Method for increasing alkaloid content in goji berry pulp by kefir fermentation and its application in improving colitis

Abstract

This application belongs to the field of biotechnology, and specifically relates to a method for improving the efficacy of wolfberry puree by fermentation with kefir grains and the application of kefir-grain-fermented wolfberry puree in alleviating and treating colitis. The fermented wolfberry puree prepared in this application can significantly improve the inflammatory symptoms and various inflammatory indicators of colitis, and its efficacy is close to or even better than that of the existing mesalazine.

Description

Technical Field

[0001] This application belongs to the field of microbial fermentation, and specifically relates to a method for fermenting wolfberry raw pulp with kefir and enhancing the functional components therein, as well as the application of the fermented wolfberry raw pulp in relieving and treating colitis. Background Art

[0002] Wolfberry is rich in various active ingredients such as polysaccharides, polyphenols, flavonoids, carotenoids, and alkaloids, and is widely used at home and abroad as a food homologous to medicine. The total contents of phenols, flavonoids, carotenoids, betaine, and polysaccharides in wolfberry are 0.690%-0.825%, 0.318%-0.614%, 1.293%-2.535%, 0.85%-1.13%, and 2.362%-4.245% of wolfberry fruits respectively. Among them, betaine is the most abundant and representative component in wolfberry alkaloids. Betaine, also known as trimethylglycine, is a non-toxic and stable natural product. Modern medicine shows that betaine has the effects of anti-tumor, blood pressure lowering, anti-peptic ulcer, and improving gastrointestinal function. Therefore, the content of betaine is also used as an important standard for evaluating the quality of wolfberry and is included in the Chinese Pharmacopoeia. Polysaccharides, zeaxanthin dipalmitate, vitamins, betaine, etc. in wolfberry are considered to have anti-aging, vision-improving, anti-fatigue, and other beneficial effects.

[0003] Research shows that the content of betaine in wolfberry is between 8.5mg / g - 11.3mg / g, but for wolfberry wine directly processed from wolfberry, the content of betaine is only 0.90mg / mL - 3.07mg / mL. Guo Jintao et al. found that after fermentation of wolfberry by lactic acid bacteria, the content of betaine in the wolfberry ferment can be increased. Therefore, using microorganisms to ferment wolfberry is an effective means to increase the content of functional components such as betaine in deep-processed wolfberry products. In this application, kefir, this natural symbiotic mixed bacteria, is used as the fermentation strain to ferment wolfberry raw pulp, so as to increase the content of functional components such as betaine. At the same time, the fermented product has the effect of relieving and improving ulcerative colitis, and the effect is significant. Summary of the Invention

[0004] The applicant found that by using kefir to ferment wolfberry raw pulp, the content of betaine and others in the wolfberry raw pulp can be significantly increased, and the inflammatory symptoms and various inflammatory indicators of colitis can be significantly improved, and its efficacy is close to or even better than that of the existing mesalazine.

[0005] This application first provides a method for fermenting wolfberry raw pulp, and the method includes the following steps:

[0006] 1) Activation culture of kefir;

[0007] 2) Inoculate the activated kefir into wolfberry raw pulp for fermentation.

[0008] In step 1), the activation of kefir is to inoculate kefir in sterilized skim milk for activation culture.

[0009] Furthermore, in step 1), the fermentation conditions are as follows: inoculate the activated kefir into the wolfberry puree at an inoculation amount of 1%-8% (w / w), and ferment at 20-35°C for 30-60 h.

[0010] Even further, in step 1), the fermentation conditions are as follows: inoculate the activated kefir into the wolfberry puree at an inoculation amount of 3%-5% (w / w), and ferment at 25-30°C for 48-60 h.

[0011] Still further, in step 1), the fermentation conditions are as follows: inoculate the activated kefir into the wolfberry puree at an inoculation amount of 5% (w / w), and ferment at 25°C for 48 h.

[0012] Furthermore, the wolfberry puree is obtained by the following method: pick wolfberries, wash them, air-dry them until semi-dry, grind them, and filter them through a filter screen to obtain the wolfberry puree.

[0013] This application also provides a method for increasing the alkaloid content in kefir-fermented wolfberry puree. The method includes any of the above fermentation methods, and further includes the following steps:

[0014] 3) Mix the fermented wolfberry puree with an organic solvent for multiple extractions, and perform membrane treatment after vacuum evaporation.

