Probiotic fermented plant beverage capable of improving immunity and preparation method of probiotic fermented plant beverage

Abstract

The invention relates to a probiotic fermented plant beverage capable of improving immunity and a preparation method thereof, and belongs to the technical field of medicated food. The preparation method comprises the following steps: crushing American ginseng, astragalus membranaceus, rhizoma polygonati, poria cocos and malt, adding functional components and selenium-enriched water, mixing and stirring, and sterilizing to obtain a fermentation substrate; the preparation method comprises the following steps: respectively carrying out strain activation, centrifugation and freeze drying on dry casei, lactobacillus acidophilus, casei rhamnosus and lactobacillus plantarum, and then mixing to obtain composite probiotic powder; culturing the obtained composite probiotic powder in an MRS culture medium to obtain a composite probiotic liquid; and inoculating the composite probiotic liquid into the obtained fermentation substrate for fermentation, carrying out solid-liquid separation, taking a liquid phase, and sterilizing to obtain the probiotic fermented plant beverage capable of improving immunity. Aiming at the problems of immune function decline and blood sugar fluctuation after high-intensity exercises of sports people, the activity and absorption efficiency of nutritional ingredients are enhanced through probiotic fermentation, and accurate nutritional support is provided for body recovery after exercises.

Description

Technical Field

[0001] This invention relates to the field of special dietary food technology, and in particular to a probiotic fermented plant beverage for enhancing immunity and its preparation method. Background Technology

[0002] In recent years, the integration of probiotic fermentation technology with food-medicine homologous raw materials has become a core development direction in the functional food field. Probiotics can transform large-molecule active ingredients in plant raw materials into smaller molecules through metabolism, significantly improving bioavailability and endowing products with health benefits such as gut microbiota regulation. This technology has already achieved large-scale application in niche markets such as liquid beverages, solid snack foods, and health foods. With consumers' increasing demand for natural health and precise functional benefits, probiotic-fermented plant products targeting specific groups (such as those with sub-health conditions, the elderly, and those with specific functional needs) have become a research hotspot. However, current technology still faces bottlenecks in areas such as synergistic effects of bacterial strains, matching process parameters, and targeted release of active ingredients.

[0003] Current technologies mainly focus on the fermentation applications of probiotics and food-medicine homologous ingredients, but they have the following drawbacks:

[0004] (1) The application of strains has problems of “insufficient synergy” or “single specificity”: In the existing technology, liquid beverages mostly use a simple combination of 2-3 types of lactobacilli (such as Lactobacillus plantarum + Lactobacillus reuteri), without designing the functional division of strains according to the raw material components, resulting in incomplete enzyme system and inability to fully decompose macromolecules such as cellulose and pectin in plant matrix; solid food mostly uses a single strain (such as Lactobacillus helveticus OPB102), which is targeted at a specific function (weight loss), but cannot achieve the synchronous conversion of multiple active ingredients, such as the conversion efficiency of a single strain of astragaloside A in Astragalus membranaceus is only 35%-40%.

[0005] (2) Lack of systematic optimization of process parameters leads to an imbalance between viable cell count and active ingredients: Existing technologies all set fermentation parameters based on experience, without conducting multi-parameter synergistic optimization through orthogonal experiments or other methods. For example, liquid beverages use a short fermentation time of 12-24 hours, which reduces energy consumption, but the cells do not reach the stable period, and the viable cell count in the finished product is generally only 1×10⁻⁶. 8 -1×10 7 CFU / mL, far below the high viable bacteria standard (≥1×10⁻⁶). 9 (CFU / mL); For solid food products, the fermentation time was extended to 24 hours, but the stirring speed was not optimized. Static fermentation resulted in insufficient dissolved oxygen, which reduced the viable bacteria proliferation efficiency by 20%-30% and also resulted in insufficient release of active ingredients.

