CN114304335A - Method for enriching active ingredients of dendrobium leaves through fermentation and application of method - Google Patents
Method for enriching active ingredients of dendrobium leaves through fermentation and application of method Download PDFInfo
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Abstract
The invention discloses a method for enriching active ingredients of dendrobium leaves by fermentation and application thereof, comprising the following steps: A. cleaning fresh dendrobium leaves, drying and grinding the dendrobium leaves into powder, and adding water into the obtained powder product for size mixing to obtain dendrobium leaf pulp; B. heating the dendrobe leaf pulp for sterilization, cooling, inoculating and fermenting, wherein the microorganism used for inoculating and fermenting is the compound of lactobacillus plantarum, saccharomyces cerevisiae, aspergillus niger and aspergillus oryzae; C. after fermentation is completed, performing ultrasonic-assisted extraction on the obtained dendrobium leaves, and then performing centrifugal treatment to obtain the dendrobium leaf extract. According to the invention, the screened lactobacillus plantarum, saccharomyces cerevisiae, aspergillus niger and aspergillus oryzae are used for carrying out compound fermentation on dendrobium nobile leaves, so that the content of active substances such as polysaccharide, flavone and the like in the extract is increased, and the oxidation resistance is obviously improved; meanwhile, the freeze drying technology is adopted, so that the nutritional active ingredients of the product are reserved, the solubility and the taste of the product are improved, and a scientific basis is provided for the comprehensive utilization of the dendrobium leaves.
Description
Technical Field
The invention relates to the technical field of fermented tea preparation, and particularly relates to a method for enriching active ingredients of dendrobium leaves by fermentation and application thereof.
Background
Dendrobium officinale (Dendrobium officinale Kimura et Migo) is a rare medicinal plant of the genus Dendrobium of Orchidaceae, and enjoys the reputation of "life saving Mesona" in China. Wild dendrobium officinale is not cold-resistant and favors warm and humid climate, and in China, medicinal dendrobium officinale is mainly distributed in certain danxia geomorphologic regions of Fujian, Zhejiang, Guangdong, Yunnan and other provinces at first, and the danxia geomorphologic region is the first main habitat of dendrobium officinale.
The dendrobium officinale as a Chinese medicine variety is collected in the 2010 edition of Chinese pharmacopoeia, is sweet in taste and slightly cold in nature, and has the effects of promoting the production of body fluid, nourishing the stomach, moistening the lung and tonifying the kidney. Contains dendrobium polysaccharide, dendrobine, phenols, flavonoids, amino acid and other active ingredients, and the modern pharmacological research shows that the dendrobium officinale has the medicinal values of reducing blood pressure, improving immunity, resisting tumors, resisting aging, reducing blood fat and the like, and is widely used in cosmetics, health care products and common foods. In Chinese pharmacopoeia, dendrobium officinale takes whole herbs as medicinal parts, but in most cases, people use dendrobium stems as medicines or prepare other products, which causes certain waste in utilization of leaves and flowers. In recent years, the research on dendrobium is more and more abundant and the research range is more and more extensive due to the massive planting of dendrobium. In recent years, most researchers have found that in the same plant, 90% or more of plant organs are related to each other, for example, the types of components are relatively consistent, and the effects of functional characteristics are consistent, in the aspects of phytochemistry, pharmacological experiments thereof and the like. The main active ingredients in the dendrobium leaves are polysaccharide (stem > leaf > flower), flavonoid and phenols (flower > leaf > stem), alkaloid (stem upper segment > leaf > stem middle segment, 1 year more than or equal to 2 years more than or equal to wild type), amino acid (essential amino acid total amount stem > leaf > flower, essential amino acid account for leaf > stem > flower), and the dendrobium leaves have the effects of oxidation resistance, inflammation resistance, immunoregulation and the like. However, the study and processing of the leaves of dendrobium are less at present, and the utilization rate of the leaves is lower.
