Preparation method of collagen micromolecule active peptide and bioactive peptide for inhibiting cancer cell proliferation and improving intracellular environment

Abstract

The invention discloses a preparation method of collagen micromolecule active peptide and bioactive peptide for inhibiting cancer cell proliferation and improving intracellular environment, and belongs to the technical field of bioactive peptide preparation and biological functional raw materials. According to the method, bone collagen peptide, cartilage peptide (hydrolyzed II-type collagen peptide), collagen peptide (German Jilida), non-denatured II-type collagen peptide and yak bone marrow peptide are taken as core raw materials, and optional ingredients such as bovine bone marrow peptide powder, shark cartilage peptide and Belgian imported inulin can be compounded as required; through a step-by-step preparation process comprising the steps of graded purification pretreatment of raw materials, precise proportioning and mixing, low-temperature activation and synergism and stable micro-capsule embedding, structure protection and efficacy synergy of each collagen small-molecule active peptide and bioactive components are realized; the prepared bioactive peptide product can significantly inhibit cancer cell proliferation, regulate intracellular oxidation-reduction balance and improve intracellular microenvironment, the preparation process is mild and free of high-temperature damage, the raw material biological activity retention rate is high, the raw material source is controllable, the large-scale production feasibility is high, and the bioactive peptide product can be widely applied to preparation in the fields of functional food, bioactive raw materials and the like.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide preparation and biofunctional raw material technology, specifically to a method for preparing small molecule active peptides of collagen that inhibit cancer cell proliferation and improve the intracellular environment. Background Technology

[0002] Disruption of intracellular homeostasis is a significant contributing factor to cancer cell proliferation and metastasis. Collagen-based small molecule bioactive peptides, with their high bioavailability, strong targeting, and good biocompatibility, have become a research hotspot for regulating the cellular microenvironment and inhibiting abnormal cell proliferation. Small molecule bioactive peptides from different sources, such as bone collagen peptides, cartilage peptides, and yak bone marrow peptides, can achieve anti-proliferative effects by regulating cell signaling pathways, scavenging excess free radicals within cells, and inhibiting abnormal cell metabolism. German-made Jialida collagen peptides and non-denatured type II collagen peptides, due to their unique structures, can enhance intercellular connections and improve the intracellular metabolic environment.

[0003] Currently, most existing technologies involve the preparation of single collagen peptides or simple compounding, which presents the following problems: First, the structural compatibility of different peptides is not considered during compounding, and direct mixing easily leads to peptide chain structure destruction and loss of biological activity. Second, the preparation process often uses high-temperature treatment, which cannot preserve the activity of small molecule peptides, and no activity protection treatment is performed, making the product prone to oxidation and inactivation during storage. Third, the raw material ratio is not optimized for the dual effects of "inhibiting cancer cell proliferation + improving the intracellular environment," and the components do not have a synergistic effect, resulting in a single function. Furthermore, existing peptide preparation methods do not consider the stability of water-soluble vitamins, plant extracts, and other ingredients, which can easily interact with peptides after addition, reducing the overall efficacy.

[0004] Based on this, the present invention achieves structural protection and synergistic efficacy of various small molecule active peptides of collagen by designing a mild preparation process of low-temperature pretreatment, precise compounding, inert environment activation, and microencapsulation. At the same time, it is compatible with the stable addition of a variety of functional optional ingredients. The resulting product can achieve the dual effects of inhibiting cancer cell proliferation and improving the intracellular environment. It solves the problems of peptide activity loss, poor compounding synergy, and low ingredient compatibility in the prior art, and provides a new solution for the industrial preparation of highly active collagen bioactive peptides. Summary of the Invention

[0005] Purpose of the invention The purpose of this invention is to provide a method for preparing small molecule active peptides and bioactive peptides of collagen that inhibit cancer cell proliferation and improve the intracellular environment. This method is mild, can efficiently retain the bioactivity of raw materials, and the components of the obtained product have strong synergistic effects. It has the dual effects of inhibiting cancer cell proliferation and regulating intracellular homeostasis. In addition, the raw materials have high compatibility and functional ingredients can be flexibly added, making it highly feasible for industrialization. Technical solution

