Preparation method of dendrobium composite enzyme

Abstract

The preparation method comprises the following steps: picking stems and flowers of Dendrobium officinale, Dendrobium huoshanense and Dendrobium nobile, screening the picked stems and flowers, and carrying out pretreatment on the screened stems and flowers; the method comprises the following steps: mixing pretreated stems and flowers of the same variety of dendrobium nobile, respectively adding a first carbohydrate, uniformly stirring, putting into a fermentation container, adding saccharomyces cerevisiae, performing first-stage fermentation, and filtering to correspondingly obtain stock solutions and solid residues of three kinds of dendrobium nobile; mixing the stock solutions of the three dendrobium nobile lindl, adding lactic acid bacteria, mixing the solid residues of the three dendrobium nobile lindl, adding a second carbohydrate, adding lactic acid bacteria, and respectively carrying out second-stage fermentation to correspondingly obtain stock solution fermentation ferment and a solid fermentation product; adding the stock solution obtained by filtering the solid fermentation product into stock solution fermentation enzyme, uniformly stirring, and carrying out third-stage fermentation; and filtering to obtain the dendrobium composite enzyme stock solution. According to the method, the characteristic functional substances of various dendrobium nobile are integrated for synergistic interaction, so that the active ingredients are more balanced and abundant, and the functions are more remarkable.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of processing of medicinal and edible plants, and particularly relates to a preparation method of a Dendrobium compound enzyme. BACKGROUND

[0002] Dendrobium is one of the large genera of Orchidaceae, and has various species. Among them, Dendrobium nobile Lindl., Dendrobium huoshanense C. Z. Tang et S. J. Cheng and Dendrobium officinale Kimura et Migo have great development value as medicinal Dendrobium species in history.

[0003] Modern pharmacological studies have shown that Dendrobium officinale and other Dendrobium plants have the effects of protecting the gastrointestinal tract, regulating immunity, anti-tumor, antioxidant, anti-liver damage and the like, and these functional effects are based on effective components such as polysaccharides, alkaloids and flavonoids in Dendrobium plants. However, the types and contents of chemical components are different in Dendrobium plants of different species and from different origins. For example, Jiang Ronghui et al. (reference: Jiang Ronghui, Luo Hong, Xu Zhigao, et al. Analysis and comparison of four active components of Dendrobium nobile and Dendrobium officinale [J / OL]. Guizhou Forestry Science and Technology, 1-7 [2025-08-26]. https: / / doi.org / 10.16709 / j.cnki.gzlykj.20250515.002.) analyzed the active components in nearly wild-cultivated Dendrobium nobile and Dendrobium officinale, and the experimental results showed that the total alkaloid and total flavonoid contents of Dendrobium officinale were 0.357% and 0.3517% respectively, which were significantly higher than those of Dendrobium nobile. Li Yungong et al. (reference: Li Yungong, Zhou Yuanrun. Research on pharmacology of Dendrobium huoshanense and its application in health food [J]. Journal of Yichun University, 2022, 44(06): 25-27+107.) reported that the total alkaloid content in Dendrobium huoshanense was 0.043%, which was lower than that in Dendrobium nobile.

[0004] Studies have shown that enzymes produced by probiotic fermentation promote the release of natural products by degrading plant cell walls, or cause hydrogenation, isomerization, lipidization, cracking and the like through metabolism, thereby producing new chemical components and significantly improving the diversity, availability and functionality of active substances in raw materials. In recent years, the fermentation of Dendrobium officinale, Dendrobium huoshanense and the like to prepare functional foods has gradually become a research hotspot. However, the current application of Dendrobium fermentation in food processing is mainly concentrated in Dendrobium vinegar, Dendrobium wine, Dendrobium yogurt and the like, and the added value is generally low. The types and health effects cannot meet the market demand.

