Freeze-dried composition and method for preserving biomolecules

Abstract

Provided in some embodiments of the present disclosure are methods of preserving biomolecules comprising: providing a brasenia schreberi extract wherein the brasenia schreberi extract comprises a branched polysaccharide; providing a biomolecule; mixing the brasenia schreberi extract, biomolecules and a stabilizer to obtain a preserving fluid; and freeze-drying the preservation solution. Also provided in some embodiments of the present disclosure are lyophilized compositions comprising a brasenia schreberi extract, a biomolecule, and a stabilizer. In the method, the brasenia schreberi extract is matched with the stabilizer and is blended with the biomolecules to form the preserving fluid, and then the preserving fluid is freeze-dried, so that the biomolecules are preserved in a freeze-drying manner, the tolerance to the environment during preservation can be improved, the preservation convenience is improved, the stability of the biomolecules is improved, and the preservation time is prolonged.

Description

[Technical field]

[0001] The present disclosure relates to lyophilized compositions and methods of preserving biomolecules. [Background technology]

[0002] Biomolecules are the key active materials in skin care products at this stage. In current technology, the formula for storing biomolecules is mainly preserved in the form of solutions. However, compared with traditional small molecule drugs, biomolecules with more complex structures maintain a stable tertiary structure, which is the key to exhibiting biological functions. However, their structure is easily deformed by environmental factors, thereby reducing the activity of biomolecules and shortening their shelf life.

[0003] The current problems faced by storing biomolecules in solution include: biomolecule aggregation and degradation, high environmental sensitivity, and storage condition restrictions. The following is a description of each of these issues:

[0004] Regarding the aggregation and degradation of biomolecules: As the storage time increases, the activity of biomolecules may be reduced due to oxidation, aggregation, precipitation or degradation.

[0005] Regarding environmental sensitivity: When the storage solution is exposed to high temperature (for example, above 30°C), light, or pH changes (for example, left at room temperature), biological molecules are easily denatured or inactivated.

[0006] Storage condition restrictions: Storing biomolecules in solution usually requires a refrigerated or even frozen storage environment to preserve them for a long time, but such storage environments often lead to increased costs.

[0007] Therefore, how to provide a storage method that improves the stability and storage convenience of biomolecules is a problem to be solved. [Summary of the invention]

[0008] In some embodiments of the present disclosure, a method for preserving biomolecules is provided, comprising: providing a Brasenia schreberi extract (scientific name: Brasenia schreberi), wherein the Brasenia schreberi extract comprises branched polysaccharides; providing a biomolecule; mixing the Brasenia schreberi extract, the biomolecule, and a stabilizer to obtain a preservation solution; and freeze-drying the preservation solution.

[0009] In some embodiments, the step of providing a water shield extract comprises: providing water shield; crushing the water shield to obtain water shield fragments; heating the water shield fragments to obtain a heat-treated liquid; filtering the heat-treated liquid to remove water shield residues to obtain a water shield filtrate; and freeze-drying the water shield filtrate to obtain a water shield extract.

[0010] In some embodiments, the method further comprises adjusting the pH value of the water shield filtrate to between 5 and 6.5.

[0011] In some embodiments, the Brix of the water shield filtrate is 4 to 6 degrees.

[0012] In some embodiments, in the step of mixing the Brasenia schrenkiana extract, the biomolecule and the stabilizer, the weight percentage of the preservation solution is 100%, and the weight percentage of the Brasenia schrenkiana extract is 0.001% to 0.01%.

[0013] In some embodiments, in the step of mixing the Brasenia schrenkiana extract, the biomolecule and the stabilizer, the concentration of the biomolecule is 0.25 mg / mL to 1.25 mg / mL.

[0014] In some embodiments, the step of mixing the Brasenia schrenkiana extract, the biomolecule, and the stabilizer comprises mixing the biomolecule and the stabilizer and then adding the Brasenia schrenkiana extract.

[0015] In some embodiments, the step of mixing the Brasenia schrenkiana extract, the biomolecule, and the stabilizer comprises mixing the Brasenia schrenkiana extract, the biomolecule, the stabilizer, and a preservative additive.

[0016] In some embodiments, the step of mixing the Brasenia schrenkiana extract, the biomolecule, the stabilizer, and the preservative additive comprises mixing the biomolecule, the stabilizer, and the preservative additive and then adding the Brasenia schrenkiana extract.

[0017] In some embodiments, the step of mixing the Brasenia schrenkiana extract, the biomolecule, the stabilizer and the preservative additive is divided into a basic structure forming stage and a network structure forming stage.

[0018] In some embodiments, the base structure forming stage comprises mixing biomolecules, stabilizers, and preservative additives to form a primary protection mixture, and the network structure forming stage comprises adding Brasenia schrenkiana extract to the primary protection mixture to form a dual protection mixture.

