Lactobacillus paracasei DT33 and application of lactobacillus paracasei DT33 in preparation of products with microplastic adsorption, removal and defecation promotion functions

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus paracasei DT33 and application of the lactobacillus paracasei DT33 in preparation of products with microplastic adsorption, removal and defecation promotion functions. The lactobacillus paracasei DT33 disclosed by the invention can be used for producing various SCFAs at high yield, has better acid-resistant and cholate-resistant capabilities and safety, and can be developed into edible probiotics; the strain has the function of promoting defecation, and can improve intestinal health and immune function; in addition, the strain can adsorb micro-plastics, accelerate discharge of the micro-plastics, reduce micro-plastic residues in intestinal tracts, reduce oxidative damage and inflammatory response caused by accumulation of the micro-plastics, and provide great benefits for intestinal health of human bodies and animals.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly to a Lactobacillus paracasei DT33 and its application in the preparation of products with functions of microplastic adsorption, removal and promoting defecation. Background Art

[0002] Short-chain fatty acids (SCFAs) are a class of organic acids produced by the fermentation of intestinal bacteria, consisting of 1-6 carbon atoms, among which acetate, propionate and butyrate have the highest contents, and the total amount exceeds 95% of the SCFA content. As one of the important metabolites produced by the intestinal flora, SCFAs play an important role in regulating human health.

[0003] Specifically, SCFAs can provide energy for the intestinal flora and intestinal epithelial cells, promote the proliferation and differentiation of epithelial cells, and regulate the expression of tight junction proteins, thereby enhancing the intestinal barrier function, maintaining the structural stability of the intestinal flora and the normal physiological functions of the intestine; in addition, SCFAs play a role in reducing the inflammatory response in the intestinal mucosa by activating G protein-coupled receptors in intestinal epithelial cells and immune cells, or inhibiting the activity of histone deacetylases, and reducing the production of inflammatory factors such as IL-6, IL-8, IL-10, TNF-α. Therefore, obtaining probiotics that can efficiently produce SCFAs is of great significance for maintaining the normal functions of the human intestine, regulating the human immune system and other aspects.

[0004] On the other hand, probiotics are increasingly widely used to regulate intestinal health. A number of studies have shown that probiotics such as Streptococcus thermophilus, Bifidobacterium lactis, Lactobacillus casei can safely and effectively promote defecation and relieve constipation. Discovering new probiotics that can promote defecation is also one of the research hotspots in this field.

[0005] In addition, microplastic pollution has also become a serious environmental problem and poses a potential threat to human health. Microplastics refer to tiny particles with a particle size less than 5 mm that are formed when plastic products discarded into the environment are decomposed under physical, chemical, and biological actions. Their materials include polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyethylene terephthalate (PET), etc. Microplastics are widely present in air, water bodies, and soil, can be ingested by plankton, fish, birds, etc., and ultimately enter the human body through the food chain. It is estimated that the weekly microplastic intake by the human body can reach 5 g, and microplastics are present in human feces, blood, lung tissue, and placenta. Although there is currently no direct evidence to confirm the damage of microplastics to human health, a large number of studies have confirmed that microplastics can cause damage to the digestive system, respiratory system, immune system, nervous system, and reproductive system of rodents and aquatic organisms. The microplastics accumulated in tissues cannot be removed, which can cause a large increase in reactive oxygen species, thus causing oxidative stress and producing toxic effects. Therefore, removing microplastics from the human body and reducing the content of microplastics are of great significance for the long-term health of the human body.

[0006] Currently, there is no method to remove microplastics from the human body, and only a few reports use biological methods to reduce plastic pollution in the environment and water bodies. For example, some bacteria and fungi can secrete cutinase, protease, esterase, lipase, etc. to decompose polymers into monomers or oligomers to achieve the purpose of degrading plastics; in addition, some bacteria have the ability to capture microplastics. They can attach to the surface of microplastics and form a sticky biofilm. This sticky matrix can capture free microplastics, resulting in the bioaccumulation of microplastics, thereby achieving the separation and removal of microplastics.

[0007] However, the existing bacteria or fungi that can degrade or adsorb microplastics are all non-edible strains, and such bacteria or fungi are also difficult to tolerate the gastrointestinal environment, so it is difficult to apply them to the removal of human body microplastics. In order to reduce the accumulation of microplastics in the human body and reduce the health damage caused by microplastics to humans, there is a need in this field to discover probiotics that can tolerate the gastrointestinal environment and adsorb and remove microplastics. Summary of the Invention

[0008] To solve the above-mentioned multiple technical problems, the present invention provides a strain of Lacticaseibacillus paracasei DT33 that can produce high yields of multiple SCFAs, promote defecation, improve immunity, and adsorb microplastics. This strain was isolated from French cheese and has good acid and bile salt tolerance. After colonizing in the organism, it can promote defecation and regulate immunity. Moreover, this strain can effectively adsorb microplastics, accelerate the excretion of microplastics, reduce the residual microplastics in the intestine, reduce the oxidative damage and inflammatory response caused by the accumulation of microplastics, and promote intestinal health. In addition, this strain has good safety and can be developed into an edible probiotic. Based on this, the present invention provides the following invention content.

