A preparation method of small molecule peptides based on synergistic degradation of eggshell membranes by molds and small molecule peptides

Abstract

The application discloses a preparation method of small molecule peptides based on synergistic degradation of chicken eggshell membranes by molds and the small molecule peptides, and relates to the field of small molecule peptide preparation. The method comprises the following steps: mixing pretreated chicken eggshell membranes with mold fermentation liquor, and performing synergistic degradation; inactivating and centrifuging the degraded mixture, and collecting supernatant; purifying small molecule peptides in the supernatant through membrane separation technology; drying the purified small molecule peptide solution through a low-temperature spray drying method to obtain small molecule peptide powder; and the molds include Trichoderma reesei and Aspergillus niger. Through synergistic degradation of the two molds, the conversion of the chicken eggshell membranes is more thorough, the yield of the small molecule peptides is significantly improved, the total nitrogen recovery rate can reach 85.2%, and the product molecular weight distribution is concentrated.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide preparation technology, specifically to a method for preparing small molecule peptides based on the synergistic degradation of eggshell membrane by mold to form small molecule peptides, and the small molecule peptides themselves. Background Technology

[0002] Eggshell membranes are rich in protein and are a potential source of polypeptides and amino acids, possessing significant edible and medicinal value. The eggshell membrane is a thin membrane between the inner surface of the eggshell and the egg white, consisting of a highly cross-linked, water-insoluble fibrous protein. It contains various endogenous proteins, including lysine oxidase-like protein 2 (28%), cysteine-rich shell membrane proteins (27%), fibronectin, histones, chitin-36 and ovotransferrin (23%), lysozyme (12%), and collagen (10%). The enzymatic peptides produced after hydrolysis of these proteins exhibit rich biological functional diversity, possessing various physiological functions such as promoting wound healing, anti-inflammation, antioxidation, antibacterial activity, and hypoglycemia.

[0003] Currently, the main methods for preparing eggshell membrane peptides include chemical hydrolysis and enzymatic hydrolysis. Chinese patent CN111000790A discloses a subcritical method for preparing eggshell membrane peptides, achieving a decomposition rate of over 95% within one hour without the need for expensive enzymes. However, this method requires high-temperature and high-pressure equipment, resulting in high energy consumption and potential damage to certain bioactive components. Research from the Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sciences, shows that enzymatic hydrolysis of eggshell membranes yields enzymatically hydrolyzed peptides with molecular weights ranging from 700 to 3000 Da. However, enzymatic hydrolysis is costly, and the hydrolysis efficiency of a single enzyme system is limited. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing small molecule peptides based on the synergistic degradation of eggshell membrane by molds, and the small molecule peptides themselves. Through the synergistic degradation effect of a specific combination of molds, the eggshell membrane is efficiently converted. The preparation process is green, environmentally friendly, and low in cost. The resulting mixture of small molecule peptides has a specific molecular weight distribution range (700-1500 Da) and biological activity.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for preparing small molecule peptides based on the synergistic degradation of eggshell membrane by mold includes the following steps:

[0007] S100. Mix the pretreated eggshell membrane with the mold fermentation liquid for synergistic degradation.

[0008] S200. Inactivate and centrifuge the degraded mixture, and collect the supernatant;

[0009] S300: Purification of small molecule peptides in the supernatant using membrane separation technology;

[0010] S400. The purified small molecule peptide solution is dried using a low-temperature spray drying method to obtain small molecule peptide powder.

[0011] Among them, molds in S100 include Trichoderma reesei and Aspergillus niger.

[0012] This invention utilizes the synergistic mechanism of *Trichoderma reesei* and *Aspergillus niger* to achieve efficient biodegradation of eggshell membranes through a complex enzyme system produced during their fermentation process. Eggshell membranes, a dense, insoluble network structure composed of various fibrous proteins (including type I, V, and X collagen) and mucopolysaccharides, have a stable chemical composition that makes them difficult to effectively process using traditional decomposition methods. In the degradation system of this invention, *Trichoderma reesei*, as a highly efficient producer of cellulase, secretes cellulase, hemicellulase, and chitinase that can first act on the fibrous skeleton of the eggshell membrane, disrupting its overall structure by hydrolyzing β-1,4 glycosidic bonds and effectively breaking down its tight network structure, creating favorable conditions for subsequent degradation. Following this, acidic and neutral proteases produced by *Aspergillus niger* target exposed protein peptide bonds, specifically cleaving large protein molecules into smaller peptide fragments. This synergistic mechanism allows the enzyme systems of the two fungi to coordinate efficiently in both time and space, significantly improving the degradation efficiency of the substrate. The entire process system precisely controls the molecular weight distribution of the product through subsequent membrane separation technology, and combined with low-temperature spray drying, it ensures product purity while maximizing the preservation of the bioactivity of small molecule peptides.

[0013] Existing single-enzyme hydrolysis or single-strain fermentation methods, due to limitations in enzyme systems or metabolic pathways, struggle to achieve efficient degradation of complex substrates like eggshell membranes. This invention innovatively combines *Trichoderma reesei* and *Aspergillus niger*, constructing a functionally complementary degradation system based on a deep understanding of their enzyme characteristics: the cellulase system of *Trichoderma reesei* is responsible for breaking down structural barriers, while the protease system of *Aspergillus niger* specializes in peptide bond cleavage, forming a highly efficient division of labor and cooperation mechanism. In terms of process design, this invention focuses on targeted optimization around the dual cores of "efficient degradation" and "activity protection." By precisely controlling key parameters such as degradation temperature, pH, and time, the enzyme activities of both molds can be simultaneously utilized. Simultaneously, it innovatively combines membrane separation technology within a specific molecular weight range with low-temperature spray drying, constructing a full-process activity protection system from degradation to drying. This technological path of transforming agricultural waste into high-value-added products not only solves the environmental challenges of eggshell membrane treatment but also creates significant economic value.

