CN114438133B - Inulin fermentation product, its preparation method and use in preventing and treating animal colon cancer - Google Patents

Abstract

The invention discloses application of inulin fermented product in preparing medicines for preventing and/or treating colorectal cancer. The inulin ferment is a product obtained by fermenting inulin serving as a substrate by using bacteroides ovatus. The inulin ferment has improved nutrition characteristics for human and mammals, has no intestinal side effect, can be used for regulating intestinal microorganism composition of human and mammals, has the effect of preventing colorectal cancer, and has remarkable inhibition effect on the growth of tumors, so that the inulin ferment can provide a new idea and selection for preventing and treating colorectal cancer, in particular for treating and/or preventing colonitis related colon cancer, and has wide application prospect.

Description

Inulin fermentation product, its preparation method and use in preventing and treating animal colon cancer

Technical field

The invention specifically relates to an inulin fermentation product, its preparation method and its use in preventing and treating animal colon cancer, and belongs to the field of bioengineering.

Background technique

Colorectal cancer is a common malignant tumor in modern life. Its incidence and mortality rates rank third to fourth among all malignant tumors in the world, and its incidence rate is increasing year by year. The formation of colon cancer is related to heredity, diet, living habits and environmental factors, and often takes the form of ulcers, polyps and other forms. There are currently four known methods for treating colon cancer, namely: (1) surgical resection, which directly removes the diseased area; (2) radiotherapy, which kills cancer cells through radiation; (3) drug treatment, which mainly includes chemical drugs and targeted drugs. However, these treatments will have great toxic side effects on the body, and while killing tumor cells, they will also cause damage to normal cells and destroy the body's immune system. Therefore, it is of great significance to develop drugs for the treatment of colon cancer with low side effects.

Contents of the invention

The main purpose of the present invention is to provide an inulin fermentation product for preventing and treating animal colon cancer, so as to overcome the shortcomings of the prior art.

Another object of the present invention is to provide a method for preparing the inulin fermentation product.

Another object of the present invention is to provide the use of the inulin fermentation product in preventing and treating colon cancer in animals.

In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

An inulin fermentation product provided by one aspect of the present invention is obtained by fermenting inulin by Bacteroides ovatus.

Further, the Bacteroides ovatus is Bacteroides ovatus ATCC 8343.

Another aspect of the present invention provides a method for preparing inulin fermentation product including:

1) Mix inulin and sterilized medium at a mass ratio of 1 to 10:100, and adjust the pH value to 6 to 7 to obtain inulin fermentation medium;

2) Inoculate Bacteroides ovatus into the inulin fermentation medium, wherein the inoculation amount of Bacteroides ovale is 0.1% to 10%, and ferment anaerobically for 12 to 36 hours at 35 to 38°C to obtain inulin fermentation. things.

In a more preferred embodiment, the preparation method specifically includes:

1) Mix inulin and sterilized medium at a mass ratio of 1:20, and adjust the pH value to 7 to obtain inulin fermentation medium;

2) Inoculate Bacteroides ovatus into the inulin fermentation medium, in which the inoculation amount of Bacteroides ovale is 10%, and ferment anaerobically for 24 hours at 37°C to obtain an inulin fermentation product.

In one embodiment, the Bacteroides ovatus ATCC 8343 is Bacteroides ovatus ATCC 8343.

In one embodiment, the sterilized culture medium contains 1.5 parts by weight of tryptone, 0.5 parts by weight of soy peptone, 0.5 parts by weight of sodium chloride, 0.5 parts by weight of inulin, 1-10 parts by weight of defibrinated sheep blood and 100 parts by weight of distilled water.

In one embodiment, the inulin fermentation product is inulin fermentation broth.

In the present invention, the inulin, also known as inulin, is a fructan widely present in plants in nature. Its structure is composed of D-fructofuranose connected by β-(1,2) glycosidic bonds. The end of each molecule A glucose residue is connected with an α-(1,2) glycosidic bond, usually with a degree of polymerization of 2-60 fructose, and an average degree of polymerization of 10 fructose.

In the present invention, the Bacteroides ovatus is a common gram-negative anaerobic bacterium among intestinal Bacteroidetes. It is capsule, non-spores, non-motile, sensitive to light, and can be metabolized and utilized. Polysaccharide, involved in bile and cholesterol metabolism.

Another aspect of the present invention provides an inulin fermentation product, which is prepared by any of the aforementioned methods.

Yet another aspect of the present invention provides the use of the inulin fermentate in the preparation of medicaments for preventing and/or treating colon cancer.

In one embodiment, the colon cancer is colitis-associated colon cancer.