[0005] Edible plant enzymes are enzyme products containing specific bioactive components and suitable for human consumption, produced through microbial fermentation using plants suitable for food processing as the main raw material, with or without the addition of auxiliary materials (QB / T 5323-2018 "Plant Enzymes"). Preliminary research has been conducted by domestic and international scholars on the preparation of edible plant enzymes from fermented Dendrobium officinale and Dendrobium huoshanense. The development of Dendrobium enzymes provides new impetus for expanding the field of deep processing of Dendrobium officinale and promoting industrial quality improvement and efficiency enhancement. Existing technologies are as follows:

[0006] Chinese patent CN109691670A discloses a method for preparing Dendrobium officinale enzyme liquid and its application: Dendrobium officinale plants aged 2-3 years are selected; leaves are removed, stem segments are taken, chopped, dried, and powdered; after boiling and cooling with water, a compound bacteria are inoculated for fermentation; the fermentation broth is hydrolyzed with a compound enzyme, filtered, and the filtrate is collected to obtain Dendrobium officinale enzyme. In this method, the dried Dendrobium officinale powder is boiled with water at high temperatures, which may damage or lose some enzyme systems and active ingredients in the raw material, thus affecting the fermentation process and the functional effects of the fermentation liquid.

[0007] Chinese patent CN108157945A discloses a method for preparing Dendrobium officinale enzyme: Dendrobium officinale raw materials are washed and cut into sections. The sections are then added to water and brown sugar, mixed thoroughly, and sealed at room temperature for 50-80 days, releasing gas every 10 days. After fermentation, the fermentation liquid is filtered, and the filtrate is used to obtain Dendrobium officinale enzyme. This method does not inoculate probiotics but uses traditional natural fermentation. The microbial community during enzyme fermentation is complex, and natural fermentation is easily contaminated by other microorganisms, making it difficult to control the fermentation process and resulting in uncontrollable product quality.

[0008] In summary, current development of Dendrobium enzyme products typically involves fermentation of single Dendrobium species such as Dendrobium officinale or Dendrobium huoshanense. Furthermore, the different species and origins of Dendrobium result in variations in the types and amounts of chemical components, limiting the functional effects of the products. Therefore, this paper proposes a method for preparing a Dendrobium-based compound enzyme. Summary of the Invention

[0009] The purpose of this invention is to address the above-mentioned problems by proposing a method for preparing a Dendrobium-based compound enzyme. By comprehensively combining the synergistic effects of various characteristic functional substances of Dendrobium, the active ingredients are more balanced and abundant, and the functional effects are more significant.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] The present invention proposes a method for preparing a Dendrobium-based compound enzyme, comprising the following steps:

[0012] S1. Harvest and select the stems and flowers of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile;

[0013] S2. Pre-treat the selected stems and flowers separately;

[0014] S3. After mixing the pretreated stems and flowers of the same variety of Dendrobium according to the first mass ratio, add the first sugar substance separately, stir evenly, put into a fermentation container and add brewing yeast, carry out the first stage of fermentation, filter, and obtain the original liquid and solid residue of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile.

[0015] S4. After the first stage of fermentation, the second stage of fermentation is carried out, as follows:

[0016] The original liquids of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed in a second mass ratio and then lactic acid bacteria were added. The solid residues of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed in a third mass ratio, a second sugar substance was added, and then lactic acid bacteria were added. The second stage of fermentation was carried out to obtain the original liquid fermentation enzyme and the solid fermentation product respectively.

[0017] S5. Add the filtered solid fermentation liquid to the original liquid fermentation enzyme, stir well, and carry out the third stage of fermentation.

[0018] S6. After the third stage of fermentation is completed, filter to obtain Dendrobium complex enzyme stock solution.

[0019] Preferably, the first mass ratio is 1:0.1~0.5, the amount of brewing yeast added is 0.6%~10%, the fermentation temperature of the first stage of fermentation is 20℃~38℃, stirring is performed once every 3 days for 2 minutes~5 minutes each time, and the fermentation time is 30 days~60 days.