[0014] This invention successfully transforms waste eggshell membranes into high-value small molecule peptides by combining Trichoderma reesei and Aspergillus niger with downstream fine separation, purification and drying processes. The preparation process is green, environmentally friendly, low-cost and highly efficient.

[0015] Further, in S100, the degradation conditions are: temperature 35~50℃, pH 5.0~7.0, degradation time 12~48 hours; the mass ratio of eggshell membrane to mold fermentation broth is 1:4~6; in the mold fermentation broth, the volume ratio of Trichoderma reesei fermentation broth to Aspergillus niger fermentation broth is 1:1~1.5.

[0016] At temperatures of 35–50°C, both *Trichoderma reesei* and *Aspergillus niger* maintain high catalytic efficiency and exhibit synergistic effects. A pH of 5.0–7.0 maintains the stability of *Trichoderma reesei* cellulase while meeting the activity requirements of *Aspergillus niger* protease. A material-to-substrate ratio of 1:4–6 ensures sufficient contact between the fermentation broth and substrate, providing a suitable liquid-phase environment for the enzymatic reaction. Particularly noteworthy is the first discovery and verification of the enzyme complementarity effect between *Trichoderma reesei* and *Aspergillus niger* at a specific volume ratio (1:1–1.5). At this ratio, the structural degrading enzyme and the proteolytic enzyme can form a highly efficient relay catalysis, achieving efficient connection of the degradation process.

[0017] This parameter combination enables the degradation rate of eggshell membrane to reach over 88%, significantly improving the yield of small molecule peptides. The resulting small molecule peptides have a more concentrated molecular weight distribution and maintain complete biological activity, avoiding the problems of over-degradation or under-degradation. The material ratio of 1:4 to 6 ensures both reaction efficiency and control of production costs; the 12 to 48-hour time window provides flexibility for industrial production and exhibits good process stability.

[0018] Furthermore, the method for preparing Trichoderma reesei fermentation broth includes the following:

[0019] Inoculate the Trichoderma reesei seed culture into the fermentation medium at an inoculation rate of 5% to 10%; ferment for 60 to 72 hours at 28 to 30°C, an aeration rate of 0.8 to 1.2 vvm, and a stirring speed of 180 to 220 rpm.

[0020] Through extensive experimental optimization, this invention has discovered that while *Trichoderma reesei* exhibits optimal cell growth rate at approximately 25°C, increasing the temperature and controlling it within the range of 28-30°C during its enzyme production phase significantly enhances the specific enzyme activity (i.e., enzyme activity per unit cell) and total yield of cellulase and chitinase. This is likely because this temperature is more conducive to the proper folding, assembly, and secretion of enzyme proteins. Therefore, the primary fermentation temperature of this invention is selected as 28-30°C.

[0021] Further, by mass percentage, the fermentation medium comprises the following components: 2.0%~4.0% pretreated corn stalk microcrystals, 1.0%~2.0% soybean meal, 0.2%~0.5% ammonium sulfate, 0.3%~0.6% potassium dihydrogen phosphate, 0.05%~0.1% magnesium sulfate heptahydrate, 0.1%~0.2% Tween-80, and the balance being water;

[0022] The pretreated corn stalk microcrystals are obtained by treating corn stalks with dilute acid and steam explosion.

[0023] The fermentation medium uses corn stalk microcrystals (2.0%–4.0%) pretreated with dilute acid and steam explosion as the main carbon source. Its loose, porous structure facilitates cell utilization and effectively induces the synthesis of cellulase systems. Soybean meal (1.0%–2.0%) serves as the organic nitrogen source, providing essential amino acids and containing various growth factors that promote rapid mycelial growth. Ammonium sulfate (0.2%–0.5%) and potassium dihydrogen phosphate (0.3%–0.6%) are used as inorganic salt components, playing key roles in maintaining cellular nitrogen and energy metabolism, respectively. Magnesium ions in magnesium sulfate heptahydrate (0.05%–0.1%) act as cofactors for various enzymes, while Tween-80 (0.1%–0.2%) promotes the secretion and release of extracellular enzymes by altering cell membrane permeability.

[0024] Unlike traditional methods that use pure chemicals as inducers, this invention creatively employs pretreated corn stalk microcrystals, an agricultural waste, as the primary inducer. This achieves both resource utilization of the waste and a significant reduction in production costs. During pretreatment, the synergistic effect of dilute acid and steam explosion significantly alters the fiber structure of the corn stalks, exposing more reaction sites and greatly improving its induction efficiency for enzyme production by *Trichoderma reesei*. The content of each component was systematically optimized, with 2.0%–4.0% corn stalk microcrystals and 1.0%–2.0% soybean meal forming the optimal carbon-nitrogen balance, which effectively induces enzyme synthesis while avoiding substrate inhibition.

[0025] A specific combination of aeration rate and stirring speed effectively solves the problem of dissolved oxygen transfer during fermentation. This invention combines low-cost agricultural waste with refined fermentation technology, significantly reducing production costs while ensuring high enzyme activity.

[0026] The specific preparation method of pretreated corn stalk microcrystals is as follows:

[0027] Step 1: Mix dried and pulverized corn stalks with a 2.0%–3.0% sulfuric acid solution at a solid-liquid ratio of 1:10, and treat in a closed reactor at 121–150°C for 50–90 minutes. After treatment, separate the solid and liquid phases by pressure filtration. Wash the solid components repeatedly with deionized water until neutral to remove residual acid and fermentation inhibitors (such as furfural and hydroxymethylfurfural).