In one embodiment, the colitis-associated colon cancer is induced by azoxymethane and dextran sodium drug (AOM/DSS).

Among them, the mechanism of the inulin fermentation product to prevent and/or treat colon cancer in animals mainly lies in: inhibiting the number of tumors, the expression of proto-oncogene (C-MYC) and the proliferation of cancer cells, and repairing the intestinal mucosal barrier and colon length. .

Compared with the existing technology, the advantages of the present invention include:

(1) The inulin fermentation product provided is easy to prepare, has good stability, and has significantly improved performance compared with products containing a single inulin and Bacteroides ovatus preparation;

(2) The inulin fermentation product provided has improved nutritional properties for humans and mammals, such as a significant increase in small molecule polysaccharides (xylofuranose) and organic acids (lactic acid, formic acid and butyric acid), without intestinal side effects, It can be used to regulate the composition of intestinal microorganisms in humans and mammals, and effectively repair the intestinal barrier. It has the effect of preventing colorectal cancer and has a significant inhibitory effect on the growth of tumors. Therefore, it can be used to prevent and treat colorectal cancer. In particular, it provides new ideas and options for the treatment and/or prevention of colitis-related colon cancer, and has broad application prospects.

Description of the drawings

Figure 1 is a metabolomics diagram of inulin fermentation products in the embodiment of the present invention.

Figure 2 is an anatomical view of the colorectum of mice in the blank group, model group, inulin group, and inulin metabolism (fermented product) group in the embodiment of the present invention.

Figure 3 shows the number of tumors in mice in the blank group, model group, inulin group, and inulin metabolism group in the embodiment of the present invention.

Figure 4 shows the colorectal length of mice in the blank group, model group, inulin group, and inulin metabolism group in the embodiment of the present invention.

Figure 5 is a HE staining diagram of the intestinal mucosa of mice in the blank group, model group, inulin group, and inulin metabolism group in the embodiment of the present invention.

Figure 6 is a hot spot map of intestinal microbial abundance of mice in the blank group, model group, inulin group, and inulin metabolism group in the embodiment of the present invention.

Figure 7 is a graph showing the proliferation of colon cancer cells (HT29) in the blank group, medium group, inulin metabolism group, and inulin group in the embodiment of the present invention.

Figure 8 shows the intracellular glucose content of colon cancer cells (HT29) in the blank group, medium group, inulin metabolism group, and inulin group in the embodiment of the present invention.

Figure 9 is an oil red staining diagram of colon cancer cells (HT29) in the blank group, culture medium group, inulin metabolism group, and inulin group in the embodiment of the present invention.

Figure 10 is a western blot diagram of the proto-oncogene C-MYC protein in the colon of mice in the blank group, model group, inulin group, and inulin metabolism group in the example of the present invention.

Figures 11a-11b are respectively western blot of proto-oncogene C-MYC and regulatory gene β-catenin protein in the blank group, culture medium group, inulin metabolism group, and inulin group colon cancer cells (HT29) in the embodiment of the present invention. picture.

Detailed ways

The technical solution of the present invention will be further described below with reference to the embodiments and drawings. It should be pointed out that, unless otherwise specified, all types of reagents used in the following examples can be obtained from the market, and all types of production and testing equipment and methods used in the following examples are prepared by those skilled in the art. learned.

Example 1

1. The preparation method of inulin fermentation product includes: mixing 1.5g tryptone, 0.5g soybean peptone, 0.5g sodium chloride, 0.5g inulin (source leaf, S11143), and adding 100mL distilled water to prepare Liquid culture medium, sterilized at 120°C for 20 minutes, cooled and then added 5% (solaibo, TX0030) defibrinated sheep blood), inoculated with Bacteroidesovatus, cultured anaerobically at 37°C for 48 hours, centrifuged at 5000r/min for 5 minutes , take the supernatant liquid as inulin metabolite, that is, inulin fermentation product (also known as inulin fermentation liquid).

2. The preparation method of bacterial culture medium solution includes:

Mix 1.5g tryptone, 0.5g soy peptone, and 0.5g sodium chloride, and add 100 mL distilled water to prepare a liquid culture medium. Sterilize at 120°C for 20 minutes. After cooling, add 5% (solaibo, TX0030) defibrinated sheep blood. ), inoculated with Bacteroides ovatus, cultured anaerobically at 37°C for 48 hours, centrifuged at 5000 r/min for 5 minutes, and the supernatant was taken as the bacterial culture medium solution.