[0006] (3) Single functional positioning, failing to cover the synergistic needs of immunity and metabolism across scenarios: Existing technologies all focus on single-scenario functions and are not designed to address the core dual needs of immune regulation and metabolic improvement in the population. For example, for sub-healthy people, existing products can only solve a single problem and cannot achieve the synergistic effect of intestinal health, immune enhancement and metabolic optimization through probiotic fermentation, resulting in incomplete functional coverage. Summary of the Invention

[0007] To address the technical problems of low viable cell counts, insufficient synergy among strains, unreasonable process parameters, and inadequate release of active ingredients in existing probiotic fermented plant beverages, this invention provides a probiotic fermented plant beverage for enhancing immunity and its preparation method. The probiotic fermented plant beverage of this invention uses American ginseng, astragalus, polygonatum, poria cocos, and malt as core raw materials, combined with functional ingredients such as xylooligosaccharides and d-ribose. It is prepared by fermentation using a compound probiotic strain (Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus plantarum, all purchased from the China Industrial Microbial Culture Collection Center (CICC)). Through the combination of compound strains and optimization of key process parameters, this invention achieves a viable cell count ≥1×10⁻⁶ in the fermented product. 9 CFU / mL, simultaneously enhancing the content of active ingredients and product stability. Through orthogonal experiments, the fermentation process was optimized to determine the optimal parameters. Fermentation significantly improves the functional indicators of the product, with a marked increase in the content of total saponins, amino acids, and other nutrients. This invention addresses the issues of decreased immune function and blood sugar fluctuations after high-intensity exercise in athletes. By enhancing the activity and absorption efficiency of nutrients through probiotic fermentation, it provides precise nutritional support for post-exercise recovery.

[0008] The first objective of this invention is to provide a method for preparing a probiotic fermented plant beverage that enhances immunity, comprising the following steps:

[0009] (1) Grind American ginseng, astragalus, polygonatum, poria cocos and malt into powder, add functional ingredients and selenium-enriched water, mix and stir, sterilize to obtain fermentation substrate;

[0010] (2) The bacteria of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus and Lactobacillus plantarum were activated, centrifuged and freeze-dried and then mixed to obtain compound probiotic powder; the obtained compound probiotic powder was cultured in MRS medium to obtain compound probiotic liquid.

[0011] (3) Inoculate the compound probiotic liquid into the fermentation substrate obtained in step (1) for fermentation, separate the solid and liquid phases, sterilize and obtain the probiotic fermented plant beverage that enhances immunity.

[0012] Further, in step (1), the selenium content in the selenium-enriched water is 0.10~0.25 mg / L.

[0013] Further, in step (1), the functional component is one or more of xylooligosaccharides, d-ribose, taurine, pyrrolidinequinoline quinone (PQQ), honey, and D-aloxose.

[0014] Further, in step (2), the Lactobacillus casei is CICC 6114;

[0015] And / or, the Lactobacillus acidophilus is CICC 6087;

[0016] And / or, the Lactobacillus rhamnosus is CICC 6133;

[0017] And / or, the Lactobacillus plantarum is CICC 21790.

[0018] Furthermore, the mass ratio of the mixed *Lactobacillus casei*, *Lactobacillus acidophilus*, *Lactobacillus rhamnosus*, and *Lactobacillus plantarum* is 1:1:1:1.

[0019] Furthermore, in step (2), the activation conditions are 37°C for 12-24 hours.

[0020] Furthermore, in step (2), the viable count of the compound probiotic solution is >1×10⁻⁶. -8 CFU / mL.

[0021] Furthermore, in step (3), the inoculation volume ratio of the compound probiotic liquid is 1-3%, preferably 3%.

[0022] Furthermore, in step (3), the fermentation temperature is 20~40℃ and the time is 24~72h, preferably 48h.

[0023] Furthermore, in step (3), the fermentation speed is 30~100 r / min, preferably 60 r / min.

[0024] A second objective of this invention is to provide a probiotic fermented plant beverage that enhances immunity, prepared by the aforementioned method.

