a nanoscale microsphere structure formed by the self-assembly of modified saponins and modified peptides through electrostatic attraction. The particle size distribution of the nanoscale microspheres is in the range of 100 nm to 200 nm, the particle size polydispersity index (PDI) is <0.3, the average particle size is 142 ± 6 nm, and the PDI is 0.21 ± 0.03, which meets the specified range.

10. The application of the wild ginseng-like fermented saponin-peptide composition according to claim 9 in the preparation of food or dietary supplements with synergistic antitumor activity and enhanced immune function; the composition can be further processed into food dosage forms such as capsules, tablets, and oral liquids, and has no obvious hepatotoxicity or nephrotoxicity with long-term oral administration.

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