[0020] Preferably, the pretreated stems and flowers of the same variety of Dendrobium are mixed according to a first mass ratio, and then a first sugar substance is added separately. The first sugar substance accounts for 40% to 60% of the total weight, and is specifically as follows:

[0021] For the pretreated stems and flowers of Dendrobium officinale, the first sugar added includes 30%~70% white sugar, 10%~30% brown sugar, and 20%~50% oligosaccharide aqueous solution, with the oligosaccharide aqueous solution having a mass fraction of 50%.

[0022] For the pretreated stems and flowers of Dendrobium huoshanense, the first sugar added includes 30%~60% white sugar, 10%~30% brown sugar, and 20%~60% oligosaccharide aqueous solution;

[0023] For the pretreated stems and flowers of Dendrobium nobile, the first sugar added includes 30%~70% white sugar, 10%~30% brown sugar, and 20%~50% oligosaccharide aqueous solution.

[0024] Preferably, the second and third mass ratios are both 2~4:1:1, the amount of lactic acid bacteria added is 0.8%~12%, the fermentation temperature of the second stage fermentation is 18℃~37℃, and the fermentation time is 30 days~60 days.

[0025] Preferably, the solid residues of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile are mixed in a third mass ratio and a second sugar substance is added. The second sugar substance accounts for 10% to 40% of the total weight and includes 20% to 40% white sugar and 50% to 80% oligosaccharide aqueous solution, with the oligosaccharide aqueous solution having a mass fraction of 50%.

[0026] Preferably, the lactic acid bacteria are one or more of Lactobacillus plantarum, Lactobacillus bulgaricus, and Lactobacillus acidophilus.

[0027] Preferably, the fermentation temperature for the third stage of fermentation is 18℃~37℃, and the fermentation time is 30 days~60 days.

[0028] Preferably, the fermentation container is sterilized and then cleaned and dried before use.

[0029] Preferably, after the third stage of fermentation, filtration is performed using a 400-800 mesh filter cloth, and the obtained Dendrobium compound enzyme stock solution is aseptically dispensed and stored at -20℃ to -10℃.

[0030] Preferably, the pretreatment involves washing, drying at room temperature, and cutting into segments, with the segments being 0.5cm to 1.5cm in length.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] This method uses the stems and flowers of fresh Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile as the main raw materials. Through screening, pretreatment, staged fermentation at room temperature, and filtration, functional processed products of Dendrobium, namely Dendrobium compound enzyme stock solution, are obtained. Specifically:

[0033] 1) By making full use of the stems and flowers of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile, especially by adding the flowers of Dendrobium nobile, Dendrobium huoshanense, and Dendrobium officinale as one of the raw materials for co-fermentation, we can give full play to the role of Dendrobium flowers, which are currently under-processed, and realize the high-value utilization of the whole Dendrobium resources.

[0034] 2) By inoculating beneficial bacteria for staged fermentation at room temperature, the medicinal components in Dendrobium stems and flowers are preserved to the greatest extent. This process enhances the diversity and utilization of active substances in the raw materials, and synergistically combines several characteristic functional substances of Dendrobium to maximize the retention and full release of active ingredients. These active ingredients are then metabolized and transformed by beneficial microorganisms to produce small-molecule functional substances with excellent antioxidant activity, immune regulation, and gastrointestinal protection, resulting in significant health benefits. Simultaneously, the product's flavor is improved, and the active ingredients are more balanced and abundant, leading to more pronounced functional effects. Furthermore, the product production process is simple and free of chemical components, providing a new avenue for the diversified and high-value development of Dendrobium fermentation products, with broad application prospects and significant economic benefits.

[0035] 3) By introducing multi-strain fermentation in stages to match the raw materials, contamination by miscellaneous bacteria is avoided, while ensuring the growth of beneficial microorganisms. Through metabolism, small molecule functional substances are accumulated, which are beneficial to human absorption and enhance the nutritional and health benefits of the product. Attached Figure Description

[0036] Figure 1 This is a flowchart of the preparation method of the Dendrobium compound enzyme of the present invention. Detailed Implementation

[0037] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0038] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the application.