[0019] In some embodiments, the step of mixing the Brasenia schrenkiana extract, the biomolecule, the stabilizer, and the preservative additive comprises a final step, wherein the Brasenia schrenkiana extract is added last.

[0020] In some embodiments of the present disclosure, a freeze-dried composition is provided, comprising a Brasenia schrenkiana extract, a biomolecule, and a stabilizer. The Brasenia schrenkiana extract comprises branched polysaccharides.

[0021] In some embodiments, the branched polysaccharide comprises mannose, galactose, fucose, arabinose, rhamnose, or a combination thereof.

[0022] In some embodiments, the lyophilized composition further comprises a preservative additive, wherein the preservative additive comprises a salt, a polymer, a filler, or a combination thereof.

[0023] In some embodiments, the filler comprises mannitol, and the crystal form of the mannitol is β-crystalline form.

[0024] In some embodiments, the stabilizer comprises a disaccharide.

[0025] In some embodiments, the biomolecule comprises a peptide.

[0026] In some embodiments, the weight ratio of the Brasenia schrenkiana extract to the stabilizer is 1:100 to 1:200.

[0027] The present disclosure is to mix a water shield extract, a stabilizer and a biomolecule to form a preservation solution, and then freeze-dry the preservation solution to preserve the biomolecule in a freeze-dried composition. The charged characteristics of the polysaccharide functional groups in the water shield extract can be used in combination with a stabilizer to stabilize the freeze-dried biomolecule structure and extend the shelf life. In addition, the polysaccharides and carbohydrates in the water shield extract are not easily deformed and atrophied after freeze-drying, which can improve the formability of the freeze-dried composition. Moreover, polysaccharides have interfacial dispersion capabilities and can stabilize biomolecules. At the same time, the storage mode of the biomolecule is set to a freeze-dried mode, which is less sensitive to the environment, can be stored at room temperature, and maintains excellent stability, which can improve the convenience of storage.

Brief Description of the Drawings

[0028] In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the attached drawings are described as follows: Figure 1 A flow chart presenting a method of preserving biomolecules in some embodiments of the present disclosure. Figure 2A The PDGF-BB levels of the DU-1 group (the water shield solution was added first) and the DU-2 group (the water shield solution was added last) added with the water shield solution in some embodiments of the present disclosure were stored at room temperature (25° C.) for different days. Figure 2B The PDGF-BB content of the non-DU group without adding the water shield complex solution, the non-DU+T group without adding the water shield complex solution (non-DU group + trehalose), the DU-1 group with adding the water shield complex solution (non-DU group + trehalose + adding the water shield complex solution first), and the DU-2 group with adding the water shield complex solution (non-DU group + trehalose + adding the water shield complex solution last) after being stored at room temperature (25°C) for different days is exemplified in some embodiments of the present disclosure. Figure 2CThe PDGF-BB content of the non-DU group without adding the water shield solution, the non-DU+T group without adding the water shield solution, and the DU-2 group with adding the water shield solution in some embodiments of the present disclosure is shown after being stored at 45° C. for different days. FIG. 3A to FIG. 3D In some embodiments of the present disclosure, the non-DU group ( Figure 3A )、non-DU+T group( Figure 3B ), DU-1 group ( Figure 3C ) and DU-2 group ( Figure 3D ) electron microscope image. [Specific implementation method]

[0029] In order to make the description of the present invention more detailed and complete, the following describes in detail the implementation methods and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The various embodiments disclosed below can be combined or replaced with each other in beneficial situations, and other embodiments can be added to one embodiment without further description or explanation. In the following description, many specific details will be described in detail to enable the reader to fully understand the following embodiments. However, the embodiments of the present invention can be practiced without these specific details.

[0030] In this document, unless the context specifically limits the article, "a", "an" and "the" may refer to a single or multiple. It will be further understood that the words "include", "comprise", "have" and similar words used in this document specify the features, regions, integers, steps, operations, elements and / or components described therein, but do not exclude other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0031] As used herein, the term "about" means a value of a given amount that varies within 5% of the value (e.g., ±1%, ±2%, ±3%, ±4%, 5%). These values ​​are examples only and are not intended to be limiting. It should be understood that the term "about" may mean a percentage of the value of a given amount as interpreted by one of ordinary skill in the art in view of the teachings herein.

[0032] As used herein, "biomolecule" refers to a molecule derived from an organism or cell. In some embodiments, it refers to a biomolecule with a molecular weight greater than 6 kilodaltons.

[0033] Although a series of operations or steps are used below to illustrate the method disclosed herein, the order shown in these operations or steps should not be construed as a limitation of the present invention. For example, certain operations or steps may be performed in different orders and / or simultaneously with other steps. In addition, it is not necessary to perform all operations, steps and / or features to implement the embodiments of the present invention. Furthermore, each operation or step described herein may include several sub-steps or actions.