[0015] Furthermore, step 3) is specifically as follows: mix the fermented wolfberry puree with methanol, perform ultrasonic extraction at room temperature, centrifuge, collect the supernatant, add methanol to the precipitate for multiple extractions again; combine the supernatants, perform vacuum evaporation, dissolve in acetonitrile again, and obtain alkaloids after filtering through a membrane.

[0016] Even further, step 3) is specifically as follows: mix the fermented wolfberry puree with methanol at a ratio of 1:3-5 (v:v), perform ultrasonic extraction at room temperature for 20-30 min, centrifuge, take the supernatant, add 3-5 times the volume of methanol to the precipitate and extract according to the above method, and extract 3-5 times in total. Combine the supernatant liquids, perform vacuum evaporation at 40-45°C, dissolve in acetonitrile again, and obtain alkaloids after passing through a filter membrane.

[0017] Furthermore, the alkaloids include betaine, trigonelline, capsaicin, and ophiopogonin D'.

[0018] This application also provides a kefir-fermented wolfberry puree, which is obtained by fermenting wolfberry puree with kefir, or is prepared by any of the above methods.

[0019] The present application also provides the use of the above-mentioned kefir-fermented wolfberry raw pulp in improving colitis, or the use in preparing a product for improving colitis.

[0020] Furthermore, the product is a medicine.

[0021] Even further, the preparation form of the medicine includes tablets, granules or oral liquids, etc.

[0022] The present application also provides the use of kefir in increasing the alkaloid content in wolfberry raw pulp;

[0023] Furthermore, the application is to inoculate kefir into wolfberry raw pulp for fermentation; even further, the alkaloids include betaine, trigonelline, capsaicin and ophiopogonin D'.

[0024] Compared with the prior art, the present application has at least the following advantages:

[0025] 1) The method of the present application first uses kefir for the fermentation of wolfberry raw pulp, improving the fermentation activity. In particular, the content of alkaloids (including betaine, trigonelline, capsaicin and ophiopogonin D', etc.) is significantly increased, and at the same time, the quality of wolfberry raw pulp is significantly improved (the volatile components after fermentation increased by 3.59 times compared with before fermentation).

[0026] 2) Animal experiments show that the fermentation product prepared by the method of the present application has significant significance in relieving and treating colitis, and its efficacy is close to or even better than the existing mesalazine drug, exceeding expectations.

[0027] 3) The kefir fermentation process of the present application is simple. Fermentation can be achieved by directly inoculating wolfberry raw pulp, and by optimizing the parameters of the whole fermentation process, the optimal fermentation system is established. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 Effect of inoculation amount on the betaine content in wolfberry raw pulp.

[0030] Figure 2 Effect of fermentation time on the betaine content in wolfberry raw pulp.

[0031] Figure 3 Effect of fermentation temperature on the betaine content in wolfberry raw pulp.

[0032] Figure 4 Fermentation enhances various alkaloid functional components in wolfberry puree.

[0033] Figure 5 Results of the body weight changes (A) and disease index (B) of mice in each group.

[0034] Figure 6 Results of the colon length (A) and histopathological observation of colon tissue (B) of mice in each group.

[0035] Figure 7 MDA, MPO, T-AOC indexes (A-C) of the colon of mice in each group, LPS level (D) in the serum of mice, and levels of IL-6, IL-1β, TNF-α and IFN-γ in the colon of mice (E-H). Detailed implementation manners

[0036] The technical solutions of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without making creative efforts shall fall within the protection scope of the present application.

[0037] The following terms or definitions are provided only to assist in understanding the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0038] Unless otherwise defined hereinafter, the meanings of all technical terms and scientific terms used in the specific implementation manners of the present application are intended to be the same as those generally understood by those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0039] As used in the present application, the terms "comprise", "include", "have", "contain" or "involve" are inclusive or open-ended and do not exclude other unenumerated elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined hereinafter as including at least a certain number of embodiments, this should also be understood as disclosing a group preferably consisting only of these embodiments.

[0040] The indefinite article or definite article used when referring to a singular form noun, such as "a" or "an", "the", includes the plural form of the noun.

[0041] The terms "about" and "substantially" in this application indicate an accuracy range that those skilled in the art can understand and still ensure the technical effect of the feature being discussed. This term generally represents a deviation of ±10% from the indicated value, preferably ±5%.