[0028] Step 2: Adjust the moisture content of the washed wet straw to about 10%, place it in a steam explosion device, and introduce saturated steam to raise the system pressure to 2.2 MPa. Maintain this pressure for 200 seconds, then release the pressure instantly to complete the explosion process. The exploded material is then dried, crushed, and passed through an 80-mesh sieve to obtain pretreated corn straw microcrystals.

[0029] The pretreated corn stalk microcrystals have a specific surface area ≥200 m² / g and a moisture content controlled below 8%. This pretreatment process, through the synergistic effect of dilute acid hydrolysis and physical explosion, effectively reduces the cellulose crystallinity of corn stalks and significantly improves the enzymatic conversion rate, providing an ideal inducing substrate for Trichoderma reesei enzyme fermentation.

[0030] Furthermore, the method for preparing Aspergillus niger fermentation broth includes the following:

[0031] Inoculate the Aspergillus niger seed culture into the fermentation medium at an inoculation rate of 5% to 10%, and ferment for 48 to 60 hours at 28 to 30°C, an aeration rate of 0.6 to 1.0 vvm, and a stirring speed of 150 to 200 rpm.

[0032] Further, by mass percentage, the fermentation medium comprises the following components: 1.5% to 3.0% corn cob powder, 2.0% to 3.5% defatted soybean meal, 0.5% to 1.5% glucose, 0.2% to 0.4% potassium dihydrogen phosphate, 0.05% to 0.1% magnesium sulfate heptahydrate, 0.02% to 0.05% calcium chloride, with the balance being water, and an initial pH of 5.5 to 6.0.

[0033] Corn cob powder (1.5%~3.0%) serves as the main carbon source, and its rich lignocellulose structure can continuously induce the synthesis of protease systems. Defatted soybean meal (2.0%~3.5%) serves as a complex nitrogen source, providing both the amino acids required for cell growth and acting as a protein substrate to induce protease secretion. Glucose (0.5%~1.5%) serves as a readily available carbon source, ensuring the energy needs of the cells in the early stages of growth. Among the inorganic salt components, potassium dihydrogen phosphate (0.2%~0.4%) maintains the balance of cellular phosphorus metabolism, magnesium sulfate heptahydrate (0.05%~0.1%) provides essential cofactors for enzymatic reactions, while calcium chloride (0.02%~0.05%) significantly enhances the thermal stability and catalytic efficiency of proteases.

[0034] An inoculum size of 5% to 10% ensures rapid establishment of bacterial dominance; a culture temperature of 28 to 30°C is optimal for bacterial metabolism; an aeration rate of 1:0.6 to 1.0 vvm combined with a stirring speed of 150 to 200 rpm creates a suitable dissolved oxygen environment; and a fermentation cycle of 48 to 60 hours fully covers the dynamic processes of bacterial growth, protease synthesis, and secretion.

[0035] Unlike traditional methods that use a single nitrogen source or expensive inducers, this invention innovatively employs a combined formulation of corn cob powder and defatted soybean meal, which complement each other functionally: the complex fibrous structure of corn cob powder continuously induces enzyme synthesis, while defatted soybean meal simultaneously provides nitrogen nutrition and a protein-inducing substrate. In particular, the addition of calcium chloride, although in a small amount, significantly enhances the stability of the protease.

[0036] Further, in S200, the degraded mixture is inactivated at 85~95℃ for 5~10 minutes, then centrifuged at 8000~12000r / min for 10~20 minutes, and the supernatant is collected.

[0037] After co-degradation, a large number of active proteases and cellulases remain in the fermentation system. If not inactivated in time, these enzymes will continue to act on the generated small peptides, causing changes in product composition and affecting the homogeneity and stability of the final product. Treating the product at 85–95°C for 5–10 minutes rapidly induces irreversible denaturation of the higher-order structure of the enzyme protein, thus completely terminating the enzymatic reaction. This temperature window was rigorously verified: temperatures below 85°C may lead to incomplete inactivation, while temperatures above 95°C may cause excessive thermal denaturation of the small peptides, impairing their biological activity. Subsequent centrifugation at 8000–12000 rpm for 10–20 minutes is based on a comprehensive consideration of solid-phase particle size and liquid-phase viscosity. Under this centrifugal force, cell debris, incompletely degraded eggshell membrane particles, and other insoluble impurities can settle efficiently, while ensuring that the target small peptides in the supernatant are retained to the maximum extent in the liquid phase, laying the foundation for subsequent purification steps.

[0038] Furthermore, in S300, the supernatant is first pre-filtered using a 0.2~0.45μm filter membrane to obtain a clear small molecule peptide solution; then, it is concentrated by ultrafiltration using a 3~10kD filter membrane to obtain a concentrated and clear small molecule peptide solution.

[0039] After inactivation and centrifugation, the supernatant, in addition to the target small molecule peptides, still contained bacterial fragments, residual proteins, and polysaccharides. Initial filtration was performed using a microfiltration membrane with a pore size of 0.2–0.45 μm. This pore size range effectively retained suspended particles and microbial residues larger than 0.2 μm, while ensuring complete permeation of small molecule peptides with molecular weights below several thousand Daltons, achieving primary clarification. Subsequent ultrafiltration membrane separation, ranging from 3 to 10 kDa, constituted the core of the process. This carefully designed molecular weight cutoff effectively retained the target small molecule peptides while efficiently removing residual salts, monosaccharides, and peptides with excessively small molecular weights. This staged separation strategy not only significantly improved product purity but also simultaneously concentrated and enriched the material, creating favorable conditions for subsequent drying processes.