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for preparing small molecule active peptides and bioactive peptides of collagen that inhibit cancer cell proliferation and improve the intracellular environment includes four steps: raw material pretreatment, precise raw material compounding, low-temperature inert environment activation, microencapsulation, and finished product preparation, as detailed below: 1. Raw material pretreatment: The core raw materials, namely bone collagen peptide, cartilage peptide (hydrolyzed type II collagen peptide), collagen peptide (Germany's Jialida), non-denatured type II collagen peptide, and yak bone marrow peptide, were sieved through a 100-120 mesh sieve to remove impurities and dried in a low-temperature vacuum drying oven at 3-6℃ until the moisture content was ≤4.5%, thus obtaining the pretreated core raw materials. The core raw materials are all small molecule active peptides with a molecular weight of 500-1000 Da and a purity of ≥92%. Peptides in this molecular weight range can be directly absorbed by cells, have high bioavailability, and high purity can avoid the influence of impurities on peptide activity. 2. Precise Raw Material Compounding: By weight, take 22-40 parts of pretreated core raw materials: bone collagen peptide, 16-32 parts of cartilage peptide (hydrolyzed type II collagen peptide), 9-20 parts of collagen peptide (Germany, Jialida), 7-16 parts of non-denatured type II collagen peptide, and 6-13 parts of yak bone marrow peptide. Place them in a three-dimensional mixer and mix for 15-25 minutes at a speed of 180-220 r / min until homogeneous to obtain the core compound. If optional ingredients are required, add 1-7 parts by weight of the optional ingredients and mix with the core compound. The ingredients are uniform and can be selected from one or more combinations of bovine bone marrow peptide powder, shark cartilage peptide, inulin (imported from Belgium), acerola cherry powder, vitamin B1, vitamin B2, taurine, and vitamin C. The preferred core ingredients by weight are 30 parts of bone collagen peptide, 22 parts of cartilage peptide (hydrolyzed type II collagen peptide), 15 parts of collagen peptide (Germany's Galida), 10 parts of non-denatured type II collagen peptide, and 8 parts of yak bone marrow peptide. Under this ratio, the peptides have the strongest synergistic effect and the best effect on inhibiting cancer cell proliferation and improving the intracellular environment. 3. Low-temperature inert environment activation: Transfer the compound to a sealed activation vessel, purge the air inside the vessel with nitrogen or argon, control the inert gas purging rate to 0.4–1.2 L / min, maintain the activation environment temperature at 30–36℃ and the stirring rate at 120–160 r / min, and activate at this temperature for 50–90 min to obtain the activated compound. When the added optional ingredients include heat-sensitive components such as vitamin C and acerola cherry powder, the activation time is shortened to 50–60 min, and the activation temperature is controlled at 30–32℃ to avoid inactivation of heat-sensitive components. The inert environment can prevent peptide oxidation, and low-temperature activation can promote the gentle binding between peptide molecules, enhance synergistic effects, and at the same time, not damage the peptide chain structure. 4. Microencapsulation and finished product preparation: The activated compound and the embedding agent are mixed evenly at a mass ratio of 10:(1-3.5), and microencapsulation is performed using spray granulation. The embedding agent is one or more combinations of cyclodextrin, maltodextrin, and gum arabic. It is preferred to use a composite embedding agent composed of β-cyclodextrin and maltodextrin at a mass ratio of 1:(2-4), as the composite embedding agent has a better embedding effect and can simultaneously improve the water solubility and stability of the product. The spray granulation process parameters are: feed rate 7-13 mL / min, atomization pressure 0.2-0.35 MPa, inlet air temperature 115-135℃, and outlet air temperature 48-62℃. After collecting the embedded particles, they are sieved through an 80-100 mesh sieve and then dried in a low-temperature drying oven until the moisture content is ≤3%, thus obtaining the collagen bioactive peptide product.

