Application of clostridium tomentosum in preparation of functional product for preventing and / or relieving and / or repairing intestinal injury caused by microplastics

Abstract

The invention discloses a functional product for preventing and / or relieving and / or repairing intestinal injury caused by intake of microplastics. The method comprises the following steps: establishing an intestinal injury model mouse induced by micro-plastic gavage, and measuring the structural integrity of the intestinal barrier, the oxidative stress level, the antioxidant substance release level, the anti-oxidative inflammation capability and the lipid metabolism condition of the mouse by respectively applying yoghourt prepared by fermenting clostridium dahliae CGMCC (China General Microbiological Culture Collection Center) 27789 before and after modeling. Results show that the yoghourt prepared by fermentation of the clostridium allicum can protect and repair the intestinal barrier of a mouse, the damaged intestinal barrier is basically recovered to be normal when a mouse colon slice shows that the damaged intestinal barrier is recovered to be normal, and the expression level of the colon tight junction proteins ZO-1, Occudin and Claudin-1 is recovered to be normal. In conclusion, the clostridium dalendum is suitable for being used for developing functional products for preventing and / or relieving and / or repairing intestinal injury caused by intake of the microplastics, and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of Clostridium daliensis in the preparation of functional products for preventing and / or alleviating and / or repairing intestinal injuries caused by microplastics. Background Art

[0002] Microplastics (MPs) refer to plastic particles with a particle size less than 5 mm, which are widely distributed in soil, water bodies and the atmosphere through industrial emissions, plastic degradation and other channels. Research shows that the average content of microplastics in bottled drinking water is 325 particles / L; the average content of microplastics in marine biological shellfish is 1.5 - 7.2 particles / g, and the daily intake of microplastics by humans can reach 0.1 - 5 g, among which the intestine is the main accumulation organ of microplastics. This bioaccumulation characteristic across the food chain makes microplastic pollution have become a global public health crisis.

[0003] Multiple studies have proved that the accumulation of microplastics in the intestine can trigger multiple toxic effects. 1) Physical damage: Microplastic particles with a particle size < 100μm will embed in the basement membrane of intestinal villi, damage goblet cells, and reduce their number by 50% - 70%; 2) Chemical toxicity: Bisphenol A and polycyclic aromatic hydrocarbons adsorbed on the surface of microplastics will induce intestinal damage, increasing the ROS level by 3 - 5 times; 3) Biological barrier disruption: Microplastic exposure reduces the expression levels of tight junction proteins Occludin and ZO-1 by 60% - 80%, leading to increased intestinal permeability, and then triggering systemic inflammation, and the levels of inflammatory factors IL-6 and TNF-α in the intestine increase by 2 - 3 times. Epidemiological studies show that the risk of inflammatory bowel disease in people exposed to microplastics for a long time increases significantly.

[0004] Current prevention and treatment strategies for microplastic-induced intestinal injuries all have certain defects. 1) Physical adsorption methods such as activated carbon or montmorillonite can reduce 20% - 30% of microplastics, but may interfere with the absorption of nutrients and are difficult to prevent the physiological damage caused by them; 2) Intake of antioxidants such as vitamin C can reduce the ROS level, but cannot repair the structural damage of tight junction proteins; 3) Intake of existing probiotic preparations such as Bifidobacterium can repair 40% of specific microplastic injuries, but lack targeted regulation of the Nrf2 / NF-κB antioxidant and anti-inflammatory pathways. Therefore, developing microbial intervention means with multi-mechanism synergistic effects has become an urgent need to solve the toxicological challenges of microplastics. The present invention is committed to exploring the preventive and / or alleviating and / or repairing effects of Clostridium daliensis on microplastic-induced intestinal injuries. Summary of the Invention