[0040] Compared to traditional single-membrane separation or centrifugation techniques, this two-stage membrane separation system constructs a progressive purification path: the microfiltration stage prioritizes the removal of physical impurities, creating a stable operating environment for subsequent ultrafiltration; the ultrafiltration stage precisely controls the molecular weight limit to achieve selective enrichment of the target product. Particularly noteworthy is the determination of the 3–10 kDa molecular weight cutoff range. This is not a standard specification for conventional ultrafiltration membranes, but rather an optimized range specifically selected based on the molecular weight distribution characteristics of the target small peptide. This avoids product loss due to an excessively small molecular weight cutoff, while also preventing impurity residues introduced by an excessively large molecular weight cutoff.

[0041] Furthermore, in the S400, the inlet air temperature of the low-temperature spray drying method is 55~75℃, and the outlet air temperature is 30~50℃.

[0042] The inlet air temperature is 55~75℃, which provides sufficient heat to rapidly evaporate moisture from the surface of the atomized droplets, forming dry powder particles. Simultaneously, this temperature is far below the denaturation critical point of most proteins, effectively preventing damage to the peptide chain structure. The outlet air temperature is controlled at 30~50℃, ensuring the complete preservation of the stereoconformation and biological activity of small molecule peptides.

[0043] A small molecule peptide prepared by the method described above based on the synergistic degradation of eggshell membrane by mold to form a small molecule peptide, wherein the molecular weight of the small molecule peptide is 700~1500 Da.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] 1. This invention utilizes microbial fermentation to replace traditional chemical methods or expensive pure enzymatic hydrolysis, fundamentally avoiding the use of strong acids and alkalis and the resulting environmental pollution problems. It also significantly reduces production costs, meeting the requirements of green and sustainable development. Furthermore, through the synergistic degradation of two types of mold, this invention achieves a more thorough transformation of the eggshell membrane, significantly increasing the yield of small molecule peptides and achieving a total nitrogen recovery rate of up to 85.2%. The product has a concentrated molecular weight distribution of 700-1500 Da, exhibiting excellent absorption and bioavailability. Simultaneously, the yield of mucopolysaccharides (calculated as D-glucuronic acid) is 19.7-22.5 mg / g, and it also possesses strong antioxidant activity, making it applicable to products for repair and anti-oxidation.

[0046] 2. This invention utilizes mild process conditions throughout, ensuring the full retention of bioactive components in the product and guaranteeing its functional value. The unit operations employed in this process, such as fermentation, membrane separation, and spray drying, are all mature technologies with well-defined and stable process parameters, facilitating large-scale production. Detailed Implementation

[0047] The present invention will now be further described.

[0048] Example 1

[0049] The fermentation medium for Trichoderma reesei, by mass percentage, comprises the following components: 3.0% pretreated corn stalk microcrystals, 1.5% soybean meal, 0.3% ammonium sulfate, 0.5% potassium dihydrogen phosphate, 0.08% magnesium sulfate heptahydrate, 0.15% Tween-80, with the balance being water;

[0050] The preparation method of Trichoderma reesei fermentation broth includes the following steps:

[0051] Step 1: Inoculate *Trichoderma reesei* onto PDA slant agar and incubate at 25°C for 6 days until spores are fully developed. Wash the spores with sterile physiological saline, remove residual hyphae using sterile absorbent cotton, prepare a spore suspension, and adjust the concentration to 5 × 10⁻⁶. 7 CFU / mL. Inoculate with seed culture medium (PDB medium) at an inoculation rate of 8% and culture at 25°C and 200 rpm for 42 hours to obtain seed solution.

[0052] Step 2: Inoculate the seed culture into the fermentation medium at an inoculation rate of 7%, and ferment for 65 hours at 28°C, an aeration rate of 1 vvm, and a stirring speed of 200 rpm.

[0053] Step 3: After fermentation, remove mycelium and insoluble impurities by centrifugation (6000 rpm, 18 min), and collect the supernatant to obtain the Trichoderma reesei fermentation broth. Store at 4℃ for later use.

[0054] Example 2

[0055] The fermentation medium for Trichoderma reesei, by mass percentage, comprises the following components: 2.0% pretreated corn stalk microcrystals, 1.0% soybean meal, 0.2% ammonium sulfate, 0.3% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.1% Tween-80, with the balance being water;

[0056] The preparation method of Trichoderma reesei fermentation broth includes the following steps:

[0057] Step 1: Inoculate *Trichoderma reesei* onto PDA slant agar and incubate at 26°C for 5 days until spores are fully developed. Wash the spores with sterile physiological saline, remove residual hyphae with sterile absorbent cotton, prepare a spore suspension, and adjust the concentration to 1×10⁻⁶. 7 CFU / mL. Inoculate with seed culture medium (PDB medium) at a 7% inoculum and culture at 26℃ and 180 rpm for 36 hours to obtain seed solution.

[0058] Step 2: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 5%, and ferment for 60 hours at 29°C, an aeration rate of 0.8 vvm, and a stirring speed of 180 rpm.

[0059] Step 3: After fermentation, remove mycelium and insoluble impurities by centrifugation (4000 rpm, 15 min), and collect the supernatant to obtain the Trichoderma reesei fermentation broth. Store at 4℃ for later use.

[0060] Example 3

[0061] The fermentation medium for Trichoderma reesei, by mass percentage, comprises the following components: 4.0% pretreated corn stalk microcrystals, 2.0% soybean meal, 0.5% ammonium sulfate, 0.6% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, 0.2% Tween-80, with the balance being water;

[0062] The preparation method of Trichoderma reesei fermentation broth includes the following steps:

[0063] Step 1: Inoculate *Trichoderma reesei* onto PDA slant agar and incubate at 25°C for 7 days until spores are fully developed. Wash the spores with sterile physiological saline, remove residual mycelia using sterile absorbent cotton, and finally prepare a spore concentration of 1×10⁻⁶. 8 A spore suspension of CFU / mL was used as the inoculum for seed culture preparation. The inoculum was inoculated into seed culture medium (PDB medium) at a rate of 9% and cultured at 26°C with shaking at 220 rpm for 48 hours to obtain the seed culture.