Chinese patent CN112042781A discloses a preparation method of dendrobium officinale health tea, which is approximately characterized in that: pulping the leaves and stems of Dendrobium officinale to obtain tea pulp, sterilizing, inoculating tea fungus mycoderm and Bacillus natto, performing aerobic fermentation at 28-30 deg.C in sterile and dark environment to obtain fermented tea pulp, and spray drying the fermented tea pulp to obtain the final product. The patent technology avoids the problem of nutrient component loss caused by the fact that traditional tea needs to be aired, withered and the like in the preparation process, but the patent technology does not give the content of active components of polysaccharide and flavone in the obtained dendrobium officinale health-care tea, meanwhile, the problems that a plurality of active substances in the dendrobium officinale are inactivated due to overhigh temperature and the like due to spray drying are solved, special research and processing are not carried out on dendrobium officinale leaves, and the reference value is limited.
Chinese patent CN112006143A discloses a Mei Teng fruit leaf dendrobium leaf Kangpu tea with high polysaccharide content and a preparation method thereof, wherein the process is approximately as follows: taking the Mei Teng fruit leaves and the Dendrobium officinale leaves, rolling, fermenting, drying to obtain a dry matter, and boiling the dry matter to obtain a mixed culture solution; compounding lactobacillus plantarum, bacillus lactis DU-106 and saccharomyces cerevisiae freeze-dried powder to obtain bacterium powder, adding the bacterium powder into the mixed culture solution, culturing, and filtering for sterilization to obtain the lactobacillus plantarum and lactobacillus lactis DU-106 strain. The patent technology is prepared into liquid beverage, is inconvenient to transport and has limited shelf life.
Disclosure of Invention
The invention aims to: aiming at the existing problems, the invention provides a method for fermenting and enriching the active ingredients of the dendrobium leaves and application thereof, the screened lactobacillus plantarum, saccharomyces cerevisiae, aspergillus niger and aspergillus oryzae carry out compound fermentation on the dendrobium leaves, and the contents of active substances such as polysaccharide, flavone and the like in the extract are increased and the oxidation resistance is obviously improved by ultrasonic-assisted extraction; meanwhile, the freeze drying technology is adopted, so that the nutritional active ingredients of the product are reserved, the solubility and the taste of the product are improved, scientific basis can be provided for comprehensive utilization of the dendrobium leaves, and the defects in the prior art are overcome.
The technical scheme adopted by the invention is as follows: a method for fermenting and enriching active ingredients of dendrobium leaves is characterized by comprising the following steps:
A. cleaning fresh dendrobium leaves, drying and grinding the dendrobium leaves into powder, and adding water into the obtained powder product for size mixing to obtain dendrobium leaf pulp;
B. heating the dendrobe leaf pulp for sterilization, cooling, inoculating and fermenting, wherein the microorganism used for inoculating and fermenting is the compound of lactobacillus plantarum, saccharomyces cerevisiae, aspergillus niger and aspergillus oryzae;
C. after fermentation is completed, performing ultrasonic-assisted extraction on the obtained dendrobium leaves, and then performing centrifugal treatment to obtain the dendrobium leaf extract.
Further, the mass ratio of the lactobacillus plantarum to the saccharomyces cerevisiae to the aspergillus niger to the aspergillus oryzae is 1: (1-2): (1-2): (1-2). Specifically, Lactobacillus plantarum CICC194165, Saccharomyces cerevisiae GIM2.43, Aspergillus niger CICC 2041, and Aspergillus oryzae CICC 2011 are included. Of course, other strains can be selected, such as one or more of Lactobacillus acidophilus (Lactobacillus) CICC 6091, Lactobacillus fermentum (Lactobacillus fermentum) CICC 6233, Pichia kluyveri (Pichia kluyveri) CICC 32845 and Bacillus subtilis (Bacillus subtilis) CICC 22459, but the use effect of the strains is not good compared with that of the compound strains screened by the test method.
