Dendrobium officinale composite beverage with immunocompetence

Abstract

The invention belongs to the field of health-care food, and particularly relates to a dendrobium officinale compound beverage with immunocompetence, which comprises the following components: dendrobium officinale juice, pomegranate concentrated juice, red date juice, medlar juice, rock candy liquid and soybean peptide powder. The dendrobium officinale composite beverage capable of regulating the immunity of the human body is developed by compounding the dendrobium officinale, the Chinese wolfberry fruits, the red dates and other raw materials. Animal experiment results show that the dendrobium officinale composite beverage has a remarkable immunoregulation effect on CTX-induced immunosuppressive mice by improving immune organ indexes, repairing mouse tissues after immune injury, improving the proliferation capacity of splenic lymphocytes and promoting the generation of immune globulins and cell factors.

Description

Technical Field

[0001] The invention belongs to the field of health food, and in particular relates to a Dendrobium officinale compound beverage with immune activity. Background Art

[0002] Dendrobium officinale belongs to the genus Dendrobium of the Orchidaceae family and has long been used as a healthy tea, beverage or cooking spice. The leaves of the Dendrobium genus are bright green, ribbon-shaped, slightly leathery, and alternate at the nodes along most of the length of the stem. The erect stems are fleshy and fertile, slightly compressed cylindrical, 10-60 cm long and up to 1.3 cm in diameter. It contains a variety of effective active ingredients such as polysaccharides, flavonoids, polyphenols, and alkaloids. It has the effects of improving immunity, lowering blood sugar, protecting the liver, anti-inflammatory, and anti-oxidation, and is widely used in functional health foods. It has a wide geographical distribution, mainly including India, Australia, the United States and Japan abroad, and is mainly distributed in Zhejiang, Yunnan and Fujian in China. Dendrobium officinale is a traditional Chinese medicine with medicinal value, usually added to beverages, teas, and soups as a healthy complementary food. At present, many products aimed at improving the body's immunity have been launched, but in-depth research is still needed in terms of breadth and depth. In recent years, the research on the effective ingredients of Dendrobium candidum has gradually deepened, and its development and application have gradually attracted the attention of the market. Dendrobium candidum is a medicinal material that can improve the body's immunity. In addition to being eaten fresh, it can also be made into health products, such as functional drinks.

[0003] As a functional beverage, its sensory evaluation such as color, flavor, aroma and its health care function effects determine the degree of consumers' liking for it. At present, the functional beverage of Dendrobium officinale has not been well recognized by the market. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a Dendrobium officinale compound beverage with immune activity.

[0005] The technical effects of the present invention are as follows: a dendrobium officinale compound beverage with immune activity, comprising the following components: dendrobium officinale juice, pomegranate concentrated juice, red date juice, wolfberry juice, rock sugar liquid, and soybean peptide powder; The preparation process of the Dendrobium officinale juice comprises the following steps: taking fresh Dendrobium officinale strips, sorting, removing leaves, removing roots, rinsing, cutting into small sections, adding purified water for pulping, filtering with gauze, and adding water to the original weight to obtain the Dendrobium officinale juice; The preparation process of the red date juice comprises the following steps: removing the pits of the red dates, washing, cutting and boiling the red dates; filtering the obtained red date pulp with gauze, and adding water to the original weight to obtain the red date juice; The preparation process of wolfberry juice comprises the following steps: washing and drying wolfberries and boiling them; filtering the obtained wolfberry pulp with gauze and adding water to the original weight to obtain wolfberry juice.

[0006] Further preferably, the Dendrobium officinale composite beverage having immunological activity comprises the following components: Dendrobium officinale juice 5.36%; Pomegranate juice concentrate 12%; Red date juice 2%; Wolfberry juice 4.55%; Rock sugar liquid 4.93%; Soy peptide powder 0.6%; When preparing the pulping of Dendrobium officinale juice, the ratio of fresh Dendrobium officinale strips to water is 1:6; When preparing and boiling red date juice, the material-liquid ratio of red dates to water is 1:8; When preparing wolfberry juice, the ratio of wolfberry to water is 1:5; The rock sugar liquid is prepared by mixing rock sugar and water in a material-liquid ratio of 1:2.

[0007] The beneficial effects of the present invention are as follows: The present invention uses raw materials such as Dendrobium officinale, wolfberry and red dates to compound and develop a Dendrobium officinale compound beverage that regulates human immunity. Animal experimental results show that the Dendrobium officinale compound beverage exhibits significant immunoregulatory effects on CTX-induced immunosuppressive mice by improving immune organ index, repairing mouse tissues after immune damage, increasing spleen lymphocyte proliferation capacity, and promoting the production of immunoglobulins and cytokines. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0009] Figure 1 The results of a single factor test are as follows: (A) the amount of Dendrobium officinale juice added; (B) the amount of pomegranate juice added; (C) the amount of red date juice added; (D) the amount of wolfberry juice added; (E) the amount of rock sugar solution added; (F) the amount of soybean peptide powder added; Figure 2 The amount of rock sugar solution added ( x 2 ) and wolfberry juice addition ( x 3 ) Response surface (A) and contour plot (B); Figure 3 The amount of rock sugar solution added ( x 2 ) and the amount of Dendrobium officinale juice added (x 4 )’s response surface (A) and contour plot (B); Figure 4 For (A) DOCD to ABTS + Scavenging ability; (B) DPPH scavenging ability of DOCD; Figure 5 For spleen histopathological observation; Figure 6 The effect of DOCD on the proliferation of spleen lymphocytes in immunosuppressed mice; Note: ** p <0.01 level is extremely significant, * p <0.05 level significant; Figure 7 The effect of DOCD on serum immunoglobulin levels in immunosuppressed mice; Note: ** p <0.01 level is extremely significant, * p <0.05 level significant; Figure 8 The effect of DOCD on the relative expression level of immune-related cytokine mRNA in the spleen tissue of mice, (A) is the expression of iNOS mRNA, (B) is the expression of Cox-2 mRNA; Note: ** p <0.01 level is extremely significant, * p The level was <0.05. DETAILED DESCRIPTION