[0034] Please refer to Figure 1 Some embodiments of the present disclosure provide a method 100 for preserving biomolecules, comprising step S110, providing a Brasenia extract, wherein the Brasenia extract comprises a branched polysaccharide; step S120, providing a biomolecule; step S130, mixing the Brasenia extract, the biomolecule, and a stabilizer to obtain a preservation solution; and step S140, freeze-drying the preservation solution. Through method 100, a freeze-dried composition for preserving biomolecules is obtained to preserve the biomolecules.

[0035] In the method 100 of the present disclosure, a Brasenia schreberi extract (Brasenia schreberi scientific name) extracted from a natural product can be selected and matched with a stabilizer, mixed with a biomolecule to form a storage solution, and then freeze-dried to store the biomolecule in a freeze-dried form. Compared with a solution form, a freeze-dried composition can improve the tolerance to the environment during storage (for example, it can be stored at room temperature) and improve the convenience of storage. In addition, by matching the Brasenia schreberi extract with a stabilizer, the storage time can be further extended and the stability of the biomolecule can be improved.

[0036] Specifically, in method 100, polyelectrolyte complexes (PEC) and multilayer films are formed through the charged properties of polysaccharides (such as branched polysaccharides or glucans) in the water shield extract to protect biomolecules, and the water shield extract can be used with a stabilizer to stabilize the structure of freeze-dried biomolecules, thereby improving the storage stability of biomolecules. It is further explained that the branched polysaccharide in the water shield extract is a highly branched polysaccharide with many carboxyl groups. Its structure consists of a main chain and many branches. The main chain is based on mannose (D-mannose), and the branches include galactose (D-galactose), fucose (L-Fucose), rhamnose (L-Rhamnose), arabinose (L-arabinose) and other sugars. The high degree of branching and specific sugar residue components make the branched polysaccharides in the water shield extract have unique properties. Compared with other straight-chain polysaccharides, the branched polysaccharides in water shield extract have more functional groups (carboxyl groups) and can interact with surrounding charged ions.

[0037] In addition, the polysaccharides and carbohydrates in the water shield extract have the property of not easily deforming and shrinking after freeze-drying, which can improve the shaping effect of the preservation solution after freeze-drying. In addition, polysaccharides have interfacial dispersion ability, which can stabilize biomolecules and further replace conventional liquid surfactants, such as polysorbate, to avoid the shrinkage problem after freeze-drying when conventional liquid surfactants are used.

[0038] First, referring to step S110 , a Brasenia schrenkiana extract is provided.

[0039] In some embodiments, the water shield extract comprises branched polysaccharides (long chains with branches), the main components of which include mannose, galactose, fucose, arabinose, rhamnose or a combination thereof. In some embodiments, the water shield extract further comprises glucan and carbohydrates.

[0040] In some embodiments, the step S110 of providing a water shield extract comprises: providing water shield; crushing the water shield to obtain water shield fragments; heating the water shield fragments to obtain a heat-treated liquid; filtering the heat-treated liquid to remove water shield residues to obtain a water shield filtrate; and freeze-drying the water shield filtrate to obtain a water shield extract.

[0041] In some embodiments, the water shield is stored in acetic acid, and when it is used, it is filtered from the acetic acid to obtain the water shield. The acetic acid can maintain the freshness of the water shield and prevent it from spoiling. As for the filtered filtrate, it can be added to the subsequent water shield filtrate to increase the content of the subsequent water shield extract. In some embodiments, the water shield is selected from water shield buds and leaves, and a higher content of water shield polysaccharides can be obtained. In some embodiments, the water shield fragments are heated at a temperature of 40°C to 60°C (e.g., 40°C, 45°C, 50°C, 55°C, 60°C or a value in the aforementioned interval) for 20 minutes to 40 minutes, so that the water shield cell wall is broken and the intracellular substances (e.g., polysaccharides and carbohydrates) are released. If the temperature is too low or the time is too short, the release efficiency of the intracellular substances is poor, which affects the preservation effect of the subsequent biological molecules. If the temperature is too high or the time is too long, the molecular structure inside the water shield is destroyed, which also reduces the preservation effect of the subsequent biological molecules. In some embodiments, the step of filtering the heat-treated liquid comprises filtering with a 200-mesh to 300-mesh screen to remove the water shield residue. In some embodiments, after the step of filtering the heat-treated liquid, the water shield filtrate is centrifuged to obtain a water shield supernatant; and the water shield supernatant is dialyzed, for example, using a dialysis membrane with a pore size of 7.5 kilodaltons to 12.5 kilodaltons to dialyze the water shield supernatant for 60 hours to 84 hours to remove residual impurities or acetic acid. If the pore size of the dialysis membrane is too small or the dialysis time is too short, the impurities or acetic acid residues are high, and if the pore size of the dialysis membrane is too large or the dialysis time is too long, the water shield components may be filtered out, reducing the content of the subsequent water shield extract.