[0042] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessary for describing an order or temporal sequence. It should be understood that the terms so applied can be interchanged in appropriate circumstances, and the embodiments described in this application can be implemented in an order different from that described or illustrated in this application.

[0043] The method for fermenting wolfberry raw pulp in this application includes the following steps:

[0044] 1) Activated culture of kefir grains;

[0045] 2) Inoculate the activated kefir grains into the wolfberry raw pulp for fermentation.

[0046] In step 1), the activation of the kefir grains is to inoculate the kefir grains into sterilized skim milk for activated culture.

[0047] In some embodiments, in step 1), the fermentation conditions are: inoculate the activated kefir grains into the wolfberry raw pulp at an inoculation amount of 1%-8% (w / w), and ferment at 20-35°C for 30-60 h.

[0048] In some specific embodiments, in step 1), the fermentation conditions are: inoculate the activated kefir grains into the wolfberry raw pulp at an inoculation amount of 3%-5% (w / w), and ferment at 25-30°C for 48-60 h.

[0049] In some more specific embodiments, in step 1), the fermentation conditions are: inoculate the activated kefir grains into the wolfberry raw pulp at an inoculation amount of 5% (w / w), and ferment at 25°C for 48 h.

[0050] The method for increasing the alkaloid yield by fermenting wolfberry raw pulp with kefir grains in this application further includes mixing the fermented wolfberry raw pulp with an organic solvent for multiple extractions, and performing membrane treatment after vacuum evaporation.

[0051] In some specific embodiments, the fermented wolfberry raw pulp is ultrasonically extracted with methanol at room temperature in proportion, the supernatant is taken after centrifugation, and the precipitate is added with methanol again for multiple extractions; the supernatant liquids are combined and vacuum evaporated, then dissolved in acetonitrile, and alkaloids are obtained after filtering through a membrane.

[0052] In some more specific embodiments, the fermented wolfberry pulp is ultrasonically extracted with methanol at a ratio of 1:3 - 5 (v:v) at room temperature for 20 - 30 min. After centrifugation, the supernatant is taken, and the precipitate is extracted again with 3 - 5 times the volume of methanol according to the above method, and the extraction is carried out 3 - 5 times in total. The combined supernatant is evaporated under reduced pressure at 40 - 45 °C, redissolved in acetonitrile, and alkaloids are obtained after passing through a filter membrane. In some embodiments, the alkaloids include betaine, trigonelline, capsaicin, and ophiopogon saponin D', especially betaine.

[0053] The Tibetan kefir fermented wolfberry pulp of the present application is obtained by fermenting wolfberry pulp with Tibetan kefir or prepared by any of the above - mentioned methods.

[0054] The following are specific embodiments.

[0055] Example 1 Tibetan kefir fermented wolfberry pulp

[0056] The applicant found in the previous research on Tibetan kefir that it can effectively ferment wolfberry pulp, so the present technology was proposed. The specific steps of the previous experiment are as follows: Take 5 g of Tibetan kefir granules and inoculate them into 100 mL of sterilized skim milk, culture at 25 °C for 24 h, filter with a sterilized filter mesh after continuous culturing 5 times to obtain activated Tibetan kefir bacteria. The activated Tibetan kefir is inoculated into wolfberry pulp at a ratio of 2% (w / v) and fermented at 25 °C for 36 h. The production process of wolfberry pulp is as follows: Pick wolfberries, wash them, and air - dry them to semi - dryness, then finely grind them with a grinder, and filter the ground wolfberry pulp with a filter mesh to obtain wolfberry pulp. The fermented wolfberry pulp is ultrasonically extracted with methanol at a ratio of 1:3 (v:v) at room temperature (100 W, 20 kHz) for 30 min. After centrifugation, the supernatant is taken, and the precipitate is extracted again with 3 times the volume of methanol according to the above method, and the extraction is carried out 3 times in total. The combined supernatant is evaporated under reduced pressure at 45 °C, redissolved in 5 mL of acetonitrile, and after passing through a 0.22 - μm filter membrane, it is detected by high - performance liquid chromatography. The detection conditions are as follows: Use a Zorbax NH2 column (250 mm×4.6 mm, 5 μm, Agilent), detection wavelength 210 nm, the mobile phase is composed of ultrapure water and acetonitrile (25:75), isocratic elution, flow rate 1 mL / min, column temperature 25 °C, injection volume 10 μL, and the external standard method is used to quantify the content of betaine.