[0009] First of all, the present invention provides a strain of Lacticaseibacillus paracasei, which is Lacticaseibacillus paracasei DT33.

[0010] This strain was deposited at the Guangdong Provincial Culture Collection of Microorganisms on July 5, 2023. The address of the depositary institution: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou; Postal Code: 510070. The taxonomic designation is Lacticaseibacillus paracasei, and the deposit number is GDMCC No: 63622.

[0011] The morphological characteristics of Lacticaseibacillus paracasei DT33 include: Gram staining is positive, rod-shaped under a light microscope, round at both ends, and can exist in pairs or chains. After culturing in MRS solid medium for 24 hours, it forms round, convex in the middle, smooth at the edges, and bright white colonies on the surface.

[0012] The physiological characteristics of Lacticaseibacillus paracasei DT33 include: Lacticaseibacillus paracasei DT33 can grow in acidic or bile salt-containing media. After colonizing in animals, it can promote defecation and regulate immunity. This strain can effectively adsorb microplastics, accelerate the excretion of microplastics, reduce the residual microplastics in the intestine, and reduce the oxidative damage and inflammatory response caused by the accumulation of microplastics.

[0013] Lacticaseibacillus paracasei is widely present in the human intestine and has a long and extensive history of use in food fermentation, especially in dairy fermentation. Lacticaseibacillus paracasei has been included in the list of strains that can be used in foods. It has also obtained the GRAS (generally recognized as safe) certification from the US Food and Drug Administration (FDA) and is listed in the Qualified Presumption of Safety (QPS) list issued by the European Food Safety Authority (EFSA). It is widely used in various probiotic foods and dietary supplements globally. In addition, genomic research data, mouse experiments, and human clinical trials have all proven the safety of Lacticaseibacillus paracasei.

[0014] Preferably, Lactobacillus paracasei DT33 is cultured in MRS medium.

[0015] Preferably, the MRS medium comprises the following components:

[0016] Casein peptone 10 g / L, beef extract powder 10 g / L, yeast extract powder 4 g / L, ammonium citrate tribasic 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, and Tween 80 1 g / L. Preferably, pH = 5.7 ± 0.2.

[0017] Preferably, the culture temperature is anaerobic culture at 37°C.

[0018] Preferably, the culture time is 24 h.

[0019] Furthermore, the present invention provides a microbial inoculum, which contains the above-mentioned Lactobacillus paracasei DT33.

[0020] Furthermore, the present invention provides a reagent or a kit, which contains the above-mentioned Lactobacillus paracasei DT33 or the above-mentioned microbial inoculum.

[0021] Preferably, the reagent or the kit is used for adsorbing, removing, or detecting microplastics in the environment.

[0022] Furthermore, the present invention provides a food, which contains the above-mentioned Lactobacillus paracasei DT33 or the above-mentioned microbial inoculum.

[0023] Adding the Lactobacillus paracasei of the present invention as a probiotic to food can promote defecation, improve intestinal health and immune status, and effectively remove microplastics in the body.

[0024] Furthermore, the present invention provides a feed or a feed additive, which contains the above-mentioned Lactobacillus paracasei DT33 or the above-mentioned microbial inoculum.

[0025] The above-mentioned feed or feed additive can promote animal defecation, improve animal intestinal health and immune status, effectively remove microplastics in animals, and promote the healthy growth and development of animals.

[0026] Furthermore, the present invention provides a medicine, which contains the above-mentioned Lactobacillus paracasei DT33 or the above-mentioned microbial inoculum.

[0027] The medicine of the present invention can promote defecation, adsorb microplastics or accelerate the removal of microplastics, be acid-resistant or bile-salt-resistant, promote the production of high levels of short-chain fatty acids, improve immune function, reduce oxidative damage or intestinal inflammation, and have an antioxidant effect.

[0028] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0029] Preferably, the excipients include fillers, excipients, lubricants, wetting agents, and diluents.

[0030] In the specific implementation process, the preparation types of the drug include but are not limited to powders, granules, capsules, tablets, and oral liquids.

[0031] Furthermore, the present invention provides the use of the above-mentioned Lactobacillus paracasei DT33 or the above-mentioned microbial agent in adsorbing, removing, or detecting microplastics in the environment.

[0032] Preferably, the environment includes air, water bodies, and soil environments.