[0042] In some embodiments, method 100 further includes adjusting the pH value of the water shield filtrate to between 5 and 6.5, such as 5, 5.5, 6.0, 6.5, or values ​​in the aforementioned intervals. For example, after filtering the heat-treated liquid, an alkaline solution can be added to the water shield filtrate to adjust the pH value. It is worth emphasizing that water shield polysaccharides have polycarboxylic acids (poly (carboxylic acid)s). When the pH value is 3 to 6, as the pH increases, the polycarboxylic acids will be deprotonated to increase the total negative charge, and the carboxyl group (-COOH) will dissociate, thereby having better electrophoretic mobility and higher viscosity. Since water shield polysaccharide has a highly branched structure, as the pH value increases to between 5 and 6.5, the degree of carboxyl dissociation increases, so that water shield polysaccharide can interact with other preservation solution components to form a complex structure that protects biological molecules (for example, via hydrogen bonds or Coulomb attraction), for example, water shield polysaccharide and mannitol have more interactions. Secondly, the increase in viscosity can improve the protective effect of biological molecules after freeze-drying. If the pH value is too high (for example, higher than 6.5), it is easy to cause the native chemical substances of water shield to change and not be easy to remove, and hydrolyze the chemical bonds of polysaccharides, and precipitate some dissolved components in the water shield filtrate. If the pH value is too low, it is not conducive to the preservation of biological molecules after freeze-drying. In some embodiments, the step of adjusting the pH value of the water shield filtrate to between 5 and 6.5 includes centrifuging the water shield filtrate and filtering it again, and adjusting the pH value of the filtered water shield filtrate to 5 to 6.5.

[0043] In some embodiments, the Brix (usually expressed as Bx) of the water shield filtrate is 4 to 6 degrees, such as 4, 5, 6, or a value in the aforementioned range. If the Brix is ​​too low, it means that the sugars in the water shield are excessively lost during the extraction process. If the Brix is ​​too high, it means that the sugars in the water shield are higher than the standard state, and it is necessary to confirm whether there is a problem with the source of the water shield material or the operation process. In some embodiments, the water shield filtrate is centrifuged and then the Brix is ​​measured.

[0044] Next, referring to step S120 , biomolecules are provided.

[0045] In some embodiments, the biomolecule comprises a peptide (e.g., a growth factor) or a polymer. For example, it comprises fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) (e.g., platelet-derived growth factor-BB (PDGF-BB)), transforming growth factor Beta (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF). In some embodiments, the biomolecule comprises a protein, such as an antibody. In some embodiments, the biomolecule comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA, such as messenger RNA (mRNA)), or a combination thereof. In some embodiments, the biomolecule comprises a flavonoid, such as catechins.

[0046] Next, in step S130, the water shield extract, the biomolecules and the stabilizer are mixed to obtain a preservation solution.

[0047] In some embodiments, stabilizers can be used to inhibit degradation of biomolecules and include disaccharides (eg, sucrose, trehalose, lactose, maltose, or a combination thereof).

[0048] In some embodiments, in step S130 of mixing the water shield extract, the biomolecules, and the stabilizer, the weight percentage of the preservation solution is 100%, and the weight percentage of the water shield extract is 0.001% to 0.01%, such as 0.001%, 0.002%, 0.004%, 0.006%, 0.008%, 0.01%, or values ​​in the aforementioned range. If the weight percentage is too low, the preservation effect of the biomolecules is limited, and if the weight percentage is too high, the required extraction cost is too high. In some embodiments, in step S130 of mixing the water shield extract, the biomolecules and the stabilizer, the weight percentage of the preservation solution is 100%, and the concentration of the biomolecules is 0.25 mg / ml to 1.25 mg / ml, such as 0.25 mg / ml, 0.35 mg / ml, 0.45 mg / ml, 0.55 mg / ml, 0.65 mg / ml, 0.75 mg / ml, 0.85 mg / ml, 0.95 mg / ml, 1.05 mg / ml, 1.15 mg / ml, 1.25 mg / ml or values ​​in the aforementioned ranges. If the concentration is too high, the content is excessive, the preservation effect is limited, and the stability is poor. If the concentration is too low, the preservation cost is too high.