[0025] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0026] (1) The active ingredients of medicinal materials are comprehensive and significantly improved.

[0027] This invention utilizes fermentation with four types of compound probiotics and optimized processes to achieve efficient release of multiple active ingredients from raw materials such as Astragalus membranaceus and American ginseng. Compared with the comparative method of soaking and extraction, the core indicators are significantly improved: polysaccharide content reaches 3.45 mg / mL (an increase of 12.9%), free amino acids 0.401 mg / mL (an increase of 20.1%), total phenols 0.069 mg / mL (an increase of 35.3%), and total flavonoids 0.730 mg / mL (an increase of 18.9%). Among them, the improvement of characteristic components of medicinal materials is even more prominent, with astragaloside A 0.309 mg / mL (an increase of 33.2%), verbascoside 0.208 mg / mL (an increase of 84.1%), and ginsenoside series (Re1, Rg1, Rb) increased by 34.5%, 40.0%, and 42.3%, respectively, fully verifying the efficient decomposition ability of the compound strain on the medicinal material matrix.

[0028] (2) Significantly enhanced antioxidant capacity

[0029] The synergistic effect of the active ingredients in this invention significantly enhances the antioxidant performance of the product compared to existing technologies. In the examples, the DPPH free radical scavenging rate reached 88.5% (an increase of 17.1% compared to Comparative Example 1), the hydroxyl free radical scavenging rate was 42.2% (an increase of 19.2%), and the superoxide anion free radical scavenging rate was 87.3% (an increase of 9.9%). Short-chain fatty acids produced during fermentation (such as butyric acid and acetic acid) can enhance the activity of antioxidant enzymes (SOD, GSH-Px) in intestinal epithelial cells, scavenging superoxide anion free radicals through enzymatic reactions and reducing their accumulation and chain reactions. The synergistic effect of polysaccharides, total phenols, and total flavonoids: polysaccharides provide antioxidant active sites, while total phenols and total flavonoids directly scavenge free radicals. These three components form an "enzymatic-non-enzymatic" synergistic antioxidant system, significantly improving overall scavenging capacity.

[0030] (3) A surge in the production of short-chain fatty acids

[0031] Short-chain fatty acids are products of probiotic metabolism of carbohydrates, mainly including acetic acid, propionic acid, butyric acid, and isobutyric acid. The synergistic metabolism of compound probiotics leads to a significant increase in the content of beneficial short-chain fatty acids (SCFAs) in the intestine. In the example, acetic acid was 17.09 μg / mL (an increase of 656.2% compared to Comparative Example 1), propionic acid was 4.98 μg / mL (an increase of 99.2%), butyric acid was 4.23 μg / mL (an increase of 73.5%), and isobutyric acid was 7.40 μg / mL (an increase of 2287.1%).

[0032] (4) Optimization of process economy

[0033] The probiotic fermented plant beverage of this invention, which enhances immunity, has an 8-hour fermentation cycle that is 33% shorter than the traditional 72-hour process, reduces energy consumption by 25%, and reduces bacterial cell damage rate by 15% with a stirring speed of 60 rpm, thus balancing efficiency and product quality. Attached Figure Description

[0034] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0035] Figure 1 The active components of the fermentation broth are shown in the diagrams of the fermented Chinese medicinal materials fermented with probiotics according to the present invention, using the optimal fermentation conditions of Example 3 and the conditions of Comparative Example 1.

[0036] Figure 2 The graph shows the fatty acid content of the fermentation broth when the probiotic fermented Chinese medicinal materials of the present invention are fermented under the optimal fermentation conditions of Example 3 and Comparative Example 1.

[0037] Figure 3 The graph shows the antioxidant capacity of the fermentation broth when the probiotic fermented Chinese medicinal materials of the present invention are fermented under the optimal fermentation conditions of Example 3 and Comparative Example 1. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0040] Lactobacillus casei CICC 6114, Lactobacillus acidophilus CICC 6087, Lactobacillus rhamnosus CICC 6133, and Lactobacillus plantarum CICC 21790 were all purchased from the China Industrial Microbial Culture Collection Center (CICC).