[0064] Step 2: Inoculate the seed culture into the fermentation medium at an inoculation rate of 10%, and ferment for 72 hours at 30°C, aeration rate of 1.2 vvm, and stirring speed of 220 rpm.

[0065] Step 3: After fermentation, remove mycelium and insoluble impurities by centrifugation (8000 rpm, 20 min), and collect the supernatant to obtain the Trichoderma reesei fermentation broth. Store at 4℃ for later use.

[0066] In Examples 1-3, the pretreated corn stalk microcrystals were prepared using the following specific methods:

[0067] Step 1: Mix dried and pulverized corn stalks with a 2.5% sulfuric acid solution at a solid-liquid ratio of 1:10, and treat in a closed reactor at 135°C for 70 minutes. After treatment, separate the solid and liquid phases by pressure filtration. The solid component is repeatedly washed with deionized water until neutral to remove residual acid and fermentation inhibitors.

[0068] Step 2: Adjust the moisture content of the washed wet straw to 10%, place it in a steam explosion device, and introduce saturated steam to raise the system pressure to 2.2 MPa. Maintain this pressure for 200 seconds, then release the pressure instantly to complete the explosion process. The exploded material is then dried, crushed, and passed through an 80-mesh sieve to obtain pretreated corn straw microcrystals.

[0069] The specific surface area of ​​the pretreated corn stalk microcrystals is 220 m² / g, and the moisture content is 7.5%.

[0070] Example 4

[0071] The fermentation medium for Aspergillus niger, by mass percentage, comprises the following components: 2.2% corn cob powder, 2.8% defatted soybean meal, 1.0% glucose, 0.3% potassium dihydrogen phosphate, 0.07% magnesium sulfate heptahydrate, 0.04% calcium chloride, with the balance being water, and an initial pH of 5.8.

[0072] The preparation method of Aspergillus niger fermentation broth includes the following steps:

[0073] Step 1: Inoculate Aspergillus niger onto PDA slant agar and incubate at 28°C for 4.5 days. Wash off the spores with sterile physiological saline, remove residual hyphae with sterile absorbent cotton, and prepare a spore suspension (concentration 5×10⁻⁶). 7 (CFU / mL). Inoculate the seed culture medium at a rate of 7% and incubate at 29°C with shaking at 200 rpm for 42 hours to obtain the seed solution.

[0074] Step 2: Inoculate the seed culture into the fermentation medium at an inoculation rate of 8%, and ferment for 55 hours at 29°C, an aeration rate of 0.8 vvm, and a stirring speed of 180 rpm.

[0075] Step 3: After fermentation, remove mycelium and insoluble impurities by centrifugation (5500 rpm, 17 min), and collect the supernatant to obtain the Aspergillus niger fermentation broth. Store at 4℃ for later use.

[0076] Example 5

[0077] The fermentation medium for Aspergillus niger, by mass percentage, comprises the following components: 1.5% corn cob powder, 2.0% defatted soybean meal, 0.5% glucose, 0.2% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.02% calcium chloride, with the balance being water, and an initial pH of 5.5.

[0078] The preparation method of Aspergillus niger fermentation broth includes the following steps:

[0079] Step 1: Inoculate Aspergillus niger onto PDA slant agar and incubate at 28°C for 4 days. Wash off the spores with sterile physiological saline, remove residual hyphae with sterile absorbent cotton, and prepare a spore suspension (concentration 1×10⁻⁶). 7 (CFU / mL). Inoculate the seed culture medium at a 6% inoculum and incubate at 28°C with shaking at 180 rpm for 36 hours to obtain the seed solution.

[0080] Step 2: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 5%, and ferment for 48 hours at 28℃, aeration rate of 0.6 vvm, and stirring speed of 150 rpm.

[0081] Step 3: After fermentation, remove mycelium and insoluble impurities by centrifugation (4000 rpm, 15 min), and collect the supernatant to obtain the Aspergillus niger fermentation broth. Store at 4℃ for later use.

[0082] Example 6

[0083] The fermentation medium for Aspergillus niger, by mass percentage, comprises the following components: 3.0% corn cob powder, 3.5% defatted soybean meal, 1.5% glucose, 0.4% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, 0.05% calcium chloride, with the balance being water, and an initial pH of 6.0.

[0084] The preparation method of Aspergillus niger fermentation broth includes the following steps:

[0085] Step 1: Inoculate Aspergillus niger onto PDA slant agar and incubate at 30°C for 5 days. Wash off the spores with sterile physiological saline, remove residual hyphae with sterile absorbent cotton, and prepare a spore suspension (concentration 1×10⁻⁶). 7 (CFU / mL). Inoculate the seed culture medium at an 8% inoculum and culture at 30°C with shaking at 220 rpm for 48 hours to obtain the seed solution.

[0086] Step 2: Inoculate the seed culture into the fermentation medium at an inoculation rate of 10%, and ferment for 60 hours at 30°C, an aeration rate of 1.0 vvm, and a stirring speed of 200 rpm.

[0087] Step 3: After fermentation, remove mycelium and insoluble impurities by centrifugation (8000 rpm, 20 min), and collect the supernatant to obtain the Aspergillus niger fermentation broth. Store at 4℃ for later use.

[0088] Example 7

[0089] A method for preparing small molecule peptides based on the synergistic degradation of eggshell membrane by mold includes the following steps:

[0090] S100. Clean the eggshell membrane with deionized water, dry it at 50°C to constant weight, and then crush it to 80 mesh using a pulverizer; mix the pretreated eggshell membrane with the mold fermentation liquid for synergistic degradation.

