Method for stabilizing anthocyanin in dendrobium officinale beverage and application

Abstract

The invention belongs to the technical field of food industry, and provides a stabilizing method and application of anthocyanin in a dendrobium officinale beverage, and the method comprises the following steps: selecting fresh Xiaoshan dendrobium officinale stems without plant diseases and insect pests and mildew and rot, removing leaves, and cutting into slices of 1-3mm; fully mixing the sliced dendrobium officinale with water, then adding proanthocyanidin, fully and uniformly mixing again, and carrying out heating treatment on the mixed solution to obtain a dendrobium officinale extracting solution; the dendrobium officinale extracting solution obtained in the second step is subjected to centrifugal separation, supernate is taken and filtered, and the stable dendrobium officinale beverage which is clear and free of sediment is obtained. The stability of the anthocyanin in the dendrobium officinale beverage is improved through the color assisting effect of the proanthocyanidin, so that the problem that the anthocyanin is easy to degrade and fade in the prior art is solved. According to the method, the color stability of the dendrobium officinale beverage can be remarkably improved, the violet color characteristic of the dendrobium officinale beverage is guaranteed, the shelf life of the beverage is prolonged, and the method has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of food industry, and particularly relates to a method and application of stabilizing anthocyanins in a dendrobium officinale beverage. Background Art

[0002] Dendrobium officinale belongs to the genus Dendrobium of the orchid family. It contains active substances such as polysaccharides and polyphenols, and has the effects of regulating intestinal flora and improving immunity. Because it contains natural pigments (mainly anthocyanins) on its surface and between the stems and nodes, it is also called black knot grass. After the anthocyanins are dissolved, they give the Dendrobium officinale beverage a unique violet color, increasing the commercial value of the product.

[0003] Anthocyanins are a class of water-soluble pigments widely found in plants. They not only give foods bright colors, but also have a variety of health functions such as anti-oxidation, neuroprotection and anti-aging. However, the phenolic hydroxyl groups of anthocyanins are easily affected by external factors such as light, temperature, oxidants and pH value, leading to degradation and fading. This has led to the current problem of quality degradation of Dendrobium officinale beverages, which cannot meet consumers' demand for high-quality Dendrobium officinale beverages.

[0004] Therefore, how to effectively improve the stability of Dendrobium officinale beverage products is a problem that urgently needs to be solved. Summary of the invention

[0005] In order to overcome the problems of the prior art, the present invention proposes a method for stabilizing anthocyanins in a Dendrobium officinale beverage and its application, which solves the problem that anthocyanins in the existing Dendrobium officinale beverage are easily degraded and discolored, and the product has a short shelf life.

[0006] The object of the present invention is achieved in that:

[0007] The first aspect of the present invention provides a method for stabilizing anthocyanins in a Dendrobium officinale beverage, comprising the following steps:

[0008] Step 1: Raw material pretreatment:

[0009] Select fresh, pest-free, and mildew-free stems of Yandang Mountain Dendrobium officinale, remove the leaves and cut into 1-3 mm thin slices.

[0010] Step 2: Steady-state extraction:

[0011] The sliced ​​Dendrobium officinale is fully mixed with water, and then proanthocyanidins are added and fully mixed again. The mixed solution is heated to obtain a stabilized Dendrobium officinale extract.

[0012] Step 3: Filtration and separation:

[0013] The Dendrobium officinale extract obtained in step 2 is centrifuged, and the supernatant is taken and filtered to obtain a clear, precipitate-free, stabilized Dendrobium officinale beverage.

[0014] Furthermore, in step 2, the sliced ​​Dendrobium officinale is mixed with water at a solid-liquid ratio of 1:15 to 1:30.

[0015] Furthermore, in step 2, the proanthocyanidin is selected from grape seed proanthocyanidin, and the added amount thereof is 1 to 10 g / L; preferably, the added amount of the proanthocyanidin is 3 to 6 g / L.

[0016] Furthermore, in step 2, the heating treatment condition is heating at 60-80° C. for 30-40 minutes.

[0017] Furthermore, in step three, the Dendrobium officinale beverage is filtered using 100-200 mesh diatomaceous earth.

[0018] Furthermore, in step 3, a mixed diatomaceous earth is used for filtering, wherein the mixed diatomaceous earth is obtained by mixing 100 mesh diatomaceous earth and 200 mesh diatomaceous earth in a ratio of 2:1;

[0019] First, 100-mesh diatomaceous earth is laid as the bottom layer, and then 200-mesh diatomaceous earth is placed on the upper layer. The centrifuged Dendrobium officinale extract is finely filtered through the mixed diatomaceous earth filter layer to remove impurities.

