Application of lactobacillus paracasei WQ-WX2011 in fermentation of scutellaria baicalensis

Abstract

The invention discloses application of lactobacillus paracasei WQ-WX2011 in fermentation of scutellaria baicalensis, and belongs to the technical field of micro-ecological probiotic preparations. Through research, screening and verification of the Scutellaria baicalensis fermented by the Pseudomonas paracasei, the invention provides the Scutellaria baicalensis fermentation broth capable of reducing the uric acid level of mice, and the Pseudomonas paracasei can effectively prevent hyperuricemia of mice.

Description

Technical Field

[0001] The invention relates to the technical field of microecological probiotic preparations, and in particular to application of Lactobacillus paracasei WQ-WX2011 in scutellaria baicalensis fermentation. Background Art

[0002] As a treasure of the Chinese nation, Chinese medicine has a long history and unique advantages in disease treatment and prevention. As a commonly used Chinese herbal medicine, Scutellaria baicalensis has the effects of clearing away heat and dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus, and is widely used in clinical treatment. However, the traditional processing method of Scutellaria baicalensis has some limitations, which restricts the full play of its efficacy and the further expansion of its application scope.

[0003] Traditional methods of processing Scutellaria baicalensis include wine processing and charcoal-frying. Although wine-processed Scutellaria baicalensis can enhance its function of clearing heat from the head and eyes, impurities such as alcohol may be introduced during the processing, which is not suitable for some special groups such as those who are allergic to alcohol and patients with liver disease. Charcoal-frying Scutellaria baicalensis is mainly used to stop bleeding, but during the frying process, high temperatures may destroy some of the active ingredients in Scutellaria baicalensis and reduce its efficacy. In addition, the traditional processing methods are complex and difficult to control quality, and the product quality varies greatly between different batches.

[0004] With the continuous development of biotechnology, the application of microbial fermentation technology in the preparation of traditional Chinese medicine has attracted more and more attention. Microbial fermentation can change the chemical composition of traditional Chinese medicine through the metabolism of microorganisms, increase the content of active ingredients, enhance the efficacy, and reduce toxic side effects. At the same time, microbial fermentation can also improve the taste and smell of traditional Chinese medicine and improve patient compliance.

[0005] Paracasei is a common probiotic that is widely found in the natural environment and the human intestine. Paracasei has multiple physiological functions, such as regulating intestinal flora, enhancing immunity, anti-oxidation, and lowering blood lipids. In recent years, studies have found that Paracasei can also change the chemical composition of traditional Chinese medicine through fermentation and improve the efficacy of traditional Chinese medicine.

[0006] Scutellaria contains a variety of effective ingredients, such as flavonoids such as baicalin, baicalein, wogonin, wogonin, as well as volatile oils, polysaccharides, etc. These ingredients have multiple pharmacological effects such as antibacterial, antiviral, antioxidant, anti-inflammatory, and anti-tumor. However, the content of effective ingredients in Scutellaria is low, and the bioavailability of some ingredients is not high, which limits the performance of its drug efficacy. Therefore, developing a preparation method of Scutellaria fermented by Paracasei has important practical significance and application value. Summary of the invention

[0007] The purpose of the present invention is to provide application of Lactobacillus paracasei WQ-WX2011 in Scutellaria baicalensis fermentation to solve the problems existing in the above-mentioned prior art.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] One of the technical solutions of the present invention is a method for preparing a scutellaria baicalensis fermentation liquid, which comprises fermenting scutellaria baicalensis by using Lactobacillus paracasei WQ-WX2011.

[0010] The second technical solution of the present invention is the scutellaria baicalensis fermentation broth prepared by the preparation method.

[0011] The third technical solution of the present invention is the use of the Scutellaria baicalensis fermentation liquid in the preparation of a drug for reducing uric acid.

[0012] A fourth technical solution of the present invention is a drug for reducing uric acid, which is prepared from the scutellaria baicalensis fermentation liquid.

[0013] A fifth technical solution of the present invention is the application of Lactobacillus paracasei WQ-WX2011 in the fermentation of Scutellaria baicalensis.