[0091] Temperature 42℃, pH 6.0, degradation time 30 hours; the mass ratio of eggshell membrane to mold fermentation broth is 1:5; in the mold fermentation broth, the volume ratio of Trichoderma reesei fermentation broth (prepared by the method of Example 1) to Aspergillus niger fermentation broth (prepared by the method of Example 4) is 1:1.2.

[0092] S200. Inactivate the degraded mixture at 90°C for 8 minutes, then centrifuge at 10000 r / min for 15 minutes and collect the supernatant.

[0093] S300: First, the supernatant is preliminarily filtered using a 0.2μm filter membrane to obtain a clear small molecule peptide solution; then, it is concentrated by ultrafiltration using a 6kD filter membrane to obtain a concentrated and clear small molecule peptide solution.

[0094] S400: The purified small molecule peptide solution is dried using a low-temperature spray drying method. The inlet air temperature is 65℃, the outlet air temperature is 40℃, the solid content is 15%, and the feed rate is 3.5L / h to obtain small molecule peptide powder.

[0095] Example 8

[0096] A method for preparing small molecule peptides based on the synergistic degradation of eggshell membrane by mold includes the following steps:

[0097] S100. Clean the eggshell membrane with deionized water, dry it at 55℃ to constant weight, and then crush it to 100 mesh using a pulverizer; mix the pretreated eggshell membrane with the mold fermentation liquid for synergistic degradation.

[0098] Temperature 35℃, pH 5.0, degradation time 12 hours; the mass ratio of eggshell membrane to mold fermentation broth is 1:4; in the mold fermentation broth, the volume ratio of Trichoderma reesei fermentation broth (prepared by the method of Example 1) to Aspergillus niger fermentation broth (prepared by the method of Example 4) is 1:1.

[0099] S200. Inactivate the degraded mixture at 85°C for 5 minutes, then centrifuge at 8000 r / min for 10 minutes and collect the supernatant.

[0100] S300: First, the supernatant is initially filtered using a 0.45μm filter membrane to obtain a clear small molecule peptide solution; then, ultrafiltration is performed using a 3kD filter membrane to obtain a concentrated and clear small molecule peptide solution.

[0101] S400: The purified small molecule peptide solution is dried using a low-temperature spray drying method. The inlet air temperature is 55℃, the outlet air temperature is 30℃, the solid content is 10%, and the feed rate is 4.5L / h to obtain small molecule peptide powder.

[0102] Example 9

[0103] A method for preparing small molecule peptides based on the synergistic degradation of eggshell membrane by mold includes the following steps:

[0104] S100. Clean the eggshell membrane with deionized water, dry it at 60℃ to constant weight, and then pulverize it to 120 mesh using a pulverizer. Mix the pretreated eggshell membrane with the mold fermentation broth for synergistic degradation.

[0105] Temperature 50℃, pH 7.0, degradation time 48 hours; the mass ratio of eggshell membrane to mold fermentation broth is 1:6; in the mold fermentation broth, the volume ratio of Trichoderma reesei fermentation broth (prepared by the method of Example 1) to Aspergillus niger fermentation broth (prepared by the method of Example 4) is 1:1.5.

[0106] S200. Inactivate the degraded mixture at 95°C for 10 minutes, then centrifuge at 12000 r / min for 20 minutes and collect the supernatant.

[0107] S300: First, the supernatant is preliminarily filtered using a 0.2μm filter membrane to obtain a clear small molecule peptide solution; then, it is concentrated by ultrafiltration using a 10kD filter membrane to obtain a concentrated and clear small molecule peptide solution.

[0108] S400: The purified small molecule peptide solution is dried using a low-temperature spray drying method. The inlet air temperature is 75℃, the outlet air temperature is 50℃, the solid content is 20%, and the feed rate is 2.5L / h to obtain small molecule peptide powder.

[0109] Comparative Example 1

[0110] In S100, only the Trichoderma reesei fermentation broth (prepared by the method in Example 1) was used, and its volume was the same as the total volume of the two fermentation broths in Example 7. Other steps and parameters were the same as in Example 7.

[0111] Comparative Example 2

[0112] In S100, only Aspergillus niger fermentation broth (prepared by the method in Example 1) was used, and its volume was the same as the total volume of the two fermentation broths in Example 7. Other steps and parameters were the same as in Example 7.

[0113] Comparative Example 3

[0114] In S100, no mold fermentation broth is used. The pretreated eggshell membrane is mixed with phosphate buffer (pH 6.0, 1:5 by mass), and 2% commercially available neutral protease (enzyme activity 100,000 U / g) is added. The mixture is then degraded at 42°C for 30 hours. Other steps and parameters are the same as in Example 7.

[0115] Comparative Example 4

[0116] In S100, mold fermentation broth is not used. The pretreated eggshell membrane is mixed with a 1 mol / L NaOH solution (mass ratio 1:10), hydrolyzed at 80°C for 4 hours, and then neutralized with hydrochloric acid to pH 7.0. Other steps and parameters are the same as in Example 7.

[0117] Comparative Example 5

[0118] The volume ratio of Trichoderma reesei fermentation broth to Aspergillus niger fermentation broth in S100 is 1:0.5, and the rest is the same as in Example 7.

[0119] Comparative Example 6

[0120] The volume ratio of Trichoderma reesei fermentation broth to Aspergillus niger fermentation broth in S100 is 1:2, and the rest is the same as in Example 7.

[0121] Comparative Example 7

[0122] The Aspergillus niger fermentation broth was prepared according to the method of Example 4, but calcium chloride was not added to the fermentation medium. The other components and preparation method were the same as in Example 4.