[0010] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0011] Example 1 Configuration of Dendrobium officinale composite beverage (DOCD) 1. Materials and Methods 1 Materials and instruments 1.1 Experimental Materials Dendrobium officinale: Zhejiang Tiefengtang Biotechnology Co., Ltd.; wolfberry, red dates, and rock sugar: Huadong Medicine Co., Ltd. Medicinal Materials Ginseng and Antler Branch; Soybean peptide powder: Jiangsu Jiujia Biotechnology Co., Ltd.; Pomegranate concentrate juice (hereinafter also referred to as red pomegranate juice): Xi'an Mixianer Biotechnology Co., Ltd.; KM mice: provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, half each, license number [SCXK (Gan) 2020-0002].

[0012] 1.2 Instruments and Equipment JYL-C2012 juicer: Joyoung Co., Ltd.; MC-22EB15 induction cooker: Midea Group Co., Ltd.; HH-4 digital constant temperature water bath: Shanghai Jiecheng Experimental Instrument Co., Ltd.; SW-32D digital refractometer: Guangzhou Suwei Electronic Technology Co., Ltd.; WZB-170 portable turbidity meter: Shanghai Yidian Scientific Instrument Co., Ltd.; ME203E / 02 electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd.; KZ-Ⅲ-F high-speed tissue grinder: Wuhan Sevier Biotechnology Co., Ltd.; D3024R desktop high-speed refrigerated centrifuge: Dalong Biotechnology Co., Ltd.; RT-3100 fully automatic plate washer: Raydu Life Science Co., Ltd.; Epoch ELISA detector: BioTek; JB-P5 embedding machine: Wuhan Junjie Electronic Co., Ltd.; RM2016 pathology slicer: Shanghai Leica Instrument Co., Ltd.; KD-P tissue slide machine: Jinhua Kedi Instrument Equipment Co., Ltd., Zhejiang Province.

[0013] 2 Methods 2.1 DOCD configuration process Fresh Dendrobium officinale strips → sorting → removing leaves and roots → cleaning → pulping → centrifugation → Dendrobium officinale juice Fresh dried red dates → select the fruit → wash → cook → remove the core → filter → red date juice Fresh wolfberry → select fruit → wash → cook → filter → wolfberry juice Pomegranate juice concentrate → add water → pomegranate juice Soy peptide powder → sugar adjustment → blending → homogenization → compound beverage → filling → sterilization → inspection → cooling 2.2 Operation points (1) Preparation of Dendrobium officinale juice: Select fresh Dendrobium officinale strips, sort, remove leaves, remove roots, rinse, cut into small pieces about 5 cm long, add purified water for pulping, the solid-liquid ratio (m / m) of pulping to water is 1:6, filter through 3 layers of gauze, add water to the original weight, and obtain Dendrobium officinale juice.

[0014] (2) Preparation of red date juice: Select high-quality red dates with purple-red skin, large and uniform grains, short, strong and round fruit rows, few wrinkles, shallow marks, no diseases and insect pests, and no rot as raw materials, remove the core, weigh, wash, cut into pieces, and cook at a solid-liquid ratio of 1:8; filter the obtained red date pulp with 3 layers of gauze, add water to the original weight, and obtain red date juice.

[0015] (3) Preparation of wolfberry juice: Select high-quality wolfberries, wash, dry and weigh them, and cook them with a solid-liquid ratio of 1:5 (mashing them with a spoon while cooking). Filter the obtained wolfberry pulp through 3 layers of gauze, add water to the original weight, and obtain wolfberry juice.

[0016] (4) Preparation of rock sugar solution: Accurately weigh a certain amount of rock sugar, add purified water at a material-liquid ratio of 1:2 and boil, stir until the rock sugar is completely melted, cool, and add water to the original weight to obtain rock sugar solution.

[0017] 2.3 Single-factor experimental design Single factor experiments were conducted on different addition amounts of Dendrobium officinale juice, pomegranate juice, red date juice, wolfberry juice, rock sugar solution and soybean peptide powder to determine the factors that had a more significant effect on the sensory experience of DOCD.

[0018] like Figure 1 A, the group with 5.5% of Dendrobium officinale juice added was the optimal addition group. This group was brown in color, slightly layered, clear on top and turbid on the bottom, with the unique beany smell of soybean peptide powder, which partially or completely masked the flavors of Dendrobium officinale, red dates and pomegranates, with the aftertaste of red dates, a suitable ratio of sour and sweet, and a slightly sticky taste.

[0019] like Figure 1 B. The group with 12% pomegranate juice added was the optimal addition group. The optimal addition group was reddish brown, with a slight beany smell unique to soybean peptide powder, which partially masked the flavors of Dendrobium officinale, red dates and pomegranates. The sour-sweet ratio was appropriate and the taste was relatively refreshing.

[0020] like Figure 1 C, the group with 2% red date juice added was the optimal addition group. The optimal addition group was brown in color with slight stratification, which was less obvious than that of the other test groups. It did not have the beany smell of soybean peptide powder, and did not mask the flavors of Dendrobium officinale, red dates and pomegranate. It had a suitable sour and sweet taste, with the aftertaste of red dates and a slightly sticky taste.