[0057] The results show that in the wolfberry pulp fermented by Tibetan kefir, the content of betaine increases from 5.36 ± 0.35 mg / mL to 11.22 ± 0.17 mg / mL, indicating that Tibetan kefir fermentation can significantly increase the content of betaine - type functional components in wolfberry pulp.

[0058] Example 2 Single - factor analysis of Tibetan kefir fermented wolfberry pulp

[0059] Activate kefir according to the method of Example 1, and then investigate the effects of the inoculation amount of kefir (1%, 3%, 5%, 7%, 9%), fermentation time (36h, 48h, 60h, 72h, 84h), and fermentation temperature (20°C, 25°C, 30°C, 35°C) on the betaine content in wolfberry puree. When conducting single-factor experiments, other non-determined factors were fixed at the levels of fermentation temperature 25°C, kefir inoculation amount 3%, and fermentation time 48h respectively, and then use the method of Example 1 to detect the betaine content.

[0060] The results show that, as Figures 1 - 3 : When the inoculation amount is between 1% and 3%, the betaine content increases with the increase of the inoculation amount; when the inoculation amount is 3%, it can reach the highest 15.83 mg / mL, which is 194% higher than that before fermentation. The content of betaine first increases and then decreases with the prolongation of fermentation time, and reaches the highest 15.25 mg / mL at about 48h, which is 183% higher than that before fermentation. With the increase of fermentation temperature, the betaine content first increases and then decreases, which may be related to the fact that too low or too high fermentation temperature is not suitable for the growth of kefir, resulting in a slowdown in its metabolic rate. When the fermentation temperature is 30°C, the betaine content in wolfberry puree reaches the highest 16.95 mg / mL.

[0061] Orthogonal optimization of kefir-fermented wolfberry puree in Example 3

[0062] Based on the basic conditions established by single-factor experiments, further optimize the process of kefir-fermented wolfberry puree through orthogonal experiments. The parameter settings for orthogonal experiments are shown in Table 1, and analyze the effects of various factors on kefir-fermented wolfberry puree. The fermentation process and the detection method of betaine are the same as those in Example 1.

[0063] Table 1 Orthogonal level design of kefir wolfberry puree conditions

[0064]

[0065] The experimental results are shown in Tables 2 and 3 below.

[0066] The orthogonal experimental design and results of kefir-fermented wolfberry puree are shown in Table 2. By comparing the R values of various factors, it can be seen that the order of the influence of various factors on the betaine content in wolfberry puree is fermentation temperature, fermentation time, and inoculation amount.

[0067] Table 2 Orthogonal test results

[0068]

[0069] Table 3 Analysis of variance of orthogonal test

[0070]

[0071]

[0072] The variance analysis of the Tibetan mushroom-fermented wolfberry puree is shown in Table 3. It can be seen that the fermentation temperature has a significant effect on the betaine content in the wolfberry puree (p<0.05). In summary, when the inoculation amount of the Tibetan mushroom is 3-5%, the fermentation time is 48-60 h, and the fermentation temperature is 25-30 °C, the effect is better; among them, the optimal fermentation conditions are A2B2C1, that is, the inoculation amount of the Tibetan mushroom is 5%, the fermentation time is 48 h, and the fermentation temperature is 25 °C. Under these conditions, a verification test was carried out, and the betaine content in the fermented wolfberry puree was measured to be 18.95 mg / mL (n = 3).

[0073] Example 4 Enhancement of alkaloid functional components in Tibetan mushroom-fermented wolfberry puree

[0074] Ferment the wolfberry puree according to the results of orthogonal optimization, and then measure the content of alkaloids in it. The methods for strain activation, fermentation, and alkaloid detection are the same as those in Example 1. A total of six alkaloid components were monitored, namely betaine, trigonelline, capsaicin, isoleucine, ophiopogonin D', and oxymatrine. After fermentation with the Tibetan mushroom, the results are as Figure 4 shown. The contents of betaine, trigonelline, capsaicin, and ophiopogonin D' increased by 3.54, 7.94, 33.74, 0.68, and 16.19 times respectively. This increase has a very significant advantage, and oxymatrine is a product after fermentation.

[0075] The results show that the fermentation of the Tibetan mushroom can significantly enhance various alkaloid functional components in the wolfberry puree.