[0008] This invention also protects the collagen-based bioactive peptides prepared by the above-described method, and the application of these bioactive peptides in the preparation of functional foods that inhibit cancer cell proliferation and bioactive raw materials that improve the intracellular environment.

[0009] Mechanism of synergistic effect of raw materials The core ingredients of this invention, when precisely proportioned, exhibit a significant synergistic effect, achieving the dual benefits of inhibiting cancer cell proliferation and improving the intracellular environment. The specific mechanism is as follows: 1. Bone collagen peptides + yak bone marrow peptides: synergistically inhibit angiogenesis in cancer cells, reduce the nutrient supply to cancer cells, while clearing excess free radicals in cells, regulating the redox balance in cells, and improving the cellular metabolic environment; 2. Chondroitin peptide (hydrolyzed type II collagen peptide) + non-denatured type II collagen peptide: The two have complementary structures, which can inhibit the adhesion and proliferation signaling pathways of cancer cells, while enhancing the structural stability of normal cells and repairing the intracellular homeostasis. 3. Collagen peptides (Germany's Jialida): As a core connecting ingredient, it enhances the cell absorption efficiency of various peptides, while regulating intracellular osmotic pressure, improving the intracellular microenvironment, and strengthening the anti-proliferative effects of other peptides. 4. Synergistic effects of optional ingredients: Shark cartilage peptides can enhance the inhibitory effect on cancer cell proliferation; inulin (imported from Belgium) can regulate the intestinal microecology and indirectly improve the whole body's cellular environment; B vitamins, vitamin C, and taurine can scavenge free radicals and enhance the bioactivity of peptides; acerola cherry powder can supplement natural antioxidants and work synergistically with peptides to improve the intracellular environment. Detailed Implementation

[0011] The present invention will be further described in detail below with reference to specific embodiments. The scope of protection of the present invention is not limited to the following embodiments, and all equivalent transformations based on the technical solutions of the present invention are within the scope of protection of the present invention.

[0012] Example 1: Preparation of basic collagen-based bioactive peptides (no optional ingredients) 1. Raw material pretreatment: Take bone collagen peptides, chondroitin peptides (hydrolyzed type II collagen peptides), collagen peptides (Germany's Jialida), non-denatured type II collagen peptides, and yak bone marrow peptides (all with molecular weights of 500-1000 Da and purity of 92%), sieve them through a 100-mesh sieve, and dry them at a low temperature of 3°C under vacuum until the moisture content is 4.5% to obtain the pretreated core raw materials; 2. Raw material compounding: By weight, take 30 parts of pretreated core raw materials, 22 parts of cartilage peptides, 15 parts of German Jialida collagen peptides, 10 parts of non-denatured type II collagen peptides, and 8 parts of yak bone marrow peptides, and mix them in a three-dimensional mixer at 200 r / min for 20 min to obtain the core compound. 3. Low-temperature activation: The core compound is transferred to an activation tank, nitrogen gas is introduced at a rate of 0.8 L / min, and the mixture is activated at 32℃ and 140 r / min for 70 min to obtain the activated compound; 4. Microencapsulation: The activated compound and the β-cyclodextrin + maltodextrin composite encapsulating agent (mass ratio 1:3) are mixed at a ratio of 10:2, spray granulation (feed rate 10mL / min, atomization pressure 0.25MPa, inlet air temperature 125℃, outlet air temperature 55℃), granulated through an 80-mesh sieve, and dried at low temperature to a moisture content of 2.8% to obtain the finished product.