Preferably, the mass ratio of the lactobacillus plantarum to the saccharomyces cerevisiae to the aspergillus niger to the aspergillus oryzae is 1:2:1: 2.
further, the fermentation conditions of the dendrobium stem leaf pulp are as follows: the ratio of the material to the liquid is 1:5-25, the fermentation temperature is 28-38 ℃, the fermentation time is 24-120h, and the inoculation amount of the microbial strains in the fermentation liquid is 6-10% of the volume of the dendrobium leaf pulp.
Preferably, the fermentation conditions of the dendrobium stem leaf pulp are as follows: the ratio of the material to the liquid is 1:10, the fermentation temperature is 32-35 ℃, the fermentation time is 46-48h, and the inoculation amount of the microbial strains in the fermentation liquid is 8-9% of the volume of the dendrobium leaf pulp.
Further, the extraction conditions of the ultrasonic-assisted extraction are as follows: the ultrasonic treatment time is 28-30min, and the ultrasonic treatment temperature is 62-65 ℃.
Preferably, the centrifugation conditions after the ultrasonic extraction are: rotating speed 4000r/min, and centrifuging for 10 min.
The invention also comprises a method for preparing the dendrobium tea by applying the method, in particular to a method for preparing the dendrobium officinale leaf instant tea, which comprises the following steps:
s1, preparing the dendrobe leaf extract according to the method for later use;
s2, mixing the raw tea and the dendrobium leaf extract according to a certain proportion, concentrating, and freeze-drying to obtain the dendrobium tea.
Further, the mass ratio of the dendrobium leaf extract to the raw tea is 1: 1-5.
further, pre-freezing treatment is carried out before freeze drying, and the pre-freezing conditions are as follows: refrigerating at-80 deg.C for 24-48 h, and freeze drying under the following conditions: freezing at-40- (-50) deg.C for 24-26 h.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. at present, most researchers pay attention to the development and utilization of stem of dendrobium, research on effective active ingredients of the stem, often discard dendrobium leaves as waste, pollute the environment and occupy the land, because the same plant has 90% homology, and more active ingredients also exist in the dendrobium leaves, the invention utilizes the dendrobium leaves to ferment and extract the dendrobium leaves under the condition of not using auxiliary raw materials, in the fermentation stage, the invention obtains a composite strain which is beneficial to obtaining more active ingredients through screening tests (the enrichment effect of single strain active ingredients is not obvious), the invention overcomes the problem that the fermentation effect is greatly different under the same fermentation condition by controlling the mass ratio among strains, and simultaneously, a set of fermentation process and parameters suitable for the composite strain are obtained through research on the influence of the fermentation process and parameters on the composite strain and experimental summary analysis, the compound strain is developed towards the most favorable direction as far as possible, the contents of polysaccharide and flavone in the dendrobium leaf extract are improved, the utilization efficiency of the dendrobium leaf is improved, the obtained dendrobium extract can be directly applied to the fields of food, medicines, health products and the like, and the application field of the dendrobium leaf is widened;
2. the dendrobe leaf extract obtained by the enrichment method has high content of effective components such as polysaccharide, flavone and the like, the content of polysaccharide is not lower than 35%, the content of flavone is not lower than 2.8%, the dendrobe leaf extract has good solubility, the antioxidation and other effects can be fully exerted, meanwhile, the effective components are enriched and increased by adopting microbial fermentation, part of components influencing the taste quality of a product are degraded, the extraction rate is increased and the active components are retained by adopting an ultrasonic auxiliary extraction technology, meanwhile, the solvent residue is avoided, and the quality of the fermented extract is improved;
3. after the dendrobium extract obtained by fermentation and the raw tea are compounded according to a specific proportion, the taste of the obtained product is improved, a part of components influencing the taste quality of the product in dendrobium are reduced by the way of compounding the dendrobium extract and the raw tea, the obtained instant dendrobium officinale leaf tea has good taste, has health-care effects of oxidation resistance, bacteriostasis and the like to a certain extent, and provides a scientific basis for comprehensive utilization of dendrobium leaves.
