Dendrobium officinale extract, preparation method thereof and preparation

Abstract

This application belongs to the field of fermentation technology and discloses an extract of Dendrobium officinale and its preparation method and formulation. The preparation method includes the following steps: Dendrobium officinale is crushed and mixed with water to obtain a mixed solution; citrate is added to the mixed solution, and the mixture is stirred under weakly acidic conditions to obtain a pretreated solution; a polyol stabilizer is then added, and the mixture is stirred to obtain an ion-treated solution; a flocculant is added to the ion-treated solution, and the mixture is stirred and filtered to obtain a supernatant and a filter residue; an enzyme inactivating agent is added to the supernatant, and the mixture is stirred and allowed to stand under acidic conditions to obtain an enzyme-treated solution; the filter residue is washed with deionized water to obtain a fermentation substrate; calcium salt is added to the enzyme-treated solution, and the mixture is stirred to obtain a pre-fermentation solution; fermentation inoculum and fermentation substrate are added to the pre-fermentation solution, and the mixture is subjected to anaerobic fermentation, inactivation, centrifugation, and filtration to obtain the Dendrobium officinale extract. The above method effectively increases the content of polysaccharides and polypeptides in the Dendrobium officinale extract.

Description

Technical Field

[0001] This application relates to the field of fermentation technology, specifically to an extract of Dendrobium officinale and its preparation method and formulation. Background Technology

[0002] Dendrobium officinale, a perennial herb belonging to the genus Dendrobium in the Orchidaceae family, is rich in various active ingredients such as polysaccharides, polypeptides, amino acids, alkaloids, and flavonoids. It possesses high nutritional and medicinal value and is widely used in health foods, functional beverages, and pharmaceuticals. Among the extraction methods for Dendrobium officinale, fermentation has gradually become an important method because it can promote cell wall disruption through microbial metabolism, thereby increasing the release efficiency of active ingredients and improving the absorption and utilization rate and flavor quality of the extract. Compared to traditional hot water extraction, fermentation extraction typically offers advantages such as high extraction efficiency, lower energy consumption, and stronger conversion of active ingredients.

[0003] However, during the fermentation and extraction process, the iron, copper and other metal ions naturally present in Dendrobium officinale raw materials participate in the oxidation reaction during fermentation, generating active free radicals, which leads to polysaccharide chain breakage and polypeptide oxidative degradation. Furthermore, the endogenous hydrolytic enzymes such as polysaccharide enzymes and proteases naturally present in Dendrobium officinale tissue are easily activated under fermentation conditions, causing polysaccharides and polypeptides to undergo self-degradation, resulting in a decrease in the content of effective ingredients, thereby affecting the quality and activity of the final Dendrobium officinale extract. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Dendrobium officinale extract and its preparation method and formulation, which aims to solve the problem that the content of effective components in the extract decreases due to oxidative degradation and enzymatic hydrolysis during the fermentation and extraction process of Dendrobium officinale extract.

[0005] To address the aforementioned technical problems, a method for preparing Dendrobium officinale extract is proposed, characterized by the following steps: S1. After crushing Dendrobium officinale, mix it with water to obtain a mixture. Then add citrate to the mixture and stir under weak acid conditions to obtain a pretreatment solution. Then add polyol stabilizer to the pretreatment solution and stir to obtain an ion treatment solution. S2. Add the flocculant to the ion treatment liquid, stir and filter to obtain the supernatant and filter residue. Then add the enzyme inactivator to the supernatant, stir and let stand under acidic conditions to obtain the enzyme treatment liquid. Wash the filter residue to obtain the fermentation substrate. The enzyme inactivator contains boric acid groups. S3. Add calcium salt to the enzyme treatment solution, stir to obtain pre-fermentation liquid, add fermentation inoculum and fermentation substrate to pre-fermentation liquid, anaerobic fermentation, inactivation, centrifugation, and filtration to obtain Dendrobium officinale extract.

[0006] In addition, a Dendrobium officinale extract is provided, which is prepared by the above-described method for preparing a Dendrobium officinale extract.

[0007] In addition, a formulation is provided that contains the Dendrobium officinale extract as described above.

[0008] As can be seen from the above technical solutions, the exemplary embodiments disclosed herein possess at least the following advantages and positive effects: On the one hand, free metal ions are treated synergistically by citrate and polyol stabilizers. Specifically, multiple carboxyl and hydroxyl groups in the citrate molecule react with Fe released from the Dendrobium officinale raw material. 3+ Cu 2+ The formation of stable, soluble chelate structures by isooxidizing metal ions encapsulates them within the chelate ring, thereby reducing their ability to participate in free radical generation and oxidative chain reactions. Simultaneously, the polyol stabilizer utilizes its multiple hydroxyl groups to form a hydrogen bond network with the surface of the metal-citrate chelate, constructing a stable hydration protective layer around the chelate. This reduces the probability of the chelate dissociating and releasing metal ions, maintaining the oxidizing metal ions in the system at a low activity level for an extended period. This solves the problem of free metal ions continuously catalyzing the oxidative degradation of polysaccharides, peptides, and other active ingredients during the fermentation and extraction of Dendrobium officinale in existing technologies. This reduces the oxidative loss of active ingredients and improves the stability and retention rate of active ingredients during the pretreatment stage and subsequent fermentation process.

[0009] On the other hand, by adding a flocculant after the metal ion treatment, the metal-chelate complex formed in step S1 aggregates into larger particles through charge neutralization and adsorption bridging, and is then separated and removed, preventing the re-release of metal ions in the subsequent system. Subsequently, endogenous enzymes are treated in a synergistic manner using an enzyme inactivator containing boric acid groups. The acidic environment promotes the spatial conformation unfolding and denaturation of endogenous enzymes such as polysaccharides and proteases, while the boric acid groups further form a binding structure with the hydroxyl sites in the enzyme active center, continuously inhibiting residual enzyme activity, thereby achieving deep inactivation of endogenous enzymes. Furthermore, calcium salt is added before fermentation, causing calcium ions to complex with the residual boric acid groups in the system, reducing the concentration of free boric acid and eliminating its impact on the growth and metabolic activity of fermenting bacteria. This solves the problems in the prior art of continuous hydrolysis of active ingredients such as Dendrobium officinale polysaccharides and polypeptides by endogenous enzymes and the impact of residual enzyme inactivators on the fermentation process. It achieves effective inhibition of enzymatic hydrolysis and restoration of the biological activity of the fermentation system, improving the stability of the fermentation process and the preservation of effective components. Attached Figure Description

[0010] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure. It is obvious that the drawings described below are merely some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort. In the drawings: Figure 1 This is a schematic diagram of a method for preparing Dendrobium officinale extract in one embodiment; Figure 2 This is a schematic diagram of step S1 in the preparation method of Dendrobium officinale extract in one embodiment; Figure 3 This is a schematic diagram of step S2 in the preparation method of Dendrobium officinale extract in one embodiment; Figure 4 This is a schematic diagram of step S3 in the preparation method of Dendrobium officinale extract in one embodiment. Detailed Implementation

[0011] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the examples set forth herein; rather, these embodiments are provided so that this disclosure will be more thorough and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art.