[0020] A second aspect of the present invention provides a stabilized Dendrobium officinale beverage, wherein the stabilized Dendrobium officinale beverage is prepared by the method of the first aspect.

[0021] The third aspect of the present invention provides application of the stabilization method described in the first aspect in the processing of Dendrobium officinale beverage.

[0022] The advantages and beneficial effects of the present invention are:

[0023] 1. The method for stabilizing anthocyanins in the Dendrobium Officinale beverage of the present invention utilizes the auxiliary color effect between anthocyanins and proanthocyanidins - a "π-π stacking" sandwich structure is formed between anthocyanins and co-pigments, thereby protecting the flavanoid cations of anthocyanins and preventing hydration, thereby significantly improving the stability of anthocyanins in the Dendrobium Officinale beverage.

[0024] 2. The Dendrobium officinale beverage prepared by the method of the present invention is clear and has no precipitation, and has bright color and violet characteristics.

[0025] 3. The present invention determines the optimal amount of proanthocyanidins added to the Dendrobium officinale beverage.

[0026] 4. The color stability of the Dendrobium officinale beverage obtained by the present invention is greatly improved compared with the prior art, and the thermal stability and storage stability of the Dendrobium officinale beverage are also significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0028] Figure 1 This is a comparison chart of the anthocyanin retention rates of the Dendrobium officinale beverage in Example 1 and Comparative Example 1 after adding H2O2;

[0029] Figure 2 The result comparison diagram of anthocyanin retention rate of Dendrobium officinale beverage in Example 1 and Comparative Example 1 after heating;

[0030] Figure 3 This is a comparison chart of the anthocyanin retention rates of the Dendrobium officinale beverage in Example 1 and Comparative Example 1 under room temperature storage conditions;

[0031] Figure 4 This is a comparison chart of the color difference of anthocyanins in the Dendrobium officinale beverage in Example 1 and Comparative Example 1 under room temperature storage conditions;

[0032] Figure 5 The Fourier transform infrared spectroscopy test result diagram for test example 3;

[0033] Figure 6 This is the X-ray diffraction test result diagram for Test Example 3;

[0034] Figure 7 This is a diagram of the molecular docking test results for Test Example 3. DETAILED DESCRIPTION

[0035] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0036] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not used to limit the present invention.

[0038] In the following examples, Dendrobium officinale was from No. 1 Dendrobium officinale in Yandang Mountain, Yueqing City, Zhejiang Province, and food-grade grape seed extract proanthocyanidins were purchased from Jiangxi Huayuyuan Biotechnology Co., Ltd.

[0039] Example 1: Stabilized Dendrobium officinale beverage with added proanthocyanidins

[0040] This embodiment provides a method for stabilizing anthocyanins in a Dendrobium officinale beverage, comprising the following steps:

[0041] Step 1: Raw material pretreatment:

[0042] Select fresh, pest-free, and mildew-free stems of Dendrobium candidum from Yandang Mountain to ensure their high quality. Remove the leaves from the stems and cut them into 1mm slices.

[0043] Step 2: Steady-state extraction:

[0044] The sliced ​​Dendrobium officinale was mixed with water at a solid-liquid ratio of 1:20, and then 5 g / L of food-grade grape seed extract proanthocyanidins was added and mixed again to ensure full dissolution. Then, the mixed solution was heated to 80°C and maintained at this temperature for 30 minutes to extract the active ingredients in the Dendrobium officinale to obtain a Dendrobium officinale extract.

[0045] Step 3: Filtration and separation:

[0046] The Dendrobium officinale extract obtained in step 2 is centrifuged to separate the supernatant. 100 mesh and 200 mesh diatomaceous earth are mixed in a ratio of 2:1 to obtain mixed diatomaceous earth. The mixed diatomaceous earth is used for filtration. First, 100 mesh diatomaceous earth is laid as the bottom layer, and then 200 mesh diatomaceous earth is placed on the upper layer. The Dendrobium officinale beverage is finely filtered through the filter layer to remove impurities, improve the clarity and stability of the beverage, and finally obtain a stabilized Dendrobium officinale beverage.

[0047] Final product: After the above steps, a high-quality stabilized Dendrobium officinale beverage is obtained, which has good color stability and clarity. The food-grade grape seed extract proanthocyanidins can effectively enhance the color stability of the beverage while retaining the nutritional components of Dendrobium officinale.