Drawings
FIG. 1 is a schematic diagram of a process route for extracting active ingredients from leaves of Dendrobii caulis by fermentation and ultrasonic-assisted extraction;
FIG. 2 is a route chart of a preparation process of the instant dendrobium leaf tea;
FIG. 3 is a graph showing the effect of strain species on polysaccharide content and flavone content;
FIG. 4 is a graph showing the effect of different strain ratios on polysaccharide content and flavone content;
FIG. 5 is a graph showing the effect of fermentation process on polysaccharide content and flavone content;
FIG. 6 is a graph showing the effect of ultrasound temperature on polysaccharide content and flavone content;
FIG. 7 is a graph showing the effect of ultrasound time on polysaccharide content and flavone content;
FIG. 8 is a graph showing the measurement of antioxidant capacity of the fermentation broth of Dendrobii nobile Lindl;
FIG. 9 is a graph showing the comparison of the oxidation resistance of the fermentation liquid of the leaves of Dendrobii nobile and the extract of Dendrobii nobile;
FIG. 10 is a graph showing the effect of the development process of instant dendrobium leaf tea powder on sensory evaluation.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Dendrobium leaf fermentation process
Strain: lactobacillus acidophilus (Lactobacillus) CICC 6091, Lactobacillus fermentum (Lactobacillus fermentum) CICC 6233, Lactobacillus plantarum (Lactobacillus plantarum) CICC194165, Pichia kluyveri (Pichia kluyveri) CICC 32845, Bacillus subtilis (Bacillus subtilis) CICC 22459, Saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.43, Aspergillus niger (Aspergillus niger) CICC 2041, and Aspergillus oryzae (Aspergillus oryzae) CICC 2011.
(1) Strain activation
And (3) activating bacteria: inoculating the strain into 200mL of sterilized nutrient broth culture medium, and culturing at 28 ℃ in a constant-temperature incubator for 24h to obtain a first-generation bacterial suspension; inoculating the first generation bacterial suspension into a nutrient broth culture medium according to the inoculation amount of 12%, and culturing at the constant temperature of 28 ℃ for 18h to obtain a second generation bacterial suspension; and repeating the steps for constant humidity culture at 28 ℃ for 12h, carrying out third activation to obtain third generation activated bacteria suspension, and storing in a refrigerator at 4 ℃ for later use.
Activating lactic acid bacteria: inoculating the strain into 200mL sterilized MRS broth culture medium, and culturing at 37 deg.C in a constant temperature incubator for 24h to obtain first generation bacterial suspension; inoculating the first generation bacterial suspension into MRS broth culture medium according to the inoculation amount of 12%, and culturing at the constant temperature of 37 ℃ for 18h to obtain second generation bacterial suspension; and repeating the steps at 37 ℃ for constant humidity culture for 12h, carrying out third activation to obtain third generation activated bacteria suspension, and storing in a refrigerator at 4 ℃ for later use.
Activation of fungi: inoculating the strain to potato glucose agar (PDA) slant culture medium, and culturing at 30 deg.C for 6 days in constant temperature incubator to activate the strain.
(2) Strain and proportion screening
Strains are screened by measuring the crude polysaccharide content and flavone of the dendrobium leaf fermentation liquor, and strains capable of well fermenting the dendrobium leaves are screened.
(3) Dendrobium leaf fermentation process screening
Taking the yield of crude polysaccharide and the content of flavone in the dendrobium leaf fermentation liquor as indexes, fermenting the dendrobium leaves by using 4 strains of screened bacteria according to (lactobacillus plantarum: saccharomyces cerevisiae: aspergillus niger and aspergillus oryzae: 1:2), and inspecting the influence of different fermentation temperatures (28 ℃, 30 ℃, 33 ℃, 35 ℃, 38 ℃) and fermentation times (24 hours, 48 hours, 72 hours, 96 hours and 120 hours) and strain inoculation amounts (6%, 7%, 8%, 9% and 10% of the volume of dendrobium pulp) on the content of polysaccharide and flavone in the fermentation liquor according to the material-liquid ratio (1:5, 1:10, 1:15, 1:20 and 1:25 g/mL).
And analyzing the polysaccharide content and the flavone content in the dendrobium leaves before and after fermentation.