[0012] Furthermore, the described features or characteristics may be combined in any suitable manner in one or more embodiments. Numerous specific details are provided in the following description to give a thorough understanding of embodiments of this disclosure. However, those skilled in the art will recognize that the technical solutions of this disclosure may be practiced without one or more of the specific details, or other methods, steps, etc. may be employed. In other instances, well-known methods, implementations, or operations are not shown or described in detail to avoid obscuring various aspects of this disclosure.

[0013] The flowcharts shown in the accompanying drawings are merely illustrative and do not necessarily include all content and operations / steps, nor do they necessarily have to be performed in the described order. For example, some operations / steps can be broken down, while others can be combined or partially combined; therefore, the actual execution order may change depending on the specific circumstances.

[0014] Please refer to Figure 1 This invention proposes a method for preparing Dendrobium officinale extract, the preparation method comprising the following steps: S1. After crushing Dendrobium officinale, mix it with water to obtain a mixture. Then add citrate to the mixture and stir under weak acid conditions to obtain a pretreatment solution. Then add polyol stabilizer to the pretreatment solution and stir to obtain an ion treatment solution.

[0015] The citrate is sodium citrate and / or potassium citrate, and the polyol stabilizer includes at least one of glycerol, sorbitol, D-mannitol, and erythritol.

[0016] By crushing Dendrobium officinale and mixing it with water, the cell tissue is broken down, causing free metal ions to be released into the liquid phase. Then, the carboxyl and hydroxyl groups in the citrate react with Fe... 3+ Cu 2+ Free metal ions form stable, soluble chelate structures, thereby reducing their catalytic ability to oxidize polysaccharides, polyphenols, and other active ingredients. Simultaneously, under weakly acidic conditions, partial oxidation reactions can be further inhibited, and the chelation efficiency of metal ions can be improved. Subsequently, a polyol stabilizer is added. Multiple hydroxyl groups in the polyol molecule can form hydrogen bonds with the surface of the metal-citrate chelate, constructing a stable hydration protective layer around it. This reduces the possibility of chelate dissociation and the re-release of metal ions, thus maintaining the oxidized metal ions in the system at a low activity level and reducing the oxidative degradation of active ingredients catalyzed by metal ions during fermentation extraction. Furthermore, because the oxidation reaction is effectively inhibited, the induction and promotion effects of oxidation products on endogenous enzyme activity are reduced, lowering the risk of further degradation of active ingredients in subsequent processing. This improves the stability and retention rate of active ingredients such as Dendrobium officinale polysaccharides and peptides, creating favorable conditions for obtaining high-content active ingredients in subsequent fermentation extraction.

[0017] refer to Figure 2 Step S1 includes: S1.1 Wash and crush Dendrobium officinale with deionized water to obtain material particles. Then mix the material particles with deionized water and stir at 25~35℃ for 10~30 minutes to obtain a mixture. The particle size of the material particles is 1~3mm, the mass ratio of material particles to deionized water is 1:(8~10), and the stirring speed is 200~500rpm.

[0018] Washing Dendrobium officinale with deionized water removes surface dust, impurities, and soluble ionic impurities, reducing interference with subsequent processing. Crushing the Dendrobium officinale material into particles of 1-3 mm helps disrupt cell wall structure, increases the specific surface area of ​​the material, and allows water molecules to penetrate more fully into the cells, promoting the release of active ingredients and metal ions. Subsequently, it is mixed with deionized water at a mass ratio of 1:(8-10) and stirred at 25-35℃ for 10-30 minutes. This ensures uniform contact between the material particles and the aqueous phase, guaranteeing the full dissolution and dispersion of active ingredients in the liquid phase. Simultaneously, controlling the stirring speed at 200-500 rpm maintains a homogeneous liquid state, preventing particle sedimentation and agglomeration, thus obtaining a uniform and stable mixture.

[0019] S1.2 Add citrate to the mixture and adjust the pH of the mixture to 6.0~6.5 using a regulator. Stir at 25~45℃ for 20~60 min to obtain a pretreated solution. The amount of citrate added is 0.1~1wt% of the mass of Dendrobium officinale, and the stirring speed is 300~600 rpm.

[0020] The regulators include at least one of citric acid, malic acid, lactic acid, tartaric acid, and gluconic acid.

[0021] By adding citrate to the mixture, the carboxyl and hydroxyl groups in citrate can selectively chelate free metal ions in the liquid phase, such as Fe³⁺. + Cu² + The process involves various steps to form stable, soluble chelates, thereby reducing the oxidative catalytic effect of metal ions on Dendrobium officinale polysaccharides, polypeptides, and other active ingredients. Simultaneously, adjusting the pH of the mixture to 6.0–6.5 using citric acid, malic acid, lactic acid, tartaric acid, or gluconic acid optimizes the chelation efficiency of metal ions under weakly acidic conditions. Stirring at 25–45°C for 20–60 min at a speed of 300–600 rpm ensures sufficient reaction between citrate and metal ions, promotes solution homogeneity, and enhances the stability of the chelates and active ingredients, resulting in a homogeneous, stable pretreatment solution with low oxidation risk.

[0022] S1.3 Add the polyol stabilizer to the pretreatment solution and continue stirring at 20~35℃ for 15~40 min to obtain the ion treatment solution. The amount of polyol stabilizer added is 0.1~0.5wt% of the mass of the pretreatment solution.

[0023] By adding a polyol stabilizer to the pretreatment solution, the abundant hydroxyl groups in the polyol molecules form a stable hydration layer with the surface of the metal-citric acid complex formed in the pretreatment solution through hydrogen bonds. This creates a hydrophilic steric hindrance, preventing the collision aggregation or dissociation of metal ion complexes, thereby further inhibiting the oxidative degradation reaction catalyzed by free metal ions. Simultaneously, the molecular structure of the polyol can interact weakly with polysaccharide and peptide molecules in the system, providing molecular-level protection, reducing the degradation or structural changes of active ingredients in subsequent operations, and improving the retention rate and stability of active ingredients such as polysaccharides and peptides in the extract.

[0024] In one embodiment, step S1 further includes: S1.1 Wash and crush Dendrobium officinale with deionized water to obtain material particles. Then mix the material particles with a sodium bicarbonate solution with a concentration of 0.05~0.1% and soak at 4~10℃ for 30~60 minutes. Then add a citric acid solution with a concentration of 0.05~0.1% to adjust the pH to 6.0~6.5 to obtain a treated slurry. Then mix the treated slurry with deionized water and stir at 25~35℃ for 10~30 minutes to obtain a mixed liquid. The particle size of the material particles is 1~3mm, and the mass ratio of material particles to sodium bicarbonate solution and deionized water is 1:(1~2):(7~8). The stirring speed is 200~500rpm.