[0048] Comparative Example 1: Dendrobium officinale beverage without adding proanthocyanidins

[0049] In order to explore the effect of proanthocyanidins on the stability of Dendrobium officinale beverage, this comparative example provides a method for preparing a Dendrobium officinale beverage without adding proanthocyanidins, which is different from Example 1 in that no "proanthocyanidins" are added, and specifically comprises the following steps:

[0050] Step 1: Raw material pretreatment:

[0051] Select fresh, pest-free, and mildew-free stems of Dendrobium candidum from Yandang Mountain to ensure their high quality. Remove the leaves from the stems and cut them into 1mm slices.

[0052] Step 2: Extraction:

[0053] The sliced ​​Dendrobium officinale and water are fully mixed at a solid-liquid ratio of 1:20, heated to 80° C., and the temperature is maintained and heated for 30 minutes to extract the effective ingredients in the Dendrobium officinale to obtain a Dendrobium officinale extract.

[0054] Step 3: Filtration and separation:

[0055] The Dendrobium officinale extract obtained in step 2 is centrifuged to separate the supernatant. 100-mesh and 200-mesh diatomaceous earth are mixed in a ratio of 2:1 to obtain mixed diatomaceous earth. The mixed diatomaceous earth is used for filtration. First, 100-mesh diatomaceous earth is laid as the bottom layer, and then 200-mesh diatomaceous earth is placed on the upper layer. The Dendrobium officinale beverage is finely filtered through the filter layer to remove impurities and improve the clarity and stability of the beverage.

[0056] Final product: After the above steps, a Dendrobium officinale beverage without adding proanthocyanidins is obtained. Test Example 1: Stability test of Dendrobium officinale beverage

[0057] In order to explore the effect of the addition of proanthocyanidins on the stability of the Dendrobium officinale beverage, the following oxygen stability and thermal stability tests were conducted in this test example.

[0058] Oxygen stability test: Using the Dendrobium officinale beverage provided in Example 1 and Comparative Example 1 as samples, different concentrations of H2O2 (0.01, 0.02, 0.04, 0.08 and 0.1%) were added to the sample solutions, stored in the dark at 25°C for 7 days, and the anthocyanin retention rates before and after heating were calculated.

[0059] Thermal stability test: The Dendrobium officinale beverage provided in Example 1 and Comparative Example 1 was used as a sample, placed in a 90° C. water bath and heated for 5 h, and the anthocyanin retention rate before and after heating was calculated.

[0060] The content of anthocyanin was determined by pH differential method. 1 mL of sample solution was added with 4 mL of pH 1.0 and pH 4.5 The buffer solution was equilibrated at room temperature for 20 min, and the absorbance of the sample at 520 nm and 700 nm was measured respectively. The anthocyanin content in the sample solution was calculated according to formula (1) and (2).

[0061] A=(A 520nm A 700nm ) pH1.0 -(A 520nm -A 700nm ) pH4.5 (1)

[0062]

[0063] Wherein: V is the volume of the extract, mL; n is the dilution multiple; M is the relative molecular mass of cyanidin-3-glucoside, 449.4; ε is the molar extinction coefficient of cyanidin-3-glucoside, 26900; m is the sample mass, g.

[0064] In the presence of H2O2, the oxidative degradation of anthocyanins is accelerated. Figure 1 As shown, the anthocyanin retention rates of the Dendrobium Officinale beverage provided by Example 1 and Comparative Example 1 under different concentrations of H2O2. Under low concentrations of H2O2 (0.01% and 0.02%), the retention rate of Comparative Example 1 is relatively low, especially at a concentration of 0.02%, which is only 53.81%. After adding proanthocyanidins, the retention rate of Example 1 was significantly improved to 84.99%. As the concentration of H2O2 increased, the retention rates of each group decreased. Under high concentrations of H2O2 (0.10%), the retention rate of Comparative Example 1 dropped to 27.74%, but Example 1 still maintained a relatively high retention rate. Example 1 significantly improved the retention rate in the Dendrobium Officinale beverage under different concentrations of H2O2, showing a strong antioxidant protection effect.