[0033] Furthermore, the present invention provides the use of the above-mentioned Lactobacillus paracasei DT33 or the above-mentioned microbial agent in the preparation of a drug; the drug has at least one of the following functions:

[0034] (1) Promote defecation;

[0035] (2) Adsorb microplastics or accelerate the removal of microplastics;

[0036] (3) Be acid-resistant or bile-salt-resistant;

[0037] (4) Produce short-chain fatty acids;

[0038] (5) Improve immune function;

[0039] (6) Reduce oxidative damage or intestinal inflammation;

[0040] (7) Antioxidation.

[0041] Furthermore, the present invention provides the use of the above-mentioned Lactobacillus paracasei DT33 or the above-mentioned microbial agent in the preparation of a product for promoting the excretion of microplastics.

[0042] Furthermore, the present invention provides the use of the above-mentioned Lactobacillus paracasei DT33 or the above-mentioned microbial agent in the preparation of a product for adsorbing microplastics.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] The Lactobacillus paracasei DT33 of the present invention can produce a variety of SCFAs in high yield, and at the same time has good acid and bile-salt resistance and safety, and can be developed into an edible probiotic; this strain has the function of promoting defecation and can improve intestinal health and immune function; in addition, this strain can adsorb microplastics, accelerate the excretion of microplastics from the intestine, thereby reducing the oxidative damage and inflammatory response caused by the accumulation of microplastics, and providing great benefits for the intestinal health of humans and animals. Description of the Drawings

[0045] Figure 1 It is the colony morphology diagram of Lactobacillus paracasei DT33 on MRS medium.

[0046] Figure 2 It is the microscopic examination diagram of Lactobacillus paracasei DT33.

[0047] Figure 3 It is the statistical chart of the survival rate of Lactobacillus paracasei DT33 in acidic medium; the statistical analysis method is ttest, ns indicates that the p-value is greater than 0.05, the data are from three repeated experiments, and the error bars represent the standard deviation.

[0048] Figure 4 It is the statistical chart of the survival rate of Lactobacillus paracasei DT33 in medium containing bile salts; the statistical analysis method is ttest, ns indicates that the p-value is greater than 0.05, the data are from three repeated experiments, and the error bars represent the standard deviation.

[0049] Figure 5 It is the statistical chart of the acetic acid concentration in the fermentation broth of Lactobacillus paracasei DT33; the data are from three repeated experiments, the error bars represent the standard deviation, the statistical analysis method is t test, and *** indicates that the p-value is less than 0.001.

[0050] Figure 6 It is the effect diagram of the determination of the activated carbon transport rate after colonizing Lactobacillus paracasei DT33.

[0051] Figure 7 It is the statistical chart of the determination of the TNF-α content in the ileum tissue after colonizing Lactobacillus paracasei DT33, the error bars represent the standard deviation, the statistical analysis method is t test, and *** indicates that the p-value is less than 0.001.

[0052] Figure 8 It is the photo of the adsorption and aggregation of Lactobacillus paracasei DT33 and PS fluorescent microspheres in the solution.

[0053] Figure 9 It is the statistical chart of the adsorption rate of Lactobacillus paracasei DT33 to PS fluorescent microspheres in the solution, the error bars represent the standard deviation, the data are from three repeated experiments, and the statistical analysis method is t test, **** indicates that the p-value is less than 0.0001.

[0054] Figure 10 It is the electron microscopy image of the adsorption of Lactobacillus paracasei DT33 and PS fluorescent microspheres, with a magnification of 50,000 times.

[0055] Figure 11 It is the statistical chart of the scavenging rate of Lactobacillus paracasei DT33 to DPPH, the error bars represent the standard deviation, and the data are from three repeated experiments.

[0056] Figure 12 It is an electron micrograph of the adsorption of Lactobacillus paracasei DT33 and mixed microplastic particles, with a magnification of 2000 times.

[0057] Figure 13 It is a fluorescence imaging of the adsorption of Lactobacillus paracasei DT33 and mixed microplastic particles; among them, the green signal represents Lactobacillus paracasei DT33, the red signal represents microplastic particles, and the arrow indicates that Lactobacillus paracasei DT33 is adsorbed on the surface of the microplastic particles, with a magnification of 1000 times.

[0058] Figure 14 It is a detection chart of the residual amount of PS fluorescent microspheres in the mouse colon. The error bars represent the standard deviation. The statistical analysis method is ANOVA. * indicates that the p-value is less than 0.05, and **** indicates that the p-value is less than 0.0001.

[0059] Figure 15 It is the detection of immune factors in mouse serum (A) and ileum tissue (B - D). The error bars represent the standard deviation. The statistical analysis method is ANOVA. ** indicates that the p-value is less than 0.01, *** indicates that the p-value is less than 0.001, and **** indicates that the p-value is less than 0.0001. Detailed implementation manners

[0060] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0061] For those not specifying specific technologies or conditions in the embodiments, they are all conventional methods or are carried out according to the technologies or conditions described in the literature in this field, or according to the product specifications. For those reagents, instruments, etc. not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0062] The trace element solution and vitamin solution in the formula of the in vitro simulated intestinal environment culture medium are both purchased from Coolaber Company. The product number of the trace element solution is SL0120, and the product number of the vitamin solution is SL0110.