[0025] The material is soaked in a low-concentration sodium bicarbonate solution at 4-10℃, causing moderate swelling of the pectin and hemicellulose structures in the cell walls, promoting intercellular expansion, and improving tissue permeability. Subsequently, a low-concentration citric acid solution is used to adjust the pH of the system to 6.0-6.5, restoring the tissue from the initial alkaline treatment to a suitable weakly acidic environment and preventing the degradation of active ingredients such as polysaccharides, phenols, and amino acids from *Dendrobium officinale* under continuous alkaline conditions. During this process, the carbon dioxide microbubbles generated by the reaction of sodium bicarbonate and citric acid create a micro-diffusion effect within the plant tissue, further promoting cell structure loosening and improving the subsequent release efficiency of active ingredients into the liquid phase. The treated slurry formed after this pretreatment has higher cell wall disruption efficiency, a more uniform mass transfer environment, and a lower risk of active ingredient loss. When mixed with deionized water, it can more fully release polysaccharides, oligosaccharides, and other functional components from *Dendrobium officinale*.

[0026] S2. Add the flocculant to the ion treatment liquid, stir and filter to obtain the supernatant and filter residue. Then add the enzyme inactivator to the supernatant, stir and let stand under acidic conditions to obtain the enzyme treatment liquid. Wash the filter residue to obtain the fermentation substrate. The enzyme inactivator contains boric acid groups.

[0027] The flocculant includes at least one of alum, ammonium aluminum sulfate, calcium chloride, and calcium acetate, and the enzyme inactivator includes at least one of boric acid, sodium tetraborate, sodium metaborate, and potassium metaborate.

[0028] The ion treatment solution is flocculated using flocculants such as alum, ammonium aluminum sulfate, calcium chloride, or calcium acetate. This causes the metal ion complex products and suspended impurities formed in step S1 to aggregate and be separated, thereby reducing the content of active components that may participate in oxidation reactions in the system and reducing their continuous oxidative consumption of Dendrobium officinale polysaccharides, phenols, and other active ingredients. At the same time, the supernatant obtained by filtration retains a large number of soluble effective components. After adding an enzyme inactivating agent containing borate groups under acidic conditions, borate ions can interact with the ortho-hydroxyl groups, serine residues, or glycoprotein structures in enzyme protein molecules, changing the spatial conformation of enzyme molecules. This inhibits the catalytic activity of polyphenol oxidase, peroxidase, and some glycoside hydrolases, reducing the risk of enzymatic oxidation and enzymatic hydrolysis of active components in the pre-fermentation stage, resulting in an enzyme treatment solution with higher stability. On the other hand, washing the filter residue removes soluble flocculant residue and some free metal ions adhering to its surface, reducing their cumulative effect in the subsequent fermentation system. Simultaneously, it retains Dendrobium officinale cell wall fragments, unreleased polysaccharides, and other nutrients, allowing them to re-participate in the subsequent fermentation process as a substrate, providing a continuous carbon and nutrient source for the fermenting bacteria. Through the synergistic effect of the enzyme treatment solution and the fermentation substrate, oxidative degradation and enzymatic hydrolysis are inhibited, while the release and conversion efficiency of effective components during fermentation are improved, thereby enhancing the retention rate and extraction stability of active ingredients in the final Dendrobium officinale extract.

[0029] refer to Figure 3 Step S2 includes: S2.1 Add the flocculant to the ion treatment liquid, stir at 4~10℃ for 10~20min, filter to obtain supernatant and filter residue, wherein the mass concentration of flocculant in the ion treatment liquid is 0.1~0.2wt%, and the stirring speed is 200~400rpm.

[0030] By adding a flocculant to the ion treatment solution and stirring at 4-10℃ for 10-20 minutes, metal ion complexes and suspended impurities in the solution aggregate to form flocculent precipitates through charge neutralization and adsorption bridging. After filtration, these potentially oxidizing active components and suspended particles are removed from the supernatant, thereby reducing the risk of oxidative degradation of Dendrobium officinale polysaccharides, phenols, and other active ingredients in the system, while retaining soluble active ingredients. At the same time, the resulting filter residue is rich in unreleased cell wall fragments and some active components, which can be reused as fermentation substrate after subsequent washing and treatment, providing a stable carbon source and nutrients for the fermentation bacteria, which is beneficial to improving the release efficiency of active ingredients during the fermentation process and the content of active ingredients in the final extract.

[0031] S2.2 After adjusting the pH of the supernatant to 2.5-3.0 using a regulator, add the enzyme inactivator and antioxidant, stir at 4-10℃ for 5-10 min to obtain the enzyme pretreatment solution, cool the enzyme treatment solution to 4-10℃, and let it stand for 30-60 min to obtain the enzyme treatment solution. The concentrations of the enzyme inactivator and antioxidant in the supernatant are 0.05-0.1wt% and 0.02-0.05wt%, respectively, and the stirring speed is 100-300 rpm.

[0032] The regulators include at least one of citric acid, malic acid, lactic acid, tartaric acid, and gluconic acid, and the antioxidants include at least one of ascorbic acid, sodium ascorbate, calcium ascorbate, and potassium ascorbate.

[0033] The pH of the supernatant was adjusted to 2.5-3.0 using regulators such as citric acid, malic acid, lactic acid, tartaric acid, or gluconic acid, creating a strongly acidic environment that reduced the activity of polyphenol oxidase, peroxidase, and some glycoside hydrolases, thus creating favorable conditions for subsequent enzyme inactivation. Subsequently, an enzyme inactivating agent containing boric acid groups was added. Boric acid substances can interact with the ortho-hydroxyl structures in the enzyme protein molecule, causing a conformational change in the enzyme molecule, thereby further weakening the enzyme's catalytic activity and reducing enzymatic hydrolysis and oxidation of Dendrobium officinale polysaccharides, oligosaccharides, phenols, and other active ingredients in subsequent processes. Simultaneously, antioxidants such as ascorbic acid, sodium ascorbate, calcium ascorbate, or potassium ascorbate were added. By preferentially consuming dissolved oxygen and reactive oxygen free radicals in the system, these antioxidants blocked the oxidation chain reaction, inhibiting oxidative browning and structural degradation of the active ingredients. Then, by low-temperature static treatment, the enzyme inactivating agent is allowed to come into full contact with the residual enzyme molecules, thereby increasing the degree of enzyme inactivation. On the other hand, the activity of residual enzyme and the rate of oxidation reaction are reduced under low-temperature conditions, thus obtaining an enzyme treatment solution with higher stability.

[0034] S2.3 Wash the filter residue 1-3 times with a citric acid washing solution of 0.2-0.5wt%, then wash it 1-2 times with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is (2-5):(2-5):1 for each washing. The washing temperature is 4-15℃, the washing time is 5-15min, and the stirring speed during the washing process is 100-300rpm.

[0035] The filter residue was washed with a low-concentration citric acid washing solution. The carboxyl groups in the citric acid molecules reacted with the Al³⁺ residue remaining on the surface of the filter residue during the flocculation process. + Ca² +When metal ions undergo complexation, the metal ion complexes, flocculation byproducts, and some metal bridging structures attached to the filter residue surface gradually dissociate and transfer to the washing liquid for removal. Subsequent washing with deionized water further removes soluble metal-citric acid complexes, residual citric acid, and other soluble impurities generated during the washing process, preventing these substances from affecting the normal growth and metabolism of the fermenting bacteria during subsequent fermentation. Simultaneously, because the washing process is carried out at 4-15℃ with a low citric acid concentration, it effectively removes flocculation byproducts while minimizing the loss of Dendrobium officinale polysaccharides, oligosaccharides, and other active ingredients, thus retaining cell wall fragments, unreleased polysaccharides, and nutrients in the filter residue.