[0005] In view of the problem of intestinal injuries caused by microplastic intake, the present invention provides a Clostridium daliensis based on the preservation number CGMCC27789 ( Clostridium dalinumProbiotic products thereof and their applications. By establishing a mouse model of intestinal injury induced by intragastric administration of polystyrene microplastics, the present invention verified the multi-dimensional efficacy of the product prepared by fermenting with this strain (specifically, yogurt fermented by a single strain of Clostridium daliensis) in preventing, alleviating, and repairing intestinal injuries caused by microplastics. The specific manifestations are as follows: Through repairing the structural integrity of the intestinal barrier, restoring the expression of tight junction proteins ZO-1, Occludin, and Claudin-1 to normal levels, reducing oxidative stress to scavenge the ROS level in the intestine, inhibiting the release of inflammatory factors TNF-α and IL-1β, and significantly reducing the metabolic levels of lipid substances such as TC and LDL-C and other synergistic mechanisms, systematic protection of intestinal health is achieved.

[0006] In the technical solution, the Clostridium daliensis can be prepared into a functional product in the form of a single strain or in combination with short-chain fatty acids metabolized by it. The core production process of its fermented product includes: using pure cow's milk as the matrix, adding 7% (w / v) granulated sugar, inoculating 1% by volume of the bacterial solution, fermenting at 42 °C for 4 hours, and ripening at 4 °C for 48 hours, finally obtaining a yogurt preparation with a viable bacteria concentration of 1×10 4 to 1×10 15 CFU / g.

[0007] In some embodiments, the research method of the present invention is as follows: The mice are divided into a blank control group, a microplastic group, a yogurt prepared by fermenting Clostridium daliensis (abbreviated as Clostridium daliensis yogurt) pre-protection group, and a Clostridium daliensis yogurt repair group. The mice in the blank control group are fed sterilized milk every day during the entire experimental period (90 days); the mice in the microplastic group are fed milk every day from day 1 to day 30, intragastrically administered with a polystyrene microplastic suspension every day from day 31 to day 60, and continue to be fed milk every day from day 61 to day 90; the mice in the Clostridium daliensis yogurt pre-protection group are fed yogurt every day from day 1 to day 30, intragastrically administered with a microplastic suspension uniformly every day from day 31 to day 60. The microplastics fed are polystyrene (PS) microspheres with a particle size of 1 μm, the suspension concentration is 30 mg / mL, and the intragastric administration dose for each mouse is 30 μg / g. They continue to be fed milk every day from day 61 to day 90; the mice in the Clostridium daliensis yogurt repair group are fed milk every day from day 1 to day 30, fed with a polystyrene microplastic suspension every day from day 31 to day 60, and fed yogurt every day from day 61 to day 90. All mice are uniformly sampled within 12 hours after the last intervention to obtain serum, intestine, colon tissue, and contents respectively. The experimental results show that long-term intake (30 days) of Clostridium daliensis yogurt can significantly reverse the colon gland disorder, goblet cell reduction, and mucosal fibrosis caused by microplastics, and the pre-protection group and the repair group achieve differential regulation by upregulating the Nrf2 pathway (the activity of HO-1 increases by 64.41%) and inhibiting the NF-κB pathway (TNF-α decreases by 91.17%) respectively.

[0008] Compared with the prior art, the positive and beneficial effects of the present invention are as follows:

[0009] 1. The present invention uses an animal experimental model to establish a mouse model of intestinal injury caused by microplastics, and explores the effect of long-term administration of yogurt prepared by fermenting Clostridium taiiense on preventing and / or alleviating and / or repairing intestinal injury caused by microplastics, including the effects on the integrity of the intestinal barrier structure, antioxidant stress capacity, antioxidant stress capacity, antioxidant substance release level, anti-oxidative inflammation capacity, and lipid metabolism of animals.

[0010] 2. The present invention provides an application of a fermentation product of Clostridium taiiense in preventing / alleviating / repairing the integrity of the intestinal barrier structure, antioxidant stress capacity, antioxidant stress capacity, antioxidant substance release level, anti-oxidative inflammation capacity, and lipid metabolism of the intestines of mice with intestinal injury induced by microplastics, providing a new idea for the preparation of functional foods for preventing and / or alleviating and / or repairing diseases caused by intestinal injury induced by microplastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a diagram of the experimental design.

