Lactobacillus brevis MPA01 capable of adsorbing microplastics and degrading uric acid and application of lactobacillus brevis MPA01

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus brevicornu MPA01 capable of adsorbing microplastics and degrading uric acid and application of the lactobacillus brevicornu MPA01. The preservation number of the lactobacillus brevis MPA01 is CGMCC (China General Microbiological Culture Collection Center) No. 31977. According to the present invention, the Lactobacillus brevicornu is the marine-derived natural strain, and has significant adsorption effects on the micro-plastics PS and PE, and the Lactobacillus brevicornu MPA01 and the plant lactobacillus MPs-68 are compounded so as to provide the synergistic effect on the adsorption effects on the micro-plastics PS and PE; besides, the strain has very high DPPH free radical scavenging rate and antioxidant capacity, is resistant to gastric acid and cholate, has better intestinal colonization capacity, also has certain inhibition capacity on xanthine oxidase, has remarkable capacity of degrading uric acid, and can be applied to the field of intestinal colonization. The method has important application value in the fields of food or health care products and the like for assisting adsorption of micro-plastics and reduction of uric acid.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Lactobacillus curvatum MPA01 capable of adsorbing microplastics and degrading uric acid and an application thereof. Background Art

[0002] Microplastics (MPs) are tiny plastic particles with a diameter less than 5 mm, primarily including polystyrene (PS), polyethylene (PE), and polypropylene (PP). With their widespread use in daily life, MPs have entered marine, terrestrial, and freshwater systems through various pathways, subsequently accumulating in organisms through the food chain. Due to their poor biodegradability and inadequate recycling, microplastics have caused widespread environmental pollution. Therefore, it is crucial to find a method that can effectively adsorb MPs while being harmless to humans and beneficial to health.

[0003] Hyperuricemia (HUA), caused by purine metabolism disorders, is a common metabolic disease characterized by elevated serum uric acid levels. Hyperuricemia, along with hypertension, hyperglycemia, and hyperlipidemia, is collectively referred to as the "four highs" metabolic disease. The key to treating hyperuricemia is inhibiting xanthine oxidase and reducing uric acid concentrations. Current treatment options, in addition to medication, generally require strict dietary restrictions to minimize the intake of exogenous purines. However, patient compliance with medication and a strict diet is low. Therefore, the use of lactic acid bacteria to prevent and treat hyperuricemia has become a hot topic of research.

[0004] However, existing technologies do not yet have a strain that can significantly degrade both PS and PE microplastics while also significantly degrading uric acid. Furthermore, existing strains have poor tolerance to gastric and intestinal fluids and are unable to colonize, survive, and function in the human intestine.

[0005] In summary, how to develop a natural and safe bacterial strain that has a significant adsorption effect on microplastics PS and PE, can significantly degrade uric acid, and has good tolerance to gastric juice and intestinal juice is the technical difficulty that researchers in this field are committed to solving. Summary of the Invention

[0006] In order to solve the deficiencies of the prior art mentioned in the above background, the present invention provides a Lactobacillus curvatum MPA01 capable of adsorbing microplastics and degrading uric acid and its application.

[0007] The present invention provides a Lactobacillus curvatus MPA01 capable of adsorbing microplastics and degrading uric acid, the Latin name of which is: Latilactobacillus curvatus, deposited in the General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: September 14, 2024, deposit number: CGMCCNo.31977.

[0008] The present invention also provides the use of the Lactobacillus curvatum MPA01 capable of adsorbing microplastics and degrading uric acid as described above in probiotic products.

[0009] The present invention also provides the use of the above-mentioned Lactobacillus curvatum MPA01 with the ability to adsorb microplastics and degrade uric acid in functional foods, the components of which include the above-mentioned Lactobacillus curvatum MPA01 with the ability to adsorb microplastics and degrade uric acid.

[0010] Furthermore, the functional food includes one or more of functional foods that can adsorb microplastics, antioxidant foods, acid-resistant and bile-resistant foods, and functional foods that can degrade uric acid.

[0011] The present invention also provides a fermentation product, the components of which include the above-mentioned Lactobacillus curvatum MPA01 capable of adsorbing microplastics and degrading uric acid.

[0012] The present invention also provides a bacterial agent, including the above-mentioned Lactobacillus curvatum MPA01 capable of absorbing microplastics and degrading uric acid, wherein the number of viable bacteria is ≥1×10 11 CFU / g or ≥1×10 11 CFU / mL.