The polysaccharide content is measured by a phenol-sulfuric acid method, and the flavone content is measured by the following method:
drawing a rutin standard curve
A volume of rutin sample is aspirated and configured as a standard solution at mass concentration of 0.30, 0.60, 0.90, 1.20, 1.50, 1.80 mg/mL. Taking 1mL of standard solution, 4mL of 50% ethanol solution, and 0.4mL5% NaNO2The solution was added to 5 prepared 10mL volumetric flasks and then mixed well. After standing for 6min, 0.4mL of 10% Al (NO) was added to the flask3)3The solution was shaken well and shaken well. Then, the mixture is fully mixed. After standing for 6min, 0.4mL of 10% Al (NO) was added to the flask3)3The solution was shaken well and shaken well. Meanwhile, taking a 50% ethanol solution as a control group, drawing different standard curves corresponding to rutin mass concentration X (mg/mL) and absorbance Y, and obtaining a regression curve equation through linear regression: Y1.1563X +0.0009, R2 0.9998.
Measurement of Dendrobium total flavone
Putting 1mL of the extracting solution (supernatant obtained by centrifuging the fermentation liquor after ultrasonic extraction) into a 10mL volumetric flask, measuring according to the method, and calculating the total flavone yield of the dendrobium leaves according to the following formula.
In the formula: a is the yield of the total flavonoids in the dendrobium leaves, mg/g; c is the mass concentration of the total flavone, mg/mL; v is the volume of the solution, mL; n is the dilution multiple; m is raw material mass, g.
The fermentation process of the dendrobe leaves comprises the following steps:
s1, cleaning fresh dendrobium leaves, drying and grinding the dendrobium leaves at 55-65 ℃, adding water into the obtained powdery product, and mixing to obtain dendrobium leaf pulp;
s2, heating the dendrobe leaf pulp to 121 ℃ for sterilization, cooling after sterilization for 10min, and then inoculating and fermenting;
C. after fermentation is completed, performing ultrasonic-assisted extraction on the obtained dendrobium leaves, and then performing centrifugal treatment to obtain the dendrobium leaf extract.
Example 2
Ultrasonic-assisted extraction process for extracting active ingredients from dendrobium leaves
Taking the yields of crude polysaccharide and flavone in the fermentation liquid of the dendrobium leaves as indexes, and investigating the influence of ultrasonic time (10min, 20min, 30min, 40min and 50min) and ultrasonic temperature (50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃) on the contents of polysaccharide and flavone in the dendrobium leaves. The process route is shown in figure 1.
The centrifugation conditions after ultrasonic extraction were as follows: centrifuging at 4000r/min for 10 min. Performing oxidation resistance comparison on the supernatant and the dendrobium leaf extracting solution (water extraction);
measurement of hydroxyl radical scavenging ability: 0.2mL of 9mmol/L FeSO is added into the reaction system4And 0.2mL of 9mmol/L salicylic acid-ethanol solution, adding centrifuged and ultrasonically extracted herba Dendrobii leaf fermentation liquid 0.2mL, and adding 0.2mL of 8.8mmol/L H2O2The reaction was started. Keeping the temperature at 37 deg.C for 30min, and measuring absorbance at 510nm with pure water as reference solution.
OH clearance (%) - (a)0-(A1-A2))/A2]×100％
In the formula: a 0-blank control absorbance, a 1-sample absorbance; A2-No color developer H2O2And (3) absorbance of the dendrobium leaf fermentation liquor extracted by ultrasonic waves.
② measuring the total oxidation resistance. Sucking centrifugal dendrobium leaf fermentation liquor 0.5mL extracted by ultrasonic wave, adding 3.8mL ABTS + diluted working solution, reacting for 6min at room temperature, measuring absorbance at 734nm, replacing dendrobium leaf fermentation liquor extracted by ultrasonic wave with pure water as blank, and replacing ABTS + diluted working solution with pure water for zero setting.