[0123] The preparation method of small molecule peptides is the same as in Example 7.

[0124] The method for determining the molecular weight distribution in this invention is as follows:

[0125] The molecular weight distribution of small peptides was determined by high-performance gel filtration chromatography (HPLC). Chromatographic conditions: TSKgel G2000SWXL column (7.8 mm × 300 mm), mobile phase: acetonitrile-water-trifluoroacetic acid (40:60:0.1, v / v / v), flow rate: 0.5 mL / min, detection wavelength: 220 nm, column temperature: 30 °C. A standard curve was plotted using cytochrome C (12500 Da), aprotinin (6500 Da), bacitracin (1450 Da), oxidized glutathione (612 Da), and reduced glutathione (307 Da) as standards, as well as the logarithm of molecular weight versus retention time. Small peptide samples prepared in Examples 7-9 and Comparative Examples 1-7 were diluted with the mobile phase and injected for analysis. The relative content of each molecular weight range was calculated based on the standard curve.

[0126] The performance of the small molecule peptides prepared by the methods in Examples 7-9 and Comparative Examples 1-7 is shown in Table 1.

[0127] Table 1. Performance of small molecule peptides prepared by the methods of Examples 7-9 and Comparative Examples 1-7

[0128]

[0129] As shown in Table 1, the yields of small molecule peptides in Examples 7-9 ranged from 80.5% to 85.2%, with the proportion of peptides with a molecular weight <1000 Da ranging from 85.1% to 89.2%, indicating that the eggshell membrane conversion of the present invention was thorough and the target product was highly enriched. The yield of mucopolysaccharides (calculated as D-glucuronic acid) was 19.7-22.5 mg / g, and the strong antioxidant activity further demonstrates that the process conditions of the present invention maximized the yield of the main product while optimally preserving the structure and function of the bioactive components.

[0130] In Comparative Examples 1 and 2, a single mold was used to degrade the eggshell membrane, and the yields of small molecule peptides were 69.6% and 64.2%, respectively, which were far lower than the synergistic degradation by Trichoderma reesei and Aspergillus niger in Example 7. This indicates that the two molds in this invention have a synergistic effect on the degradation of the eggshell membrane.

[0131] Example 7 outperformed Comparative Example 3 (enzymatic hydrolysis) and Comparative Example 4 (chemical hydrolysis) in all aspects. In particular, compared to Comparative Example 4, Example 7 showed a yield approximately 25% higher and an antioxidant activity (IC50) more than doubled. This demonstrates that the technical approach of this invention combines high efficiency with high activity retention. While the enzymatic hydrolysis method (Comparative Example 3) is better than the chemical method, it is costly. The chemical hydrolysis method (Comparative Example 4) involves harsh conditions that not only destroy the product's activity but also cause environmental pollution. This invention utilizes microbial fermentation to achieve a green, low-cost, and highly efficient balance.

[0132] The key performance indicators of Example 7 (ratio 1:1.2) were all superior to those of Comparative Examples 5 (1:0.5) and 6 (1:2), which had unbalanced ratios. In Comparative Example 5, the proportion of *Aspergillus niger* was too low, resulting in insufficient protease production and making protein cleavage a bottleneck. In Comparative Example 6, the proportion of *Trichoderma reesei* was too low, leading to insufficient breakdown of the fiber structure and limiting the protease's activity. The ratio in Example 7 achieved an optimal balance between the production and activity of the two enzyme systems, maximizing degradation efficiency.

[0133] Even using the same dual-strain synergistic process, the comparative example 7, which did not contain calcium chloride in the Aspergillus niger fermentation medium, still showed significantly lower performance indicators than Example 7. This demonstrates that trace amounts of calcium chloride play a crucial role in enhancing protease stability and catalytic efficiency.

[0134] Example 10

[0135] A facial mask liquid containing small molecule peptides prepared by the method of Example 7 and its efficacy verification:

[0136] 1. Preparation of the facial mask liquid:

[0137] By mass percentage, it includes the following components:

[0138] Small molecule peptide (prepared by the method in Example 7) 3.0%; glycerol 8.0%; butylene glycol 5.0%; sodium hyaluronate 0.2%; xanthan gum 0.3%; 1,2-pentanediol 3.0%; p-hydroxyacetophenone 0.5%; balance: deionized water.

[0139] The preparation method is as follows:

[0140] Add glycerin, butylene glycol, sodium hyaluronate, and xanthan gum to deionized water, heat to 80°C and stir until dissolved; cool to below 40°C, add the small molecule peptide from Example 7, 1,2-pentanediol, and p-hydroxyacetophenone, and stir until dissolved; adjust the pH to 6.0±0.5 to obtain the mask solution.

[0141] 2. Verification of antioxidant efficacy:

[0142] The determination was performed according to ISO 11930:2019, "Determination of antioxidant activity in cosmetics – DPPH free radical scavenging method". The specific steps are as follows:

[0143] Prepare a 0.2 mmol / L DPPH ethanol solution;

[0144] Mix 2.0 mL of sample solution with 2.0 mL of DPPH solution and react at room temperature in the dark for 30 min.

[0145] The absorbance was measured at 517 nm, and the DPPH scavenging rate was calculated according to the formula.

[0146] The experimental group consisted of the mask solution from Example 7 (diluted 10 times with deionized water, with a small molecule peptide concentration of 3.0 mg / mL); the blank group consisted of the mask solution without added small molecule peptides (the formula was the same as the experimental group, but deionized water was added to bring the concentration to 100%); the positive control group consisted of a 0.1 mg / mL vitamin C solution. The results are shown in Table 2.

[0147] Table 2. Results of antioxidant performance determination of the small molecule peptide facial mask liquid prepared by the method in Example 7.