[0013] The present invention also provides a probiotic preparation, the components of which include the Lactobacillus curvatum MPA01 having the ability to adsorb microplastics and degrade uric acid as described above.

[0014] In one embodiment, the probiotic preparation includes the fermentation broth of the Lactobacillus curvatus MPA01 that has the ability to adsorb microplastics and degrade uric acid as described above and / or the bacterial powder of the Lactobacillus curvatus MPA01 that has the ability to adsorb microplastics and degrade uric acid as described above; the fermentation broth of the Lactobacillus curvatus MPA01 that has the ability to adsorb microplastics and degrade uric acid includes one or more of fermentation supernatant, cell-free extract, and bacterial cell metabolites; the bacterial powder of the Lactobacillus curvatus MPA01 that has the ability to adsorb microplastics and degrade uric acid is vacuum freeze-dried bacterial powder and / or spray-dried bacterial powder.

[0015] In one embodiment, the bacterial powder of Lactobacillus curvatum MPA01 capable of absorbing microplastics and degrading uric acid has a viable bacterial count of not less than 1×10 11 CFU / g.

[0016] The microecological preparation has the function of adsorbing microplastics polystyrene and / or polyethylene.

[0017] The microecological preparation has the function of degrading uric acid.

[0018] The present invention also provides a microplastic adsorbent, the components of which include Lactobacillus curvatum MPA01 or a composite bacteria including Lactobacillus curvatum MPA01 and Lactobacillus plantarum MPs-68; The number of viable Lactobacillus curvatus MPA01 is ≥1×10 9 CFU / mL or ≥1×10 9 CFU / g; The number of viable bacteria of the composite bacteria is ≥1×10 9 CFU / mL or ≥1×10 9 CFU / g; The Lactobacillus curvatum MPA01 is the Lactobacillus curvatum MPA01 having the ability to adsorb microplastics and degrade uric acid as described above; The deposit number of the Lactobacillus plantarum MPs-68 is CGMCC No.31976, and its Latin name is: Lactiplantibacillus plantarum , deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is: September 14, 2024.

[0019] Preferably, the ratio of the viable counts of Lactobacillus curvatus MPA01 and Lactobacillus plantarum MPs-68 is 1: 1. Those skilled in the art can compound Lactobacillus curvatus MPA01 and Lactobacillus plantarum MPs-68 in different ratios according to the effects of the present invention to meet corresponding requirements.

[0020] The present invention also provides the use of the microplastic adsorbent as described above in adsorbing microplastic polystyrene and / or polyethylene.

[0021] Based on the above characteristics, the Lactobacillus curvatum MPA01 provided by the present invention, which has the ability to adsorb microplastics and degrade uric acid, has the following beneficial effects: The Lactobacillus curvatum MPA01 of the present invention, which has the ability to adsorb microplastics and degrade uric acid, is a natural marine bacterial species with high food safety. It has a significant adsorption effect on microplastics polystyrene (PS) and polyethylene (PE), and can be used to adsorb microplastics polystyrene and / or polyethylene, breaking through the current dilemma of a single adsorption object for microplastic adsorption functional bacteria.

[0022] The Lactobacillus curvatum MPA01 capable of adsorbing microplastics and degrading uric acid of the present invention has a very high DPPH free radical scavenging rate and significant antioxidant capacity.

[0023] The Lactobacillus curvatum MPA01 of the present invention, which has the ability to adsorb microplastics and degrade uric acid, is resistant to gastric acid and bile salts and has good intestinal colonization ability, which is conducive to its preparation as a probiotic product.

[0024] The Lactobacillus curvatum MPA01 of the present invention, which has the ability to adsorb microplastics and degrade uric acid, also has good inhibitory ability against xanthine oxidase and has significant ability to degrade uric acid. It has important application value in the fields of assisting in the adsorption of microplastics and lowering uric acid, as well as in the fields of food or health products.

[0025] The Lactobacillus curvatum MPA01 bacterial agent, probiotic product, functional food, fermented product and microecological preparation provided by the present invention contain Lactobacillus curvatum MPA01, which has the ability to adsorb microplastics and degrade uric acid. It is a natural bacterial species of marine origin with high food safety. It has excellent adsorption capacity for microplastics PS and PE and uric acid degradation ability, strong antioxidant capacity, resistance to gastric acid and bile salts, and high intestinal colonization efficiency.