[0076] Example 5 Quality evaluation of Tibetan mushroom-fermented wolfberry puree

[0077] GC-MS was used to determine the volatile components in the Tibetan mushroom-fermented wolfberry puree. 4.5 μL of 2-octanol solution (0.40 mg / mL) was added as an internal standard to 5 mL of the sample, and headspace microextraction was carried out at 40 °C using 50 / 30 μm PDMS / DVB. A DB-1MS capillary column (30 m×0.25 mm, 0.25 μm) was used, the carrier gas was helium, and the flow rate was 1 mL / min. The mass spectrometry was scanned in the range of 35–500 m / z, the ion source was in EI ionization mode, and the energy was 70 eV. The temperatures of both the ion source and the quadrupole were 230 °C.

[0078] There were 41 kinds of aroma substances in the wolfberry raw pulp before fermentation. Among them, the relative contents of alcohols, ketones and acids were relatively high. The typical volatile component was (2R,3R)-(-)-2,3-butanediol, with a content of 3.45 mg / L. There were 65 kinds of volatile components in the wolfberry raw pulp after fermentation. Among them, the relative contents of alcohols, esters and acids were relatively high. The typical volatile component was 3-methyl-4-oxopentanoic acid, with a content as high as 10.35 mg / L. The total content of volatile components after fermentation was 39.76 mg / L, which was 3.59 times higher than that before fermentation. This indicated that the fermentation with kefir increased the types and total content of volatile components in the wolfberry raw pulp, and improved the sensory quality of the wolfberry raw pulp (Table 4).

[0079] Table 4 GC-MS determination results of wolfberry raw pulp and fermented wolfberry

[0080]

[0081]

[0082]

[0083]

[0084] Example 6 Improvement of colitis by kefir-fermented wolfberry raw pulp

[0085] The mice were fed in a mouse room at a temperature of 22 - 25 °C and a humidity of 50 - 60%, and were allowed to freely drink purified water and commercially available ordinary feed. When making the model, dextran sulfate sodium (DSS) was dissolved in physiological saline to prepare a 2.5% (w / v) solution, and the mice were allowed to freely drink it for 7 days to construct a colitis mouse model. After 7 days of adaptive feeding, the mice were randomly divided into 5 groups, with 10 mice in each group, and the experiment lasted for 21 days. The grouping was as follows:

[0086] Control group: Intragastric administration of physiological saline from day 1 to day 21, and at the same time, freely drinking sterile water.

[0087] Model group: Intragastric administration of physiological saline from day 1 to day 14, and at the same time, freely drinking sterile water. From day 15 to day 21, freely drinking 2.5% DSS solution.

[0088] Positive drug group: Intragastric administration of physiological saline from day 1 to day 14, and at the same time, freely drinking sterile water. From day 15 to day 21, freely drinking 2.5% DSS solution and intragastric administration of 200 mg / kg / d mesalazine (5-aminosalicylic acid) at the same time.

[0089] Fermented wolfberry raw pulp group: Intragastric administration of 100 mg / kg / d fermented wolfberry alkaloids from day 1 to day 21. At the same time, freely drinking sterile water from day 1 to day 14, and freely drinking 2.5% DSS solution from day 15 to day 21.

[0090] Unfermented goji berry puree group: Gavage with 100 mg / kg / d of unfermented goji berry alkaloids from day 1 to day 21, and at the same time, freely drink sterile water from day 1 to day 14, and freely drink 2.5% DSS solution from day 15 to day 21.

[0091] Continuously measure the body weight of mice, and at the same time use the Disease Activity Index (DAI) to evaluate the effect of different treatments in inhibiting colitis.

[0092] The DAI assessment mainly consists of three parts: body weight loss, fecal characteristics, and fecal occult blood. The specific scoring rules refer to Table 5. Fecal occult blood is detected by the o-tolidine method. The calculation formulas for body weight loss and DAI are as follows:

[0093] Percentage of body weight loss = (daily body weight - initial body weight) / initial body weight × 100%

[0094] DAI = body weight loss score + fecal characteristics score + fecal occult blood score

[0095] Table 5 Disease Index (DAI) Scoring Table

[0096]

[0097] The results are as Figure 5 shown. Body weight loss is the main apparent symptom of ulcerative colitis. Among all treatment groups, the body weight loss in the drug administration group is the least, only 7.11 ± 4.72%, followed by the Tibetan mushroom-fermented goji berry puree group, where the body weight loss is only 8.10 ± 3.09%. There is no significant difference between this group and the drug administration group. Both of these two treatments are significantly better than the goji berry puree group and the colitis model group. Fecal blood in the stool is also one of the main symptoms of the colitis model. In the DAI results, compared with the control group, as the treatment time prolongs, the fecal blood in the model group becomes more and more serious, and the DAI score can reach 9.0 points on the seventh day. The DAI of the drug administration group and the Tibetan mushroom-fermented goji berry puree group is significantly lower than that of the model group and the unfermented goji berry puree group, and the DAI score of the Tibetan mushroom-fermented goji berry puree group is close to that of the drug administration group. These results all indicate that taking Tibetan mushroom-fermented goji berry puree can significantly improve the symptoms of ulcerative colitis.