[0063] Example 1 Isolation and identification of Lactobacillus paracasei DT33

[0064] 1. Isolation and identification of Lactobacillus paracasei DT33

[0065] 1.1 Sample source

[0066] The strain Lacticaseibacillus paracasei DT33 used in this example was isolated from French cheese.

[0067] 1.2 Preparation of culture medium

[0068] The culture medium used for sample isolation and strain screening was MRS medium, and the culture medium used for culturing Lacticaseibacillus paracasei DT33 was MRS medium. The composition of MRS medium is shown in Table 1, and the final pH of MRS medium was 5.7 ± 0.2; adding 1.5% agar makes it MRS solid medium.

[0069] Table 1 Formulation of MRS medium

[0070] Formula Content (per liter) Caseinase digest 10.0g Beef extract powder 10.0g Yeast extract powder 4.0g Ammonium citrate 2.0g Sodium acetate 5.0g <![CDATA[Magnesium sulfate (MgSO4·7H2O)]]> 0.2g <![CDATA[Manganese sulfate (MnSO4·4H2O)]]> 0.05g Dipotassium hydrogen phosphate 2.0g Glucose 20.0g Tween-80 1.0g

[0071] 1.3 Isolation of the strain

[0072] Put 1 g of French cheese sample into 10 mL of the MRS liquid medium prepared in step 1.2, mix well and culture at 36 °C for 24 h. Then, in a laminar flow hood, aspirate 1 mL of the enrichment solution and perform ten-fold serial dilution. Select 10 -4 、10 -5 、10 -6 、10 -7 100 μL of the bacterial suspensions at four dilution gradients and spread them on petri dishes containing sterile MRS solid medium. Incubate statically at 36 °C under anaerobic conditions for 48 h - 72 h. After obvious single colonies are formed, use a high-throughput automated platform to automatically pick typical colonies from the petri dishes and culture them in MRS liquid medium. The isolated strain is identified by 16S rRNA sequencing to determine its species information.

[0073] 2. Identification of Lacticaseibacillus paracasei DT33

[0074] 2.1 Colony characteristics

[0075] After culturing Lacticaseibacillus paracasei DT33 in MRS solid medium for 24 h, milky white colonies that are round, convex in the middle, smooth at the edges, and bright on the surface are formed, as shown in Figure 1 .

[0076] 2.2 Morphology under the microscope

[0077] Smear of Lacticaseibacillus paracasei DT33 colonies: Gram staining is positive. Under a light microscope, it is rod-shaped, with round ends, and can exist in pairs or chains, as shown in Figure 2 .

[0078] 2.3 16S rRNA identification

[0079] Identification unit: Tsingke Biotechnology Co., Ltd.

[0080] The identification sequence is shown in SEQ ID No. 1.

[0081] Identification result: The sequencing result was compared with the NCBI database, and combined with the physiological and biochemical results, it was determined that the strain was Lactobacillus paracasei.

[0082] Example 2 Acid Tolerance Detection of Lactobacillus paracasei DT33

[0083] The overall pH condition in the human gastric environment is strongly acidic. Therefore, the acid tolerance of the strain is an important indicator to evaluate whether it can survive and colonize in the gastric acid environment. The commercial strain Lactobacillus rhamnosus GG (LGG) is a probiotic widely used at present and has strong acid tolerance.

[0084] In this example, MRS medium with pH = 2.5 was used to verify the acid tolerance of Lactobacillus paracasei DT33. The specific steps include:

[0085] Take 1 mL of the bacterial solution, centrifuge at 4000 rpm for 10 min, discard the supernatant, then add 1 mL of PBS for washing once, after centrifuging at 4000 rpm for 10 min, the precipitate was resuspended with MRS medium with pH = 2.5. Incubate at 37 °C for 3 h, and sample at 0 h and 3 h respectively. After centrifuging the samples, they were resuspended with PBS and diluted in gradients. The diluted samples were spread on MRS agar plates and anaerobically cultured at 37 °C for 16 h, and then colony counting was performed. The survival rate calculation formula is: Acid tolerance survival rate (%) = C1 / C0 × 100% (C0: counting result at 0 h; C1: counting result at 3 h). The control strain was Lactobacillus rhamnosus LGG.

[0086] After 3 hours of culture in the acidic medium, the survival rate of the control strain Lactobacillus rhamnosus LGG was 84.56%, and the survival rate of Lactobacillus paracasei DT33 was 87.78% ( Figure 3 ), which was equivalent to the acid tolerance of the control strain, indicating that the strain had strong acid tolerance and could survive in the gastric environment.