[0012] Figure 2 It shows that the yogurt prepared by fermenting Clostridium taiiense can prevent and repair intestinal tissue injury caused by microplastics.

[0013] Figure 3 It shows that the yogurt prepared by fermenting Clostridium taiiense can prevent and repair intestinal barrier protein injury caused by microplastics.

[0014] Figure 4 It shows that the yogurt prepared by fermenting Clostridium taiiense can prevent and repair oxidative stress and inflammatory injury caused by microplastics.

[0015] Figure 5 It shows that the yogurt prepared by fermenting Clostridium taiiense can prevent and repair the injury of the antioxidant substance release pathway caused by microplastics.

[0016] Figure 6 It shows that the yogurt prepared by fermenting Clostridium taiiense can prevent and repair lipid metabolism disorders caused by microplastics. DETAILED DESCRIPTION OF THE INVENTION

[0017] The following further elaborates the present invention in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0018] Clostridium taiiense was inoculated into MRS liquid medium and cultured with shaking at 37 °C and 120 rpm for 24 h. Then, it was taken out, and the bacterial liquid was poured into a sterile 50 mL centrifuge tube and centrifuged at 8000 rpm for 10 min. The supernatant was discarded. Sterile PBS was added for washing, and it was centrifuged at 8000 rpm for 10 min. The supernatant was discarded, and this washing process was repeated 3 times. An appropriate volume of PBS was added to adjust the OD value of the bacterial liquid to 0.60 ± 0.02 to obtain the Clostridium taiiense bacterial liquid.

[0019] According to the "General Experimental Course of Microbiology", 1000 mL of pure milk was taken, 7% (w / v) of granulated sugar was added, and 1% (v / v) of the live bacterial liquid of Clostridium taiiense was inoculated. It was placed in an environment at 42 °C for fermentation culture for 4 h, and then put into a 4 °C refrigerator for 48 h to obtain fermented yogurt.

[0020] After the mice were sacrificed by cervical dislocation, an incision was made along the "V" shape in the abdomen to expose the abdominal cavity, and the colon was excised. The samples were processed as follows: 3 mice were randomly selected from each group, and the intestinal segments were cut 1 cm below the cecum and fixed in 4% paraformaldehyde for storage at room temperature for tissue section preparation. Hematoxylin-Eosin (HE) staining was used to observe the tissue sections. Figure 1 The H&E results of the colon of mice in different groups under microplastic infection.

[0021] It can be seen from Figure 2 that in the microplastic group, the arrangement of colon glands was disordered, the goblet cells were reduced, and the submucosa was fibrotic. In the pre-protection group of Clostridium taiiense yogurt, the arrangement of colon glands was close to normal, the goblet cells were dense, and the mucus layer was intact. In the repair group of Clostridium taiiense yogurt, the glandular morphology and submucosal thickness were almost completely restored, indicating that it can not only effectively prevent the acute injury of microplastics, but also promote the deep repair of chronic injury.

[0022] Paraffin sections of mouse colon tissues were successively dewaxed and dehydrated with absolute ethanol, and then hydrated with distilled water. When performing antigen retrieval, the sections were microwaved to boiling in citrate buffer (pH 6.0), cooled, and washed with PBS. Subsequently, an immunohistochemical pen was used to draw a circle and 3% hydrogen peroxide was added dropwise to block endogenous peroxidase, and incubated at room temperature in the dark for 25 min, and then washed with PBS. Next, serum blocking was performed, and 10% rabbit serum or 3% BSA was used to block for 30 min. After the first round of incubation with the primary antibody (overnight at 4°C), HRP-labeled secondary antibody was added and incubated for 50 min, and the signal was amplified with iF555-TSA, and incubated in the dark for 10 min. After removing the antibody complex by microwave treatment, the above steps were repeated for the second and third rounds of labeling. In the second round, the signal was amplified with iF488-TSA, and in the third round, a fluorescent secondary antibody labeled with iF647 was used. Finally, the sections were stained with DAPI for nuclei (10 min), the autofluorescence was quenched for 5 min, rinsed with running water, and then mounted. The fluorescence signals were panoramically imaged by a digital scanning system, and the expression intensity and distribution of positive cells or regions were analyzed. Figure 3 Immunohistochemical results of the colon of mice in different groups under microplastic infection.

