Kombucha capable of improving ulcerative colitis as well as preparation method and application of kombucha

Abstract

The invention discloses kombucha capable of improving ulcerative colitis as well as a preparation method and application of the kombucha, and belongs to the technical field of fermented foods. The preparation method comprises the following steps: S1, inoculating 5% of kombucha pellicle into black tea sugar tea water for fermentation culture, and activating to prepare Kangpu tea fermentation liquor; and S2, inoculating the Kangpu tea fermentation liquor in the S1 into black tea sugar tea water according to the inoculum size of 1%, and performing fermentation culture for 4 days to obtain the kombucha. According to the kombucha capable of improving ulcerative colitis as well as the preparation method and the application of the kombucha, the kombucha can effectively relieve colon injury caused by dextran sulfate sodium (DSS), regulate the intestinal barrier and inflammation level and effectively relieve colon oxidative injury at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of fermented foods, and in particular to a kombucha fungus capable of improving ulcerative colitis, and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by diarrhea, bloody stools, shortened colon, mucosal damage, and colonic ulcers. Numerous medications, such as infliximab, aminosalicylic acid, corticosteroids, and thiopurines, are currently used to treat UC. However, these medications are expensive and have been shown to cause severe side effects, leading to burdensome long-term complications (such as hepatotoxicity, autoimmune diseases, and even malignancies), significantly impacting patients' lives. In recent years, UC management strategies have evolved significantly, moving toward preventing disease progression and improving long-term outcomes. A variety of environmental factors and dietary habits can influence the risk of UC, but the underlying mechanisms remain unclear. If a causal relationship is indeed present, lifestyle and dietary modifications may become a future trend in disease prevention strategies. Therefore, the search for new and improved alternative treatments for UC is necessary, and the potential to alleviate UC through dietary interventions has become a research hotspot.

[0003] Kombucha, a traditional microbial fermented beverage, contains a large number of bioactive ingredients and exhibits excellent therapeutic effects in terms of antibacterial, antioxidant, anti-inflammatory, liver protection, detoxification, anti-cancer, cholesterol-lowering, and blood pressure-lowering properties. Studies have shown that drinking kombucha beverages may benefit our health and support the immune system. Its potential anti-inflammatory and immunomodulatory effects can prevent and reduce the severity of allergies, infections, and some inflammatory diseases.

[0004] Studies have shown that the physicochemical properties of kombucha and the bioactive compounds it produces are determined by the interactions between the microorganisms within the fungus and the microbial composition of the culture medium. Therefore, identifying kombucha with superior properties and exploring its health benefits are crucial for promoting the industrialization of kombucha fermentation. Summary of the Invention

[0005] The purpose of the present invention is to provide a kombucha tea having the effect of improving ulcerative colitis and a preparation method and application thereof, so as to solve the problems in the background technology.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing kombucha for improving ulcerative colitis, comprising the following steps:

[0007] S1, inoculating the kombucha fungus biofilm at an inoculum rate of 5% into black tea sugar tea water for fermentation, culturing, and activation to prepare kombucha fermentation liquid;

[0008] S2. The kombucha fermentation liquid in S1 is inoculated into black tea sugar tea water at an inoculum rate of 1%, and fermented and cultured for 4 days to obtain kombucha fungus.

[0009] Preferably, the preparation of black tea sugar tea includes:

[0010] Soak the black tea in 85-90℃ hot water for 15 minutes. Filter the tea water and add sugar. After the sugar is completely dissolved, sterilize it in a 99℃ water bath for 10 minutes.

[0011] The ratio of black tea to hot water is 5g:500mL; the ratio of white sugar to black tea water is 50g:500mL.

[0012] Preferably, in S1, the fermentation culture conditions are: static fermentation at 30°C in the dark, a fermentation cycle of 4 days, and 3 consecutive activations.

[0013] Preferably, in S2, the fermentation culture conditions are: static fermentation at 30° C. in the dark, and the fermentation cycle is 4 days.

[0014] In a second aspect, the present invention provides a kombucha prepared by the above-mentioned preparation method.

[0015] In a third aspect, the present invention provides a use of the aforementioned kombucha for improving ulcerative colitis for non-therapeutic purposes.