[0049] In some embodiments, the step S130 of mixing the water shield extract, the biomolecules and the stabilizer comprises mixing the water shield extract, the biomolecules, the stabilizer and the additive for preservation. In some embodiments, the additive for preservation comprises salts, polymers, fillers or a combination thereof. In some embodiments, the salts can be used as pH buffers to maintain a suitable pH environment, such as Tris (hydroxymethyl) aminomethane (Tris for short), or citric acid, etc. In some embodiments, the salts comprise sodium chloride, which can be used to regulate the osmotic pressure of the preservation solution to isotonic pressure to simulate the physiological environment in the body. After freeze-drying, the salt crystals also have the function of an excipient and can support the shape of the freeze-dried composition. In some embodiments, the polymer is used to disperse the biomolecules to avoid aggregation, such as comprising polyvinylpyrrolidone. In some embodiments, the filler can be used to improve the stability of the structure after freeze-drying and enhance the stability of the biomolecules, such as comprising sugar alcohols, mannitol, glycine, hydroxyethyl starch, serum albumin or a combination thereof.

[0050] In some embodiments, the step S130 of mixing the water shield extract, the biomolecules and the stabilizer includes adding the water shield extract after mixing the biomolecules and the stabilizer. Similarly, the step of mixing the water shield extract, the biomolecules, the stabilizer and the preservative additive includes adding the water shield extract after mixing the biomolecules, the stabilizer and the preservative additive. Specifically, this step is to delay the addition of the water shield extract after the other components in the solution are evenly distributed through stirring. It is worth emphasizing that compared to adding the water shield extract first, or adding it with each component at the same time, the method of mixing each component first and adding the water shield extract after each component is evenly distributed and fully reacted can utilize the charged water shield extract to disperse and encapsulate the components of the mixture, thereby preparing a preservation solution with better protection for biomolecules after freeze-drying. For example, when the preservation additive includes mannitol, after freeze-drying the preservation solution to which the Brasenia schrenkiana extract is first added, the crystal form of mannitol is the δ crystal form with poor thermal stability, whereas after freeze-drying the preservation solution to which the Brasenia schrenkiana extract is later added, the crystal form of mannitol is the β crystal form with better thermal stability. Therefore, the stability of biomolecules can be improved by selecting the order of adding the Brasenia schrenkiana extract later.

[0051] In some embodiments, the step of mixing a water shield extract, a biomolecule, a stabilizer, and a preservation additive (e.g., mannitol) is divided into a basic structure formation stage and a network structure formation stage. In some embodiments, the basic structure formation stage includes mixing biomolecules, stabilizers, and preservation additives to form a preliminary protection mixture, and the network structure formation stage includes adding the water shield extract to the preliminary protection mixture to form a double protection mixture. Through the charge carried by the water shield extract, it can interact with the preliminary protection mixture to change the structure of the preliminary protection mixture and form a double protection mixture that further stabilizes the biomolecule. In some embodiments, the step of mixing the water shield extract, biomolecules, stabilizers, and preservation additives includes a final procedure, and the final procedure is to add the water shield extract at the end. That is, no matter what component is added in this step, the water shield extract is set to be added last to ensure that double protection can be formed for the biomolecule.

[0052] Next, please refer to step S140, freeze-drying the preservation solution. Through step S140, a freeze-dried composition is obtained.

[0053] In some embodiments, the lyophilized composition comprises a Brasenia schrenkiana extract, a biomolecule, and a stabilizer.

[0054] In some embodiments, the freeze-dried preservation solution comprises a first drying step and a second drying step, wherein the first drying step comprises rapid freezing to -40°C to -60°C (e.g., -40°C, -45°C, -50°C, -55°C, -60°C, or values ​​in the aforementioned range), and the second drying step comprises gradual temperature increase to 20°C to 30°C, and continuous drying of the preservation solution. Compared to the freeze-drying method of slow cooling, the drying method of rapid cooling followed by warming can avoid structural changes in biomolecules and improve the stability of biomolecules in the freeze-dried composition.

[0055] In some embodiments, the weight ratio of the biomolecule to the Brasenia schrenkiana extract is 10:1 to 20:1, such as 10:1, 12.5:1, 15:1, 17.5:1, 20:1 or values ​​in the aforementioned intervals. If the weight ratio is too high, the protective effect on the biomolecule is limited. If the weight ratio is too low, the amount of Brasenia schrenkiana extract is too high, and the cost is high. The weight ratio of the Brasenia schrenkiana extract to the stabilizer is 1:100 to 1:200, such as 1:100, 1:125, 1:150, 1:175, 1:200 or values ​​in the aforementioned intervals. If the weight ratio is too high or too low, the protective effect on the biomolecule will be reduced. In some embodiments, when the freeze-dried composition contains a preservation additive, the weight ratio of the preservation additive to the water shield extract is 2000:1 to 3500:1, for example 2000:1, 2225:1, 2525:1, 2725:1, 3000:1, 3250:1, 3500:1, or values ​​in the aforementioned ranges. If the weight ratio is too high, the protective effect of the biological molecules is reduced, and if the weight ratio is too low, the amount of water shield extract used is too high, and the cost is high. In some embodiments, when the preservation additive includes mannitol, the weight ratio of mannitol to water shield extract is 1500:1 to 2500:1 (e.g., 1500:1, 1750:1, 2000:1, 2250:1, 2500:1 or values ​​in the aforementioned range). Since water shield extract affects the crystal form of mannitol, if the weight ratio is too high, the efficiency of water shield extract is limited, thereby reducing the protective effect on biological molecules. If the weight ratio is too low, the amount of water shield extract used is too high, resulting in high cost.