[0026] The microplastic adsorbent provided by the present invention, wherein the Lactobacillus curvatum MPA01 has a significant adsorption effect on microplastics PS and PE, among which the adsorption effect on microplastics PS is more significant, and the combination of Lactobacillus plantarum MPs-68 and Lactobacillus curvatum MPA01 further improves the microplastic adsorption rate, and in particular, has a significant synergistic effect on the adsorption of microplastics PE. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are 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 work.

[0028] Figure 1 This is a diagram showing the adsorption effect of Lactobacillus curvatus on microplastic PS according to Example 1 of the present invention; Figure 2 This is a diagram showing the adsorption effect of Lactobacillus curvatus on microplastic PE according to Example 1 of the present invention; Figure 3 This is a colony morphology diagram of Lactobacillus curvatus according to Example 2 of the present invention; Figure 4 This is a Gram staining display of Lactobacillus curvatus according to Example 2 of the present invention; Figure 5 This is a schematic diagram of the phylogenetic tree of Lactobacillus curvatus based on 16S rRNA sequences in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] Considering the existing technology, there is no bacterial strain that can have a significant adsorption and degradation effect on microplastics PS and PE while also having a significant degradation effect on uric acid and being able to tolerate gastric acid and bile salts.

[0031] The present invention provides the following operation examples and embodiments: The specific process of isolating and screening Lactobacillus curvatum MPA01 from oysters in the waters of Xiamen, Fujian Province is as follows: Oyster samples were collected aseptically from fishermen in the waters of Xiamen, Fujian Province. Using the spread plate method, the oyster samples were placed in sterile homogenizing bags, marked, and 30 mL of 0.85% saline was added and thoroughly patted to mix.

[0032] Then, 1 mL of the sample was drawn for 10-fold gradient dilution. 3 , 10 4 , 10 5 200 μL of the sample was spread onto an MRS plate containing 2.5% CaCO₃ and incubated inverted at 37°C for 48 h. Well-growing colonies with large calcium-dissolving zones were selected and repeatedly isolated and purified by streak plate separation until a single colony was obtained. This isolate was designated MPA01 and stored in a glycerol stock at -80°C in a bacterial library.

[0033] The formula of MRS solid medium is as follows: glucose 20.0 g, agar powder 15.0 g, tryptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, anhydrous sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, ammonium citrate 2.0 g, magnesium sulfate 0.5 g, manganese sulfate monohydrate 0.25 g, Tween-80 1.0 mL, deionized water 1 L, pH 6.5 (MRS liquid medium is obtained by removing 15.0 g of agar powder).

[0034] Example 1 Screening of strains that efficiently adsorb microplastics polystyrene (PS) and polyethylene (PE) Twenty-five strains of lactic acid bacteria isolated from marine samples and stored in glycerol tubes were activated for two generations and inoculated into MRS liquid medium at a 3% (v / v) inoculum. The culture was cultured at 37°C for 24 h. The culture was centrifuged at 5000 r / min and 4°C for 10 min, the supernatant was discarded, and 500 μL of sterile PBS buffer was added for washing twice. PBS was then added to resuspend the culture to adjust the bacterial concentration to 1×10 9 CFU / mL.

[0035] 1. Determination of microplastic polystyrene (PS) adsorption For the blank control group A, 100 μL of PBS and 900 μL of PS fluorescent microsphere working solution (0.16 mg / mL, particle size 5 μm, Aladdin) were added to a 1.5 mL EP tube and mixed and incubated. For the experimental group B, 100 μL of each test bacterial suspension was added to a 1.5 mL EP tube, 900 μL of PS fluorescent microsphere working solution was added and mixed, and then placed on a shaker in the dark and incubated for 4 hours at 37°C and 300 rpm. After the incubation, the incubation solution was observed as follows: Figure 1 .

[0036] The incubation solutions of blank control group A and experimental group B were centrifuged at 2000 rpm for 10 minutes. 100 μL of the supernatant was then placed in a 96-well microplate. The fluorescence intensity was measured using a microplate reader. The results are shown in Table 1. The microplate reader parameters were: excitation wavelength: 488 nm; detection wavelength: 518 nm. The adsorption rate was calculated based on the fluorescence intensity values ​​using the following formula: Adsorption rate (%) = (AB) / A × 100% (1) (A: fluorescence value of blank control group, B: fluorescence value of experimental group).