[0039] Dendrobium plants, as valuable medicinal and edible resources, have seen their development in the functional food sector become a significant growth point in the health industry. Faced with the current development opportunities in the medicinal and edible industry and rapidly growing market demand, the research and development of high-value functional foods and new, sophisticated processing technologies has become a crucial issue for the Dendrobium industry. While the stems of common Dendrobium species are the primary medicinal part, the flowers and leaves also possess high medicinal value. Studies have found that Dendrobium leaves and flowers contain active ingredients such as flavonoids, polysaccharides, and alkaloids, exhibiting numerous pharmacological effects. However, in recent years, the application of Dendrobium flowers in functional foods has been relatively limited, mainly concentrated in primary processed products, failing to achieve high-value, full utilization. This application aims to achieve high-value, full utilization of Dendrobium resources through co-fermentation of stems and flowers from several Dendrobium species.

[0040] like Figure 1 As shown, a method for preparing a Dendrobium-based compound enzyme includes the following steps:

[0041] S1. Harvest and select the stems and flowers of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile. Specifically, harvest fresh stems and flowers of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile grown in a semi-wild environment, and select those that are free from rot, pests, diseases, and mechanical damage.

[0042] S2. The selected stems and flowers are pretreated separately.

[0043] In one embodiment, the pretreatment involves washing, drying at room temperature, and cutting into segments of 0.5cm to 1.5cm in length. The selected stems and flowers are repeatedly rinsed with running water to remove surface impurities, dried at room temperature (i.e., air-dried at room temperature), and cut into segments of 0.5cm to 1.5cm each for later use.

[0044] S3. After pretreatment, the stems and flowers of the same Dendrobium species are mixed according to the first mass ratio, and the first sugar substance is added separately. The mixture is stirred evenly, placed in a fermentation container, and brewing yeast is added. After the first stage of fermentation, the mixture is filtered to obtain the original liquid and solid residue of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile. After the first stage of fermentation, the mixture can be filtered through a 400-600 mesh filter cloth.

[0045] In one embodiment, the first mass ratio is 1:0.1~0.5, the amount of brewing yeast added is 0.6%~10%, the fermentation temperature of the first stage of fermentation is 20℃~38℃, the stirring is carried out once every 3 days for 2 minutes~5 minutes each time, and the fermentation time is 30 days~60 days.

[0046] In one embodiment, pretreated stems and flowers of the same Dendrobium species are mixed according to a first mass ratio, and then a first sugar substance is added separately. The first sugar substance accounts for 40% to 60% of the total weight, and is specifically as follows:

[0047] For the pretreated stems and flowers of Dendrobium officinale, the first sugar added includes 30%~70% white sugar, 10%~30% brown sugar, and 20%~50% oligosaccharide aqueous solution, with the oligosaccharide aqueous solution having a mass fraction of 50%.

[0048] For the pretreated stems and flowers of Dendrobium huoshanense, the first sugar added includes 30%~60% white sugar, 10%~30% brown sugar, and 20%~60% oligosaccharide aqueous solution;

[0049] For the pretreated stems and flowers of Dendrobium nobile, the first sugar added includes 30%~70% white sugar, 10%~30% brown sugar, and 20%~50% oligosaccharide aqueous solution.

[0050] In one embodiment, the fermentation vessel is sterilized and then cleaned and dried before use.

[0051] S4. After the first stage of fermentation, the second stage of fermentation is carried out, as follows:

[0052] The original liquids of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed at a second mass ratio and then lactic acid bacteria were added. The solid residues of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed at a third mass ratio, a second sugar substance was added, and then lactic acid bacteria were added. The second stage of fermentation was carried out separately to obtain the original liquid fermentation enzyme and the solid fermentation product.

[0053] In one embodiment, the second mass ratio and the third mass ratio are both 2~4:1:1, the amount of lactic acid bacteria added is 0.8%~12%, the fermentation temperature of the second stage fermentation is 18℃~37℃, and the fermentation time is 30 days~60 days.