[0014] The sixth technical solution of the present invention is the use of the Scutellaria baicalensis fermentation liquid in the preparation of a medicine for treating hyperuricemia.

[0015] The seventh technical solution of the present invention is a medicine for treating hyperuricemia, which is made from the Scutellaria baicalensis fermentation liquid.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] The invention provides a scutellaria baicalensis fermentation liquid capable of reducing hyperuricemia. The scutellaria baicalensis fermented by paracasei can increase the total flavonoid content and reduce hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The figure is the result of biological characteristic test, where A is the bile salt tolerance test, B is the growth curve, and C is the pH test.

[0020] Figure 2 The effects of different material-liquid ratios on baicalein content are shown in Table 1. Different lowercase letters indicate significant differences among the groups (P<0.05), the same below.

[0021] Figure 3 This is the effect of different bacterial concentrations on baicalein content.

[0022] Figure 4The effect of different rotation speeds on the baicalein content.

[0023] Figure 5 The effect of different temperatures on the content of baicalein.

[0024] Figure 6 The effect of different pH on the content of baicalein.

[0025] Figure 7 This is the effect of different days on the baicalein content.

[0026] Figure 8 To determine the total flavonoids content in fermented Scutellaria baicalensis and fermented Scutellaria baicalensis liquid.

[0027] Fig. 9 The blood index levels of mice, A is uric acid (UA), B is urea nitrogen (BUN), C is creatinine (CR), and D is xanthine oxidase (XOD).

[0028] Fig.10 This is a pathological section of kidney tissue (HE staining, 200×).

[0029] Fig.11 The blood index levels of goslings are shown in Figure 1. A is uric acid (UA), B is urea nitrogen (BUN), C is creatinine (CR), and D is xanthine oxidase (XOD). DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0036] The embodiment of the present invention provides a method for preparing a scutellaria baicalensis fermentation liquid, and the scutellaria baicalensis is fermented by using Lactobacillus paracasei WQ-WX2011.

[0037] In some specific embodiments, the fermentation conditions include: the ratio of scutellaria baicalensis to culture medium is 1:30, the inoculation amount of bacteria is 10% (v:v), the concentration of bacterial solution is 1×10 6 CFU / mL, the shaking speed was 180r / min, the fermentation temperature was 37℃, the pH was 7, and the fermentation days were 4 days.

[0038] The embodiment of the present invention also provides the scutellaria baicalensis fermentation liquid prepared by the preparation method.

[0039] The embodiment of the present invention also provides the use of the scutellaria baicalensis fermentation liquid in the preparation of a medicament for reducing uric acid.

[0040] The embodiment of the present invention also provides a medicine for reducing uric acid, which is prepared from the scutellaria baicalensis fermentation liquid.

[0041] The embodiment of the present invention also provides the application of Lactobacillus paracasei WQ-WX2011 in the fermentation of Scutellaria baicalensis.

[0042] The embodiment of the present invention also provides the use of the scutellaria baicalensis fermentation liquid in preparing a medicine for treating hyperuricemia.

[0043] The embodiment of the present invention also provides a medicine for treating hyperuricemia, which is prepared from the scutellaria baicalensis fermentation liquid.

[0044] By fermenting scutellaria baicalensis with paracasei bacteria, the metabolism of paracasei bacteria can be utilized to convert more effective ingredients in scutellaria baicalensis into flavonoid compounds, thereby improving the drug efficacy of scutellaria baicalensis. At the same time, paracasei bacteria fermentation can also reduce the content of toxic components in scutellaria baicalensis, thereby improving the safety of scutellaria baicalensis. The fermentation method provided by the invention can improve the drug efficacy of scutellaria baicalensis, reduce toxic and side effects, improve taste and smell, improve patient compliance, and provide new approaches and methods for the clinical application of scutellaria baicalensis.

[0045] As a traditional Chinese medicine, Scutellaria baicalensis has the effects of clearing away heat and drying dampness. Fermentation of Scutellaria baicalensis with Bacillus paracasei can produce a series of beneficial metabolites and enhance its biological activity.