[0036] S3. Add calcium salt to the enzyme treatment solution, stir to obtain pre-fermentation liquid, add fermentation inoculum and fermentation substrate to pre-fermentation liquid, anaerobic fermentation, inactivation, centrifugation, and filtration to obtain Dendrobium officinale extract.

[0037] The calcium salts include at least one of calcium lactate, calcium gluconate, calcium acetate, calcium chloride, and calcium sulfate, and the fermentation strains include at least one of lactic acid bacteria, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus acidophilus.

[0038] By adding calcium salts to the enzyme treatment solution, calcium ions can complex and precipitate with residual impurities such as borate ions in the system, thereby reducing the interference of these components on the subsequent fermentation process and optimizing the ion balance and microenvironment stability of the fermentation system. Simultaneously, calcium ions can also form weak interactions with functional groups such as carboxyl and hydroxyl groups in polysaccharide molecules, improving the stability of the polysaccharide structure to a certain extent and reducing the loss of active ingredients during subsequent fermentation. Based on this, the fermentation substrate and fermentation strains are added to the pre-fermentation liquid for anaerobic fermentation. The fermentation bacteria gradually disrupt the cell walls and intercellular matrix structure of *Dendrobium officinale* through metabolic activity, promoting the release of intracellular embedded polysaccharides and bound peptides into the liquid phase system, improving the extraction efficiency of active ingredients. At the same time, the anaerobic environment effectively reduces oxygen-mediated oxidation reactions, lowering the risk of structural damage or decreased activity of polysaccharides and peptides due to oxidation. Since the preceding steps have already inactivated the endogenous enzymes in Dendrobium officinale, the enzymatic hydrolysis of polysaccharides and peptides by endogenous enzymes such as glycosidases and proteases during fermentation can be avoided, effectively separating the release and degradation processes of active ingredients. Finally, the inactivation treatment promptly terminates microbial metabolism and extracellular enzyme activity, and centrifugation and filtration remove cell residues and insoluble impurities, preventing the loss of active ingredients due to continuous metabolism in the later stages of fermentation, thereby obtaining Dendrobium officinale extract rich in polysaccharides and peptides.

[0039] refer to Figure 4 Step S3 includes: S3.1 Add calcium salt to the enzyme treatment solution, stir at 20~30℃ for 15~30 min, filter to obtain the pre-fermentation liquid, wherein the amount of calcium salt added is 0.05~0.30wt% of the enzyme treatment solution mass, and the stirring speed is 200~400 rpm.

[0040] By adding calcium salts to the enzyme treatment solution and stirring, calcium ions can complex and precipitate residual impurities such as borate in the solution, separating them from the liquid phase. Subsequently, filtration removes these impurities and their precipitates, reducing the content of antibacterial factors and non-target components in the fermentation system, thus minimizing their impact on the growth, reproduction, and metabolic activity of the fermenting bacteria. Simultaneously, calcium ions can form weak coordination with active groups such as carboxyl and hydroxyl groups in Dendrobium officinale polysaccharide molecules, improving the conformational stability of the polysaccharide molecules and reducing the risk of structural degradation caused by changes in the acidic environment during subsequent fermentation. The pre-fermentation broth obtained after calcium salt purification and filtration has lower impurity content, better fermentation adaptability, and a more stable polysaccharide and peptide system, creating favorable conditions for the subsequent fermentation strains to efficiently utilize the fermentation substrate, promote the release and enrichment of active ingredients, and ultimately help improve the retention rate and content of polysaccharides and peptides in Dendrobium officinale extract.

[0041] S3.2 Add fermentation substrate to the pre-fermentation broth and inoculate with fermentation bacteria. Perform anaerobic fermentation at 30-38℃ for 24-72 hours to obtain the fermentation broth. The inoculation amount of fermentation bacteria is 1×10⁻⁶. 6 ~1×10 8 CFU / mL.

[0042] The purified pre-fermentation broth and fermentation substrate were used together as the fermentation system, and an appropriate amount of fermentation bacteria were inoculated for anaerobic fermentation. On the one hand, the cell walls, fibrous structures, and undissolved active ingredients of *Dendrobium officinale* retained in the fermentation substrate continuously provided substrates for microorganisms, allowing the fermentation bacteria to gradually destroy the plant cell wall structure through metabolic processes, promoting the release of polysaccharides and peptides originally embedded in the cell tissue into the liquid phase system. On the other hand, lactic acid bacteria and other fermentation bacteria produced organic acids, various metabolic enzymes, and small molecule active substances under anaerobic conditions, which could carry out mild biotransformation of *Dendrobium officinale* tissue, improving the dissolution efficiency and bioavailability of polysaccharides and peptides. At the same time, the anaerobic fermentation environment reduced the oxidation reaction involving oxygen, reducing the oxidative loss of polysaccharides and peptides during fermentation. The inactivation treatment of endogenous enzymes in the previous step prevented the continuous degradation of polysaccharides and peptides by endogenous hydrolytic enzymes, thus ensuring that the release rate of active ingredients during fermentation was greater than the degradation rate. Finally, a fermentation broth rich in polysaccharides and peptides with a high retention rate of active ingredients was obtained, laying the foundation for the subsequent preparation of high-quality *Dendrobium officinale* extract.

[0043] S3.3. Heat the fermentation broth to 70~85℃ and keep it at that temperature for 10~20 min to inactivate it. Then centrifuge at 4000~8000 rpm for 10~20 min and filter it through 0.45μm and 0.22μm filter membranes in sequence to obtain Dendrobium officinale extract.

[0044] By heating the fermentation broth to 70-85℃ and maintaining this temperature, the fermentation cells and their secreted extracellular enzymes can be effectively inactivated, thus terminating the fermentation process in a timely manner. This avoids further decomposition of peptides and polysaccharides by the continuous metabolism of the fermentation cells and active enzymes such as proteases and glycosidases, thereby maximizing the retention of released active ingredients. Subsequently, centrifugation is used to remove cell debris, insoluble impurities, and polysaccharides and peptides, enriching them in the liquid phase and improving the purity of the extract. Further gradient filtration through 0.45 μm and 0.22 μm filter membranes effectively removes residual particles, microorganisms, and colloidal impurities, resulting in a clear and stable extract. Through the synergistic effect of inactivation, centrifugation, and fine filtration, not only can the loss of active ingredients caused by microbial growth and enzymatic degradation during subsequent storage be reduced, but the stability, purity, and content of polysaccharides and peptides in the Dendrobium officinale extract can also be improved, resulting in a Dendrobium officinale extract with more stable quality and higher retention of active ingredients.

[0045] In addition, a Dendrobium officinale extract is provided, which is prepared by the above-described method for preparing a Dendrobium officinale extract.