[0098] Example 7 Improvement of Colitis by Tibetan Mushroom-Fermented Goji Berry Puree

[0099] The mice in Example 6 were fasted 12 h in advance, anesthetized and sacrificed with 4% chloral hydrate solution, blood was taken from the orbital cavity, and the serum was stored at -80 °C. The part of the mouse cecum to the anus was taken as the colon, which was placed on white paper and naturally stretched to measure its length. The colon tissue was fixed in 4% paraformaldehyde solution for about 24 h. Then, the tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Observation was performed using an inverted fluorescence microscope at a magnification of 200 times. The histological scoring was referred to Table 6. The remaining colon part was removed of its contents, washed clean with physiological saline and then stored.

[0100] Table 6 Histopathological scoring

[0101]

[0102] The results showed ( Figure 6 ), although the colon lengths of the mice in the drug administration group, fermented wolfberry puree group and wolfberry puree group were shorter than those in the control group, they were all better than those in the colitis model group, indicating that taking fermented wolfberry puree or wolfberry puree was helpful for improving the colon shortening symptoms caused by colitis; meanwhile, the histopathological scoring results also showed that, compared with the model group, the colon tissue damage of the mice in the other three treatment groups was alleviated to varying degrees. Especially in the fermented wolfberry puree group, the histopathological scoring results were significantly better than those in the drug administration group and wolfberry puree group, indicating that the wolfberry puree fermented by kefir could effectively relieve the symptoms of ulcerative colitis.

[0103] Example 8 Improvement of colitis by kefir-fermented wolfberry puree

[0104] The stored mouse colon tissue in Example 7 was weighed, added with PBS buffer at a ratio of 1:9 (w / v), and thoroughly ground on ice with a hand-held homogenizer, centrifuged (4 °C, 3500 r / min) for 15 min, the supernatant was aspirated, and its antioxidant indexes were measured according to the instructions of the malondialdehyde (MDA), myeloperoxidase (MPO) and total antioxidant capacity (T-AOC) kits. At the same time, the LPS level in the mouse serum stored in Example 6 was measured with an LPS kit. And kits were used to measure the levels of interleukin 6 (IL-6), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) to reflect the verification indexes.

[0105] Among various indicators, MDA is the main end product of lipid peroxidation. The MDA index of mice taking Tibetan mushroom fermented wolfberry puree (fermentation group) was significantly improved compared with that of mice taking unfermented wolfberry puree (unfermentation group), and there was no significant difference in the improvement effect compared with the drug-treated group; excessively high MPO levels can cause inflammation, and taking mesalazine, fermented wolfberry puree and wolfberry puree can help the MPO of the mouse colon return to the level of the control group; and taking fermented wolfberry puree can also significantly improve the total antioxidant capacity (T-AOC) of the mouse colon to the level of the control group, and the effect is equivalent to that of mesalazine; LPS is called serum endotoxin, which can promote the production of inflammatory factors. Taking fermented wolfberry puree can significantly reduce LPS in the mouse blood to normal levels, and the effect is better than unfermented wolfberry puree and no less than mesalazine.

[0106] For the main validation factor indicators, taking mesalazine, fermented wolfberry puree and unfermented wolfberry puree all helped to reduce the inflammatory indicators of the colon tissue of colitis mice. And the validation indicators of mice taking fermented wolfberry puree all returned to normal levels, and even slightly better than mesalazine in the IL-6 indicator.

[0107] The above results all show that the use of Tibetan mushroom fermented wolfberry puree can prepare a fermented product that can relieve and improve the symptoms of colitis, and the improvement effect on the main apparent indicators, pathological indicators, antioxidant indicators and inflammatory indicators of colitis is not inferior to the mainstream drug mesalazine for the treatment of colitis. This shows that Tibetan mushroom fermented wolfberry puree is a fermented product with great potential for improving colitis.