[0148]

[0149] As shown in Table 2, the mask liquid of Example 7 still had a DPPH scavenging rate of 72.5% after being diluted 10 times, which was significantly higher than that of the blank mask, confirming that the mask liquid has good antioxidant activity.

[0150] 3. Verification of repair efficacy:

[0151] The repair activity of the samples was detected using a cell scratch assay.

[0152] Cell culture: HaCaT human skin keratinocytes were seeded into 24-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator until the cell confluence reached more than 90%.

[0153] Scratch preparation: Use a 200μL sterile pipette tip to vertically scratch a cell-free area on the surface of a monolayer of cells, and gently wash three times with PBS to remove detached cells.

[0154] Grouped sampling:

[0155] Example 7 Mask Liquid Group: Add serum-free culture medium containing 0.5 mg / mL (based on peptides) of the Example 7 mask liquid.

[0156] Blank mask group: Add an equal volume of serum-free culture medium containing diluted blank mask solution (without small molecule peptides).

[0157] Blank control group: only an equal volume of serum-free culture medium was added.

[0158] Culture and observation: Continue culture for 24 hours, observe and photograph under an inverted microscope at 0h and 24h respectively, measure the scratch area using ImageJ software, and calculate the scratch healing rate.

[0159] Scratch healing rate (%) = (0h scratch area - 24h scratch area) / 0h scratch area × 100%, and the measurement results are shown in Table 3.

[0160] Table 3. Results of the repair efficacy test of the small molecule peptide facial mask liquid prepared by the method in Example 7.

[0161]

[0162] As shown in Table 3, the scratch healing rate of the mask liquid group in Example 7 (78.5%) was significantly higher than that of the blank mask group (45.3%) and the blank control group (42.6%), while there was no significant difference between the blank mask group and the blank control group. The results indicate that the small molecule peptides of Example 7 have activity in promoting cell migration and repair, while the mask matrix has no significant repair effect. This suggests that the small molecule peptides of Example 7 can be used to prepare mask products with repairing effects.

[0163] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing small molecule peptides based on the synergistic degradation of eggshell membrane by mold, characterized in that, Includes the following steps: S100. Mix the pretreated eggshell membrane with the mold fermentation liquid for synergistic degradation. S200. Inactivate and centrifuge the degraded mixture, and collect the supernatant; S300: Purification of small molecule peptides in the supernatant using membrane separation technology; S400. The purified small molecule peptide solution is dried using a low-temperature spray drying method to obtain small molecule peptide powder. Among them, molds in S100 include Trichoderma reesei and Aspergillus niger.

2. The preparation method according to claim 1, characterized in that, In S100, the degradation conditions are: temperature 35~50℃, pH 5.0~7.0, and degradation time 12~48 hours; the mass ratio of eggshell membrane to mold fermentation broth is 1:4~6; and the volume ratio of Trichoderma reesei fermentation broth to Aspergillus niger fermentation broth in the mold fermentation broth is 1:1~1.

5.

3. The preparation method according to claim 2, characterized in that, The preparation method of Trichoderma reesei fermentation broth includes the following: inoculating Trichoderma reesei seed liquid into the fermentation medium at an inoculation rate of 5% to 10%; fermenting at 28 to 30°C, an aeration rate of 0.8 to 1.2 vvm, and a stirring speed of 180 to 220 rpm for 60 to 72 hours.

4. The preparation method according to claim 3, characterized in that, The fermentation medium comprises the following components by weight percentage: The pretreatment consists of 2.0%–4.0% corn stalk microcrystals, 1.0%–2.0% soybean meal, 0.2%–0.5% ammonium sulfate, 0.3%–0.6% potassium dihydrogen phosphate, 0.05%–0.1% magnesium sulfate heptahydrate, 0.1%–0.2% Tween-80, with the balance being water. The pretreated corn stalk microcrystals are obtained by treating corn stalks with dilute acid and steam explosion.

5. The preparation method according to claim 2, characterized in that, The preparation method of Aspergillus niger fermentation broth includes the following: Inoculate the Aspergillus niger seed culture into the fermentation medium at an inoculation rate of 5% to 10%, and ferment for 48 to 60 hours at 28 to 30°C, an aeration rate of 0.6 to 1.0 vvm, and a stirring speed of 150 to 200 rpm.

6. The preparation method according to claim 5, characterized in that, The fermentation medium comprises the following components by mass percentage: 1.5%~3.0% corn cob powder, 2.0%~3.5% defatted soybean meal, 0.5%~1.5% glucose, 0.2%~0.4% potassium dihydrogen phosphate, 0.05%~0.1% magnesium sulfate heptahydrate, 0.02%~0.05% calcium chloride, with the balance being water, and an initial pH of 5.5~6.

0.

7. The preparation method according to claim 1, characterized in that, In S200, the degraded mixture is inactivated at 85-95℃ for 5-10 minutes, then centrifuged at 8000-12000 r / min for 10-20 minutes, and the supernatant is collected.

8. The preparation method according to claim 1, characterized in that, In the S300 process, the supernatant is first pre-filtered using a 0.2~0.45μm filter membrane to obtain a clear small molecule peptide solution; then, it is concentrated by ultrafiltration using a 3~10kD filter membrane to obtain a concentrated and clear small molecule peptide solution.

9. The preparation method according to claim 1, characterized in that, In the S400, the inlet air temperature for the low-temperature spray drying method is 55~75℃, and the outlet air temperature is 30~50℃.

10. A small molecule peptide prepared by the method according to any one of claims 1 to 9, based on the synergistic degradation of eggshell membrane by mold to form small molecule peptides, characterized in that, The molecular weight of the small molecule peptide is 700~1500 Da.

Images