[0021] like Figure 1 D. The group with 5% wolfberry juice added was the optimal addition group. The optimal addition group was brown in color, with no obvious stratification and a slight beany smell unique to soybean peptide powder, which partially or completely masked the flavors of Dendrobium officinale, red dates and pomegranates. The sour-sweet ratio was appropriate, the smell was harmonious, and there was an aftertaste of red dates.

[0022] like Figure 1 E, the group with 4.5% rock sugar solution added is the optimal addition amount group. The optimal addition amount group is reddish brown, slightly stratified, clear on the top and turbid on the bottom. It does not have the beany smell of soybean peptide powder, and does not cover the flavors of Dendrobium officinale, red dates and pomegranate. The smell is harmonious, the wolfberry flavor is stronger, the sour and sweet taste is more appropriate, and there is an aftertaste of red dates.

[0023] like Figure 1 F, the group with 0.6% soybean peptide powder added is the optimal addition amount group. The optimal addition amount group is yellow-brown, slightly stratified, clear on the top and turbid on the bottom. It does not have the beany smell of soybean peptide powder, and does not cover the flavors of Dendrobium officinale, red dates and pomegranate. The smell is harmonious, the red dates flavor is stronger, the sour and sweet taste is more appropriate, and there is a red dates aftertaste.

[0024] 2.4 Principal component experimental design On the basis of single factor, PB design was performed on the six factors that affect the sensory evaluation results of DOCD, and the top four factors with important influence were selected through dimensionality reduction. The obtained data were processed by SAS 9.2 statistical software, and finally the multiple regression model was determined by combining the response surface experimental design to obtain the optimal process formula. In this work, six factors that affect the sensory evaluation results of DOCD, including Dendrobium officinale juice, pomegranate juice, red date juice, wolfberry juice, rock sugar liquid and soybean peptide powder, were selected as the investigation objects, and the DOCD sensory evaluation score was used as the main investigation indicator. The coding level and design scheme of PB experimental design are shown in Table 1.

[0025] Table 1 Principal component design Table 2 Results of principal component analysis The results in Table 2 were analyzed to establish a preliminary regression model for DOCD sensory evaluation: Y 1 =79.5 + 0.775 x 1 +0.075 x 2 + 0.35 x 3 +1 x 4 +1.075 x 5 -2.375 x 6 (1) Table 3 Analysis of variance of regression model From the variance analysis in Table 3, we can see that the sum of squares and mean square value of the error value are very small, indicating that the error term is not significant and the quadratic regression model can be further tested. 2 =97.15%, indicating that the regression equation can explain the influence of these six factors on DOCD sensory evaluation (Y value) at a confidence level of 97.15%. The experimental data are basically consistent with the adopted quadratic mathematical model, and the model has a good fit with the actual situation. It can be used to predict the influence of the four factors selected by Plackett-Burman principal component analysis on DOCD sensory evaluation, which has practical application significance.

[0026] It can be clearly seen from the above regression model variance analysis that the order of influence of the above six factors on DOCD sensory evaluation is: , that is, the amount of soybean peptide powder added> the amount of rock sugar liquid added> the amount of wolfberry juice added> the amount of Dendrobium officinale juice added> the amount of pomegranate juice added> the amount of red date juice added. Among them, the factors with mean square>4 are significant factors, so the first four influencing factors obtained by dimensionality reduction are the amount of soybean peptide powder added, the amount of rock sugar liquid added, the amount of wolfberry juice added and the amount of Dendrobium officinale juice added.

[0027] 2.5 Response surface design experiment In order to analyze the influence of the raw materials used on the sensory quality of DOCD, the present invention adopts response surface experimental design to optimize the formula of DOCD. The fuzzy evaluation sensory score is used as the response value, the addition amount of soybean peptide powder, crystal sugar liquid, wolfberry juice and Dendrobium officinale juice is used as the investigation factor, and the four-factor BB design is used to arrange the experiment using SAS 9.2 software. The response surface factors and level design are shown in Table 4.

[0028] Table 4 Response surface analysis factors and levels 2.6 Evaluation criteria After preparation, sensory evaluation was performed according to Table 5. The method is that a sensory evaluation team composed of 10 trained food professional technicians performed sensory scoring on each group of DOCD according to the established sensory evaluation standards (Table 3), and each calculated evaluation result was analyzed using fuzzy judgment. The score was scored in percentage, corresponding to the color and texture state, flavor, aroma and impurity evaluation standards of DOCD.

[0029] Table 5 Sensory evaluation criteria 2.7 Filling, sterilization, inspection and cooling The prepared DOCD is homogenized and quantitatively vacuum-filled into glass bottles, and sterilized for 20 minutes under normal pressure 85-90°C steam conditions in a steam boiler, and the sterilized products are subjected to physical, chemical and microbiological tests.

[0030] 2.8 Fuzzy Evaluation 2.8.1 Establishment of factor set and comment set The factor set is the set of indicators that affect the quality of DOCD, expressed as U = (u 1 ,u 2 ,…,u i ), where U represents the factor set, u i Indicates the i-th factor. For the Dendrobium officinale compound beverage, there are 4 indicators that determine its sensory quality, namely U= (u color, tissue state, u flavor, taste, u aroma, u impurities).

[0031] Comment set: Each factor is evaluated according to four levels: excellent, good, fair, and poor. For the DOCD comment set, V = {V 1, V 2 , V 3 , V 4},V 1 、V 2 、V 3 、V 4 They represent excellent, good, medium, and poor, respectively, that is, V={V 1 (Excellent), V 2 (good), V 3 (middle), V 4 (Difference)}.