[0054] In one embodiment, the solid residues of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile are mixed in a third mass ratio and a second sugar substance is added. The second sugar substance accounts for 10% to 40% of the total weight and includes 20% to 40% white sugar and 50% to 80% oligosaccharide aqueous solution, with the oligosaccharide aqueous solution having a mass fraction of 50%.

[0055] In one embodiment, the lactic acid bacteria are one or more of Lactobacillus plantarum, Lactobacillus bulgaricus, and Lactobacillus acidophilus.

[0056] S5. Add the filtered solid fermentation liquid to the original liquid fermentation enzyme, stir well, and carry out the third stage of fermentation.

[0057] In one embodiment, the fermentation temperature of the third stage fermentation is 18°C ​​to 37°C, and the fermentation time is 30 to 60 days.

[0058] S6. After the third stage of fermentation is completed, filter to obtain Dendrobium complex enzyme stock solution.

[0059] In one embodiment, after the third stage of fermentation, filtration is performed using a 400-800 mesh filter cloth, and the obtained Dendrobium complex enzyme stock solution is aseptically dispensed and stored at -20℃ to -10℃.

[0060] This method utilizes the stems and flowers of various Dendrobium species, including Dendrobium nobile, Dendrobium huoshanense, and Dendrobium officinale, as the main raw materials to prepare Dendrobium-based compound enzymes (Dendrobium-based compound enzyme stock solution) through multi-stage fermentation. By improving the diversity and utilization of active substances in the raw materials through fermentation, and by combining the synergistic effects of the characteristic functional substances of several Dendrobium species, the active ingredients are more balanced and abundant, and the functional effects are more significant. This provides a new approach for the diversified and high-value development of Dendrobium fermentation products, and has broad application prospects and significant economic benefits.

[0061] For ease of understanding, the following detailed description is provided through specific embodiments.

[0062] Example 1:

[0063] Dendrobium officinale is a traditional Chinese medicine from Zhejiang Province, and its cultivation areas are mainly distributed in Yueqing, Tiantai, Wucheng, Yiwu, etc.; Dendrobium huoshanense, commonly known as "rice dendrobium", is mainly produced in Huoshan, Anhui Province, in the Dabie Mountains; Dendrobium nobile has a long history of medicinal use in Guizhou and is the Dendrobium variety with the largest cultivation area and highest yield in Guizhou Province. The health care functions and medicinal value of these three Dendrobium species are widely recognized in the market.

[0064] In this embodiment, stems and flowers of fresh, semi-wild cultivated Dendrobium officinale (Yueqing Zhongfangrun Dendrobium Co., Ltd.), Dendrobium huoshanense (Anhui Bieshan Dendrobium Biotechnology Co., Ltd.), and Dendrobium nobile (Chishui Wanglong Fuxing Family Farm) aged 3 to 6 years were harvested. Stems and flowers of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile that were free from rot, pests, diseases, and mechanical damage were selected.

[0065] After screening, rinse the stems and flowers repeatedly with running water to remove surface impurities, dry them at room temperature, and cut them into 1.2cm sections for later use.

[0066] Pretreated Dendrobium officinale stems and flowers were mixed at a mass ratio of 1:0.2. A primary sugar content was added, comprising 45% white sugar, 25% brown sugar, and 30% oligosaccharide aqueous solution (50% by mass), accounting for 40% of the total weight. The mixture was stirred thoroughly and placed in a sterilized, clean, and dry fermentation container. 5% brewing yeast was added, and the first stage of fermentation was initiated. The fermentation temperature was controlled at 35℃, and the mixture was stirred for 3 minutes every 3 days for a total fermentation time of 45 days. After fermentation, the Dendrobium officinale extract and solid residue were filtered through a 600-mesh filter cloth.

[0067] Pretreated Dendrobium huoshanense stems and flowers were mixed at a mass ratio of 1:0.2. A primary sugar, comprising 45% white sugar, 25% brown sugar, and 30% oligosaccharide aqueous solution (50% by mass), was added, making up 40% of the total weight of the material. The mixture was stirred thoroughly and placed in a sterilized, clean, and dry fermentation container. 4% brewing yeast was added, and the first stage of fermentation was initiated. The fermentation temperature was controlled at 35℃, and the mixture was stirred for 3 minutes every 3 days for a total fermentation time of 40 days. After fermentation, the original liquid and solid residue of Dendrobium huoshanense were filtered out using a 600-mesh filter cloth.