[0046] In addition, a formulation is provided that contains the Dendrobium officinale extract as described above.

[0047] It should be noted that, depending on the specific usage requirements, this preparation can also be made into oral or topical preparations by combining Dendrobium officinale extract with other active ingredients (such as selenium-enriched mushroom powder) through fermentation, emulsification, or other suitable processes.

[0048] For example, the present invention provides the following specific embodiments to illustrate the specific preparation method: Example 1: S1.1 The Dendrobium officinale is washed and crushed with deionized water to obtain material particles. Then the material particles are mixed with deionized water and stirred at 30°C for 20 minutes to obtain a mixture. The particle size of the material particles is 2 mm, the mass ratio of material particles to deionized water is 1:9, and the stirring speed is 350 rpm. S1.2 Add sodium citrate to the mixture and adjust the pH of the mixture to 6.0~6.5 with citric acid. Stir at 35℃ for 40 min to obtain the pretreated solution. The amount of sodium citrate added is 0.5wt% of the mass of Dendrobium officinale, and the stirring speed is 450 rpm. S1.3. Add glycerol to the pretreatment solution and continue stirring at 28°C for 25 min to obtain the ion-treated solution. The amount of glycerol added is 0.3 wt% of the mass of the pretreatment solution. S2.1 Add alum to the ion treatment solution, stir at 7°C for 15 min, filter to obtain supernatant and filter residue, wherein the mass concentration of alum in the ion treatment solution is 0.15 wt%, and the stirring speed is 300 rpm; S2.2 After adjusting the pH of the supernatant to 2.5~3.0 with citric acid, add boric acid and ascorbic acid, stir at 7°C for 8 min to obtain enzyme pretreatment solution, cool the enzyme treatment solution to 7°C and let it stand for 45 min to obtain enzyme treatment solution, wherein the concentrations of boric acid and ascorbic acid in the supernatant are 0.08wt% and 0.03wt% respectively, and the stirring speed is 200 rpm; S2.3 Wash the filter residue twice with a 0.35wt% citric acid washing solution, then wash it twice with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is 4:4:1 for each washing, the washing temperature is 10℃, the washing time is 10min, and the stirring speed during the washing process is 200rpm. S3.1 Add calcium lactate to the enzyme treatment solution and stir at 25°C for 20 min to obtain the pre-fermentation solution. The amount of calcium lactate added is 0.15 wt% of the enzyme treatment solution mass, and the stirring speed is 300 rpm. S3.2 Add fermentation substrate to the pre-fermentation liquid and inoculate with lactic acid bacteria. Perform anaerobic fermentation at 35℃ for 48 hours to obtain the fermentation broth. The inoculum size is 1×10⁻⁶. 7 CFU / mL; S3.3. The fermentation broth was heated to 80℃ and kept at that temperature for 45 min to inactivate it. Then it was centrifuged at 6000 rpm for 15 min and filtered through 0.45 μm and 0.22 μm filter membranes to obtain Dendrobium officinale extract.

[0049] Example 2: S1.1 The Dendrobium officinale was washed and crushed with deionized water to obtain material particles. The material particles were then mixed with a 0.08% sodium bicarbonate solution and soaked at 7°C for 45 minutes. A 0.08% citric acid solution was then added to adjust the pH to 6.0-6.5 to obtain a treated slurry. The treated slurry was then mixed with deionized water and stirred at 30°C for 20 minutes to obtain a mixed liquid. The particle size of the material particles was 2 mm, and the mass ratio of the material particles to the sodium bicarbonate solution and deionized water was 1:1.5:7.5. The stirring speed was 350 pm. S1.2 Add sodium citrate to the mixture and adjust the pH of the mixture to 6.0~6.5 with citric acid. Stir at 35℃ for 40 min to obtain the pretreated solution. The amount of sodium citrate added is 0.5wt% of the mass of Dendrobium officinale, and the stirring speed is 450 rpm. S1.3. Add glycerol to the pretreatment solution and continue stirring at 28°C for 25 min to obtain the ion-treated solution. The amount of glycerol added is 0.3 wt% of the mass of the pretreatment solution. S2.1 Add alum to the ion treatment solution, stir at 7°C for 15 min, filter to obtain supernatant and filter residue, wherein the mass concentration of alum in the ion treatment solution is 0.15 wt%, and the stirring speed is 300 rpm; S2.2 After adjusting the pH of the supernatant to 2.5~3.0 with citric acid, add boric acid and ascorbic acid, stir at 7°C for 8 min to obtain enzyme pretreatment solution, cool the enzyme treatment solution to 7°C and let it stand for 45 min to obtain enzyme treatment solution, wherein the concentrations of boric acid and ascorbic acid in the supernatant are 0.08wt% and 0.03wt% respectively, and the stirring speed is 200 rpm; S2.3 Wash the filter residue twice with a 0.35wt% citric acid washing solution, then wash it twice with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is 4:4:1 for each washing, the washing temperature is 10℃, the washing time is 10min, and the stirring speed during the washing process is 200rpm. S3.1 Add calcium lactate to the enzyme treatment solution and stir at 25°C for 20 min to obtain the pre-fermentation solution. The amount of calcium lactate added is 0.15 wt% of the enzyme treatment solution mass, and the stirring speed is 300 rpm. S3.2 Add fermentation substrate to the pre-fermentation liquid and inoculate with lactic acid bacteria. Perform anaerobic fermentation at 35℃ for 48 hours to obtain the fermentation broth. The inoculum size is 1×10⁻⁶. 7 CFU / mL; S3.3. The fermentation broth was heated to 80℃ and kept at that temperature for 45 min to inactivate it. Then it was centrifuged at 6000 rpm for 15 min and filtered through 0.45 μm and 0.22 μm filter membranes to obtain Dendrobium officinale extract.

[0050] Comparative Example 1: S1.1 The Dendrobium officinale is washed and crushed with deionized water to obtain material particles. Then the material particles are mixed with deionized water and stirred at 30°C for 20 minutes to obtain a mixture. The particle size of the material particles is 2 mm, the mass ratio of material particles to deionized water is 1:9, and the stirring speed is 350 rpm. S1.2. Adjust the pH of the mixture to 6.0~6.5 with citric acid, stir at 35℃ for 40 min to obtain the pretreated solution, wherein the stirring speed is 450 rpm; S1.3. Add glycerol to the pretreatment solution and continue stirring at 28°C for 25 min to obtain the ion-treated solution. The amount of glycerol added is 0.3 wt% of the mass of the pretreatment solution. S2.1 Add alum to the ion treatment solution, stir at 7°C for 15 min, filter to obtain supernatant and filter residue, wherein the mass concentration of alum in the ion treatment solution is 0.15 wt%, and the stirring speed is 300 rpm; S2.2 After adjusting the pH of the supernatant to 2.5~3.0 with citric acid, add boric acid and ascorbic acid, stir at 7°C for 8 min to obtain enzyme pretreatment solution, cool the enzyme treatment solution to 7°C and let it stand for 45 min to obtain enzyme treatment solution, wherein the concentrations of boric acid and ascorbic acid in the supernatant are 0.08wt% and 0.03wt% respectively, and the stirring speed is 200 rpm; S2.3 Wash the filter residue twice with a 0.35wt% citric acid washing solution, then wash it twice with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is 4:4:1 for each washing, the washing temperature is 10℃, the washing time is 10min, and the stirring speed during the washing process is 200rpm. S3.1 Add calcium lactate to the enzyme treatment solution and stir at 25°C for 20 min to obtain the pre-fermentation solution. The amount of calcium lactate added is 0.15 wt% of the enzyme treatment solution mass, and the stirring speed is 300 rpm. S3.2 Add fermentation substrate to the pre-fermentation liquid and inoculate with lactic acid bacteria. Perform anaerobic fermentation at 35℃ for 48 hours to obtain the fermentation broth. The inoculum size is 1×10⁻⁶. 7 CFU / mL; S3.3. The fermentation broth was heated to 80℃ and kept at that temperature for 45 min to inactivate it. Then it was centrifuged at 6000 rpm for 15 min and filtered through 0.45 μm and 0.22 μm filter membranes to obtain Dendrobium officinale extract.