[0037] Table 1 Adsorption determination of microplastic PS

[0038] After the experimental data were processed by formula (1), it can be seen from Table 1 that the adsorption rate of microplastic PS by the lactic acid bacteria numbered MPA01 extracted, separated, purified and cultured from the oyster sample was 42.80%, which is relatively high. Figure 1 It can be seen that in the lactic acid bacteria blank control group numbered MPA01, the PS fluorescent microspheres at the bottom of the EP tube did not self-aggregate; in the experimental group, adsorbed and aggregated flocs of MPA01 and PS fluorescent microspheres appeared at the bottom of the EP tube, indicating that MPA01 has a certain adsorption effect on microplastic PS, so MPA01 was selected for further experimental research.

[0039] 2. Determination of microplastic polyethylene (PE) adsorption In the blank control group A, 100 μL of PBS and 900 μL of PE fluorescent microsphere working solution (0.16 mg / mL, particle size 0.5 μm, Zhongke Keyou) were added to a 1.5 mL EP tube and mixed and incubated. In the experimental group B, 100 μL of each test bacterial suspension was added to a 1.5 mL EP tube, 900 μL of PE fluorescent microsphere working solution was added and mixed, and then placed on a shaker in the dark and incubated for 4 hours at 37 ° C and 300 rpm. After the incubation, the incubation solution was observed as follows: Figure 2 .

[0040] The incubation solutions of blank control group A and experimental group B were centrifuged at 2000 rpm for 10 min. 100 μL of the supernatant was placed in a 96-well microplate and the fluorescence intensity was measured using a microplate reader. The parameters of the microplate reader were: excitation wavelength: 488 nm; detection wavelength: 520 nm. The adsorption rate was calculated based on the fluorescence intensity value. The adsorption rate calculation formula is shown in (1).

[0041] The experimental data were processed by formula (1), and the adsorption rate of MPA01 on microplastic PE was 22.13%. Figure 2 It can be seen that in the blank control group, the PE fluorescent microspheres at the bottom of the EP tube did not self-aggregate; in the experimental group, adsorbed and aggregated flocs of MPA01 and PE fluorescent microspheres appeared at the bottom of the EP tube, indicating that MPA01 has a certain adsorption effect on microplastic PE.

[0042] 3. Determination of adsorption of microplastics polystyrene (PS) and polyethylene (PE) by MPA01 and MPs-68 composite MPA01 and MPs-68 were inoculated into MRS liquid medium at a 3% (v / v) inoculum, respectively, and cultured at 37°C for 24 h. The activated second-generation fermentation broth was centrifuged at 5000 r / min and 4°C for 10 min, the supernatant was discarded, and 500 μL of sterile PBS buffer was added for washing twice. The MPA01 and MPs-68 bacterial pellets were then resuspended in PBS and mixed, shaken evenly, and the concentration of the composite bacterial solution was adjusted to 1×10 9 CFU / mL is reserved.

[0043] The composite bacterial suspensions of MPA01 and MPs-68 were subjected to the aforementioned adsorption assay for microplastics polystyrene (PS) and polyethylene (PE). The adsorption rate was calculated based on the fluorescence intensity values. The results are shown in Table 2, and the adsorption rate calculation formula is shown in (1).

[0044] Table 2 Adsorption of microplastics PS and PE by MPA01 and MPs-68 composites

[0045] According to formula (1), Table 2 shows that the adsorption rate of microplastics PS by MPA01 alone was 42.80%, and the adsorption rate of PE was 22.13%. The adsorption rate of microplastics polystyrene (PS) by the composite bacteria of MPA01 and MPs-68 was (66.25±0.49)%, and the adsorption rate of polyethylene (PE) was (52.70±0.71)%. This shows that the adsorption rate of microplastics PS and PE by the composite bacteria of MPA01 and MPs-68 was significantly improved ( p <0.05), showing a synergistic effect, especially for PE adsorption.

[0046] Example 2 Identification of strain MPA01 1. Colony morphology identification Take one tube of glycerol bacteria numbered MPA01 from the bacterial library, inoculate it into MRS liquid culture medium at a 2% (v / v) inoculation rate for activation, and culture it in a shaking incubator at 37°C and 180 rpm for 24 hours. Then, streak it on an MRS solid culture medium plate and culture it upside down in a constant temperature incubator at 37°C for 24 hours to obtain a single colony.

[0047] Its colony morphology is Figure 3 As shown, it is oval, light white, with a smooth and raised surface, neat edges, and opaque.