[0087] Example 3 Bile Salt Tolerance Detection of Lactobacillus paracasei DT33

[0088] After bacteria enter the intestine through the stomach, the high concentration of bile salts in the small intestine will kill the bacteria. The residence time of food in the small intestine is generally 1 - 4 h.

[0089] In this example, 0.1% bile salt-MRS medium was used to verify the bile salt tolerance of Lactobacillus paracasei DT33 strain. The specific steps include:

[0090] Inoculate the Lactobacillus paracasei DT33 bacterial solution into a 96-well deep-well plate containing MRS medium and incubate anaerobically at 37°C for 24 h. Take 300 μL of the cultured bacterial solution, centrifuge at 4000 rpm for 10 min, discard the supernatant, add 600 μL of MRS medium containing 0.1% bile salt, and resuspend and mix well. For the control group, take 100 μL of the resuspended solution and add 20 μL of MTT (thiazolyl blue) solution; for the treatment group, take 100 μL of the resuspended solution, incubate at 37°C for 4 h, and then add 20 μL of MTT solution. After adding the MTT solution, react in the dark at 37°C for 4 h. After the reaction is completed, centrifuge at 4000 rpm for 10 min and discard the supernatant. Add 100 μL of DMSO solution to each well, incubate at 37°C with shaking for 10 min to completely dissolve and mix the purple formazan produced by the reaction. After mixing, measure the absorbance of the solution at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the survival rate. Survival rate = A1 / A0 × 100% (A1: absorbance value of the solution in the treatment group at 570 nm, A0: absorbance value of the solution in the control group at 570 nm). Measure the survival rate of the control strain Lactobacillus rhamnosus LGG using the same method.

[0091] After 4 hours of culture in 0.1% bile salt-MRS medium, the survival rate of the control strain Lactobacillus rhamnosus LGG was 101.5%, and the survival rate of Lactobacillus paracasei DT33 was 97.28% ( Figure 4 ). This indicates that the bile salt tolerance of Lactobacillus paracasei DT33 is equivalent to that of the control strain Lactobacillus rhamnosus LGG and can survive in the small intestine.

[0092] Example 4 Detection of the SCFA-producing ability of Lactobacillus paracasei DT33

[0093] To better verify the ability of Lactobacillus paracasei DT33 to produce SCFA in vivo, an in vitro simulated intestinal environment medium was used in this example, and the medium formulation is shown in Table 2. Inoculate Lactobacillus paracasei DT33 into this medium and incubate anaerobically at 37°C for 24 h. Take the culture solution, centrifuge at 4000 rpm for 10 min, filter the supernatant through a 0.22-μm aqueous filter membrane and add it to a liquid-phase vial, and detect the SCFA content in the supernatant by HPLC. The control strain is another strain of Lactobacillus paracasei obtained from the same batch of screening experiments.

[0094] From Figure 5It can be seen that the acetic acid content in the fermentation broth of Lactobacillus paracasei DT33 is significantly higher than that of the control strain. In addition, the content of propionic acid in the supernatant of the fermentation broth of Lactobacillus paracasei DT33 was measured in this example, which was 0.038 mg / mL. Therefore, Lactobacillus paracasei DT33 is a high-yield SCFA strain isolated this time. Since the glucose content of the in vitro simulated intestinal environment medium used in this example is significantly lower than that of common microbial media such as MRS medium, the contents of acetic acid and propionic acid produced by Lactobacillus paracasei DT33 will be relatively low. If common microbial media are used, it is expected that higher yields of acetic acid and propionic acid can be obtained.

[0095] Table 2 Formula of in vitro simulated intestinal environment medium

[0096]

[0097] Example 5 Promotion of mouse excretion by Lactobacillus paracasei DT33

[0098] Six-week-old male C57 mice that had been adaptively fed for one week were randomly divided into two groups. The experimental group of mice (6 mice) were gavaged with Lactobacillus paracasei DT33, and the control group of mice were gavaged with the commercial strain Lactobacillus rhamnosus GG. The gavage days were 7 days, and the gavage dose was 10 9 CFU / day. Twenty-four hours after the last gavage of probiotics, all mice were gavaged with 2 ml of 5% activated carbon. Twenty minutes after the gavage of activated carbon, the mice were sacrificed, the abdominal cavity was dissected, the intestine from the pylorus to the ileocecal part was taken out, placed on paper, and the transmission position of the activated carbon was measured under the condition that the intestine was fully relaxed. After the measurement was completed, the colon tissue was stored at -80 °C for ELISA detection.

[0099] From Figure 6 It can be seen that compared with the colonization of Lactobacillus rhamnosus GG, the colonization of Lactobacillus paracasei DT33 accelerated the transport rate of activated carbon and better promoted the excretion of mice.