[0051] It is basically the same as Example 1, except that sodium citrate is not used in step S1.2, that is, citrate is not used in step S1.

[0052] Comparative Example 2: S1.1 The Dendrobium officinale is washed and crushed with deionized water to obtain material particles. Then the material particles are mixed with deionized water and stirred at 30°C for 20 minutes to obtain a mixture. The particle size of the material particles is 2 mm, the mass ratio of material particles to deionized water is 1:9, and the stirring speed is 350 rpm. S1.2 Add sodium citrate to the mixture and adjust the pH of the mixture to 6.0~6.5 with citric acid. Stir at 35℃ for 40 min to obtain the pretreated solution. The amount of sodium citrate added is 0.5wt% of the mass of Dendrobium officinale, and the stirring speed is 450 rpm. S1.3. Continue stirring the pretreatment solution at 28℃ for 25 min to obtain the ion treatment solution; S2.1 Add alum to the ion treatment solution, stir at 7°C for 15 min, filter to obtain supernatant and filter residue, wherein the mass concentration of alum in the ion treatment solution is 0.15 wt%, and the stirring speed is 300 rpm; S2.2 After adjusting the pH of the supernatant to 2.5~3.0 with citric acid, add boric acid and ascorbic acid, stir at 7°C for 8 min to obtain enzyme pretreatment solution, cool the enzyme treatment solution to 7°C and let it stand for 45 min to obtain enzyme treatment solution, wherein the concentrations of boric acid and ascorbic acid in the supernatant are 0.08wt% and 0.03wt% respectively, and the stirring speed is 200 rpm; S2.3 Wash the filter residue twice with a 0.35wt% citric acid washing solution, then wash it twice with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is 4:4:1 for each washing, the washing temperature is 10℃, the washing time is 10min, and the stirring speed during the washing process is 200rpm. S3.1 Add calcium lactate to the enzyme treatment solution and stir at 25°C for 20 min to obtain the pre-fermentation solution. The amount of calcium lactate added is 0.15 wt% of the enzyme treatment solution mass, and the stirring speed is 300 rpm. S3.2 Add fermentation substrate to the pre-fermentation liquid and inoculate with lactic acid bacteria. Perform anaerobic fermentation at 35℃ for 48 hours to obtain the fermentation broth. The inoculum size is 1×10⁻⁶. 7 CFU / mL; S3.3. The fermentation broth was heated to 80℃ and kept at that temperature for 45 min to inactivate it. Then it was centrifuged at 6000 rpm for 15 min and filtered through 0.45 μm and 0.22 μm filter membranes to obtain Dendrobium officinale extract.

[0053] It is basically the same as Example 1, except that glycerol is not used in step S1.3, that is, polyol stabilizer is not used in step S1.

[0054] Comparative Example 3: S1.1 The Dendrobium officinale is washed and crushed with deionized water to obtain material particles. Then the material particles are mixed with deionized water and stirred at 30°C for 20 minutes to obtain a mixture. The particle size of the material particles is 2 mm, the mass ratio of material particles to deionized water is 1:9, and the stirring speed is 350 rpm. S1.2 Add sodium citrate to the mixture and adjust the pH of the mixture to 6.0~6.5 with citric acid. Stir at 35℃ for 40 min to obtain the pretreated solution. The amount of sodium citrate added is 0.5wt% of the mass of Dendrobium officinale, and the stirring speed is 450 rpm. S1.3. Add glycerol to the pretreatment solution and continue stirring at 28°C for 25 min to obtain the ion-treated solution. The amount of glycerol added is 0.3 wt% of the mass of the pretreatment solution. S2.1 Stir the ion treatment solution at 7°C for 15 min to obtain supernatant and filter residue, wherein the stirring speed is 300 rpm; S2.2 After adjusting the pH of the supernatant to 2.5~3.0 with citric acid, add boric acid and ascorbic acid, stir at 7°C for 8 min to obtain enzyme pretreatment solution, cool the enzyme treatment solution to 7°C and let it stand for 45 min to obtain enzyme treatment solution, wherein the concentrations of boric acid and ascorbic acid in the supernatant are 0.08wt% and 0.03wt% respectively, and the stirring speed is 200 rpm; S2.3 Wash the filter residue twice with a 0.35wt% citric acid washing solution, then wash it twice with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is 4:4:1 for each washing, the washing temperature is 10℃, the washing time is 10min, and the stirring speed during the washing process is 200rpm. S3.1 Add calcium lactate to the enzyme treatment solution and stir at 25°C for 20 min to obtain the pre-fermentation solution. The amount of calcium lactate added is 0.15 wt% of the enzyme treatment solution mass, and the stirring speed is 300 rpm. S3.2 Add fermentation substrate to the pre-fermentation liquid and inoculate with lactic acid bacteria. Perform anaerobic fermentation at 35℃ for 48 hours to obtain the fermentation broth. The inoculum size is 1×10⁻⁶. 7 CFU / mL; S3.3. The fermentation broth was heated to 80℃ and kept at that temperature for 45 min to inactivate it. Then it was centrifuged at 6000 rpm for 15 min and filtered through 0.45 μm and 0.22 μm filter membranes to obtain Dendrobium officinale extract.

[0055] It is basically the same as Example 1, except that alum is not used in step S2.1, that is, flocculant is not used in step S2.