[0032] 2.8.2 Establishment of weight set The indicators that affect the quality of DOCD include color, texture, flavor, taste, aroma and impurities. Among them, flavor and taste are the most important to product quality, followed by color and texture, while aroma and impurities have a relatively small impact. According to the importance of the evaluation factors in the sensory evaluation of DOCD, the weight of each factor is determined by the forced determination method. In the comprehensive evaluation, the proportion of each factor is 0.2, 0.6, 0.1, and 0.1, respectively, so X = (0.2, 0.6, 0.1, 0.1).

[0033] 2.8.3 Fuzzy Evaluation According to the evaluation made by 10 judges on each sample comment set V, the number of comments on each factor is counted to form the evaluation matrix R. The result vector of fuzzy mathematical comprehensive evaluation of DOCD sensory factors is Y=XR, which is the synthesis of sensory factor weight X and evaluation matrix R.

[0034] 2.8.4 Evaluation results The fuzzy vectors were converted to single values ​​for comparison and sorting, and the four grades of excellent, good, medium and poor were assigned 90, 80, 70 and 60 points respectively. Each quantity in the comprehensive evaluation result set of the fuzzy evaluation was multiplied by its corresponding score and added up to obtain the sensory score of each sample.

[0035] Table 6 Four-factor Box-Behnken results Regression model building The results in Table 6 were statistically processed, and the sensory score was used as the main evaluation index to obtain the preliminary expected model affecting the sensory score of DOCD: Y 1 =83.53333+0.516667 x 1 +2.116667 x 2 -0.6 x 3 -0.8 x4 -5.083333 x 1 2 -0.3 x 1 x 2 +0.1 x 1 x 3 -0.05 x 1 x 4 -2.083333 x 2 2 -2.35 x 2 x 3 -1.4 x 2 x 4 -2.858333 x 3 2 -0.15 x 3 x 4 -3.558333 x 4 2 (2) The simulation equations were subjected to variance analysis, and the results are shown in Table 7.

[0036] Table 7 Analysis of variance of quadratic response surface regression model Note:** p <0.01 level is extremely significant, * p The level was <0.05.

[0037] Note: **p<0.01 level is very significant, *p<0.05 level is significant. From the variance analysis of the regression model in Table 7, it can be seen that the amount of rock sugar liquid added x 2 The linear term of the addition amount and the quadratic terms of all four single factors as well as the interaction term between the addition amount of rock sugar solution and wolfberry juice had a very significant effect on the DOCD sensory score ( p <0.01), amount of Dendrobium officinale juice added x 4The linear term of the addition amount of rock sugar solution and the interaction term of the addition amount of Dendrobium officinale juice had significant differences in DOCD scores ( p <0.05, and the other variables had no significant effect ( p >0.05), with no statistical difference.

[0038] The insignificant items are eliminated at the significance level of α=0.05, and the simplified regression equation is: Y 1 =83.53333+2.116667 x 2 -0.8 x 4 -5.083333 x 1 2 -2.083333 x 2 2 -2.35 x 2 x 3 -1.4 x 2 x 4 -2.858333 x 3 2 -3.558333 x 4 2 (3) The confidence level of the regression equation is 95.51%. The correlation coefficient R 2 It is 90.26%, indicating that the regression equation can explain the influence of these four factors on the DOCD sensory score (Y value) at a confidence level of 90.26%. The experimental data are basically consistent with the adopted quadratic mathematical model. The model has a good fit with the actual situation and can be used to predict the influence of these four factors on the DOCD sensory score, which has practical application significance.

[0039] One factor was fixed at zero level, and the interaction effect between the other two factors was studied. SAS 9.2 software was used to make response surface plots and contour plots.

[0040] From the amount of rock sugar liquid added ( x 2 ) and wolfberry juice addition ( x 3 ) of the interaction response surface and contour plots ( Figure 2) It can be seen that the interaction between the two is not a simple linear relationship. When the level of rock sugar liquid addition remains unchanged and is at a low level, as the level of wolfberry juice addition decreases, the DOCD sensory score first increases slightly and then decreases linearly, and the downward trend is much higher than the upward trend, presenting a wide-mouthed downward parabola shape; when the level of rock sugar liquid addition remains unchanged and is at a high level, as the level of wolfberry juice addition decreases, the DOCD sensory score first increases linearly and then decreases slightly, and the upward trend is much higher than the downward trend, also presenting a wide-mouthed downward parabola shape; when the level of wolfberry juice addition remains unchanged and is at a low level, as the level of rock sugar liquid addition decreases, the DOCD sensory score first decreases slightly and then decreases rapidly, slightly presenting a semi-saddle downward trend; when the level of wolfberry juice addition remains unchanged and is at a high level, as the level of rock sugar liquid addition decreases, the DOCD sensory score first increases linearly and then decreases linearly, and the upward trend is basically close to the downward trend, presenting a strong wide-mouthed downward parabola shape. It can be seen from the contour map that the process has an effect on the amount of rock sugar liquid addition ( x 2 ) and wolfberry juice addition ( x 3 ) is close to but slightly closer to the crystal sugar solution addition amount ( x 2 ) changes in sensitivity.