[0016] Therefore, the kombucha tea for improving ulcerative colitis and its preparation method and application of the present invention have the following beneficial effects:

[0017] (1) The kombucha in the present invention can alleviate the shortening of the colon and pathological damage of the colon caused by colitis, and reduce the levels of inflammatory factors (TNF-α, IL-6).

[0018] (2) The kombucha of the present invention can significantly reduce the content of myeloperoxidase MPO and lipid oxidation product malondialdehyde MDA, increase the activity of superoxide dismutase SOD and the content of glutathione GSH, and effectively alleviate colon oxidative damage.

[0019] (3) The kombucha in the present invention alleviates ulcerative colitis by upregulating the expression of Nrf2 and NQO-1 genes in colon tissue and downregulating the expression of HO-1 gene to relieve oxidative stress, while regulating the intestinal barrier and inflammation levels.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. 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 any creative labor.

[0022] Figure 1 is the weight change of mice during DSS intervention;

[0023] Figure 2 The changes in the disease activity index scores of mice during DSS intervention;

[0024] Figure 3 The colon lengths of mice in the blank control group (CON), DSS intervention model group (DSS), and Kombucha intervention group (GDK);

[0025] Figure 4 Effects of kombucha on intestinal tissue histopathology, where (a) is hematoxylin and eosin staining (HE), and (b) is Alcian blue staining (AB);

[0026] Figure 5 Effects of kombucha on proinflammatory cytokines in the serum of mice with DSS-induced colitis, where (a) is the concentration of TNF-α in mouse serum, and (b) is the concentration of IL-6 in mouse serum;

[0027] Figure 6 The effect of kombucha on the oxidative stress level in mouse colon tissue, where (a) is the MPO content in mouse colon tissue, (b) is the MDA content in mouse colon tissue, (c) is the SOD enzyme activity level in mouse colon tissue, and (d) is the GSH content in mouse colon tissue.

[0028] Figure 7 The effects of kombucha on Nrf2-related genes in mouse colon tissue, where (a) is the Nrf2 gene expression level, (b) is the NQO1 gene expression level, and (c) is the HO-1 gene expression level. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0030] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0031] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0032] Example 1

[0033] The preparation method of kombucha comprises the following steps:

[0034] S1. Weigh 5g of black tea (purchased from Fujian Zhengshantang Tea Co., Ltd.) into a beaker, add 500mL of 90°C hot water, and steep for 15 minutes. Filter the tea through a strainer into a 1000mL conical flask. Weigh 50g of white sugar and add it to the tea. Once the sugar is completely dissolved, sterilize in a 99°C water bath for 10 minutes.

[0035] S2. Inoculate 500 mL of prepared black tea and sugar tea with a 5% inoculum of kombucha fungus (purchased from Zhanjiang, Guangdong, Guangdong Kombucha, GDK). Seal the mixture with sterile gauze and a lid. Ferment at 30°C in the dark for 4 days, followed by 3 activation cycles to obtain kombucha fermentation broth.

[0036] S3. Take the kombucha fermentation liquid from S2 and inoculate (inoculation volume 1%) into 500 mL of the prepared black tea sugar solution. Seal the container with sterile gauze and a lid. Ferment at 30°C in the dark for 4 days to obtain kombucha fungus.

[0037] Test Example 1

[0038] Experimental Animals: Male C57BL / 6N mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The mice were housed in an ambient temperature of (22 ± 1)°C and a humidity range of 40%–60%. They received a 12-h light cycle and free access to water and food. All experimental procedures were approved by the Animal Experimentation Ethics Committee of the College of Food Science and Engineering, Ocean University of China, under the approval protocol number SPXY2023121501.

[0039] Test method:

[0040] After one week of adaptive feeding, the mice were weighed and randomly divided into three groups according to their body weight, namely, blank control group (CON), DSS intervention modeling group (DSS), and Kombucha intervention group (GDK). On days 1-14 of the formal experiment, the mice in the fermentation broth intervention group were gavaged with 0.2 mL of fermentation broth daily. The CON group and DSS group were gavaged with 0.2 mL of PBS solution daily. On days 15-22 of the experiment, except for the CON group, the other two groups of mice were given an acute enteritis model by drinking a 2.5% (w / v) concentration of DSS solution dissolved in distilled water. During the DSS intervention period, the mice were weighed every day, and the stool characteristics and occult blood were observed. The scores were scored according to Table 1, and the three scores were added together to obtain the DAI of each mouse to assess its disease activity.