[0068] Pretreated Dendrobium nobile stems and flowers were mixed at a mass ratio of 1:0.3. A primary sugar, comprising 45% white sugar, 25% brown sugar, and 30% oligosaccharide aqueous solution (50% by mass), was added, making up 40% of the total weight of the material. The mixture was stirred thoroughly and placed in a sterilized, clean, and dry fermentation container. 5% brewing yeast was added, and the first stage of fermentation was initiated at 35°C. The mixture was stirred every 3 days for 3 minutes each time, for a total fermentation time of 40 days. After fermentation, the Dendrobium nobile extract and solid residue were filtered through a 600-mesh filter cloth.

[0069] After the first stage of fermentation, the original liquids of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed in a mass ratio of 2:1:1. 5% lactic acid bacteria (Lactobacillus plantarum and Lactobacillus bulgaricus in a mass ratio of 3:1) were added for the second stage of fermentation. The fermentation temperature was controlled at 35℃ and the fermentation time was 45 days to obtain the original liquid fermented enzyme.

[0070] After the first stage of fermentation, the solid residues of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed in a mass ratio of 2:1:1. The added secondary sugars consisted of 35% white sugar and 65% oligosaccharide aqueous solution (mass fraction of 50%), accounting for 30% of the total weight. 6% lactic acid bacteria (Lactobacillus plantarum and Lactobacillus bulgaricus in a mass ratio of 3:1) were added for the second stage of fermentation. The fermentation temperature was controlled at 35℃ and the fermentation time was 45 days to obtain solid fermented products.

[0071] After filtering the solid fermentation material obtained in the second stage of fermentation, the resulting stock solution was added to the stock solution fermentation enzyme obtained in the second stage of fermentation. The mixture was stirred evenly and then the third stage of fermentation was carried out. The fermentation temperature was controlled at 35℃ and the fermentation time was 45 days.

[0072] After the third stage of fermentation, the mixture was filtered through a 600-mesh filter cloth. The resulting Dendrobium compound enzyme stock solution was then dispensed under aseptic conditions and stored at -16℃.

[0073] Experimental verification showed that the obtained Dendrobium complex enzyme stock solution was rich in various beneficial microorganisms such as Saccharomyces cerevisiae and Lactobacillus plantarum, with a total phenol content of 192.57±4.34 mg / L. Its in vitro antioxidant activity was significantly enhanced compared to before fermentation. Using vitamin C as a control, the half-maximal inhibitory concentration (IC50) of the ABTS (2,2'-adiazono-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging ability was also significantly improved. 50 The result was 1.214 mg Vc / mL.

[0074] Example 2:

[0075] Dendrobium officinale is a traditional Chinese medicine from Zhejiang Province, and its cultivation areas are mainly distributed in Yueqing, Tiantai, Wucheng, Yiwu, etc.; Dendrobium huoshanense, commonly known as "rice dendrobium", is mainly produced in Huoshan, Anhui Province, in the Dabie Mountains; Dendrobium nobile has a long history of medicinal use in Guizhou and is the Dendrobium variety with the largest cultivation area and highest yield in Guizhou Province. The health care functions and medicinal value of these three Dendrobium species are widely recognized in the market.

[0076] In this embodiment, stems and flowers of fresh, semi-wild cultivated Dendrobium officinale (Yueqing Zhongfangrun Dendrobium Co., Ltd.), Dendrobium huoshanense (Anhui Bieshan Dendrobium Biotechnology Co., Ltd.), and Dendrobium nobile (Chishui Wanglong Fuxing Family Farm) aged 3 to 6 years were harvested. Stems and flowers of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile that were free from rot, pests, diseases, and mechanical damage were selected.