[0041] From the amount of rock sugar liquid added ( x 2 ) and the amount of Dendrobium officinale juice added ( x 4 ) of the interaction response surface and contour plots ( Figure 3 ) It can be seen that the interaction between the two presents an obvious saddle surface. When the level of rock sugar solution addition remains unchanged and is at a low level, as the level of Dendrobium officinale juice addition decreases, the DOCD sensory score first rises linearly and then falls linearly, and the downward trend is higher than the upward trend, presenting a basically symmetrical downward-opening parabola shape; when the level of rock sugar solution addition remains unchanged and is at a high level, as the level of Dendrobium officinale juice addition decreases, the DOCD sensory score first rises linearly and then falls linearly, and the upward trend is much higher than the downward trend, presenting a wider-mouthed downward-opening parabola shape; when the level of Dendrobium officinale juice addition remains unchanged and is at a low level, as the level of rock sugar solution addition decreases, the DOCD sensory score first falls slightly and then falls rapidly, presenting a slightly semi-saddle downward trend; when the level of wolfberry juice addition remains unchanged and is at a high level, as the level of rock sugar solution addition decreases, the DOCD sensory score first rises linearly and then falls linearly, and the upward trend is much lower than the downward trend, presenting a strong downward-opening parabola shape. It can be seen from the contour map that the process has an effect on the addition of Dendrobium officinale juice ( x 4 ) is slightly more sensitive to changes than the amount of rock sugar solution added (x 2 ) changes in sensitivity.

[0042] Ridge Analysis It can also be seen from the response surface and contour plot that the amount of rock sugar solution added ( x 2 ) and wolfberry juice addition ( x 3 ) and the amount of rock sugar solution added ( x 2 ) and the amount of Dendrobium officinale juice added ( x 4 ) The secondary response surface of DOCD sensory score showed a saddle surface and no extreme value. Therefore, the optimal influencing factor parameters could not be directly found from the secondary response surface, and further ridge analysis was needed.

[0043] The ridge analysis results show that with the increase of the coding radius r, the estimated response value Y, i.e., the sensory score of the Dendrobium officinale compound beverage, also increases. It can be seen that the response value Y is basically related to the addition amount of soybean peptide powder ( x 1 ) and the amount of rock sugar solution added ( x 2 ) and is proportional to the amount of wolfberry juice added ( x 3 ) and the amount of Dendrobium officinale juice added ( x 4 ), that is, as the amount of soybean peptide powder added ( x 1 ) and the amount of rock sugar solution added ( x 2 ) increased, and with the addition of wolfberry juice ( x 3 ) and the amount of Dendrobium officinale juice added ( x 4 ) increases with the increase of DOCD sensory score. When the coding radius is 1.0, the response value Y, i.e., the DOCD sensory score, is the highest, about 84.6796. At this time, the corresponding soybean peptide powder addition amount ( x 1 ) is 0.604526%, the amount of rock sugar liquid added ( x 2 ) is 4.925815%, and the amount of wolfberry juice added ( x 3 ) is 4.55189%, and the amount of Dendrobium officinale juice added ( x 4 ) is 5.36453%. Keep two significant figures, and the optimal formula after optimization is the addition amount of soybean peptide powder ( x 1 ) is 0.6%, the amount of rock sugar liquid added (x 2 ) is 4.93%, and the amount of wolfberry juice added ( x 3 ) is 4.55%, and the amount of Dendrobium officinale juice added ( x 4 ) was 5.36%, the amount of red date juice added was 2%, and the amount of pomegranate juice added was 12%. At this time, the DOCD sensory score was the highest, about 84.6796. According to the optimal formula, a Dendrobium officinale compound beverage was prepared and the sensory evaluation was performed, with a score of 84.82.

[0044] Example 2 Determination of free radical scavenging activity of DOCD: The following experiment is a Dendrobium officinale compound beverage (DOCD) prepared using the optimal formula obtained in Example 1.

[0045] 1 Determination of free radical scavenging activity of DOCD DOCD and 0.2 mg / mL Vc solution were diluted to 5%, 25%, 50%, 75%, and 100% of the original concentration (i.e., the sample volume concentration was 0.05, 0.25, 0.50, 0.75, and 1.00 mL / mL) for free radical scavenging experiments. ABTS + , DPPH scavenging activity determination.

[0046] ABTS + The clearance curve is as follows Figure 4 (A) shows the ABTS + The scavenging ability of DOCD can be used as an indicator of the antioxidant activity of DOCD. + The clearance rate increased with the increase of sample concentration. When the sample concentration was 1.8 mg / mL, ABTS + The clearance rate reached 73.26%, and the half clearance rate (IC 50 ) is 0.9789 mg / mL, indicating that DOCD has a great influence on ABTS + It has certain scavenging ability and good antioxidant activity.

[0047] Depend on Figure 4 (B) It can be seen that both DOCD and VC have the ability to scavenge DPPH·. Under the experimental conditions, VC has a higher scavenging ability for DPPH· than DOCD, and the scavenging ability of both increases with the increase of concentration. As the concentration increases, the increasing trend of its scavenging ability slows down, and the concentration of antioxidant substances in DOCD approaches saturation. When the concentration is 1.8 mg / mL, the scavenging rate of DOCD for DPPH· reaches 74.50%. The half scavenging rate (IC 50) was 0.9720 mg / mL, indicating that DOCD had a strong scavenging ability for DPPH·.

[0048] 2 Determination of DOCD immunoreactivity 2.1 Animal Experiment Design Experimental Kunming mice were 4-5 weeks old, weighing (18±22) g, maintained with 12 h of alternating light per day, a constant temperature of 25 ℃, and adequate food and water supply. The experiment was conducted after adaptive feeding for 1 week in an SPF animal room. The mice were randomly divided into a normal control group (CK group), a CTX immunosuppression group (model group), a low-dose DOCD treatment group, a medium-dose DOCD treatment group, and a high-dose DOCD treatment group, with 10 mice in each group. Dosage method: intraperitoneal injection once a day for the first 3 days. The blank control group mice were intraperitoneally injected with 100 μL of normal saline, and the model group and the low-, medium-, and high-dose DOCD treatment groups were intraperitoneally injected with 100 μL of CTX (40 mg / kg m b ); Starting from the 4th day, each group was gavaged with 200 μL normal saline, low-dose DOCD (100 mg / kg m b ), medium dose DOCD (200 mg / kg m b ), high dose DOCD (400 mg / kg m b ), and then gavage was continued for 4 weeks. On the last day after gavage, the mice were killed by cervical dislocation, the spleen was removed under sterile conditions, and blood was collected and serum was separated for subsequent experiments.