ABTS · radical clearance (%) [ (s-sb)/(c-cb) ] × 100%;
in the formula: s-ABTS-free radical scavenging ability absorbance, sb-sample interference test absorbance (zero), c-ABTS-working solution absorbance, cb-pure water absorbance (zero).
And (3) reduction force determination: 1mL of polysaccharide solutions of different concentrations (0.5, 1, 1.5, 2, 3, 4mg/mL) were put in a test tube, 0.2mL of phosphate buffer (0.2, pH 6.6, PBS) and 0.5mL of 1% potassium ferricyanide were added, mixed, and then subjected to a water bath at 50 ℃ for 20 min. Taking out, rapidly cooling, adding 1mL 10% TCA, mixing, centrifuging at 4000r/min for 10min, collecting supernatant 2.5mL, adding 2.5mL distilled water and 0.5mL 1% FeCl3. At 700nm wavelengthThe absorbance was measured and zeroed with distilled water.
Blank group: the polysaccharide solution was replaced with 1mL of distilled water.
Control group: 0.5ml of distilled water was used instead of the potassium ferricyanide solution.
Calculated according to the following formula: reducing power (A)Sample (I)-AControl)-ABlank space
Fe2+Measurement of chelating force: 1mL of polysaccharide sample solution with different concentrations (0.5, 1, 1.5, 2, 3, 4mg/mL) is added with 0.1mL of 2mmol/L FeCl23.7mL of distilled water, mixed and left for 5 min. Adding 0.2m L5mmol/L Ferrozine, standing at room temperature for 10min, and measuring absorbance at 562 nm.
Blank group: the polysaccharide sample solution was replaced with distilled water.
Control group: replacement of FeCl by distilled water2。
Calculated according to the following formula: fe2+Chelating force (%) ═ aBlank space-(ASample (I)-AControl)}/ABlank space×100％
Example 4 development of instant tea powder of dendrobe leaves
The influence of the ratio (1:1, 1:2, 1:3, 1:4, 1:5) of the dendrobium leaf fermented product to the raw tea (solid content), the freezing temperature (-20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃), the freezing time (12h, 18h, 24h, 30h and 36h) on the instant tea powder is examined by taking sensory score as an index.
10 persons with a certain professional knowledge are trained to form a sensory evaluation group, the dendrobium leaf instant tea powder is scored in 4 aspects of aroma (25 points), color (25 points), taste (25 points) and tissue state (25 points), and sensory scoring standards are shown in table 1.
TABLE 1 sensory Scoring criteria
Analysis of results
Strain screening and ratio determination
The key link of fermentation is the screening of fermentation strains. Different microorganisms have different enzyme secretion systems and secondary metabolites, so that the screening of good fermentation strains is a key link of fermentation. As can be seen in FIG. 3, the active substances produced by each strain through fermentation have different contents of polysaccharides and flavones, and the relative contents are similar to those of Lactobacillus plantarum (Lactobacillus plantarum) CICC194165, Saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.43, Aspergillus niger (Aspergillus niger) CICC 2041 and Aspergillus oryzae (Aspergillus oryzae) CICC 2011. The lactobacillus plantarum can increase flavor substances of fermentation substrates and change the structural composition of the fermentation substrates; the mould can secrete a plurality of digestive enzymes, so that nutrients are promoted to be absorbed and utilized, and the fermentation effect is increased; the saccharomyces cerevisiae can decompose sugar to provide energy substances for body activities and promote the synthesis of active substances. In order to obtain more active substance polysaccharide and flavone contents, the four strains are compounded and mixed for fermentation, and the mixed fermentation effect and the fermentation effect of different ratios of the strains are shown in figure 4. In fig. 4, as a whole, the effect of mixed fermentation is not the superposition of the fermentation effects of single strains, and no synergistic result is generated, but the contents of active substances, namely polysaccharide and flavone, are coordinated and unified, the content difference between the two contents is small, and the overall trend of rising is shown, so that the effect of single strains is limited, and the strains are subjected to compound fermentation, so that a plurality of active substances can be mutually converted, and the effect of the strains is optimal. In addition, by examining the mixing ratio among strains, when the content of lactobacillus plantarum is too high, the content of active substances polysaccharide and flavone both tend to decrease, therefore, the content of lactobacillus plantarum is not suitable to be too high, and when the content of aspergillus niger is too high, the content of active substances polysaccharide and flavone also decrease, because aspergillus oryzae and aspergillus niger belong to the genus aspergillus, both have synergistic effect, and the aspergillus oryzae, which is a commonly used enzyme in the food industry, can decompose starch and protein into sugar and amino acid, promote ingredient conversion, therefore, the ratio is relatively large. As shown in FIG. 4, when the mass ratio of the four bacterial species is 1:2:1:2, the contents of polysaccharides and flavones as the active substances are the highest, and this is taken as the preferred mass ratio.