[0023] It can be seen from Figure 3 that the fluorescence intensities of tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon of mice in the microplastic group were significantly decreased, indicating that the physical integrity and permeability regulation function of the intestinal barrier were impaired. The expressions of ZO-1, Occludin, and Claudin-1 in the Clostridium butyricum yogurt pre-protection group were close to those in the blank control group, suggesting that its components may reduce the direct damage of microplastics to tight junction structures by enhancing the stability of cell-cell junctions in advance.

[0024] Oxidative stress factors, oxidative inflammatory factors, and blood lipid-related biochemical indexes were detected according to the methods of commercial kits. The kits used included: ROS, MDA, 8-OHdG, and LDH for evaluating the level of oxidative stress; NF-κB, TNF-α, IL-1β, IL-6, IL-10, and IL-17 for measuring oxidative inflammatory reactions; Nrf2, HO-1, SOD, and GSH for evaluating antioxidant capacity; TC, TG, HDL-C, and LDL-C for analyzing blood lipid metabolism status. All indexes were operated according to the kit instructions, the optical density (OD) values were measured using an enzyme-linked immunosorbent assay (ELISA) reader (450 nm), and the concentrations of each index were calculated according to the standard curve. Figure 4 、 5 Figures 5, 6, and 7 are the oxidative stress levels, antioxidant and anti-inflammatory capabilities, antioxidant substance release levels, and lipid metabolism conditions of mice in different groups under microplastic infection, respectively.

[0025] It can be seen from Figure 4It can be seen that the levels of ROS, 8-OHdG, and MDA in the serum of the microplastic group increased by 12.69% ( P <0.001), 32.61% ( P <0.001), 72.02% ( P <0.001), indicating that microplastics can cause oxidative stress in the body. After pre-protection by oral administration of Dali Clostridium yogurt, the ROS level in the serum of mice decreased by 9.10% compared with the microplastic group ( P <0.001), 8-OHdG level decreased by 19.06% ( P <0.001, P <0.001), MDA level decreased by 31.67% ( P <0.001); the ROS level in the serum of mice treated with Dali Clostridium yogurt was reduced by 10.50% compared with the microplastic group ( P <0.001), 8-OHdG level decreased by 20.37% ( P <0.001), MDA level decreased by 43.73% ( P <0.001). The experimental results showed that yogurt prepared by Clostridium Dali fermentation showed good anti-oxidative stress ability, and both pre-protection and repair treatments could effectively alleviate the oxidative stress damage caused by microplastics.

[0026] Depend on Figure 4 It can be seen that the levels of NF-κB, TNF-α and IL-1β in the microplastic group increased by 42.33% ( P <0.001), 672.53% ( P <0.001), 117.48% ( P <0.001), which indicates that microplastics induce or aggravate inflammatory responses. The serum TNF-α level of mice treated with Dali Clostridium yogurt for pre-protection was reduced by 90.59% compared with the microplastic group ( P <0.001), and IL-1β levels decreased by 60.37% ( P <0.001), and NF-κB levels decreased by 8.76% ( P <0.001); the TNF-α level of mice treated with Dali Clostridium yogurt was reduced by 91.17% compared with the microplastic group ( P <0.001), and IL-1β levels decreased by 52.81% ( P <0.001), and NF-κB level decreased by 12.22% ( P<0.001). The experimental results showed that the yogurt prepared by Clostridium taiense fermentation could prevent or repair oxidative inflammation caused by microplastics by downregulating the levels of NF-κB, TNF-α, and IL-1β, and had good anti-inflammatory effects.