[0108] In particular, the animal experimental protocols involved in Examples 6-8 were all approved by the Animal Ethics Committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, ISBN-10:0309-15396-4).

[0109] Example 9 Preparation of oral liquid for improving colitis containing Tibetan mushroom fermented wolfberry puree

[0110] After ultra-high temperature instant sterilization of the Tibetan mushroom fermented wolfberry puree prepared in Example 4, 10 mL was accurately measured, and then compounded with 2 g sucrose, 0.2 g vitamin C, 0.2 g sodium chloride and 0.8 ml patchouli oil, and purified water was added to a total volume of 25 mL, and ultrasonically dissolved until uniform. An oral liquid that can be used to improve colitis was obtained.

[0111] Example 10 Preparation of Tablets Containing Tibetan Ling Mushroom Fermented Wolfberry Puree for Improving Colitis

[0112] The Tibetan mushroom-fermented wolfberry original pulp prepared in Example 4 was subjected to pressure filtration and homogenization, then mixed evenly with maltodextrin of the same mass and fructooligosaccharide of 0.5 times the mass, dried by hot air, then ground into powder, and pressed into tablets using a tablet press to prepare tablets for improving colitis.

[0113] Preparation of Granules for Improving Colitis Containing Tibetan Mushroom-Fermented Wolfberry Original Pulp in Example 11

[0114] The volume of the Tibetan mushroom-fermented wolfberry original pulp prepared in Example 4 was concentrated to one-fifth using a multi-effect concentrator. Then, 0.5 L of the concentrated paste of the Tibetan mushroom-fermented wolfberry original pulp, 1 kg of sucrose, and 1 kg of maltodextrin were mixed evenly, and then made into granules using a shaking granulator and dried at 65 °C for 24 h to obtain granules for improving colitis.

[0115] The foregoing description of specific exemplary embodiments of the present application is for purposes of illustration and exemplification. These descriptions are not intended to limit the present application to the precise forms disclosed, and it is apparent that many changes and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the present application, as well as various different selections and changes. The scope of the present application is intended to be defined by the claims and their equivalents.

Claims

1. A method for increasing the alkaloid yield by fermenting wolfberry raw pulp with kefir grains, characterized in that, It includes the following steps: 1) Activated cultivation of kefir grains; 2) Inoculating the activated kefir grains into wolfberry puree for fermentation; In the step 1), the activation of the kefir grains is to inoculate the kefir grains into sterilized skim milk for activated cultivation; In the step 2), the fermentation is as follows: inoculating the activated kefir grains into wolfberry puree at an inoculation amount of 3%-5% (w / w), and fermenting at 25-30 °C for 48-60 h; The alkaloids include betaine, trigonelline, capsaicin and ophiopogonin D'; 2. The method according to claim 1, characterized in that, In the step 2), the wolfberry puree is obtained by the following method: picking wolfberries, washing them and drying them to semi-dryness, grinding them and filtering them with a filter screen to obtain wolfberry puree.

3. The method according to any one of claims 1-2, characterized in that, The method further includes the following steps: 3) Mixing the fermented wolfberry puree with an organic solvent for multiple extractions, followed by vacuum evaporation and membrane filtration.

4. The method according to claim 3, wherein The step 3) is specifically as follows: mixing the fermented wolfberry puree with methanol for ultrasonic extraction at room temperature, centrifuging and collecting the supernatant, adding methanol to the precipitate for multiple extractions again; combining the supernatants, performing vacuum evaporation, dissolving in acetonitrile again, and obtaining alkaloids after filtering through a membrane.

5. A Tibetan mushroom fermented wolfberry puree, characterized in that, Prepared by the method according to any one of claims 1-4.

6. Use of the kefir-fermented wolfberry puree according to claim 5 in the preparation of a medicament for improving colitis.

7. The application according to claim 6, wherein The dosage form of the medicament is tablet, granule or oral liquid.

8. Use of kefir grains in increasing the alkaloid content in wolfberry puree; the use is to inoculate kefir grains into wolfberry puree for fermentation; the fermentation is as follows: inoculating the activated kefir grains into wolfberry puree at an inoculation amount of 3%-5% (w / w), and fermenting at 25-30 °C for 48-60 h; the alkaloids include betaine, trigonelline, capsaicin and ophiopogonin D'.

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