[0041] Table 1 Disease Activity Index Scoring Criteria

[0042] Weight loss (%) Stool characteristics Degree of blood in stool Fraction 0 normal normal 0 1-5 Loose but formed stools Occult blood 1 5-10 loose stools Bleeding (mild) 2 10-15 Diarrhea (mild) Bleeding 3 >15 Diarrhea (watery) Large amounts of blood in the stool 4

[0043] On the last day of the experiment, mice were fasted for 12 hours, blood was collected from the eyeballs, and the blood was kept at room temperature for 2 hours before centrifugation at 3000 rpm for 15 minutes to obtain serum, which was then stored at -80°C. Mice were killed by cervical dislocation, and the liver and spleen were removed and weighed. The colon and cecum were used for subsequent determination of relevant indicators. The length of the mouse colon was measured at the time of killing and sampling. 1 cm of colon was fixed in 4% paraformaldehyde for histopathological analysis. The remaining intestinal tissue was temporarily stored in liquid nitrogen and transferred to -80°C for later use.

[0044] Histopathological analysis: Colon tissue 1 cm from the anus was obtained and fixed with 4% paraformaldehyde. The dehydrated colon tissue was embedded in paraffin and then sectioned and stained with hematoxylin and eosin.

[0045] The changes in mouse body weight during DSS intervention Figure 1 As shown, during the experimental period, mice in the CON group had normal urination and defecation, continued weight gain, shiny fur, and normal diet, activity, and mental state. However, mice in the DSS group, after drinking 2.5% DSS, began to decline on the fourth day of modeling, with visible blood in the stool around the anus. GDK alleviated the weight loss of mice.

[0046] Changes in the disease activity index scores of mice during DSS intervention Figure 2 As shown in the figure, during the experiment, the DAI score was calculated based on the changes in mouse body weight, stool characteristics and the degree of blood in stool. Compared with the CON group, the DAI score of mice in the DSS group gradually increased after DSS intervention; the blood in stool and weight loss of GDK mice were improved.

[0047] Colon length is an important indicator for evaluating the severity of enteritis in mice, and colon length is negatively correlated with the severity of inflammation. Figure 3 As shown, the colon length of mice in the DSS group was 5.23±0.68 cm, which was significantly shorter than that in the CON group (7.30±0.56 cm), while the GDK group was able to improve colon shortening.

[0048] HE staining and Alcian blue staining were used to detect the degree of colon tissue damage. An inverted microscope was used to observe and collect images of the results. Figure 4 As shown, (a) is the HE staining result, and (b) is the Alcian blue staining result.

[0049] Depend on Figure 4As shown in (a), the colonic epithelium of mice in the CON group was intact and continuous, with regular arrangement of villi and crypts, and a clear, intact structure. No inflammatory cell infiltration or ulcers were observed. In contrast, the colonic epithelium of mice in the DSS group was incomplete, with severely damaged villi and crypt structures and inflammatory cell infiltration. Symptoms in the GDK group were alleviated compared to the DSS group, with the mucus layer restored.

[0050] Depend on Figure 4 As shown in (b), compared with CON, the mucus layer in the DSS group was severely damaged, and the light blue mucus layer and goblet cells almost disappeared. The mucus layer in the GDK group was restored to some extent.

[0051] The levels of inflammatory factors TNF-α and IL-6 in mouse serum were determined by ELISA according to the instructions of the kit. Figure 5 As shown, (a) is the TNF-α level, and (b) is the IL-6 level. The results showed that GDK effectively reduced the TNF-α and IL-6 levels in the mouse serum compared with the DSS group.

[0052] Test Example 2

[0053] Based on the experimental animals of Test Example 1, the myeloperoxidase MPO, lipid oxidation product malondialdehyde MDA, superoxide dismutase SOD activities and glutathione GSH levels in mouse colon tissue were determined using a kit according to the instructions attached to the kit.