[0065] Increased temperature will accelerate the oxidation of anthocyanins, leading to hydrolysis or glycosylation of the 2-phenylbenzopyran structure, which will degrade into benzoic acids and aldehydes, resulting in a decrease in anthocyanin content. Figure 2 It can be seen that with the extension of heating time at 90°C, the retention rate of anthocyanins in each group of samples decreased. Within the first 4 hours of heating, proanthocyanidins significantly increased the retention rate of anthocyanins in Dendrobium officinale. Compared with Comparative Example 1 without the addition of proanthocyanidins, proanthocyanidins improved the thermal stability of the Dendrobium officinale beverage.

[0066] Test Example 2: Storage stability test of Dendrobium officinale beverage

[0067] In order to explore the effect of the addition of proanthocyanidins on the stability of the Dendrobium officinale beverage, the following storage stability test was conducted in this test example.

[0068] The storage stability test process is as follows: 45 mL of the Dendrobium officinale beverage provided in Example 1 and Comparative Example 1 is put into a glass sample bottle and stored at room temperature, each in triplicate, with sampling and testing at intervals of one week.

[0069] Use a colorimeter to measure the CIELAB color parameters of the sample solution. Using the white plate color of the photometer as the standard, measure the L* (brightness), a* (red to green) and b* (yellow to blue) of each sample; use these values ​​to calculate the color difference value ΔE before and after illumination, formula (3).

[0070]

[0071] like Figure 3 As shown, in the first 4 weeks, the anthocyanin retention rate of the Dendrobium officinale beverage in Example 1 was always higher than that of Comparative Example 1. By the 4th week, the anthocyanin retention rate of Comparative Example 1 had dropped to 50.72%, while Example 1 remained above 73.22%. By the 7th week, the anthocyanin retention rate of Comparative Example 1 was only 29.72%. In contrast, Example 1 still maintained a relatively high retention rate of 67.5%. This shows that proanthocyanidins can enhance the storage stability of the Dendrobium officinale beverage and protect it from degradation.

[0072] like Figure 4 As shown, the color difference value gradually increases with the extension of the illumination time, especially in the 3rd and 4th weeks, the color difference value of comparative example 1 is significantly higher than that of example 1. In the 3rd week, the color difference value of comparative example 1 reached 14.15, while the color difference value of example 1 was only 6.91, indicating that proanthocyanidins can effectively inhibit color changes under illumination. After long-term illumination, the color difference value of comparative example 1 exceeded 24, while that of example 1 remained at 10.57, further indicating that proanthocyanidins significantly enhanced the color stability of the Dendrobium officinale beverage.

[0073] Test Example 3: Mechanism of action of proanthocyanidins and anthocyanins from Dendrobium officinale

[0074] In order to explore the mechanism by which the addition of proanthocyanidins significantly improves the stability of Dendrobium officinale beverage, this test case used Fourier Transforminfrared spectroscopy (FT-IR), X-ray diffraction analysis and molecular simulation for detection.

[0075] FT-IR analysis: Fourier transform infrared spectrometer (Nicolet IS5, Thermo Fisher Scientific, US) was used to measure the complex at 500-4000 cm -1 Fourier transform infrared spectroscopy in the range.

[0076] FT-IR analysis results are as follows Figure 5 As shown in the figure, the OH stretching vibration peak of the complexes showed a red shift (from 3438 cm -1 Up to 3440cm -1 ), which indicates that proanthocyanidins interact with the hydroxyl groups in the anthocyanins of Dendrobium officinale through hydrogen bonds. The hydroxyl groups of proanthocyanidins may form hydrogen bonds with the hydroxyl groups in the anthocyanins molecules of Dendrobium officinale, which reduces the OH vibration frequency and causes the absorption peak to red shift. Since proanthocyanidins molecules contain multiple hydroxyl groups, they can form stronger and more dense hydrogen bonds. In addition, the aromatic ring C=C skeleton vibration peak of the complex (about 1600cm -1 ) and CO stretching vibration peak (about 1225cm -1) showed an increase in intensity and a red shift in the peak position, indicating that proanthocyanidins interacted significantly with the aromatic rings and CO bonds in anthocyanins from Dendrobium officinale. This phenomenon may be due to the π-π stacking effect between proanthocyanidins and anthocyanins from Dendrobium officinale, which reduces the free movement and internal rotation within the molecule, thereby enhancing the intermolecular binding strength.

[0077] X-ray diffraction analysis: The crystalline properties of the DOA-SA complex were analyzed using an X-ray diffractometer (Smart Lab 9kW, Rigaku, Japan) with an operating voltage of 40 kV, a current of 200 mA, a scanning speed of 0.5° per minute, and a diffraction angle (2θ) range of 5–90°.