Dendrobium leaf fermentation process screening
As can be seen from FIG. 5, the crude polysaccharide yield and the flavone content in the dendrobe leaf fermentation liquid are used as indexes, the dendrobe leaf is fermented by 4 strains of bacteria (Lactobacillus plantarum: Saccharomyces cerevisiae: Aspergillus niger: Aspergillus oryzae: 1:2), the polysaccharide and flavone contents in the dendrobe leaf are increased to a certain extent before and after fermentation, which indicates that the fermentation conditions influence the active substance enrichment, meanwhile, when the material-liquid ratio is 1:5, 1:10, 1:15, 1:20, 1:25 (unit g/mL), the polysaccharide content maximum is 1:15 in the material-liquid ratio, when the fermentation temperature is 28 ℃, 30 ℃, 33 ℃, 35 ℃ and 38 ℃, the polysaccharide content maximum is 30 ℃ in the fermentation temperature, when the fermentation time is 24h, 48h, 72h, 96h, and 120h, the polysaccharide content maximum is 48h in the fermentation time, and when the inoculation amount is 6% of the dendrobe pulp volume, When the content of the polysaccharide is 7%, 8%, 9% and 10%, the highest value of the polysaccharide content is 8% in the strain inoculation amount, the influence conditions of each condition parameter on the polysaccharide content are integrated to obtain the optimum condition parameter, the optimum fermentation temperature is 35 ℃, the fermentation time is 48h, and the strain inoculation amount is 8% of the volume of the dendrobium leaves.
Single-factor experiment for extracting process of active ingredients of dendrobe leaves by ultrasonic-assisted extraction
As can be seen from FIGS. 6-8, the optimum ultrasonic time is 30min and the ultrasonic temperature is 50 ℃ by using the yield of crude polysaccharide in the fermentation liquid of dendrobe leaves as an index.
The antioxidant activity of the fermentation broth is determined as shown in fig. 8, and as can be seen from fig. 8, the fermentation broth has strong antioxidant activity, can remove hydroxyl radicals, ABTS radicals, reducing power and the like, has a concentration-dose dependence relationship, and the antioxidant capacity is continuously increased along with the increase of the concentration.
Comparison of antioxidant Properties of fermentation liquid and extract of Dendrobii folium
As can be seen from fig. 9, the antioxidant property of the fermentation liquid of dendrobium is compared with that of the extract of dendrobium leaves, and the antioxidant property of the fermentation liquid is higher than that of the extract of dendrobium leaves, so that the cracking of xylem cell walls can be stimulated through microbial fermentation, and a plurality of active ingredients are fully released, thereby increasing the antioxidant property.