[0041] MRS medium: 10g peptone, 10g beef extract, 5g yeast extract, 1g Tween-80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, bring to a final volume of 1L.

[0042] Example 1: Preparation of Compound Probiotic Powder

[0043] Bacterial powders of *Lactobacillus casei* CICC 6114, *Lactobacillus acidophilus* CICC 6087, *Lactobacillus rhamnosus* CICC 6133, and *Lactobacillus plantarum* CICC 21790 were prepared separately. The specific preparation method was as follows: The bacterial strains were inoculated into sterilized 250 mL Erlenmeyer flasks containing 50 mL of LMRS medium and incubated at 37 °C for 24 h. The resulting bacterial solution was centrifuged at 5000 r / min for 20 min, and the precipitate was dried using a freeze dryer to obtain a viable count of 1.0 × 10⁻⁶.9 CFU / g of each strain of bacterial powder. Synergistic fermentation bacterial powder was prepared by mixing *Lactobacillus casei*, *Lactobacillus acidophilus*, *Lactobacillus rhamnosus*, and *Lactobacillus plantarum* bacterial powders in a mass ratio of 1:1:1:1.

[0044] Example 2: Preparation method of probiotic fermented plant beverage

[0045] (1) Preparation of food and medicine homology matrix: Weigh 8g of Astragalus membranaceus, 5g of American ginseng, 6g of Polygonatum sibiricum, 4g of Poria cocos and 3g of malt, grind them to 180 mesh, add 2.0g of xylooligosaccharide, 1.5g of d-ribose, 0.8g of taurine, 0.002g of PQQ, 5.0g of honey and 1.0g of D-aloxose and mix evenly. Add 190mL of selenium-enriched water (selenium content 0.15mg / L) and stir evenly to obtain the mixed matrix.

[0046] (2) Sterilization: Transfer the mixed substrate into a sterilization tank and sterilize it with high-pressure steam at 121°C for 15 minutes. After cooling, the fermentation substrate is obtained.

[0047] (3) Fermentation: Take the compound probiotic powder and inoculate it into a sterilized 250mL Erlenmeyer flask containing 50mL MRS medium. Then, seal the Erlenmeyer flask and place it in a shaker at 37℃ for static incubation until the logarithmic growth phase. Centrifuge at 4℃ and 4000rpm for 10 minutes, discard the supernatant, wash with sterile water, and prepare a bacterial suspension (bacterial concentration >1×10⁻⁶). -8 (CFU / mL), add 1% of the inoculum to the fermentation substrate, and ferment at a constant temperature of 30℃ with stirring for 72 hours at a stirring speed of 50 rpm.

[0048] (4) Post-processing: After fermentation, shake the fermentation liquid to mix well, take 5 mL for dilution and spread for later use, then perform solid-liquid separation, centrifuge at 8000 rpm for 20 min at 4℃, and collect the supernatant. Transfer the supernatant to a sterile bottle for pasteurization, sterilize at 80℃ for 30 min, and store at 4℃ for later use to obtain a probiotic fermented plant beverage that enhances immunity.

[0049] Example 3: Condition optimization of traditional Chinese medicine fermentation concentrate

[0050] (1) Orthogonal experimental design: The process conditions affecting the fermentation capacity of the compound strain were selected as factors for the orthogonal experiment. At the same time, the levels were determined and an appropriate orthogonal table was selected for the table header design. The factors required in the experiment were arranged in each column of the orthogonal table (Table 1). According to the design principles of the orthogonal table and the determined factors and levels, the orthogonal table was designed using SPSS. An L9(3³) orthogonal table was used, with 3 levels for each factor. The core evaluation index was "the number of viable cells after fermentation (CFU / mL)". The experiment was repeated 3 times and the average value was taken. The specific design and data are as follows:

[0051] Table 1. Factors and levels of the orthogonal experiment to optimize fermentation conditions

[0052]

[0053] (2) Orthogonal results analysis: The optimal fermentation parameters were A3B3C3 (fermentation time 48h, inoculum size 3.0%, rotation speed 60rpm), at which point the viable cell count reached 11.6×10⁻⁶. 8 CFU / mL (i.e., 1.16 × 10⁻⁶) 9 (CFU / mL) The key factors affecting the number of viable bacteria are ranked as follows: stirring speed > fermentation time > inoculum size: at 0 rpm, the number of viable bacteria is the lowest due to insufficient dissolved oxygen, at 60 rpm, the number of viable bacteria is the highest due to moderate dissolved oxygen, and at 100 rpm, the bacteria are destroyed due to excessively high dissolved oxygen. The peak nutrient utilization is reached at 48 h of fermentation time, and the number of viable bacteria decreases after 72 h when nutrients are depleted.

[0054] Table 2 Orthogonal Results and Analysis (See Table 1 for specific parameters)

[0055]

[0056] As can be seen from the table above, the conditions of this invention are met when the volume ratio of the inoculum is 1-3%, the temperature is 20-40℃, the rotation speed is 30-60 r / min, and the fermentation time is 24-72 h.

[0057] (3) Fermentation verification

[0058] Fermentation was repeated three times according to the A3B3C3 parameters (48h, 3.0%, 60rpm), and the viable cell counts were 11.8 × 10⁻⁶ each time. 8 CFU / mL, 11.5×10 8 CFU / mL, 11.7×10 8 CFU / mL, average value 11.6 × 10 8 The CFU / mL data is stable, proving that the optimized parameters are reliable.

[0059] 2. Unoptimized control group

[0060] Using a control group with "fermentation time 24h, inoculum size 1.0%, and rotation speed 0rpm" (standard, unoptimized parameters), the viable cell count was only 5.1×10⁻⁶. 8 The CFU / mL count after optimization increased the viable bacteria count by 127.5% compared to the control group, highlighting the optimization effect.

[0061] Comparative Example 1

[0062] This comparative example provides a method for preparing a plant-based beverage, which is similar to the optimal conditions (A3B3C3) in Example 3, except that the compound probiotic liquid is replaced with the same volume of sterilized water.

[0063] Performance testing

[0064] The antioxidant capacity of the product obtained under the optimal conditions of Example 3 (fermentation time 48 h, inoculum size 3.0%, rotation speed 60 rpm) and the product prepared in Comparative Example 1 were tested, and their polysaccharide and total phenolic flavonoid contents were also determined. The specific methods are as follows:

[0065] (1) Superoxide anion free radical scavenging ability

[0066] Take 4.5 mL of 0.05 mol / L pH 8.2 Tris-HCl buffer solution and preheat it in a 25℃ water bath for 20 min. Add 1 mL of fermentation broth and 0.4 mL of 25 mmol / L pyrogallol solution, mix well, and react in a 25℃ water bath for 5 min. Add 1.0 mL of 8 mol / L HCl to terminate the reaction. Using Tris-HCl buffer solution as a reference, measure the absorbance at 299 nm and calculate the scavenging rate. The blank control group is replaced with 1 mL of sample solvent. The formula for calculating the scavenging rate is: Superoxide anion radical scavenging rate (%) = [(A0-(A1-A2) / A0]×100%, where A0 is the absorbance of the blank control solution; A1 is the absorbance of the sample test tube; and A2 is the absorbance of the sample background tube.