[0049] 2.2 Determination of organ indexes in immunosuppressed mice The mice were weighed and then killed by cervical dislocation. The thymus and spleen were removed, the fascia was removed, the blood on the surface of the organs was absorbed with filter paper and weighed, and the ratio of spleen to body weight and the ratio of thymus to body weight were calculated.

[0050] In immunosuppressed animals, decreased organ index is generally considered an important signal of decreased immune function. As an immune organ, the spleen is the main regulatory site of immune function. Its development and maturation directly affect the immune level. The immune organ index can reflect the body's immune function.

[0051] Table 8 Effects of DOCD on organ indexes of cyclophosphamide-induced immunosuppressed mice ( ± s ) Note: ## indicates P < 0.01 compared with the CK group, ** indicates P < 0.01 compared with the Model group. # indicates P < 0.05 compared with the CK group, * indicates P < 0.05 compared with the Model group.

[0052] As shown in Table 8, compared with the blank group, the spleen index and thymus index of the model group were significantly reduced (P < 0.01). Compared with the model group, the thymus and spleen index of the DOCD-treated mice increased in a dose-dependent manner. The low-dose DOCD-treated group could slightly increase the thymus index, but there was no statistical significance ( P >0.05), the promoting effect of the middle and high dose groups on spleen index and thymus index reached a very significant level ( P <0.01). This suggests that DOCD can effectively alleviate CTX-induced immune organ damage and regulate immunity.

[0053] 2.3 Histopathological observation of immunosuppressed mice Part of the spleen tissue of the mouse was fixed in 10% formaldehyde fixative for 24 h, then repeatedly rinsed with distilled water until it was odorless, dehydrated, waxed, embedded, sliced, and subsequently HE stained. Pathological examination was then performed under a microscope.

[0054] The spleen is the main immune organ of the human body. Changes in its mass can reflect changes in the number of lymphocytes in the body, and thus indirectly reflect the degree of immune response. The spleen is the site of immune response to blood-borne antigens. When a reaction occurs, the volume and internal structure of the spleen change. When a humoral immune response occurs, the number of lymph nodes increases and the number of plasma cells in the splenic cords increases. In the cellular immune response, the periarterial lymphoid sheath is significantly thickened, indicating that cyclophosphamide can induce oxidative stress and lead to pathological changes in the spleen.

[0055] After HE staining, Figure 5 It can be seen that the structure of the spleen of mice in the CK group is basically normal, with densely distributed white pulp lymphocytes, loose red pulp cells, and obvious lymph nodes. The spleen of mice in the Model group has irregular morphology, unclear boundaries between red and white pulp, and increased macrophages; this indicates that CTX may have damaged the spleen tissue. However, after treatment with DOCD, the spleen capsule gradually became intact, the white pulp area increased significantly, the center contracted inward, the number of lymphocytes increased significantly, the lymphocytes were closely arranged, and the spleen tissue structure was well restored. This shows that DOCD has a certain repair effect on spleen damage caused by CTX. Therefore, DOCD may improve spleen tissue damage by improving the absorption of antioxidant proteins and peptides, thereby showing immunomodulatory effects.

[0056] 2.4 Determination of the proliferation capacity of spleen lymphocytes in immunosuppressed mice The MTT method was used to determine the ability of spleen lymphocyte proliferation assay. Briefly, a single spleen cell suspension was prepared after sterile spleen removal. After washing with HBSS, the cell concentration was adjusted to 3× 106 cells / mL with RPMI-1640 medium. Each spleen cell was divided into a control well and a proliferation reaction well containing Con-A (7.5 μg / mL) in a 24-well plate at 37°C and 5% CO 2 After 72 h, the relative cell number was determined by measuring the OD value at 570 nm on a microplate reader (BioTek, USA) using MTT reagent according to previous studies.

[0057] The activation of T lymphocytes plays a vital role in the body's cellular immune function. Among the various indicators for measuring the level of cellular immunity, the proliferation ability of T lymphocytes is particularly important, and the degree of its activation can be reflected by measuring the proliferation ability.

[0058] like Figure 6 As shown, compared with the CK group, the proliferation capacity of spleen lymphocytes in the Model group was significantly reduced ( p <0.01). Compared with the Model group, there was no significant difference in the Low group ( p >0.05), the proliferation capacity of mice in the Middle and High groups was significantly increased ( p <0.01), and this effect was concentration-dependent. The experimental results showed that DOCD could restore the proliferation ability of spleen lymphocytes in mice after cyclophosphamide inhibition.

[0059] 2.5 Effect of serum immunoglobulins in immunosuppressed mice The serum samples stored at -80°C were taken out and thawed at low temperature. The contents of two major immunoglobulins, IgM and IgG, in the serum of each group of mice were determined according to the requirements and operating instructions of different ELISA kits.

[0060] Immunoglobulin ( Immunoglobulin Ig) is mainly present in plasma, and is also found in other body fluids, tissues and some secretions, playing an important role in antigen recognition during the immune response. Among them, IgM is the main antibody in the initial stage of immune response, and IgG is the most abundant immunoglobulin in serum.