Single factor experiment for preparing instant dendrobium leaf tea powder
The research on the preparation process of the dendrobe raw tea powder is examined by taking sensory score as an index in fig. 10. Since spray drying causes many problems such as deactivation of active substances due to an excessively high temperature, the drying treatment is performed by using a freeze-drying technique. The raw tea and the dendrobium fermentation product are compounded, so that the taste is peculiar, and the nutritional value and the functional components of the product are increased. In the figure 10, when the ratio of the dendrobium to the raw tea is 1:2, the sensory score is highest, and the tea powder obtained by freeze drying for 24 hours at the temperature of minus 40 ℃ is highest, and comprehensively, the tea powder which contains a large amount of active substances and has strong dendrobium taste and fragrant raw tea taste can be obtained by adopting the process parameters.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for fermenting and enriching active ingredients of dendrobium leaves is characterized by comprising the following steps:
A. cleaning fresh dendrobium leaves, drying and grinding the dendrobium leaves into powder, and adding water into the obtained powder product for size mixing to obtain dendrobium leaf pulp;
B. heating the dendrobe leaf pulp for sterilization, cooling, inoculating and fermenting, wherein the microorganism used for inoculating and fermenting is the compound of lactobacillus plantarum, saccharomyces cerevisiae, aspergillus niger and aspergillus oryzae;
C. after fermentation is completed, performing ultrasonic-assisted extraction on the obtained dendrobium leaves, and then performing centrifugal treatment to obtain the dendrobium leaf extract.
2. The method for fermenting and enriching the active ingredients of the dendrobium nobile leaves as claimed in claim 1, wherein the mass ratio of the lactobacillus plantarum, the saccharomyces cerevisiae, the aspergillus niger and the aspergillus oryzae is 1: (1-2): (1-2): (1-2).
3. The method for fermenting and enriching the active ingredients of the dendrobium nobile leaves as claimed in claim 2, wherein the mass ratio of the lactobacillus plantarum, the saccharomyces cerevisiae, the aspergillus niger and the aspergillus oryzae is 1:2:1: 2.
4. the method for fermenting and enriching the active ingredients of the dendrobium leaves as claimed in claim 2, wherein the fermentation conditions of the dendrobium leaf pulp are as follows: the ratio of the material to the liquid is 1:5-25, the fermentation temperature is 28-38 ℃, the fermentation time is 24-120h, and the inoculation amount of the microbial strains in the fermentation liquid is 6-10% of the volume of the dendrobium leaf pulp.
5. The method for fermenting and enriching the active ingredients of the dendrobium leaves as claimed in claim 4, wherein the fermentation conditions of the dendrobium leaf pulp are as follows: the ratio of the material to the liquid is 1:10, the fermentation temperature is 32-35 ℃, the fermentation time is 46-48h, and the inoculation amount of the microbial strains in the fermentation liquid is 8-9% of the volume of the dendrobium leaf pulp.
6. The method for fermenting and enriching the active ingredients of the dendrobium leaves as claimed in claim 4, wherein the extraction conditions of the ultrasonic-assisted extraction are as follows: the ultrasonic treatment time is 28-30min, and the ultrasonic treatment temperature is 62-65 ℃.
7. The method for fermenting and enriching the active ingredients of the dendrobium leaves as claimed in claim 6, wherein the centrifugation conditions after ultrasonic extraction are as follows: rotating speed 4000r/min, and centrifuging for 10 min.
8. A preparation method of the dendrobium officinale leaf instant tea is characterized by comprising the following steps:
s1, preparing the dendrobe leaf extract according to the method of any one of claims 1 to 7 for later use;
s2, mixing the raw tea and the dendrobium leaf extract according to a certain proportion, concentrating, and freeze-drying to obtain the dendrobium tea.
9. The preparation method of the dendrobium officinale leaf instant tea as claimed in claim 8, wherein the mass ratio of the dendrobium officinale leaf extract to the raw tea is 1: 1-5.
10. the preparation method of the dendrobium officinale leaf instant tea as claimed in claim 9, wherein pre-freezing treatment is performed before freeze drying, and the pre-freezing conditions are as follows: refrigerating at-80 deg.C for 24-48 h, and freeze drying under the following conditions: freezing at-40- (-50) deg.C for 24-26 h.
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| CN116509783A (en) * | 2023-05-18 | 2023-08-01 | 肽源（广州）生物科技有限公司 | Dendrobium officinale extract capable of promoting collagen synthesis and preparation method and application thereof |
| CN116509783B (en) * | 2023-05-18 | 2024-03-15 | 肽源（广州）生物科技有限公司 | Dendrobium officinale extract capable of promoting collagen synthesis and preparation method and application thereof |
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