[0056] Comparative Example 4: S1.1 The Dendrobium officinale is washed and crushed with deionized water to obtain material particles. Then the material particles are mixed with deionized water and stirred at 30°C for 20 minutes to obtain a mixture. The particle size of the material particles is 2 mm, the mass ratio of material particles to deionized water is 1:9, and the stirring speed is 350 rpm. S1.2 Add sodium citrate to the mixture and adjust the pH of the mixture to 6.0~6.5 with citric acid. Stir at 35℃ for 40 min to obtain the pretreated solution. The amount of sodium citrate added is 0.5wt% of the mass of Dendrobium officinale, and the stirring speed is 450 rpm. S1.3. Add glycerol to the pretreatment solution and continue stirring at 28°C for 25 min to obtain the ion-treated solution. The amount of glycerol added is 0.3 wt% of the mass of the pretreatment solution. S2.1 Add alum to the ion treatment solution, stir at 7°C for 15 min, filter to obtain supernatant and filter residue, wherein the mass concentration of alum in the ion treatment solution is 0.15 wt%, and the stirring speed is 300 rpm; S2.2 After adjusting the pH of the supernatant to 2.5~3.0 with citric acid, add ascorbic acid and stir at 7°C for 8 min to obtain enzyme pretreatment solution. Cool the enzyme treatment solution to 7°C and let it stand for 45 min to obtain enzyme treatment solution. The concentration of ascorbic acid in the supernatant is 0.03wt% and the stirring speed is 200rpm. S2.3 Wash the filter residue twice with a 0.35wt% citric acid washing solution, then wash it twice with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is 4:4:1 for each washing, the washing temperature is 10℃, the washing time is 10min, and the stirring speed during the washing process is 200rpm. S3.1 Add calcium lactate to the enzyme treatment solution and stir at 25°C for 20 min to obtain the pre-fermentation solution. The amount of calcium lactate added is 0.15 wt% of the enzyme treatment solution mass, and the stirring speed is 300 rpm. S3.2 Add fermentation substrate to the pre-fermentation liquid and inoculate with lactic acid bacteria. Perform anaerobic fermentation at 35℃ for 48 hours to obtain the fermentation broth. The inoculum size is 1×10⁻⁶. 7 CFU / mL; S3.3. The fermentation broth was heated to 80℃ and kept at that temperature for 45 min to inactivate it. Then it was centrifuged at 6000 rpm for 15 min and filtered through 0.45 μm and 0.22 μm filter membranes to obtain Dendrobium officinale extract.

[0057] It is basically the same as Example 1, except that boric acid is not used in step S2.2, that is, no enzyme inactivating agent is used in step S2.

[0058] Comparative Example 5: S1.1 The Dendrobium officinale is washed and crushed with deionized water to obtain material particles. Then the material particles are mixed with deionized water and stirred at 30°C for 20 minutes to obtain a mixture. The particle size of the material particles is 2 mm, the mass ratio of material particles to deionized water is 1:9, and the stirring speed is 350 rpm. S1.2 Add sodium citrate to the mixture and adjust the pH of the mixture to 6.0~6.5 with citric acid. Stir at 35℃ for 40 min to obtain the pretreated solution. The amount of sodium citrate added is 0.5wt% of the mass of Dendrobium officinale, and the stirring speed is 450 rpm. S1.3. Add glycerol to the pretreatment solution and continue stirring at 28°C for 25 min to obtain the ion-treated solution. The amount of glycerol added is 0.3 wt% of the mass of the pretreatment solution. S2.1 Add alum to the ion treatment solution, stir at 7°C for 15 min, filter to obtain supernatant and filter residue, wherein the mass concentration of alum in the ion treatment solution is 0.15 wt%, and the stirring speed is 300 rpm; S2.2 After adjusting the pH of the supernatant to 2.5~3.0 with citric acid, add boric acid and ascorbic acid, stir at 7°C for 8 min to obtain enzyme pretreatment solution, cool the enzyme treatment solution to 7°C and let it stand for 45 min to obtain enzyme treatment solution, wherein the concentrations of boric acid and ascorbic acid in the supernatant are 0.08wt% and 0.03wt% respectively, and the stirring speed is 200 rpm; S2.3 Wash the filter residue twice with a 0.35wt% citric acid washing solution, then wash it twice with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is 4:4:1 for each washing, the washing temperature is 10℃, the washing time is 10min, and the stirring speed during the washing process is 200rpm. S3.1 Stir the enzyme treatment solution at 25℃ for 20 min to obtain the pre-fermentation solution, wherein the stirring speed is 300 rpm; S3.2 Add fermentation substrate to the pre-fermentation liquid and inoculate with lactic acid bacteria. Perform anaerobic fermentation at 35℃ for 48 hours to obtain the fermentation broth. The inoculum size is 1×10⁻⁶. 7 CFU / mL; S3.3. The fermentation broth was heated to 80℃ and kept at that temperature for 45 min to inactivate it. Then it was centrifuged at 6000 rpm for 15 min and filtered through 0.45 μm and 0.22 μm filter membranes to obtain Dendrobium officinale extract.

[0059] It is basically the same as Example 1, except that calcium lactate is not used in step S3.1, that is, calcium salt is not used in step S3.

[0060] Comparative Example 6: Dendrobium officinale was washed and crushed with deionized water to obtain material particles. These particles were then mixed with deionized water and stirred at 30°C for 20 minutes to obtain a mixture. The particle size was 2 mm, the mass ratio of particles to deionized water was 1:9, and the stirring speed was 350 rpm. Lactic acid bacteria were then inoculated into the mixture and anaerobic fermented at 35°C for 48 hours to obtain a fermentation broth. The inoculation amount of the fermentation bacteria was 1×10⁻⁶. 7 CFU / mL; the fermentation broth was heated to 80℃ and kept at that temperature for 45 min to inactivate the broth, then centrifuged at 6000 rpm for 15 min, and filtered through 0.45 μm and 0.22 μm filter membranes to obtain Dendrobium officinale extract.

[0061] Performance testing: Polysaccharide content detection: Take 0.5g of Dendrobium officinale extract sample, add 50mL of deionized water, heat under reflux for 2h to extract polysaccharides, filter, and precipitate the polysaccharides in the filtrate with 95% ethanol to 3 times the volume, let stand for 12h, centrifuge to remove impurities, obtain polysaccharide precipitate, dissolve in deionized water and make up to 50mL. Take 1mL of solution, add 1mL of 5% phenol solution, then quickly add 5mL of concentrated sulfuric acid, react at room temperature for 10min, and measure the absorbance at 488nm. Establish a standard curve using anhydrous glucose as the standard, perform linear regression with absorbance as the ordinate and glucose concentration as the abscissa, and calculate the polysaccharide content based on the sample absorbance substituted into the standard curve equation.

[0062] Peptide content determination: The peptide content was determined by o-phthalaldehyde (OPA) spectrophotometry. 1 mL of *Dendrobium officinale* fermentation broth was taken, 2 mL of OPA reagent was added, and the mixture was reacted at 37℃ for 2 min. The absorbance at 340 nm was measured. A standard curve (0–200 μg / mL) was established using L-leucine as the standard. Linear regression was performed with absorbance as the ordinate and standard concentration as the abscissa. The peptide content was calculated by substituting the sample absorbance into the standard curve equation.

[0063] The test results are shown in Table 1.