[0056] To further illustrate the methods for preserving biomolecules and freeze-dried compositions provided by various embodiments of the present disclosure, the following examples are performed. It should be noted that the following examples are provided for illustrative purposes only and are not intended to limit the present invention.

[0057] 1. Preparation method

[0058] 1.1. Preparation of Brasenia schrenkiana extract

[0059] First, 550 g of food-grade water shield preserved material (containing water shield sprouts and glacial acetic acid, wherein the water shield sprouts are 450 g, manufacturer: Sansui Bussan Co., Ltd.) was filtered using a 250-mesh sieve to separate the water shield sprouts and the first filtrate. Then, the water shield sprouts were crushed using a blender, and the crushed water shield sprouts were heated at 50° C. for 30 minutes to obtain a heat-treated liquid. Then, the heat-treated liquid was filtered using a 250-mesh sieve to remove the water shield sprout residue, and a second filtrate was collected.

[0060] The first filtrate and the second filtrate were combined to obtain a total filtrate. Then, the total filtrate was centrifuged at 2400 x g for 30 minutes at 22°C to separate the supernatant. Then, the supernatant was dialyzed for 72 hours using a dialysis membrane with a pore size of 10 kilodaltons, and the pH value of the supernatant was adjusted to 6 with a 1N sodium hydroxide solution. Then, the Brix of the supernatant with adjusted pH was detected using a sugar meter. The Brix was about 5 degrees. The appropriate range of Brix in this step was 4 to 6 degrees, indicating that the sugars in the water shield sprouts were successfully retained during the extraction process.

[0061] Next, the supernatant of the total filtrate was freeze-dried to obtain a Brasenia schrenkiana extract.

[0062] 1.2. Preparation of freeze-dried composition

[0063] According to the following Table 1, a preservation solution for preserving biomolecules was prepared (the biomolecules in this series of embodiments were growth factors separated from blood as examples), wherein the initial concentration of the growth factor was 30 mg / ml, and the water shield complex solution was prepared by dissolving 0.4 g of water shield extract in 100 g of deionized water (the weight percentage of the water shield extract in the water shield complex solution was 0.4%).

[0064] Table 1

[0065] In Table 1, the non-DU group is the control group 1 without adding the water shield solution and trehalose, the non-DU+T group is the control group 2 with added trehalose but without adding the water shield solution, the DU-1 group and the DU-2 group are both experimental groups with added water shield solution and trehalose, and the difference between the DU-1 group and the DU-2 group is the order of adding the water shield solution. The DU-1 group adds the water shield solution first among all the ingredients, and the DU-2 group adds the water shield solution last among all the ingredients.

[0066] Each group of preservation solution was filled into a drying bottle, and each group of preservation solution was freeze-dried for 48 hours using a shelf-type freeze dryer (Jinming Technology; model: FD12) to ensure that the moisture content of the finished product was less than 3%. The bottles were vacuum-sealed to obtain freeze-dried compositions containing different preservation ingredients (hereinafter referred to as non-DU group, non-DU+T group, DU-1 group and DU-2 group).

[0067] The components of the freeze-dried DU-2 group are shown in Table 2.

[0068] Table 2

[0069] 2. Stability of freeze-dried composition

[0070] 2.1. Room temperature stability test

[0071] In order to test the protective effect of freeze-dried compositions with different formulations on growth factors, the non-DU group, non-DU+T group, DU-1 group and DU-2 group were stored at 25°C for 90 days, and the PDGF-BB content of the freeze-dried compositions of each group was detected on the 0th day, the 30th day, the 60th day and the 90th day, and the measurement results were plotted as shown in the figure. Figure 2A (Comparison between DU-1 group and DU-2 group) and Figure 2B (Comparison of four groups: non-DU group, non-DU+T group, DU-1 group and DU-2 group). The results of each group are the average values ​​obtained after at least three repeated tests. Since PDGF is a common growth factor indicator of platelet extracts, the content of PDGF-BB in the growth factor was selected to evaluate the protective effect of the freeze-dried composition on the growth factor.

[0072] Figure 2A The results show that the content of PDGF-BB in the DU-2 group (the water shield solution was added last) was higher than that in the DU-1 group (the water shield solution was added first), indicating that the stability of PDGF-BB in the DU-2 group was better. Therefore, the order of adding the water shield solution affects the protective effect of the freeze-dried composition on the growth factor. The water shield solution was added last, which had a better protective effect on the growth factor and could achieve better stability of the growth factor.