[0100] Example 6 Reduction of intestinal inflammation in mice by Lactobacillus paracasei DT33

[0101] The ileum tissue samples stored in Example 5 were taken for ELISA detection and analysis.

[0102] The results are as Figure 7 shown. Compared with the colonization of Lactobacillus rhamnosus GG, the gavage of Lactobacillus paracasei DT33 significantly reduced the level of tumor necrosis factor (TNF-α), which indicates that the colonization of Lactobacillus paracasei DT33 has a better effect on reducing intestinal inflammation than the colonization of Lactobacillus rhamnosus GG.

[0103] Example 7 Determination of the effect of Lactobacillus paracasei DT33 on adsorbing microplastics

[0104] Lactobacillus paracasei DT33 was inoculated into MRS medium and anaerobically cultured at 37 °C for 24 h. The cultured bacterial solution was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 450 μL of sterile PBS buffer was added for washing twice. Then, it was resuspended with PBS, and the bacterial solution concentration was adjusted to 1×10 9 CFU / mL. For the experimental group, 100 μL of the Lactobacillus paracasei DT33 bacterial suspension was taken into a 1.5 mL EP tube, and 900 μL of the PS fluorescent microsphere working solution (0.16 mg / mL, particle size 0.1 μm, Besler Company) was added and mixed well. It was placed on a shaker and incubated in the dark for 4 h, and the incubation conditions were 37 °C and 800 rpm. For the blank control group, 100 μL of PBS and 900 μL of the PS fluorescent microsphere working solution were taken into a 1.5 mL EP tube and mixed well for incubation. For the bacterial solution control group, 100 μL of the Lactobacillus paracasei DT33 bacterial suspension and 900 μL of PBS were taken into a 1.5 mL EP tube and mixed well for incubation. After incubation, the incubation solution was taken for observation and photography. For the incubation solutions of the experimental group and the blank control group, they were centrifuged at 2000 rpm for 10 min, 100 μL of the supernatant was taken, and the fluorescence intensity was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The parameters of the ELISA reader were: excitation wavelength, 494 nm; detection wavelength, 518 nm. The adsorption rate was calculated based on the fluorescence intensity value. The control strain was another strain of Lactobacillus paracasei obtained in the same batch of screening experiments, and the adsorption rate of the control strain was measured in the same way. The adsorption rate calculation formula was: adsorption rate (%) = (A1 - A2) × 100 / A1 × 100% (A1: fluorescence value of the blank control group, A2: fluorescence value of the Lactobacillus paracasei group). The precipitate after centrifugation was taken, fixed with glutaraldehyde at 4 °C overnight, then dehydrated with a gradient of ethanol, and observed under an electron microscope after drying.

[0105] From Figure 8 It can be seen that in the blank control group, the PS fluorescent microspheres did not self-aggregate; in the bacterial solution control group, Lactobacillus paracasei DT33 did not self-aggregate; in the experimental group, specific adsorption and aggregation flocs of Lactobacillus paracasei DT33 and PS fluorescent microspheres appeared.

[0106] From Figure 9 It can be seen that the adsorption rate of the control strain was 15.33%, and its microplastic adsorption ability was poor, while the adsorption rate of Lactobacillus paracasei DT33 reached 72.53%, indicating a strong ability to adsorb microplastics. This shows that the adsorption effect of Lactobacillus paracasei DT33 on microplastics is strain-specific.

[0107] From Figure 10 It can be seen that under electron microscope observation, spherical PS fluorescent microspheres were adsorbed on the surface of rod-shaped Lactobacillus paracasei DT33.

[0108] Example 8 Determination of the antioxidant capacity of Lactobacillus paracasei DT33

[0109] Lactobacillus paracasei DT33 was inoculated into MRS medium and anaerobically cultured at 37 °C for 24 h. The cultured bacterial solution was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 450 μL of sterile PBS buffer was added for washing twice, and the bacterial solution concentration was adjusted to 1×10 9 CFU / mL. In the experimental group, 500 μL of the bacterial suspension was added to 500 μL of 0.2 mmol / L 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution. The antioxidant vitamin C (Vc) was used as a positive control. 500 μL of 3 μg / mL vitamin solution was taken and added to 500 μL of 0.2 mmol / L DPPH ethanol solution. In the control group, 500 μL of PBS was added to 500 μL of 0.2 mmol / L DPPH ethanol solution. In the blank group, 500 μL of the bacterial suspension was added to 500 μL of absolute ethanol solution. After mixing, it was placed on a shaker at 30 °C and reacted with light shielding and oscillation for 30 min. After the oscillation ended, the reaction solution was centrifuged at 4000 rpm for 10 min, and 100 μL of the supernatant was taken to measure the absorbance at 517 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the DPPH radical scavenging rate. The calculation formula is: DPPH scavenging rate (%) = [1 - (As - A0) / Ai] × 100% (As: fluorescence value of the experimental group; A0: fluorescence value of the blank group; Ai: fluorescence value of the control group).