[0048] 2. Physiological and biochemical tests The strain MPA01 isolated and purified by the plate streak method was selected for single colonies with good growth, and Gram staining and catalase test were performed. Its physiological and biochemical indices were measured. The test results were compared with the "Bergey's Manual of Systematic Bacteriology, 8th Edition" to make a preliminary identification of the strain species. Specifically, the strain MPA01 was examined by Gram staining microscopy as follows: Figure 4 As shown, it is purple, positive, and its cell shape is slender, curved, and slender rods. It is negative for catalase and oxidase, has no flagella, and does not form spores.

[0049] 3. 16S rRNA identification 1) Extract the strain gene DNA. The specific process is as follows: Step 1: Take 2 mL of bacterial culture medium, centrifuge at 12000 rpm for 1 min, remove the supernatant, and retain the bacteria; Step 2: Add 200 μL of lysozyme (final concentration: 20 mg / mL) to the cells and incubate at 37°C for at least 30 min (lysozyme buffer: 20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton X-100). Step 3: Add 200 μL of solution A to the cells, oscillate or pipette to fully suspend the cells, add 20 μL of RNase A (10 mg / mL) to the suspension, mix thoroughly by inversion, and let it stand at room temperature for 15-30 minutes; Step 4: Add 20 μL of proteinase K (10 mg / mL) to the tube, mix thoroughly, and digest at 55°C for 30-60 min. During the digestion period, invert the tube several times to mix until the sample is completely digested and the bacterial solution becomes clear and viscous. Step 5: Add 200 μL of solution B to the tube and mix thoroughly by inversion. If a white precipitate appears, place it at 75°C for 15-30 minutes. The precipitate will disappear and will not affect subsequent experiments. Step 6: Add 200 μL of anhydrous ethanol to the tube and mix thoroughly. Flocculent precipitates may appear at this time, which does not affect DNA extraction. Add both the solution and flocculent precipitates to the adsorption column and let it stand for 2 minutes. Step 7: Centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column into the collection tube; Step 8: Add 600 μL of rinse solution to the adsorption column (check and add 60 mL of anhydrous ethanol before use). Centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and place the adsorption column in a collection tube. Step 9: Add 600 μL of rinse solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube; Step 10: Centrifuge at 12,000 rpm for 2 minutes. Leave the adsorption column open at room temperature for 15 minutes to remove any remaining rinse solution. Otherwise, the ethanol in the rinse solution may affect subsequent experiments such as enzyme digestion and PCR. Step 11: Place the adsorption column in a clean centrifuge tube, add 100 μL of eluent preheated in a 65°C water bath to the center of the adsorption membrane, let it stand at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 1 minute; Step 12: Add the eluate obtained by centrifugation to the adsorption column, place it at room temperature for 2 minutes, and centrifuge it at 12000 rpm for 2 minutes to obtain bacterial genomic DNA.

[0050] 2) PCR amplification of the 16S rRNA gene was performed using the gene DNA as a template.

[0051] The universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') were used as amplification primers.

[0052] The PCR reaction system was: DNA 2 μL, 27F 2 μL, 1492R 2 μL, Premix Ex Taq 25 μL, and ddH2O 19 μL.

[0053] PCR reaction conditions: pre-denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 5 min.

[0054] The DNA genome of strain MPA01 was extracted according to the above method, and then PCR amplification was performed. The PCR amplification product was subjected to 16S rRNA sequencing (the sequencing company was Fuzhou Qingke Biotechnology Co., Ltd.).

[0055] The 16S rRNA sequencing results were searched for similar sequences in the NCBI database using Blast software. The strain MPA01 sequence was compared with the 16S rRNA gene sequences of related species obtained from the gene bank. The phylogenetic tree was constructed using Mega7.0 software. The phylogenetic tree results are shown in the figure. Figure 5 The gene sequence is as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1).

[0056] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0057] The results of 16S rRNA gene sequencing are as follows:

[0058] In summary, based on the comprehensive analysis of the colony morphology, cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence and other data of the strain MPA01, with reference to the "Bergey's Manual of Systematic Bacteriology, 8th Edition", the strain MPA01 was identified as Lactobacillus curvatus ( Latilactobacillus curvatus ).

[0059] Example 3 Determination of Antioxidant Capacity of Lactobacillus curvatus MPA01 Lactobacillus curvatum MPA01 was inoculated into MRS medium and activated for 2 generations at a 3% (v / v) inoculum. The culture was cultured at 37°C for 24 h. The cultured bacteria were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with 500 μL of sterile PBS buffer to adjust the bacterial concentration to 1×10 9 CFU / mL.