Example 2 Induction colon cancer test:

72 C57 male mice, weighing between 19-23g, were randomly divided into 4 groups, 18 mice in each group, namely blank group), model group, inulin group, and inulin fermentation group. After 1 week of pre-adaptation, except for the mice in the blank group, which were intraperitoneally injected with normal saline and drank normal drinking water, the mice in the other groups were intraperitoneally injected with 10 mg/kg azomethane (AOM), and one week later they drank 2.5% (wt%) azoxymethane (AOM). ) DSS drinking water for 7 consecutive days, followed by normal drinking water for 14 days. The above 21 days constitute a cycle, and a total of 3 cycles were repeated to establish a mouse colitis-related colon cancer model. Drinking 2.5% DSS in the first cycle is the first day, and is administered for 7 consecutive days. After stopping drinking DSS, the administration is stopped, and then normal food and water are given for 14 days. The above 21 days are a cycle, which is repeated in total. Perform 3 cycles. The administration method for the inulin group was to add 5% inulin to the feed, and the administration method for the inulin metabolite group was to add 25% of the inulin metabolite obtained in Example 1 to the drinking water. Observe the general living conditions of the mice.

Example 3 Treatment of test animals:

After the colon cancer induction test in Example 2 was completed, the mice in each group were first anesthetized with _CO2 , and then the mice were killed, the abdominal cavity of the mice was exposed, the colorectum was dissected and its length was measured, and the inside and outside of the colorectum were washed with physiological saline. The colorectum was slowly turned over with forceps so that the mucosal layer faced outward. Tumor formation in the colorectum was observed with the naked eye and the number of tumors was recorded. Afterwards, a part was fixed in 4% paraformaldehyde solution.

Example 4 Conventional HE staining of mouse intestine:

The fixed tissue in Example 3 was dehydrated, transparent, immersed in wax, and embedded, and then the tissue paraffin specimen sections were immersed in xylene I and xylene II in sequence for dewaxing. Use gradient alcohol method for hydration treatment. Use hematoxylin for staining, and the processing time is 1-3 minutes. Use double distilled water for cleaning, and then use hydrochloric acid and alcohol to differentiate and reverse the blue color. Then use eosin for staining for 1 minute, use gradient alcohol for dehydration, and finally use neutral gum for sealing.

Example 5 Fecal microbial 16sRNA detection:

On the last day of the mouse colon cancer model induction test in Example 2, the feces of the mice in each group were collected using sterile EP tubes and placed in a -80°C refrigerator. The delivery company then performs 16sRNA gene sequencing to determine the type and abundance of microorganisms in the feces.

Example 6 Colon cancer cell (HT29) test:

Colon cancer cells were inoculated into a 96-well plate. 8 hours later, 1/3 of PBS, bacterial culture medium solution, inulin fermentation product, and inulin solution (10 mg/mL) were added to each group, and cultured at 37°C for 48 hours, and then used Wash twice with PBS, then add DMEM medium containing 10% CCK8 reagent for 1 hour, use a microplate reader to measure the absorbance at 450nm; use a glucose detection kit (GOD-POD colorimetric method) (source leaf) to measure the content of colon cancer cells Glucose content; use Oil Red Staining Kit to stain the fat of colon cancer cells.

Description of results:

Referring to Figure 2, the anatomical structure of the colon of mice in the blank group, model group, inulin group, and inulin metabolite group is shown. It can be found that after using AOM/DSS to induce colon cancer, in each group (except the blank group) Tumors were produced in the middle and posterior segments of the colon, and compared with the model group, the number of tumors in the colon of mice fed inulin and inulin metabolites was reduced, and the group fed inulin metabolites had statistical significance, as shown in Figure 3 Show. By measuring the colon length of mice in each group, it can be found that the colon length of mice in the inulin group and inulin metabolite group was significantly longer than that of the model group, as shown in Figure 4. From the above, it can be concluded that inulin metabolites have a very significant effect on preventing colonogenesis.

Figure 5 shows the HE staining results of the colons of mice in each group. It can be found that compared with the blank group, the villus structure of the intestinal slices of mice in the model group has been completely damaged, goblet cells have disappeared, inflammatory cell infiltration is serious, and crypts disappeared, and the epithelial mucus layer almost disappeared. The intestinal sections of mice in the inulin group and inulin metabolite group were much more complete than those in the model group. In the inulin group, there was only a small amount of goblet cell damage, the degree of inflammatory cell infiltration was lighter, and the crypts and epithelial mucus layer partially disappeared. However, in the inulin metabolism group, the goblet cells, crypts, and epithelial mucus layer in the slices were very complete, which was different from that in the blank group. no difference. It can be concluded that inulin metabolites effectively maintain the integrity of the mouse colon and protect the colon structure of mice from damage by AOM/DSS.