[0061] Depend on Figure 7 It can be seen that compared with the CK group, the concentrations of immunoglobulins IgM and IgG in the serum of the Model group mice were significantly reduced ( P <0.01), indicating that the immunosuppression model was successfully established. Compared with the Model group, the Low group had no significant difference (P>0.05). The concentrations of immunoglobulins IgA and IgG in the serum of the Middle group mice were significantly increased (P <0.01), the concentrations of immunoglobulins IgM and IgG in the High group were significantly increased ( P <0.01), indicating that DOCD can restore the levels of IgM and IgG in the serum of immunosuppressed mice.

[0062] 2.6 Real-time fluorescence quantitative PCR detection Real-time fluorescence quantitative PCR was used to detect the changes in the relative expression levels of cytokines (iNOS and Cox-2) mRNA in mouse spleen tissue, with 3 parallels in each group. The detection was carried out according to the reference method. 50 mg of spleen tissue was cut into small pieces, ground into powder with liquid nitrogen, and total RNA was extracted according to the operating instructions of the magnetic bead total RNA extraction kit. According to the SYBR Premix Ex Taq The reaction system was 20 μL, and the sample tube and internal reference tube were set up in triplicate. The reaction conditions were 50°C, 10 min reverse transcription, 95°C pre-denaturation for 5 min, 1 cycle; 95°C, 10 s, 60°C, 30 s, 40 cycles. GAPDH was used as the internal reference gene, and 2 －ΔΔCt The relative expression levels of mRNA in each group were compared by the primer sequence shown in Table 8.

[0063] Table 9 Target gene primer sequences Cytokines are usually produced after activation of monocytes-macrophages, lymphocytes, interstitial cells, etc., and participate in immune inflammatory reactions. They are another important immune effector substance after immunoglobulins and complement. Many cytokines can enhance the immune effect at the right time, but will have an inhibitory effect when excessive, and this mode of action is bidirectionally regulated. iNOS is not only present in macrophages, but also induced to be expressed in other tissues. The induction process of iNOS is a natural defense system that can work with the body to resist the invasion of foreign microorganisms and pathogens. COX-2 is a key enzyme that catalyzes the rate-limiting step of converting arachidonic acid into prostaglandins, and plays a key role in the inflammatory process. iNOS and COX-2 are two important protein molecules in the NF-κB signaling pathway, and the NF-κB signaling pathway is also considered to be closely related to immune regulation.

[0064] like Figure 8 As shown in (A), compared with the CK group, the Model group strongly stimulated the expression of iNOS mRNA (P < 0.05). After DOCD treatment, the level of iNOS mRNA induced by gene release was not significant compared with the Model group in the Low group (P > 0.05), but was significant in the Middle group (P < 0.05), and extremely significant in the High group (P < 0.01). Figure 8 As shown in (B), compared with the CK group, the expression of Cox-2 mRNA in the Model group was significantly increased after cyclophosphamide treatment (P < 0.01). After DOCD treatment, the expression levels of Cox-2 mRNA in the Low group, Middle group, and High group were significantly reduced and restored to the level of normal mice. This indicates that DOCD can regulate immune activity at the gene transcription level, which may be a mechanism by which DOCD exerts its immune regulatory activity.

[0065] In recent years, Dendrobium candidum has become a mainstream product for health care consumption, and research on various aspects of it has gradually increased. Dendrobium candidum health products have the effects of enhancing immunity, reducing physical fatigue, assisting in protecting chemical liver damage, delaying aging, clearing the throat, assisting in protecting radiation hazards, assisting in lowering blood pressure, resisting mutation, and assisting in protecting gastric mucosa. The present invention uses Dendrobium candidum as the main raw material to prepare a laboratory Dendrobium candidum compound beverage (DOCD). By establishing an immunosuppressive mouse model, the immunoregulatory effect of DOCD is analyzed from various aspects such as the phenotypic recovery indicators and pathological morphological changes of mice and the determination of physiological and biochemical indicators.

[0066] Through single factor experiment, PCA dimensionality reduction experiment and response surface orthogonal experiment, DOCD sensory score was used as the main evaluation index. PCA dimensionality reduction showed that the addition amount of soybean peptide powder, rock sugar solution, wolfberry juice and Dendrobium officinale juice were significant influencing factors. Then response surface BB experiment was carried out. The variance analysis results showed that the addition amount of rock sugar solution x 2 The linear term of the addition amount and the quadratic terms of all four single factors as well as the interaction term between the addition amount of rock sugar solution and wolfberry juice had a very significant effect on the DOCD sensory score ( p <0.01), amount of Dendrobium officinale juice added x 4 The linear term of and the interaction term of the amount of rock sugar solution added and the amount of Dendrobium officinale juice added had significant differences in the DOCD sensory scores ( p <0.05, and the other variables had no significant effect ( p >0.05), there was no statistical difference. The confidence level of the simulated regression equation was 95.51%, and the correlation coefficient R 2 The regression equation can explain the influence of these four factors on the sensory score of DOCD (Y value) at a confidence level of 90.26%. The experimental data are basically consistent with the quadratic mathematical model adopted. The model has a good fit with the actual situation and can be used to predict the influence of these four factors on the sensory score of DOCD, which has practical application significance. Ridge analysis optimization, the best process parameter formula is the addition amount of soybean peptide powder ( x 1) is 0.6%, the amount of rock sugar liquid added ( x 2 ) is 4.93%, and the amount of wolfberry juice added ( x 3 ) is 4.55%, and the amount of Dendrobium officinale juice added ( x 4 ) is 5.36%, the amount of red date juice added is 2%, and the amount of pomegranate juice added is 12%. At this time, the DOCD sensory score is the highest, which is about 84.6796.