[0110] From Figure 11 It can be seen that the scavenging rate of the antioxidant vitamin C for DPPH is 28.91%. Similar to vitamin C, Lactobacillus paracasei DT33 has certain antioxidant ability, and the scavenging rate for DPPH is 19.85%. Therefore, colonizing Lactobacillus paracasei DT33 can reduce the oxidative damage brought by microplastics to the host.

[0111] Example 9 Determination of the adsorption effect of Lactobacillus paracasei DT33 on mixed microplastics

[0112] To simulate and verify the adsorption effect of Lactobacillus paracasei DT33 on microplastics in the natural environment, in this example, microplastic powders of five common materials were mixed in equal proportions (polypropylene PP, polyethylene PE, polystyrene PS, polyethylene terephthalate PET, polycarbonate PC), resuspended in PBS solution containing 0.1% Tween-80, and formulated into a 1 mg / mL mixed microplastic suspension for adsorption effect detection.

[0113] Lactobacillus paracasei DT33 was inoculated into MRS medium and anaerobically cultured at 37 °C for 24 h. The cultured bacterial solution was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 450 μL of sterile PBS buffer was added for washing twice. Then it was resuspended with PBS, and the bacterial solution concentration was adjusted to 1×10 9CFU / mL. Take 100 μL of the Lactobacillus paracasei DT33 bacterial suspension and transfer it into a 1.5 mL EP tube. Add 900 μL of the above-mentioned 1 mg / mL mixed microplastic suspension, and place it on a shaker for incubation in the dark for 4 h. The incubation conditions are 37 °C and 800 rpm. After incubation, take the precipitate for observation under an electron microscope. The results are shown in Figure 12 .

[0114] From Figure 12 It can be seen that under the observation of an electron microscope, the short rod-shaped Lactobacillus paracasei DT33 is adsorbed on the surface of microplastic particles.

[0115] Take the above-mentioned mixed microplastic powder, add 10 μg / mL nile red solution, incubate at 50 °C and 100 rpm for 1 hour, then wash three times with PBS, resuspend in PBS, and prepare a 1 mg / mL nile red-labeled mixed microplastic suspension.

[0116] Furthermore, take the overnight-cultured Lactobacillus paracasei DT33, wash it with PBS, resuspend it in 100 μM FITC solution, and incubate it on a shaker in the dark at 37 °C and 100 rpm for 0.5 h. After incubation, centrifuge, wash the bacterial precipitate with PBS and resuspend it, and adjust the bacterial solution concentration to 1×10 9 CFU / mL. Take 100 μL of the bacterial suspension and transfer it into a 1.5 mL EP tube. Add 900 μL of the above-mentioned 1 mg / mL nile red-labeled mixed microplastic suspension, and place it on a shaker for incubation in the dark for 4 h. The incubation conditions are 37 °C and 800 rpm. After incubation, take the incubation solution on a glass slide, dry it, then seal it with a mounting medium and a coverslip, and observe and photograph it with a fluorescence microscope after fixation. The results are shown in Figure 13 .

[0117] From Figure 13 It can be seen that the mixed microplastic particles labeled with nile red emit red fluorescence, and the bacteria labeled with FITC emit green fluorescence. The red fluorescence in the figure is surrounded by green signals, indicating that Lactobacillus paracasei DT33 is adsorbed on the surface of the mixed microplastics.

[0118] Example 10 Lactobacillus paracasei DT33 gavage reduces microplastic residues in vivo

[0119] Purchase 6-week-old C57 mice. After one week of adaptive feeding, the experimental group of mice is gavaged with 1 mg of PS fluorescent microspheres (10 mg / mL, particle size 5 μm, Bestlife company) every day, and at the same time, gavaged with 1*10 9CFU Lactobacillus paracasei DT33, in the control group of mice, 1 mg of PS fluorescent microspheres were intragastrically administered daily, and at the same time, an equal volume of normal saline was intragastrically administered daily. In the blank control group (NC), mice were not intragastrically administered PS fluorescent microspheres, and only an equal volume of normal saline was intragastrically administered. Intragastric administration was continued for 7 days. After the last intragastric administration of PS fluorescent microspheres, water and food were withheld for 16 h, and then the mice were sacrificed. The intestinal tissues of the mice were dissected, and the residual amount of PS fluorescent microspheres was detected. The detection method was as follows: Take the colon tissue of the mice: After weighing, add 400 μL of lysis buffer (23 g / L Na2HPO4, 4.6 g / L NaH2PO4), grind it with a tissue grinder (60 Hz, 45 s, 4 2-mm steel beads), after grinding, add 40 μL of 50 g / L SDS and mix well by oscillation, and then add 40 μL of Protein K (20 mg / mL). Incubate overnight at 37 °C, dilute with 400 μL of lysis buffer, aspirate the homogenate with a 1 mL syringe, and filter it through a 100-μm cell strainer into a 1.5 mL EP tube. Take 200 μL of the filtrate into a 96-well plate and detect the fluorescence microsphere signal with a flow cytometer. The detection parameters of the flow cytometer were FSC greater than 60000, the sample volume was 20 μL, and the detection channels were: B530, FITC-H. The results are shown in Figure 14 。