[0046] Applying fermented Scutellaria baicalensis to gosling breeding can effectively reduce the high uric acid level of goslings. Its mechanism of action may include: the active ingredients in fermented Scutellaria baicalensis are more easily absorbed and utilized by goslings, regulating the metabolic balance in the goslings, inhibiting the activity of enzymes related to uric acid production, and promoting uric acid excretion.

[0047] During the gosling breeding process, adding this fermentation product in an appropriate manner can significantly improve the health of goslings, reduce the harm caused by high uric acid, and provide a safe and effective new way for gosling breeding.

[0048] The Lactobacillus paracasei WQ-WX2011 used in the present invention was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on May 12, 2022, with a deposit number of CGMCC NO.24882. The strain has been disclosed in patent CN115975876A. The classification name is: Lactobacillus paracasei.

[0049] Example 1

[0050] 1 Isolation and purification of strains

[0051] Resuscitate the strain WQ-WX2011 stored in the laboratory: Take 100 μL of bacterial solution and streak inoculate it in agar solid medium, set the incubator temperature to 37°C, and culture it for 24 hours. After the culture is completed, pick a single colony for streak purification, and repeat the above steps 2-3 times. After purification, inoculate the single colony into LB and culture it at 37°C.

[0052] 2 Identification of Biological Characteristics After screening with specific culture medium, the obtained Lactobacillus paracasei was subjected to biological characteristic identification.

[0053] Growth curve determination: Take 5mL of LB liquid medium and put it into a test tube, and take 100μL of Lactobacillus paracasei and inoculate it into the test tube. Set the shaker temperature and speed to 37℃ and 150r / min respectively, and continue to culture for 24 hours. Within 24 hours, measure every two hours, and measure the absorbance value at 36 hours and 48 hours, and use OD 600 The absorbance value at is the ordinate, and the time is the ordinate.

[0054] Acid resistance test: Take 5 mL of LB liquid culture medium and put it into a test tube. Take 100 μL of Lactobacillus paracasei and inoculate it into the test tube. Adjust the pH in the test tube to 2.0, 2.5, 3.0, 3.5, and 4.0. Set the shaker temperature and speed to 37°C and 150 r / min, respectively, and culture continuously for 24 hours. Measure the absorbance at 600 nm and use OD 600 The absorbance value at is the ordinate and the pH value is the abscissa.

[0055] Test of bile salt tolerance: Take 5 mL of LB liquid culture medium and put it into a test tube, take 100 μL of Lactobacillus paracasei and inoculate it into the test tube, and adjust the bile salt concentration in the test tube to 0.3%, 0.6%, 0.9%, 1.2%, 1.5%, and 1.8%, respectively. Set the shaking temperature and speed to 37°C and 150 r / min, respectively, and culture continuously for 24 hours. Measure the absorbance value at 600 nm, and use the liquid culture medium without bile salt under the same inoculation conditions as the control group, and take the OD of the culture solution as the control group. 600 The bile salt tolerance test curve was drawn with the value as the ordinate and the bile salt concentration as the abscissa.

[0056] Fermentation medium: Scutellaria baicalensis powder 20 g / L, vitamin C 0.1 g / L, sodium acetate 0.1 g / L, MgSO4 0.1 g / L, KH2PO4 0.2 g / L. Sterilize at 120℃ for 20 min.

[0057] The results of biological characterization are as follows Figure 1 As shown in the figure, through the determination of the growth curve of the strain and the acid and bile salt resistance, Lactobacillus paracasei entered a rapid growth phase within 12h-24h and entered a plateau phase after 24h. From the acid resistance results, we can see that as the pH value decreases, the OD 600 The survival rate of Lactobacillus paracasei was reduced, but more than half of them survived at pH 3. At the same time, the bile salt tolerance test results showed that more than half of them still survived when the bile salt concentration increased from 0% to 1.2%, indicating that the strain also has good bile salt tolerance.