[0067] Studies have shown that the growth performance of mice in an immunosuppressive state induced by CTX is reduced, which is manifested as slower weight gain and lower organ index. In the present invention, compared with the CK group, the organ index of mice in the Model group was significantly reduced, and both the High group and the Middle group reduced the impact of CTX. Studies by Shan Zhengxin et al. have shown that Dendrobium officinale polysaccharides have the effect of antagonizing CTX-induced immunosuppression in mice, and restore the growth performance of mice after the therapeutic effect of Dendrobium officinale polysaccharides, which is consistent with the results of the present invention. Organ mass is one of the indicators of drug toxicity sensitivity, and its changes often precede morphological changes. Since the mice in the model group are continuously in an immunosuppressive state and the body is in an inflammatory environment, the spleen is damaged. Relying solely on the body's own regulation cannot improve the internal environment disorder and chronic inflammatory response. The results of HE staining showed that the spleen structure of the mice in the Model group was disordered, and the boundaries between the red pulp and the white pulp were unclear, while the spleen structure of the DOCD administration treatment group was significantly improved, and the red pulp and white pulp structures were clearer, indicating that DOCD can reduce inflammatory reactions, repair spleen damage induced by CTX, and enhance the immunity of mice. Lymphocytes are divided into T lymphocytes (CD3+) that mediate cellular immunity and B lymphocytes (CD19+) that mediate humoral immunity. Mature T lymphocytes mainly reside in peripheral immune organs and blood, and can circulate through blood, tissue fluid, etc., mainly to kill target cells or release immune-related cytokines to exert cellular immune functions, and the proliferation and differentiation of T lymphocytes is the prerequisite for the body's cellular immune response. In the present invention, compared with the CK group, the proliferation ability of splenic lymphocytes in mice injured by CTX was significantly reduced (P<0.01). After DOCD drug treatment, the proliferation ability of splenic lymphocytes in mice gradually recovered to the level of normal mice. This is consistent with the published research results. Immunoglobulin (lg) is a protein with antibody activity in serum and body fluids. It can play a certain role in the immune environment of the human body, including IgA, IgG, IgM, etc. Its secretion amount can reflect the immune status of the human body. The experimental results showed that the levels of immunoglobulins (IgG, IgM) in immunosuppressed mice increased to varying degrees after DOCD intervention, indicating that DOCD can restore CTX-induced immune dysfunction to varying degrees, indicating that DOCD has immunostimulatory activity and may have the ability to regulate related DNA expression, which needs further confirmation. Li et al. found in their study on Dendrobium officinale leaf polysaccharide (DOLP) that DOLP has the same regulatory effect on immunosuppressed mice. Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 are two downstream signaling pathways involved in inflammatory signal transduction regulated by NF-κB, which promote cellular inflammatory damage and thus enhance the body's immunity.The experimental results showed that the Model group strongly stimulated the expression of iNOS and Cox-2 mRNA. After DOCD treatment, the iNOS and Cox-2 mRNA induced at the gene level were significantly reduced, indicating that DOCD promoted the secretion of iNOS and Cox-2 in immune cells, which was probably regulated by reducing the expression of iNOS and Cox-2 mRNA, that is, DOCD could regulate immune activity at the gene transcription level, which may be a mechanism by which Dendrobium officinale exerts its immunomodulatory activity, and this mechanism of action is similar to that of some polysaccharides.

[0068] The present invention develops a Dendrobium officinale compound beverage by compounding it based on the main principle. The optimal ratio of the compound beverage was obtained through single factor experiment, principal component analysis, and response surface experiment. The results of animal experiments showed that DOCD showed significant immunoregulatory effects on CTX-induced immunosuppressive mice by improving immune organ index, repairing mouse tissues after immune damage, increasing spleen lymphocyte proliferation capacity, and promoting the production of immunoglobulins and cytokines. These results provide new ideas for the comprehensive utilization of Dendrobium officinale. Therefore, our work is conducive to the further development and utilization of Dendrobium officinale as a new type of nutritional food additive ingredient.

[0069] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A Dendrobium officinale compound beverage with immune activity, characterized in that: It includes the following components: Dendrobium officinale juice, pomegranate concentrated juice, red date juice, wolfberry juice, rock sugar liquid, soy peptide powder; The preparation process of the Dendrobium officinale juice comprises the following steps: taking fresh Dendrobium officinale strips, sorting, removing leaves, removing roots, rinsing, cutting into small sections, adding purified water for pulping, filtering with gauze, and adding water to the original weight to obtain the Dendrobium officinale juice; The preparation process of the red date juice comprises the following steps: removing the pits of the red dates, washing, cutting and boiling the red dates; filtering the obtained red date pulp with gauze, and adding water to the original weight to obtain the red date juice; The preparation process of wolfberry juice comprises the following steps: washing and drying wolfberries and boiling them; filtering the obtained wolfberry pulp with gauze and adding water to the original weight to obtain wolfberry juice.

2. The Dendrobium officinale composite beverage with immunological activity according to claim 1, characterized in that: It includes the following components: Dendrobium officinale juice 5.36%; Pomegranate juice concentrate 12%; Red date juice 2%; Wolfberry juice 4.55%; Rock sugar liquid 4.93%; Soy peptide powder 0.6%; When preparing the pulping of Dendrobium officinale juice, the ratio of fresh Dendrobium officinale strips to water is 1:6; When preparing and boiling red date juice, the material-liquid ratio of red dates to water is 1:8; When preparing wolfberry juice, the ratio of wolfberry to water is 1:5; The rock sugar liquid is prepared by mixing rock sugar and water in a material-liquid ratio of 1:2.

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