[0073] Figure 2BThe results showed that compared with the non-DU group, non-DU+T group (non-DU group + trehalose), and DU-1 group (non-DU group + trehalose + adding Brasenia schrenkiana complex solution first), the DU-2 group (non-DU group + trehalose + adding Brasenia schrenkiana complex solution last) had a higher PDGF-BB content than the other three groups when stored for 60 days and 90 days, indicating that the PDGF-BB stability of the DU-2 group was better. Therefore, among the four formulas, the DU-2 group, which added Brasenia schrenkiana complex solution last, had a better protection effect on the growth factor and could achieve better stability of the growth factor.

[0074] 2.2. Accelerated stability test

[0075] In order to clarify the protective effect of freeze-dried compositions of different formulations on growth factors under long-term storage, the non-DU group, non-DU+T group, DU-1 group and DU-2 group were placed at 45°C for 90 days, and the PDGF-BB content of each freeze-dried composition (non-DU group, non-DU+T group, DU-1 group and DU-2 group) was detected on day 0, day 30, day 60 and day 90, and the measurement results were plotted as shown in the figure. Figure 2C .

[0076] It should be noted that placing the freeze-dried composition at 45°C is an accelerated stability test, from which the stability of the freeze-dried composition placed at room temperature for four times can be estimated. Therefore, the 90-day stability test result at 45°C can correspond to the stability of the freeze-dried composition placed at room temperature for one year.

[0077] Figure 2C The results showed that compared with the non-DU group and the non-DU+T group (non-DU group + trehalose), the DU-2 group (non-DU group + trehalose + finally adding the water shield solution) was stored for 60 days and 90 days (equivalent to 8 months and 1 year at room temperature), and the PDGF-BB content was higher than that of the other three groups, indicating that the PDGF-BB stability of the DU-2 group was better. Therefore, in terms of long-term stability, among the four formulas, the DU-2 group, which finally added the water shield solution, also showed a better growth factor protection effect, and the growth factor had better stability.

[0078] In addition, since disaccharides (such as trehalose) can inhibit protein unfolding and maintain vitrification, they have the property of inhibiting protein degradation. The following further explores whether the addition of trehalose can enhance the protective effect of PDGF-BB.

[0079] Please refer again Figure 2CAccording to the results of storage until the 30th day (equivalent to storage at room temperature for 4 months), it can be found that the PDGF-BB content of the non-DU+T group (addition of trehalose) and the DU-2 group (addition of trehalose + final addition of water shield solution) is higher than that of the non-DU group (no addition of trehalose). Therefore, the addition of trehalose can improve the stability of growth factors in the short term (e.g., within 4 months at room temperature). Based on this, it can be inferred that trehalose (stabilizer) and other ingredients can form a preliminary protective mixture to improve the short-term stability period.

[0080] 3. Appearance of the freeze-dried composition

[0081] To clarify whether the better growth factor protection effect of the DU-2 group is related to the structure, the appearance of the freeze-dried non-DU group, non-DU+T group, DU-1 group, and DU-2 group was observed using a scanning electron microscope. The results are shown in FIG. 3A to FIG. 3D ,in Figure 3A For non-DU group, Figure 3B For non-DU+T group, Figure 3C For DU-1 group, and Figure 3D It is the DU-2 group.

[0082] It should be noted that, generally speaking, when mannitol and protein coexist, they tend to form block crystals, also known as the δ crystal form, which is the crystal form with the highest molecular conformational energy (energy required to form a specific structure) in mannitol, and has the characteristics of high solubility but poor thermal stability. On the other hand, mannitol also has another crystal form, the β crystal form, which has low molecular conformational energy, is needle-shaped, and has better thermal stability.

[0083] FIG. 3A to FIG. 3D The results showed that the crystals of the non-DU group and the non-DU+T group were block-shaped, which was the δ crystal form of mannitol. The DU-1 group showed a block-shaped crystal form close to the needle-shaped crystal form, which was presumably a mixture of the δ crystal form and the α crystal form. The DU-2 group, which was added with the water shield solution, showed needle-shaped crystals, which was the β crystal form of mannitol. In other words, the DU-2 group showed the β crystal form and had better thermal stability, which also corresponds to the result in point 2 that the DU-2 group had better stability.