[0058] 3. Screening of fermentation conditions

[0059] (1) Screening of material-liquid ratio The fermentation medium after high-pressure sterilization was prepared with different material-liquid ratios of 1:26, 1:28, 1:30, 1:32, and 1:34 (g / mL) of scutellaria powder to liquid volume. The liquid volume in a 250mL fermentation bottle was fixed at 80mL, and scutellaria powder was added to the fermented medium for fermentation. Three replicates were set for each group. The results are shown in Figure 2. Figure 2 shown.

[0060] (2) Screening of bacterial liquid concentration The concentration of the obtained Lactobacillus paracasei was measured using a McFarland turbidimeter tube. Lactobacillus paracasei was inoculated into the prepared culture medium at a volume ratio of 10%, and the volume of the fermentation liquid was fixed at 80 mL, and the bacterial concentration gradient was 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL. Three replicates were set for each group. Figure 3 shown.

[0061] (3) Screening of fermentation speed The effect of Lactobacillus paracasei fermentation of Scutellaria baicalensis on hyperuricemia in goslings was studied. The prepared samples were placed in a shaker and the speed was set to 160 r / min, 170 r / min, 180 r / min, 190 r / min, and 200 r / min for fermentation. Three replicates were set for each group. The results are shown in Figure 2. Figure 4 shown.

[0062] (4) Temperature screening: The prepared samples were placed in a shaker and the temperature was set at 35°C, 36°C, 37°C, 38°C, and 39°C for fermentation. Three replicates were set for each group. The results are shown in Table 1. Figure 5 shown.

[0063] (5) pH screening The pH of the prepared culture medium was adjusted with a pH meter to titrate the sample pH to five different pH values ​​of 5, 6, 7, 8, and 9 for fermentation. Three replicates were set for each group. The results are shown in the figure below. Figure 6 shown.

[0064] (6) Time screening: The prepared samples were mixed with a solid-liquid ratio of 1:30, pH 7, and bacterial liquid concentration of 1×10 6 CFU / mL was placed in a shaker at a speed of 180r / min and a temperature of 37°C for fermentation. The content of baicalin in the sample was tested every day. The test period was 6 days in total. Three replicates were set for each group. The results are as follows Figure 7 shown.

[0065] The determination method of the sample is as follows: centrifuge the treated fermentation liquid at 8000rpm for 10min, discard the supernatant, add 100mL of ethanol to the precipitate and extract overnight, filter after 80℃ water bath for 30min, draw 0.1mL of the sample, add 4mL of FeCl3, make up to 5ml with anhydrous ethanol, shake well; measure the absorbance at 490nm.

[0066] In summary, the optimal fermentation conditions for the fermentation of Scutellaria baicalensis by Lactobacillus paracasei WQ-WX2011 were as follows: the ratio of Scutellaria baicalensis powder to culture medium was 1:30, the inoculation amount of the strain was 10% (v:v), and the concentration of the bacterial solution was 1×10 6 CFU / mL, the shaking speed was 180r / min, the fermentation temperature was 37℃, the pH was 7, and the fermentation days were 4 days.

[0067] The Scutellaria baicalensis fermentation broth was prepared according to the above optimal fermentation conditions, and the total flavonoid content was determined. Figure 8 As shown, the flavonoids in the fermented Scutellaria baicalensis liquid were significantly increased compared with the unfermented Scutellaria baicalensis liquid.

[0068] Example 2

[0069] 1 Mouse validation experiment

[0070] 70 1-week-old Kunming mice with similar body weight were selected and randomly divided into a blank group, a modeling group, a fermentation low concentration group, a fermentation medium concentration group, a fermentation high concentration group, a Chinese medicine feeding group, and a negative control group, with 10 mice in each group. The fermentation product was prepared according to the optimal fermentation conditions in Example 1.