[0064] Table 1. Content of active ingredients in Dendrobium officinale extract: As shown in Table 1, compared with Comparative Example 6, the Dendrobium officinale extracts prepared in Examples 1-2 all contained higher polysaccharide and polypeptide contents. In particular, Example 2 further introduced a sodium bicarbonate pretreatment step based on Example 1, which can improve the cell wall disruption efficiency and release of effective components of Dendrobium officinale, resulting in a higher extraction rate of polysaccharides and polypeptides. Further observation of Examples 1 and Comparative Examples 1-5 shows that, compared with Example 1, Comparative Examples 1-5 lacked at least one of citrate, polyol stabilizer, flocculant, enzyme inactivator, or calcium salt, which reduced the efficiency of protection and release of effective components during the fermentation process of Dendrobium officinale. Therefore, their polysaccharide and polypeptide contents were lower than those of Example 1.

[0065] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the following claims.

Claims

1. A method for preparing Dendrobium officinale extract, characterized in that, The preparation method includes the following steps: S1. After crushing Dendrobium officinale, mix it with solvent to obtain a mixture. Then add citrate to the mixture and stir under weak acid conditions to obtain a pretreatment solution. Then add polyol stabilizer to the pretreatment solution and stir to obtain an ion treatment solution. S2. Add the flocculant to the ion treatment liquid, stir and filter to obtain the supernatant and filter residue. Then add the enzyme inactivator to the supernatant, stir and let stand under acidic conditions to obtain the enzyme treatment liquid. Wash the filter residue to obtain the fermentation substrate. The enzyme inactivator contains boric acid groups. S3. Add calcium salt to the enzyme treatment solution, stir, filter to obtain pre-fermentation liquid, add fermentation inoculum and fermentation substrate to pre-fermentation liquid, anaerobic fermentation, inactivation, centrifugation, and filtration to obtain Dendrobium officinale extract.

2. The method for preparing Dendrobium officinale extract according to claim 1, characterized in that, Step S1 includes: S1.1 Wash and crush Dendrobium officinale with deionized water to obtain material particles. Then mix the material particles with solvent and stir at 25~35℃ for 10~30min to obtain a mixture. The particle size of the material particles is 1~3mm, the mass ratio of material particles to deionized water is 1:(8~10), and the stirring speed is 200~500rpm. S1.2 Add citrate to the mixture and adjust the pH of the mixture to 6.0~6.5 using a regulator. Stir at 25~45℃ for 20~60 min to obtain a pretreated solution. The amount of citrate added is 0.1~1wt% of the mass of Dendrobium officinale, and the stirring speed is 300~600 rpm. S1.3 Add the polyol stabilizer to the pretreatment solution and continue stirring at 20~35℃ for 15~40 min to obtain the ion treatment solution. The amount of polyol stabilizer added is 0.1~0.5wt% of the mass of the pretreatment solution.

3. The method for preparing Dendrobium officinale extract according to claim 2, characterized in that, In step S1, the solvent is deionized water or an aqueous solution containing sodium bicarbonate and citric acid, the citrate is sodium citrate and / or potassium citrate, the regulator includes at least one of citric acid, malic acid, lactic acid, tartaric acid, and gluconic acid, and the polyol stabilizer includes at least one of glycerol, sorbitol, D-mannitol, and erythritol.

4. The method for preparing Dendrobium officinale extract according to claim 3, characterized in that, When the solvent is an aqueous solution containing sodium bicarbonate and citric acid, the specific steps of step S1.1 are as follows: S1.1 Wash and crush Dendrobium officinale with deionized water to obtain material particles. Then mix the material particles with a sodium bicarbonate solution with a concentration of 0.05~0.1% and soak at 4~10℃ for 30~60 minutes. Then add a citric acid solution with a concentration of 0.05~0.1% to adjust the pH to 6.0~6.5 to obtain a treated slurry. Then mix the treated slurry with deionized water and stir at 25~35℃ for 10~30 minutes to obtain a mixed liquid. The particle size of the material particles is 1~3mm, and the mass ratio of material particles to sodium bicarbonate solution and deionized water is 1:(1~2):(7~8). The stirring speed is 200~500rpm.

5. The method for preparing Dendrobium officinale extract according to claim 1, characterized in that, Step S2 includes: S2.1 Add the flocculant to the ion treatment liquid, stir at 4~10℃ for 10~20min, filter to obtain supernatant and filter residue, wherein the mass concentration of flocculant in the ion treatment liquid is 0.1~0.2wt%, and the stirring speed is 200~400rpm; S2.2 After adjusting the pH of the supernatant to 2.5-3.0 using a regulator, add the enzyme inactivator and antioxidant, stir at 4-10℃ for 5-10 min to obtain the enzyme pretreatment solution, cool the enzyme treatment solution to 4-10℃, and let it stand for 30-60 min to obtain the enzyme treatment solution. The concentrations of the enzyme inactivator and antioxidant in the supernatant are 0.05-0.1wt% and 0.02-0.05wt%, respectively, and the stirring speed is 100-300 rpm. S2.3 Wash the filter residue 1-3 times with a citric acid washing solution of 0.2-0.5wt%, then wash it 1-2 times with deionized water and filter to obtain the fermentation substrate. The mass ratio of citric acid washing solution, deionized water and filter residue is (2-5):(2-5):1 for each washing. The washing temperature is 4-15℃, the washing time is 5-15min, and the stirring speed during the washing process is 100-300rpm.

6. The method for preparing Dendrobium officinale extract according to claim 5, characterized in that, In step S2, the flocculant includes at least one of alum, ammonium aluminum sulfate, calcium chloride, and calcium acetate; the regulator includes at least one of citric acid, malic acid, lactic acid, tartaric acid, and gluconic acid; the enzyme inactivator includes at least one of boric acid, sodium tetraborate, sodium metaborate, and potassium metaborate; and the antioxidant includes at least one of ascorbic acid, sodium ascorbate, calcium ascorbate, and potassium ascorbate.

7. The method for preparing Dendrobium officinale extract according to claim 1, characterized in that, Step S3 includes: S3.1 Add calcium salt to the enzyme treatment solution and stir at 20~30℃ for 15~30 min to obtain the pre-fermentation solution. The amount of calcium salt added is 0.05~0.30wt% of the enzyme treatment solution mass, and the stirring speed is 200~400 rpm. S3.2 Add fermentation substrate to the pre-fermentation broth and inoculate with fermentation bacteria. Perform anaerobic fermentation at 30-38℃ for 24-72 hours to obtain the fermentation broth. The inoculation amount of fermentation bacteria is 1×10⁻⁶. 6 ~1×10 8 CFU / mL; S3.

3. Heat the fermentation broth to 70~85℃ and keep it at that temperature for 10~20 min to inactivate it. Then centrifuge at 4000~8000 rpm for 10~20 min and filter it through 0.45μm and 0.22μm filter membranes in sequence to obtain Dendrobium officinale extract.

8. The method for preparing Dendrobium officinale extract according to claim 7, characterized in that, In step S3, the calcium salt includes at least one of calcium lactate, calcium gluconate, calcium acetate, calcium chloride, and calcium sulfate, and the fermentation strain includes at least one of lactic acid bacteria, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus acidophilus.

9. A Dendrobium officinale extract, characterized in that, The Dendrobium officinale extract is prepared by any one of the preparation methods of Dendrobium officinale extract according to claims 1-8.

10. A formulation, characterized in that, The formulation contains the Dendrobium officinale extract as described in claim 9.

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