[0078] The X-ray diffraction analysis results are as follows Figure 6 As shown in the figure, the XRD pattern of D. officinale anthocyanin shows certain amorphous characteristics, and no obvious diffraction peaks are observed, which indicates that DOA itself has a low degree of crystallinity or a nearly amorphous structure. After the addition of proanthocyanidins, no significant sharp diffraction peaks appeared in the XRD patterns of the composites, indicating that proanthocyanidins failed to induce significant crystal structure formation, which may further enhance the amorphous characteristics of D. officinale anthocyanin. By comparing the XRD patterns of D. officinale anthocyanin and the composite, changes in peak shape and baseline can be observed, especially in the low angle range (10-30°). These changes may imply the interaction between the copigment and the D. officinale anthocyanin, thereby affecting the molecular arrangement of the D. officinale anthocyanin.

[0079] Molecular simulation: using The LigPrep module provided by the software prepares the ligand, adds hydrogen atoms to the ligand molecule, corrects the bond order, and uses Epik to determine all ionization states of the ligand under the condition of pH 7.0±2. The OPLS force field is used to generate up to 32 possible conformations for each ligand at a given pH value. The grid GRID file is generated by the Receptor GridGeneration module, and there is a grid around the ligand. Inner box and a Outer box. The receptor and ligand grids were generated using the GLIDE module for docking, and the OPLS-AA force field was used for evaluation and refinement. Finally, the best scoring ligand conformation was selected for graphical display using Pymol.

[0080] The molecular docking analysis results are as follows Figure 7 Shown: The spatial conformation between proanthocyanidins and anthocyanidins from Dendrobium officinale was simulated. Hydrogen bonds (yellow dashed lines) and π-π stacking interactions (blue dashed lines) between proanthocyanidins and anthocyanidins from Dendrobium officinale. Proanthocyanidins formed 4 hydrogen bonds and 3 π-π stacking interactions with anthocyanidins from Dendrobium officinale, respectively. These results also confirmed the analysis of XRD and FT-IR.

[0081] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for stabilizing anthocyanins in a Dendrobium officinale beverage, characterized in that: The method comprises the following steps: Step 1: Raw material pretreatment: Select fresh, pest-free, and mildew-free stems of Yandangshan Dendrobium officinale, remove the leaves and cut into 1-3 mm slices; Step 2: Steady-state extraction: The sliced ​​Dendrobium officinale is fully mixed with water, and then proanthocyanidins are added, and the mixture is fully mixed again, and the mixture is heated to obtain a Dendrobium officinale extract; Step 3: Filtration and separation: The Dendrobium officinale extract obtained in step 2 is centrifuged, and the supernatant is taken and filtered to obtain a clear, precipitate-free, stabilized Dendrobium officinale beverage.

2. The method for stabilizing anthocyanins in a Dendrobium officinale beverage according to claim 1, characterized in that: In step 2, the sliced ​​Dendrobium officinale is mixed with water in a solid-liquid ratio of 1:15 to 1:

30.

3. The method for stabilizing anthocyanins in a Dendrobium officinale beverage according to claim 1, characterized in that: In step 2, the proanthocyanidin is selected from grape seed proanthocyanidin, and the addition amount thereof is 1 to 10 g / L.

4. The method for stabilizing anthocyanins in a Dendrobium officinale beverage according to claim 1, characterized in that: In step 2, the heating treatment condition is heating at 60-80° C. for 30-40 minutes.

5. The method for stabilizing anthocyanins in a Dendrobium officinale beverage according to claim 1, characterized in that: In step three, the Dendrobium officinale beverage is filtered using diatomaceous earth with a mesh size of 100 to 200.

6. The method for stabilizing anthocyanins in a Dendrobium officinale beverage according to claim 5, characterized in that: In step 3, a filtration treatment is performed using mixed diatomaceous earth, wherein the mixed diatomaceous earth is obtained by mixing 100 mesh diatomaceous earth and 200 mesh diatomaceous earth in a ratio of 2:1; First, 100-mesh diatomaceous earth is laid as the bottom layer, and then 200-mesh diatomaceous earth is placed on the upper layer. The centrifuged Dendrobium officinale extract is finely filtered through the mixed diatomaceous earth filter layer to remove impurities.

7. A stabilized Dendrobium officinale beverage, characterized in that: The stabilized Dendrobium officinale beverage is prepared by the method according to any one of claims 1 to 6.

8. Use of the stabilization method according to any one of claims 1 to 6 in the processing of Dendrobium officinale beverage.

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