[0054] The test results are as follows Figure 6 As shown, (a) is the MPO content in mouse colon tissue, (b) is the MDA content in mouse colon tissue, (c) is the SOD enzyme activity level in mouse colon tissue, and (d) is the GSH content in mouse colon tissue.

[0055] The results showed that the GDK group could increase the levels of reactive oxygen scavenging substances such as SOD and GSH in the colon tissue of mice, and reduce the content of oxidative stress product MDA and oxidative stress level marker MPO, which suggests that kombucha can alleviate DSS-induced oxidative stress-induced intestinal inflammation in mice by increasing reactive oxygen scavengers.

[0056] Test Example 3

[0057] Based on the experimental animals of Test Example 1, qRT-PCR was used to analyze the gene expression of intestinal oxidative stress-related factors Nrf-2, NQO-1, and HO-1. Total RNA from the colon was extracted using Trizol reagent. cDNA was synthesized using a reverse transcription kit according to the manufacturer's instructions, and the gene levels of Nrf-2, NQO-1, and HO-1 expressed in colon tissue were detected using a fluorescent quantitative PCR instrument. -ΔΔCtThe relative expression of the target gene was calculated using the formula and normalized by β-actin. The primers used are shown in Table 2:

[0058] Table 2 Primers used for qRT-PCR

[0059]

[0060] The results are as follows Figure 7 As shown, (a) is the Nrf2 gene expression level, (b) is the NQO1 gene expression level, and (c) is the HO-1 gene expression level.

[0061] Results showed that the relative mRNA expression levels of Nrf2 and NQO1 in the DSS group were reduced by 94.8% and 55.53%, respectively, compared to those in the CON group. The GDK group increased the relative mRNA expression level of Nrf2 from 5.19% to 63.27%. NQO1, a downstream gene of Nrf2, showed a similar trend, with the GDH group increasing the relative mRNA expression level of NQO1 from 44.47% to 84.04%. HO-1, also a downstream gene of Nrf2, was also stimulated by DSS, leading to the production of significant amounts of HO-1 in the DSS group. The relative mRNA expression level of HO-1 increased to 2.54 times that of the CON group, while the GDK group alleviated this significant release of HO-1.

[0062] Therefore, the present invention provides a kombucha with the ability to improve ulcerative colitis, and its preparation method and application. The provided kombucha can effectively alleviate colon damage caused by dextran sulfate sodium (DSS), regulate intestinal barrier and inflammation levels, and effectively alleviate colon oxidative damage.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing kombucha for improving ulcerative colitis, characterized in that: The steps include: S1, inoculating the kombucha fungus biofilm at an inoculum rate of 5% into black tea sugar tea water for fermentation, culture, and activation to prepare kombucha fermentation liquid; S2. The kombucha fermentation liquid in S1 is inoculated into black tea sugar tea water at an inoculum rate of 1%, and fermented and cultured for 4 days to obtain kombucha fungus.

2. The method for preparing kombucha for improving ulcerative colitis according to claim 1, characterized in that: The preparation method of black tea sugar tea comprises the following steps: Soak black tea in 85-90℃ hot water for 15 minutes. Filter the tea water and add sugar. After the sugar is completely dissolved, sterilize in a 99℃ water bath for 10 minutes. The mass volume ratio of black tea to hot water is 5g:500mL; the mass volume ratio of white sugar to black tea water is 50g:500mL.

3. The method for preparing kombucha for improving ulcerative colitis according to claim 1, characterized in that: In S1, the fermentation culture conditions are: static fermentation at 30°C in the dark, a fermentation cycle of 4 days, and 3 consecutive activations.

4. The method for preparing kombucha for improving ulcerative colitis according to claim 1, characterized in that: In S2, the fermentation culture conditions are: static fermentation at 30°C in the dark, and the fermentation cycle is 4 days.

5. Kombucha prepared by the method according to any one of claims 1 to 4.

6. A use of the kombucha according to claim 5 for improving ulcerative colitis for non-therapeutic purposes, characterized in that: Improving ulcerative colitis includes: alleviating colon shortening and colon pathological loss caused by ulcerative colitis, reducing TNF-α and IL-6 levels, and alleviating colon oxidative stress.

Images