[0027] It can be seen from Figure 5 that the activities of Nrf2, SOD, GSH, and HO-1 in the microplastic group were decreased by 27.61% ( P <0.001), 33.14% ( P <0.001), 70.91% ( P <0.001), and 33.71% ( P <0.001), respectively, compared with the blank control group, indicating that microplastic exposure inhibited or weakened the expression of antioxidant-related factors and enhanced the oxidative stress response. In the mice pre-treated with intragastric administration of Clostridium taiense yogurt for pre-protection, compared with the microplastic group, the Nrf2 level in the serum was decreased by 8.76% ( P <0.001), the SOD level was increased by 90.89% ( P <0.05), the GSH level was increased by 515.74% ( P <0.001), and the HO-1 level was increased by 64.41% ( P <0.001); while in the mice treated with Clostridium taiense yogurt for repair, compared with the microplastic group, the Nrf2 level in the serum was increased by 44.50% ( P <0.001), the SOD level was increased by 126.27% ( P <0.001), the GSH level was increased by 552.87% ( P <0.001), and the HO-1 level was increased by 59.10% ( P <0.001). The experimental results showed that the yogurt prepared by Clostridium taiense fermentation could upregulate the levels of Nrf2, SOD, GSH, and HO-1 both for pre-protection and repair, so as to reduce the oxidative stress effect and reduce the body damage, and had good antioxidant stress effects.

[0028] It can be seen from Figure 5It can be seen that the activities of Nrf2, SOD, GSH, and HO-1 in the microplastic group were decreased by 27.61% (P<0.001), 33.14% (P<0.001), 70.91% (P<0.001), and 33.71% (P<0.001) respectively compared with the blank control group, indicating that microplastic exposure inhibited or weakened the expression of antioxidant-related factors and enhanced the oxidative stress response. In the mice pre-treated with intragastric administration of Clostridium taiwanense yogurt for pre-protection, compared with the microplastic group, the Nrf2 level in the serum was decreased by 8.76% (P<0.001), the SOD level was increased by 90.89% (P<0.05), the GSH level was increased by 515.74% (P<0.001), and the HO-1 level was increased by 64.41% (P<0.001); while in the mice treated with Clostridium taiwanense yogurt for repair, compared with the microplastic group, the Nrf2 level in the serum was increased by 44.50% (P<0.001), the SOD level was increased by 126.27% (P<0.001), the GSH level was increased by 552.87% (P<0.001), and the HO-1 level was increased by 59.10% (P<0.001). The experimental results show that the yogurt prepared by fermenting Clostridium taiwanense can up-regulate the levels of Nrf2, SOD, GSH, and HO-1 both for pre-protection and repair, so as to reduce the oxidative stress effect and reduce the body damage, and has a good antioxidant stress effect.

[0029] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of Clostridium taliensis with deposit number CGMCC 27789 in the preparation of a probiotic product against intestinal injury caused by microplastic ingestion. Clostridium dalinum ​ 2. The application according to claim 1, wherein The core of the probiotic product is Clostridium dalinum or its metabolites, including but not limited to fermented products of Clostridium dalinum or the compound application of Clostridium dalinum and other substances.

3. The application according to claim 1, wherein The product is used for preventing intestinal damage caused by microplastic ingestion; and / or the product is used for alleviating intestinal damage caused by microplastic ingestion; and / or the product is used for treating intestinal damage caused by microplastic ingestion.

4. The application according to claim 1, wherein The viable bacteria concentration in the functional product is 1×10 4 CFU / g to 1×10 15 CFU / g.

5. The application according to claim 3, characterized in that The product includes food, medicine, health products or animal feed.

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