[0067] (2) DPPH free radical scavenging ability

[0068] Add 40 μL of fermentation broth to 4 mL of 0.1 mmol / L DPPH-methanol solution, then add 450 μL of 50 mmol / L Tris-HCl buffer (7.4), and incubate at 25 °C for 30 min. Use deionized water as a reference solution and measure the absorbance at 517 nm. DPPH free radical scavenging rate (%) = [(A0-(A1-A2) / A0]×100%, where A0 is the absorbance of the blank control solution; A1 is the absorbance of the sample test tube; and A2 is the absorbance of the sample background tube.

[0069] (3) Hydroxyl radical scavenging ability

[0070] Add 135 μL of fermentation broth to 1.4 mL of 6 mmol / L H₂O₂, then add 0.6 mL of 20 mmol / L sodium salicylate and 2 mL of 1.5 mmol / L ferrous sulfate. Incubate at 37 °C for 1 h. Use deionized water as the reference solution. Measure the absorbance at 562 nm. Hydroxyl radical scavenging capacity (%) = [(A0 - (A1 - A2) / A0] × 100%, where A0 is the absorbance of the blank control solution; A1 is the absorbance of the sample test tube; and A2 is the absorbance of the sample background tube.

[0071] (4) Polysaccharide determination (phenol-sulfuric acid method):

[0072] a. Sample preparation: Add 5 times the volume of anhydrous ethanol to the fermentation broth and let it stand overnight at 4°C. Filter the mixture and dry the precipitate at 60°C to constant weight to obtain crude polysaccharide.

[0073] b. Construction of the standard curve: Prepare a 5% phenol solution and a 100 mg / L glucose standard solution. Accurately pipette 0, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the glucose standard solution into test tubes, and bring the volume to 2.0 mL with pure water. Add 1.0 mL of phenol solution and 5 mL of concentrated sulfuric acid solution. After reacting for 10 min, mix well and react at 30℃ for 20 min. Zero the instrument with a blank and measure the absorbance at 490 nm. Plot the glucose concentration on the x-axis and the absorbance on the y-axis to construct a standard curve.

[0074] c. Sample determination: Take 0.01g of crude polysaccharide and dilute to 50mL. The determination method is the same as that of the standard curve.

[0075] (5) Total phenols

[0076] Construction of the standard curve: Total phenols were tested using the Folin-Ciocalteu method. First, a pyrogallol standard solution was prepared. 5 mg of pyrogallol was weighed and diluted to 25 mL with methanol, yielding a concentration of 0.2 mg / mL. Accurately pipette 0, 0.01 mL, 0.02 mL, 0.05 mL, 0.1 mL, 0.12 mL, and 0.15 mL of pyrogallol solution into test tubes. The volume was brought to 0.5 mL with ethanol, and then 0.5 mL of Folin-Ciocalteu solution was added. The mixture was allowed to stand for 3 minutes, followed by 1 mL of 15% sodium carbonate solution. The mixture was then allowed to stand for 30 minutes. After standing, the mixture was centrifuged at 3500 rpm for 3 minutes. Using a reagent blank as a control, the absorbance was measured at 760 nm. A standard curve was plotted with absorbance (A) on the ordinate and pyrogallol concentration on the ordinate.

[0077] (6) Total flavonoids

[0078] Construction of the standard curve: The sodium nitrite-aluminum nitrate colorimetric method was used. First, a rutin standard solution was prepared by weighing 10.4 mg of rutin and diluting it to 25 mL with 80% ethanol. Accurately pipette 0.025 mL, 0.06 mL, 0.12 mL, 0.24 mL, and 0.48 mL of rutin solution, add ethanol to make up to 0.5 mL of reagent, mix well, add 0.15 mL of 5% sodium nitrite, and let stand for 6 min. After standing, add 0.15 mL of 10% aluminum nitrate and let stand for 6 min. Add 2 mL of 4% sodium hydroxide solution and dilute to 5 mL with water, shake to mix, and let stand for 3 min. Measure the absorbance at 508 nm. Plot the absorbance (A) on the ordinate and the rutin concentration on the abscissa to construct the standard curve.