[0077] After screening, rinse the stems and flowers repeatedly with running water to remove surface impurities, dry them at room temperature, and cut them into 0.9 cm sections for later use.

[0078] Pretreated Dendrobium officinale stems and flowers were mixed at a mass ratio of 1:0.1. A primary sugar content was added, comprising 50% white sugar, 10% brown sugar, and 40% oligosaccharide aqueous solution (mass fraction 50%), making up 50% of the total weight. The mixture was stirred thoroughly and placed in a sterilized, clean, and dry fermentation container. 4% brewing yeast was added, and the first stage of fermentation was initiated. The fermentation temperature was controlled at 37℃, with stirring every 3 days for 5 minutes each time. The fermentation period was 50 days. After fermentation, the Dendrobium officinale extract and solid residue were filtered through a 600-mesh filter cloth.

[0079] Pretreated Dendrobium huoshanense stems and flowers were mixed at a mass ratio of 1:0.1. A primary sugar, comprising 50% white sugar, 10% brown sugar, and 40% oligosaccharide aqueous solution (mass fraction 50%), was added. The primary sugar comprised 50% of the total material weight. The mixture was stirred thoroughly and placed in a sterilized, clean, and dry fermentation container. 4% brewing yeast was added, and the first stage of fermentation was initiated. The fermentation temperature was controlled at 37℃, and the mixture was stirred for 5 minutes every 3 days for a total fermentation time of 50 days. After fermentation, the original liquid and solid residue of Dendrobium huoshanense were filtered through a 600-mesh filter cloth.

[0080] Pretreated Dendrobium nobile stems and flowers were mixed at a mass ratio of 1:0.1. A primary sugar, comprising 50% white sugar, 10% brown sugar, and 40% oligosaccharide aqueous solution (mass fraction 50%), was added. The primary sugar comprised 50% of the total material weight. The mixture was stirred thoroughly and placed in a sterilized, clean, and dry fermentation container. 4% brewing yeast was added, and the first stage of fermentation was initiated at 37°C. The mixture was stirred every 3 days for 5 minutes each time, for a total fermentation time of 50 days. After fermentation, the Dendrobium nobile extract and solid residue were filtered through a 600-mesh filter cloth.

[0081] After the first stage of fermentation, the original liquids of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed in a mass ratio of 2:1:1. 5% lactic acid bacteria (Lactobacillus plantarum, Lactobacillus bulgaricus, and Lactobacillus acidophilus in a mass ratio of 3:1:1) were added for the second stage of fermentation. The fermentation temperature was controlled at 37℃, and the fermentation time was 55 days to obtain the original liquid fermented enzyme.

[0082] After the first stage of fermentation, the solid residues of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed in a mass ratio of 2:1:1. The added secondary sugars consisted of 30% white sugar and 70% oligosaccharide aqueous solution (mass fraction of 50%), accounting for 40% of the total weight. 6% lactic acid bacteria (Lactobacillus plantarum, Lactobacillus bulgaricus, and Lactobacillus acidophilus in a mass ratio of 3:1:1) were added for the second stage of fermentation. The fermentation temperature was controlled at 37℃, and the fermentation time was 55 days to obtain solid fermented products.

[0083] After filtering the solid fermentation material obtained in the second stage of fermentation, the resulting stock solution was added to the stock solution fermentation enzyme obtained in the second stage of fermentation, stirred evenly, and then carried out the third stage of fermentation. The fermentation temperature was controlled at 37℃ and the fermentation time was 55 days.

[0084] After the third stage of fermentation, the mixture was filtered through a 600-mesh filter cloth. The resulting Dendrobium compound enzyme stock solution was then dispensed under aseptic conditions and stored at -16℃.

[0085] Experimental verification showed that the obtained Dendrobium complex enzyme stock solution was rich in various beneficial microorganisms such as Saccharomyces cerevisiae and Lactobacillus plantarum, with a total phenol content of 219.52±6.35 mg / L. Its in vitro antioxidant activity was significantly enhanced compared to before fermentation. Using vitamin C as a control, the half-maximal inhibitory concentration (IC50) of the ABTS (2,2'-adiazono-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging ability was also significantly improved. 50 The result was 0.927 mg Vc / mL.