[0013] Example 2: Preparation of enhanced anti-proliferative collagen bioactive peptides (optional anti-proliferative ingredients added) 1. Raw material pretreatment: Same as in Example 1; 2. Raw material compounding: By weight, take 95 parts of the core compound from Example 1, add 3 parts of bovine bone marrow peptide powder and 2 parts of shark cartilage peptide (optional ingredients), and mix in a three-dimensional mixer at 200 r / min for 15 min to obtain the compound; 3. Low-temperature activation: The compound was transferred to an activation vessel, argon gas was introduced at a rate of 1.0 L / min, and the mixture was activated at 35℃ and 150 r / min for 80 min to obtain the activated compound; 4. Microencapsulation: The activated compound and β-cyclodextrin are mixed at a ratio of 10:1.5, spray granulation (feed rate 9 mL / min, atomization pressure 0.3 MPa, inlet air temperature 120℃, outlet air temperature 50℃), granulated through a 90-mesh sieve, and dried at low temperature to a moisture content of 2.5% to obtain the finished product.

[0014] Example 3: Preparation of a comprehensive improved collagen-based bioactive peptide (with optional antioxidant ingredients) 1. Raw material pretreatment: Same as in Example 1; 2. Raw material compounding: By weight, take 93 parts of the core compound of Example 1, add 2 parts of Belgian imported inulin, 2 parts of acerola cherry powder, 2 parts of vitamin C, and 1 part of taurine (optional ingredients), and mix in a three-dimensional mixer at 180 r / min for 25 min to obtain the compound; 3. Low-temperature activation: The compound was transferred to an activation vessel, nitrogen gas was introduced at a rate of 0.6 L / min, and the mixture was activated at 30℃ and 120 r / min for 55 min to obtain the activated compound; 4. Microencapsulation: The activated compound and β-cyclodextrin + gum arabic composite encapsulating agent (mass ratio 1:2) are mixed at a ratio of 10:3, spray granulation is performed (feed rate 8mL / min, atomization pressure 0.2MPa, inlet air temperature 115℃, outlet air temperature 48℃), granulated through a 100-mesh sieve, and dried at low temperature to a moisture content of 2.2% to obtain the finished product.

[0015] Beneficial effects

[0016] The preparation method and the obtained collagen-based bioactive peptides of the present invention have the following significant beneficial effects: 1. Mild process and high activity retention: The entire process adopts low-temperature pretreatment, low-temperature activation and low-temperature drying, combined with inert gas protection and microencapsulation technology to avoid the destruction of peptide structure by high temperature and oxidation. The bioactivity retention rate of the product is ≥85%, which is much higher than that of traditional preparation processes. 2. Scientific ingredient ratio and strong synergy: Through the precise ratio of core ingredients, the efficacy of each collagen small molecule active peptide is complementary, and the effect of inhibiting cancer cell proliferation and improving the intracellular environment is significantly better than that of single peptides or simple compound products. 3. High ingredient compatibility and expandable functions: Adaptable activation process parameters are designed for heat-sensitive and oxidizing ingredients, and anti-proliferation and antioxidant ingredients can be flexibly added to realize personalized expansion of product functions and meet the needs of different application scenarios; 4. Good product stability and long shelf life: Microencapsulation technology can effectively isolate the influence of air and moisture on peptides. The finished product can be stored at room temperature and sealed for 12 months with a bioactivity loss rate of ≤10%, which is far superior to unencapsulated peptide products. 5. Strong industrialization feasibility: The equipment used in the preparation process is all conventional equipment in the food and biological raw material fields, without the need for special customization. The source of raw materials is controllable and the ratio is fixed, which can realize large-scale and continuous production. Moreover, the product has no harmful additives, has high safety, and can be directly applied to functional foods, bioactive raw materials and other fields. Industrial applicability

[0018] The method for preparing collagen-based bioactive peptides of this invention has simple process steps and easily controllable parameters. The equipment used is conventional in the industry, and the core raw materials and optional ingredients are all commercially available. It can achieve large-scale and continuous production. The obtained products have high bioactivity, good stability, and clear efficacy. They can be directly used as core raw materials to prepare functional foods that inhibit the proliferation of cancer cells, or as bioactive raw materials to improve the intracellular environment in the fields of biological products and functional health products. It has good industrial application value and market prospects.