[0079] (7) Ginsenosides Re Rb1 Rg1

[0080] Acetonitrile and water were used as the mobile phase, and octadecylsilane-bonded silica gel was used as the packing material, with an injection volume of 10 μL. Detection was performed at a wavelength of 203 nm. The mobile phase underwent gradient elution: acetonitrile 19% from 0-35 min, acetonitrile changed from 19% to 29% from 35-55 min, acetonitrile remained at 29% from 55-70 min, and acetonitrile changed from 29% to 40% from 70-100 min.

[0081] (8) Astragaloside A

[0082] Astragaloside A was analyzed using an ELSD detector and a Kromasil 100-5-C18 column with a particle size of 5 μm, an inner diameter of 4.6 mm, a length of 250 mm, a column temperature of 35 °C, an evaporative light scattering detector (ELSD) temperature of 75 °C, a mobile phase flow rate of 1.0 mL / min, a nitrogen flow rate of 2.5 mL / min, and an injection volume of 5 μL. The analytical conditions were that the mobile phase consisted of a gradient elution of 25%–40% acetonitrile in an aqueous solution containing 0.05% trifluoroacetic acid over 30 minutes.

[0083] (9) Versicolor isoflavone glucoside

[0084] Verrucoside isoflavone glucoside was detected by high performance liquid chromatography using a C18 column at 35°C, a mobile phase flow rate of 1.0 mL / min, an injection volume of 5 μL, and a detection wavelength of UV-210 nm. Gradient elution was used, with the acetonitrile concentration increasing from 10% to 40% in 0.05% phosphoric acid aqueous solution within 30 minutes.

[0085] The experimental results are shown in Table 3 and Figures 1-3 As shown:

[0086] Table 3

[0087]

[0088] The data in the table and graph show that fermentation of medicinal materials with mixed probiotics can improve various indicators.

[0089] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a probiotic fermented plant beverage to enhance immunity, characterized in that, Includes the following steps: (1) Grind American ginseng, astragalus, polygonatum, poria cocos and malt into powder, add functional ingredients and selenium-enriched water, mix and stir, sterilize to obtain fermentation substrate; (2) The bacteria of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus and Lactobacillus plantarum were activated, centrifuged and freeze-dried and then mixed to obtain compound probiotic powder; the obtained compound probiotic powder was cultured in MRS medium to obtain compound probiotic liquid. (3) Inoculate the compound probiotic liquid into the fermentation substrate obtained in step (1) for fermentation, separate the solid and liquid phases, sterilize and obtain the probiotic fermented plant beverage that enhances immunity.

2. The preparation method according to claim 1, characterized in that, In step (1), the selenium content in the selenium-enriched water is 0.10~0.25 mg / L.

3. The preparation method according to claim 1, characterized in that, In step (1), the functional ingredient is one or more of xylooligosaccharides, d-ribose, taurine, pyrrolidinequinone, honey, and D-aloxose.

4. The preparation method according to claim 1, characterized in that, In step (2), the Lactobacillus casei is CICC6114; And / or, the Lactobacillus acidophilus is CICC 6087; And / or, the Lactobacillus rhamnosus is CICC 6133; And / or, the Lactobacillus plantarum is CICC 21790.

5. The preparation method according to claim 1, characterized in that, In step (2), the activation conditions are 37°C for 12-24 hours.

6. The preparation method according to claim 1, characterized in that, In step (2), the viable count of the compound probiotic solution is >1×10⁻⁶. -8 CFU / mL.

7. The preparation method according to claim 1, characterized in that, In step (3), the inoculation volume ratio of the compound probiotic solution is 1-3%.

8. The preparation method according to claim 1, characterized in that, In step (3), the fermentation temperature is 20~40℃ and the time is 24~72h.

9. The preparation method according to claim 1, characterized in that, In step (3), the fermentation speed is 30~60 r / min.

10. The probiotic fermented plant beverage for enhancing immunity obtained by the preparation method according to any one of claims 1-9.

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