[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The embodiments described above are merely specific and detailed examples of the embodiments described in this application, and should not be construed as limiting the scope of the application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these modifications and improvements all fall within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the appended claims.

Claims

1. A method for preparing a Dendrobium-based compound enzyme, characterized in that: Includes the following steps: S1. Harvest and select the stems and flowers of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile; S2. Pre-treat the selected stems and flowers separately; S3. After mixing the pretreated stems and flowers of the same variety of Dendrobium according to the first mass ratio, add the first sugar substance separately, stir evenly, put into a fermentation container and add brewing yeast, carry out the first stage of fermentation, filter, and obtain the original liquid and solid residue of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile. S4. After the first stage of fermentation, the second stage of fermentation is carried out, as follows: The original liquids of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed in a second mass ratio and then lactic acid bacteria were added. The solid residues of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile were mixed in a third mass ratio, a second sugar substance was added, and then lactic acid bacteria were added. The second stage of fermentation was carried out to obtain the original liquid fermentation enzyme and the solid fermentation product respectively. S5. Add the filtered solid fermentation liquid to the original liquid fermentation enzyme, stir well, and carry out the third stage of fermentation. S6. After the third stage of fermentation is completed, filter to obtain Dendrobium complex enzyme stock solution.

2. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: The first mass ratio is 1:0.1~0.5, the amount of brewing yeast added is 0.6%~10%, the fermentation temperature of the first stage of fermentation is 20℃~38℃, stirring is performed once every 3 days for 2 to 5 minutes each time, and the fermentation time is 30 to 60 days.

3. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: The process involves mixing pretreated stems and flowers of the same Dendrobium species at a first mass ratio, and then adding a first sugar substance, which accounts for 40% to 60% of the total weight, specifically as follows: For the pretreated stems and flowers of Dendrobium officinale, the first sugar substance added includes 30%~70% white granulated sugar, 10%~30% brown sugar, and 20%~50% oligosaccharide aqueous solution, wherein the mass fraction of the oligosaccharide aqueous solution is 50%. For the pretreated stems and flowers of Dendrobium huoshanense, the first sugar added includes 30%~60% white sugar, 10%~30% brown sugar, and 20%~60% oligosaccharide aqueous solution; For the pretreated stems and flowers of Dendrobium nobile, the first sugar added includes 30%~70% white sugar, 10%~30% brown sugar, and 20%~50% oligosaccharide aqueous solution.

4. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: The second and third mass ratios are both 2~4:1:1, the amount of lactic acid bacteria added is 0.8%~12%, the fermentation temperature of the second stage fermentation is 18℃~37℃, and the fermentation time is 30 days~60 days.

5. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: The solid residues of Dendrobium officinale, Dendrobium huoshanense, and Dendrobium nobile are mixed in a third mass ratio and a second sugar substance is added. The second sugar substance accounts for 10% to 40% of the total weight and includes 20% to 40% white sugar and 50% to 80% oligosaccharide aqueous solution, wherein the mass fraction of the oligosaccharide aqueous solution is 50%.

6. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: The lactic acid bacteria are one or more of Lactobacillus plantarum, Lactobacillus bulgaricus, and Lactobacillus acidophilus.

7. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: The fermentation temperature for the third stage of fermentation is 18℃~37℃, and the fermentation time is 30 days~60 days.

8. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: The fermentation container is sterilized and then cleaned and dried before use.

9. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: After the third stage of fermentation, the filter is made using a 400-800 mesh filter cloth, and the obtained Dendrobium compound enzyme stock solution is aseptically packaged and stored at -20℃ to -10℃.

10. The method for preparing Dendrobium-based compound enzymes as described in claim 1, characterized in that: The pretreatment involves washing, drying at room temperature, and cutting into segments, with the segments being 0.5cm to 1.5cm in length.

Images