[0071] Except for the blank group and the negative control group, the other five groups were intraperitoneally injected with potassium oxonate at 9 a.m. One hour after the injection, the low-concentration fermentation group was gavaged with 1 mL / kg of fermented Chinese medicine, the medium-concentration fermentation group was gavaged with 2 mL / kg of fermented Chinese medicine, the high-concentration fermentation group was gavaged with 4 mL / kg of fermented Chinese medicine, the Chinese medicine feeding group was fed with 4 mL / kg of traditional Scutellaria baicalensis, and the negative control group was fed with 4 mL / kg of fermented liquid.

[0072] Biochemical index detection: Effects of potassium oxonate and fermentation liquid on blood biochemical indexes of mice after five consecutive days of gavage.

[0073] The results are as follows Fig. 9 As shown, feeding 1×10 6 Scutellaria baicalensis liquid fermented with Bacillus paracasei at a CFU / mL concentration can reduce the levels of uric acid, urea nitrogen, creatinine and xanthine oxidase in the serum of hyperuricemia mice, thereby alleviating hyperuricemia in mice.

[0074] 2 Kidney tissue pathology sections Fig.10As shown in the figure, compared with the blank group, the model group had obvious tubular dilation, indicating that the mouse model was successfully established. Although the cavitation of renal tissue in the Chinese medicine group was reduced to a certain extent, the tubular dilation was still obvious, while the cavitation of renal tubular epithelium in the fermentation broth medium dose group was reduced, and the tissue structure was clearly arranged and complete, even close to the blank group.

[0075] The results showed that the medium-dose treatment group of Lactobacillus paracasei fermented Scutellaria baicalensis was an effective treatment group for preventing hyperuricemia in mice.

[0076] Example 3

[0077] Gosling Verification Experiment

[0078] Experimental groups: 120 one-day-old goslings of similar body weight were selected and divided into a control group, a model group, and a fermentation liquid prevention group, with 40 goslings in each group and divided into 5 replicates.

[0079] The control group was fed with a gosling basal diet with a crude protein content of 16%; the model group and the fermented liquid prevention group were fed with a diet with a crude protein content of 24% for 7 days. The fermented liquid prevention group was gavaged with a medium-dose fermented liquid at 9 o'clock every day during the feeding period. During the experiment, the goslings were free to eat and drink plenty of water.

[0080] Biochemical index detection: Effects on serum biochemical indexes of goslings after treatment at different periods and in different groups.

[0081] The results are as follows Fig.11 As shown, on the 3rd day, there were no significant differences in uric acid, urea nitrogen, creatinine and xanthine oxidase activity among the prevention group, the control group, the model group and the fermentation liquid prevention group. On the 5th and 7th days, the serum indicators of the model group increased significantly compared with those of the control group, proving that the hyperuricemia model of goslings was successfully established. At the same time, on the 5th and 7th days, there were no significant differences in the levels of blood indicators in the prevention group compared with those in the control group.

[0082] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a scutellaria baicalensis fermentation broth, characterized in that: Scutellaria baicalensis was fermented using Lactobacillus paracasei WQ-WX2011.

2. The preparation method according to claim 1, characterized in that: The fermentation conditions include: the ratio of scutellaria baicalensis to the culture medium is 1:30, the inoculation amount of the bacteria is 10% (v:v), the concentration of the bacterial solution is 1×10 6 CFU / mL, the shaking speed was 180r / min, the fermentation temperature was 37℃, the pH was 7, and the fermentation days were 4 days.

3. The scutellaria baicalensis fermentation broth prepared by the preparation method according to claim 1 or 2.

4. Use of the scutellaria baicalensis fermented liquid as claimed in claim 3 in the preparation of a medicament for reducing uric acid.

5. A drug for reducing uric acid, characterized in that: The method is prepared from the scutellaria baicalensis fermentation liquid as claimed in claim 3.

6. Application of Lactobacillus paracasei WQ-WX2011 in Scutellaria baicalensis fermentation.

7. Use of the scutellaria baicalensis fermented liquid as claimed in claim 3 in the preparation of a medicament for treating hyperuricemia.

8. A drug for treating hyperuricemia, characterized in that: The method is prepared from the scutellaria baicalensis fermentation liquid as claimed in claim 3.

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