[0084] It is worth noting that the water shield solution extracted from water shield has a variety of branched polysaccharides (branched polysaccharides, for example, include mannose, galactose, fucose, rhamnose and arabinose). Because the functional groups in the polysaccharides are charged, polyelectrolyte complexes can be formed. Freezing and drying during the freeze-drying process, as well as the subsequent rehydration stage, can reduce the denaturation and aggregation of PDGF-BB, and provide PDGF-BB with full protection. In addition, the glucan in the water shield solution has the polyelectrolyte complex characteristics of polysaccharides, can electrostatically adsorb each other with oppositely charged ions, form a multilayer film, and further enhance the structural stability of biomolecules. The water shield solution is also rich in carbohydrates, and the weight and physical formability of the freeze-dried composition can be maintained after freeze-drying to avoid atrophy due to long-term storage. On the other hand, the polysaccharides contained in the water shield solution can also replace the liquid surfactant in the conventional preservation solution to achieve uniform dispersion of the ingredients and avoid the poor molding efficiency of the liquid surfactant after freeze-drying.

[0085] It is worth mentioning that by adjusting the order of adding the water shield solution, the contact action mode of other components in the preservation solution can be changed, so that the freeze-dried composition has different crystal forms. For example, in Experimental Example 3, mannitol forms different crystal forms, thereby affecting the protective effect of the freeze-dried composition on growth factors and providing better protection for PDGF-BB.

[0086] Although the present disclosure has been disclosed in the above implementation mode, it is not intended to limit the present disclosure. Anyone skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure shall be determined by the attached claims.

Claims

1. A method for preserving biomolecules, characterized in that: Include: Providing a Brasenia schrenkiana extract, wherein the Brasenia schrenkiana extract comprises branched polysaccharides; Provide biomolecules; mixing the Brasenia schrenkiana extract, the biomolecule and a stabilizer to obtain a preservation solution; and The stock solution was freeze-dried.

2. The method of claim 1, wherein the step of providing the Brasenia schrenkiana extract comprises: Provide water shield; Crush the water shield to obtain water shield fragments; heating the broken water shield to obtain a heat-treated liquid; filtering the heat-treated liquid to remove the Brasenia schrenkiana residues and obtain a Brasenia schrenkiana filtrate; as well as The Brasenia schrenkiana filtrate is freeze-dried to obtain the Brasenia schrenkiana extract.

3. The method of claim 2, further comprising adjusting the pH value of the water shield filtrate to between 5 and 6.

5.

4. The method of claim 2, wherein the Brix of the water shield filtrate is 4 to 6 degrees. 5 . The method of claim 1 , wherein in the step of mixing the water shield extract, the biomolecule and the stabilizer, the weight percentage of the water shield extract is 0.001% to 0.01% based on 100% by weight of the preservation solution.

6. The method of claim 1, wherein in the step of mixing the water shield extract, the biomolecule and the stabilizer, the concentration of the biomolecule is 0.25 mg / ml to 1.25 mg / ml based on 100% by weight of the preservation solution.

7. The method of claim 1, wherein: The step of mixing the water shield extract, the biomolecule and the stabilizer comprises mixing the biomolecule and the stabilizer and then adding the water shield extract.

8. The method of claim 1, wherein the step of mixing the water shield extract, the biomolecule, and the stabilizer comprises mixing the water shield extract, the biomolecule, the stabilizer, and a preservative additive.

9. The method of claim 8, wherein: The step of mixing the water shield extract, the biomolecule, the stabilizer and the preservative additive comprises mixing the active ingredient, the water and the preservative additive and then adding the water shield extract.

10. The method of claim 8, wherein the step of mixing the water shield extract, the biomolecule, the stabilizer and the preservative additive is divided into a basic structure forming stage and a network structure forming stage.

11. The method of claim 10, wherein the base structure forming stage comprises mixing the biomolecule, the stabilizer and the preservative additive to form a primary protection mixture, and the network structure forming stage comprises adding the Brasenia schrenkiana extract to the primary protection mixture to form a dual protection mixture.

12. The method of claim 8, wherein the step of mixing the Brasenia schrenkiana extract, the biomolecule, the stabilizer, and the preservative additive comprises a final step of adding the Brasenia schrenkiana extract at the end.

13. A freeze-dried composition, characterized in that Include: Brasenia schreberi extract, including branched-chain polysaccharides; Biomolecules; and Stabilizer. 14 . The freeze-dried composition of claim 13 , wherein the branched polysaccharide comprises mannose, galactose, fucose, rhamnose, arabinose or a combination thereof.

15. The lyophilized composition of claim 13, wherein the stabilizer comprises a disaccharide.

16. The lyophilized composition of claim 13, wherein the biomolecule comprises a peptide.

17. The freeze-dried composition of claim 13, further comprising a preservative additive, wherein the preservative additive comprises a salt, a polymer, a filler or a combination thereof. The freeze-dried composition of claim 17 , wherein the filler comprises mannitol, and the crystal form of the mannitol is β-crystal form. 19 . The freeze-dried composition of claim 18 , wherein the weight ratio of the Brasenia schrenkiana extract to the stabilizer is 1:100 to 1:200.

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