[0019] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing collagen small molecule active peptides and bioactive peptides that inhibit cancer cell proliferation and improve the intracellular environment, characterized in that, Includes the following steps:

1. Raw material pretreatment: Core raw materials including bone collagen peptides, chondroitin peptides (hydrolyzed type II collagen peptides), collagen peptides (Germany's Jialida), non-denatured type II collagen peptides, and yak bone marrow peptides were respectively sieved through a 100-120 mesh sieve to remove impurities, and then dried in a low-temperature vacuum drying oven at 3-6℃ until the moisture content was ≤4.5%, obtaining pretreated core raw materials. All core raw materials are small molecule active peptides with a molecular weight of 500-1000 Da and a purity ≥92%.

2. Precise formulation of raw materials: By weight, take 22-40 parts of the pretreated core raw materials from step 1), 16-32 parts of cartilage peptides (hydrolyzed type II collagen peptides), 9-20 parts of collagen peptides (Germany's Galida), 7-16 parts of non-denatured type II collagen peptides, and 6-13 parts of yak bone marrow peptides. Place them in a three-dimensional mixer and mix for 15-25 minutes at a speed of 180-220 r / min to obtain the core compound. If optional ingredients need to be added, add 1-7 parts by weight of optional ingredients and mix them evenly with the core compound. The optional ingredients are one or more combinations of bovine bone marrow peptide powder, shark cartilage peptides, inulin (imported from Belgium), acerola cherry powder, vitamin B1, vitamin B2, taurine, and vitamin C.

3. Low-temperature inert environment activation: Transfer the compound into a sealed activation vessel, purge the air in the vessel with nitrogen or argon, control the inert gas purging rate to 0.4-1.2 L / min, the activation environment temperature to 30-36℃, the stirring rate to 120-160 r / min, and maintain the temperature for 50-90 min to obtain the activated compound; 4. Microencapsulation and finished product preparation: The activated compound and the encapsulating agent are mixed evenly at a mass ratio of 10:(1-3.5), and microencapsulation is performed using a spray granulation method. The feed rate is controlled at 7-13 mL / min, the atomization pressure at 0.2-0.35 MPa, the inlet air temperature at 115-135℃, and the outlet air temperature at 48-62℃. The encapsulated particles are collected and dried in a low-temperature drying oven until the moisture content is ≤3%, thus obtaining the collagen bioactive peptide product. The encapsulating agent is one or more combinations of cyclodextrin, maltodextrin, and gum arabic.

2. The preparation method according to claim 1, characterized in that, The preferred weight proportions of the core raw materials mentioned in step 2) are: 30 parts of bone collagen peptide, 22 parts of cartilage peptide (hydrolyzed type II collagen peptide), 15 parts of collagen peptide (Germany Jialida), 10 parts of non-denatured type II collagen peptide, and 8 parts of yak bone marrow peptide.

3. The preparation method according to claim 1, characterized in that, In step 3), when the optional ingredients include vitamin C and acerola cherry powder, the activation time is 50-60 minutes and the activation temperature is controlled at 30-32°C.

4. The preparation method according to claim 1, characterized in that, In step 4), the encapsulating agent is a composite encapsulating agent composed of β-cyclodextrin and maltodextrin in a mass ratio of 1:(2-4).

5. The preparation method according to claim 1, characterized in that, In step 4), after spray granulation, the encapsulated particles are granulated using an 80-100 mesh sieve to remove large particles and fine powder.

6. A collagen-based bioactive peptide prepared by the preparation method according to any one of claims 1 to 5, characterized in that, This bioactive peptide can inhibit cancer cell proliferation, regulate intracellular redox balance, improve the intracellular microenvironment, and retain ≥85% of its bioactivity.

7. The application of the collagen-based bioactive peptides according to claim 6 in the preparation of functional foods that inhibit the proliferation of cancer cells.

8. The application of the collagen-based bioactive peptides according to claim 6 in the preparation of bioactive raw materials that improve the intracellular environment.