[0120] From Figure 14 it can be seen that there was a large amount of residual PS fluorescent microspheres in the colon of the control group mice, and intragastric administration of Lactobacillus paracasei DT33 significantly reduced the residual amount of PS fluorescent microspheres in the colon of the mice; this indicates that intragastric administration of Lactobacillus paracasei DT33 reduced the residual amount of microplastics in the mice.

[0121] Example 11 Intragastric administration of Lactobacillus paracasei DT33 reduces the inflammatory response induced by microplastics

[0122] Take the serum collected before sacrificing the mice and the dissected ileum tissues in Example 10, and detect the levels of immune factors by ELISA. The results are shown in Figure 15 。

[0123] From Figure 15 it can be seen that compared with the blank control group, PS fluorescent microspheres significantly reduced the content of the anti-inflammatory cytokine IL-10 in the serum and intestine, and significantly increased the inflammatory cytokines TNF-α and IL-6 in the ileum, which indicates that PS fluorescent microspheres induced an inflammatory response in the mice. After intragastric administration of Lactobacillus paracasei DT33, the levels of inflammatory factors all significantly returned to normal levels, the content of IL-10 in the serum and intestine significantly increased, and the content of TNF-α and IL-6 in the ileum significantly decreased, indicating that intragastric administration of Lactobacillus paracasei DT33 can reduce the inflammatory response induced by microplastics.

[0124] In summary, a strain of Lactobacillus paracasei DT33 was isolated and screened in this invention. This strain can tolerate acid and bile salts, has the ability to colonize in the stomach and small intestine, and can be applied to the development of edible probiotics. Lactobacillus paracasei DT33 has a high ability to produce SCFAs. After colonizing in mice, it can increase the intestinal transport rate of mice, promote defecation in mice, and at the same time reduce the level of inflammatory factors in the intestines of mice, showing a protective ability for the intestines. This strain has a strong ability to adsorb microplastics, accelerates the excretion of microplastics from the body, reduces the residue of microplastics in the intestines, and effectively reduces the oxidative damage and inflammatory response caused by the accumulation of microplastics. The experimental data also prove the antioxidant ability of this strain. It can be seen that Lactobacillus paracasei DT33 is a strain suitable for the digestive tract environment and has broad application prospects in promoting host defecation, reducing intestinal inflammation, adsorbing microplastics, accelerating the excretion of microplastics, and reducing the oxidative damage and inflammatory response brought by microplastics.

[0125] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Lactobacillus paracasei, characterized in that, It is Lacticaseibacillus paracasei DT33, and its deposit number is GDMCC No: 63622.

2. A microbial inoculum, characterized in that, It contains the Lacticaseibacillus paracasei described in claim 1.

3. A reagent or kit, characterized in that, It contains the Lacticaseibacillus paracasei described in claim 1 or the microbial agent described in claim 2.

4. The reagent or kit according to claim 3, characterized in that, The reagent or kit is used for adsorbing, removing or detecting microplastics in the environment.

5. A food, characterized in that, It contains the Lacticaseibacillus paracasei described in claim 1 or the microbial agent described in claim 2.

6. A drug, characterized in that, It contains the Lacticaseibacillus paracasei described in claim 1 or the microbial agent described in claim 2.

7. Use of the Lacticaseibacillus paracasei described in claim 1 or the microbial agent described in claim 2 in adsorbing, removing or detecting microplastics in the environment.

8. Use of the Lacticaseibacillus paracasei described in claim 1 or the microbial agent described in claim 2 in preparing a drug; the drug has at least one of the following functions: (1) Promoting defecation; (2) Adsorbing microplastics or accelerating the removal of microplastics; (3) Being acid-resistant or bile-salt-resistant; (4) Producing short-chain fatty acids; (5) Improving immune function; (6) Alleviating oxidative damage or intestinal inflammation; (7) Antioxidation.

9. Use of the Lacticaseibacillus paracasei described in claim 1 or the microbial agent described in claim 2 in preparing a product for promoting the excretion of microplastics.

10. Use of the Lacticaseibacillus paracasei described in claim 1 or the microbial agent described in claim 2 in preparing a product for adsorbing microplastics.

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