Figure 6 shows the abundance of intestinal microorganisms in each group of mice. It can be found that compared with the model group and the inulin group, the Firmicutes and Proteobacteria in the intestinal microorganisms of the inulin fermentation group were reduced, while the pseudomicrobial phyla were reduced. The abundance of Bacilli increased significantly and returned to levels similar to those in the control group. It shows that feeding inulin fermentation can reshape the intestinal microbial composition of mice.

Figure 7 shows the results of the proliferation test of colon cancer cells in each group. It can be found that inulin fermentation material significantly reduced the proliferation of colon cancer cells, while ordinary bacterial culture medium and inulin solution had no significant inhibitory effect. By measuring the glucose content in the colon cancer cells of each group, it was found that the glucose content in the inulin fermentation product group and the bacterial culture medium group was significantly higher than that of the normal colon cancer cells and the colon cancer cells in the inulin group, as shown in Figure 8. Through oil red staining, it can be found that the fat accumulation of colon cancer cells in the inulin fermentation product group and bacterial culture medium group is significantly higher than that of normal colon cancer cells and colon cancer cells in the inulin group, as shown in Figure 9. This shows that inulin fermentation can reduce the energy metabolism rate of colon cancer cells, increase fat accumulation, and reduce the proliferation of colon cancer cells.

As shown in Figure 10 and Figure 11a-Figure 11b, in the model group of mice and HT29 colon cancer cells, the proto-oncogene C-MYC (MYC) and the regulatory gene β-catenin were highly expressed, while the proto-oncogene C-MYC (MYC ) can regulate cell proliferation and metabolism, and the β-catenin gene can activate the proto-oncogene C-MYC (MYC), indicating that proliferation and metabolic genes are highly expressed in colon cancer cells; administration of inulin fermentation can significantly reduce proto-oncogene C-MYC (MYC). The expression of oncogene C-MYC (MYC) and regulatory gene β-catenin indicates that inulin fermentation can reduce the expression of proliferation and metabolic genes in colon cancer cells by regulating the Wnt/β-catenin pathway.

To sum up, in the embodiments of the present invention, inulin is anaerobically fermented by Bacteroides ovale, and the fermented products are fed to mice that are inducing colon cancer. It can be found that the aforementioned inulin metabolites can also effectively improve the intestinal function of mammals. Microbial composition and prevention of colitis-induced colon cancer, and the tumor incidence rate in mice fed inulin metabolites is greatly reduced, indicating that fermented inulin metabolites also have significant anti-tumor effects, and the mechanism may be due to inulin Fermentation changes energy metabolism and accumulation in colon cancer cells by regulating the Wnt/β-catenin pathway.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements can be made without departing from the technical principles of the present invention. and modifications, these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (6)

1. The use of inulin fermentation product in the preparation of medicines for preventing and/or treating colon cancer, characterized in that: the inulin fermentation product is an inulin fermentation liquid obtained by fermenting inulin by Bacteroides ovatus , the Bacteroides ovatus ATCC 8343. 2. The use according to claim 1, characterized in that the preparation method of the inulin fermentation product includes: 1) Mix inulin and sterilized medium at a mass ratio of 1 to 10:100, and adjust the pH value to 6 to 7 to obtain inulin fermentation medium; 2) Inoculate Bacteroides ovatus into the inulin fermentation medium, in which the inoculation amount of Bacteroides ovale is 0.1% to 10%, and ferment anaerobically for 12 to 36 hours at 35 to 38 ^o C to obtain inulin. fermentation. 3. Use according to claim 2, characterized in that the preparation method includes: 1) Mix inulin and sterilized medium at a mass ratio of 1:20, and adjust the pH value to 7 to obtain inulin fermentation medium; 2) Inoculate Bacteroides ovatus into the inulin fermentation medium, with the inoculation amount of Bacteroides ovale being 10%, and ferment anaerobically for 24 hours at 37 ^° C to obtain the inulin fermentation product. 4. The use according to any one of claims 2-3, characterized in that: the sterilized culture medium contains 1.5 parts by weight of tryptone, 0.5 parts by weight of soy peptone, 0.5 parts by weight of sodium chloride, 0.5 parts by weight Inulin, 1-10 parts by weight of defibrinated sheep blood and 100 parts by weight of distilled water. 5. The use according to claim 1, characterized in that the colon cancer is colitis-related colon cancer. 6. The use according to claim 5, characterized in that: the colitis-related colon cancer is induced by drugs such as azomethane oxide and dextran sodium.