[0060] In the experimental group, 500 μL of bacterial suspension was added to 500 μL of a 0.2 mmol / L 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) ethanol solution. In the control group, 500 μL of PBS was added to 500 μL of a 0.2 mmol / L DPPH ethanol solution. In the blank group, 500 μL of bacterial suspension was added to 500 μL of anhydrous ethanol solution. Mix thoroughly and incubate in a 30°C shaker in the dark for 30 minutes. After shaking, the reaction mixture was centrifuged at 4000 rpm for 10 minutes. The absorbance of 100 μL of the supernatant was measured at 517 nm using a microplate reader. Repeat twice to calculate the DPPH radical scavenging rate. The test results are shown in Table 3.

[0061] The calculation formula is shown in (2): DPPH clearance rate (%) = [1-(AB) / C] × 100%. (2) (A: fluorescence value of the experimental group; B: fluorescence value of the blank group; C: fluorescence value of the control group).

[0062] Table 3 Determination of antioxidant capacity of MPA01

[0063] According to formula (2) and Table 3, the DPPH free radical scavenging rate of Lactobacillus curvatum MPA01 was (93.12±2.2)%, and its antioxidant capacity was significant.

[0064] Example 4 In vitro digestion resistance test of strain MPA01 in simulated gastric and intestinal fluids 1. Preparation of bacterial suspension of strain MPA01 After two generations of activation of strain MPA01 at a 3% (v / v) inoculum, the strain was inoculated into MRS liquid medium and cultured at 37°C for 24 hours. The strains were collected by centrifugation at 8000 rpm for 15 minutes at 4°C. After washing twice with 0.85% sterile saline, the strains were resuspended to adjust the concentration of MPA01 to 1x10 10 CFU / mL and reserve for use.

[0065] 2. Simulated gastric juice acid resistance test Preparation of simulated gastric fluid: Pepsin (p7000) was prepared into a pepsin solution with a concentration of 2 g / L using sterilized 0.85% saline, and the pH value was adjusted to 3.0 with 4 mol / L hydrochloric acid solution. The solution was then filtered through a 0.22 μm microporous filter membrane for sterilization before use.

[0066] The MPA01 bacterial suspension prepared above was shaken and 1 mL was pipetted into 9 mL of simulated gastric fluid. After thorough mixing, 1 mL of the mixture was diluted 10-fold to a certain gradient. After shaking, 1 mL was taken and the initial viable bacterial count was determined using the pour plate method. The remaining mixture was incubated at 37°C for 2 hours, and the viable bacterial count was determined using the same method. The survival rate was calculated. The results are shown in Table 4. The formula is as follows: Strain survival rate = (logN1 / logN0) × 100% (3) Where: N0 is the initial viable bacterial count after treatment with simulated gastric fluid, CFU / ml; N1 is the viable bacterial count after 2 hours of incubation in simulated gastric fluid, CFU / ml.

[0067] (3) Determination of bile salt tolerance in simulated intestinal fluid Preparation of simulated intestinal fluid: Trypsin (USP (P7545)) was prepared into a 1 mg / mL solution with sterilized 0.85% saline, 0.2% ox bile salt was added, and the pH was adjusted to 8.0 with 1 mol / L sodium hydroxide. The solution was then sterilized by filtration through a 0.22 μm microporous filter membrane before use.

[0068] After incubating in simulated gastric fluid for 2 hours, 1 mL of the MPA01 mixture was shaken and added to 9 mL of simulated intestinal fluid. Mix thoroughly and incubate at 37°C for 1 hour. Then, 1 mL of the mixture was diluted tenfold to a specific gradient. After shaking, 1 mL was taken and the viable bacterial count was determined using the pour plate method. The survival rate was calculated. The results are shown in Table 4. The formula is as follows: Strain survival rate = (logN2 / logN1) × 100% (4) Where: N1 is the number of viable bacteria after 2 hours of incubation in simulated gastric fluid, CFU / ml; N2 is the number of viable bacteria after 1 hour of incubation in simulated intestinal fluid, CFU / ml.

[0069] Table 4 MPA01 in vitro simulated gastric and intestinal fluid digestion resistance test

[0070] As shown in Table 4, strain MPA01 grew well in simulated gastric fluid, with a survival rate of 98.8% after 2 hours of incubation. This indicates that after strain MPA01 enters the gastric fluid environment, the lower pH in the gastric fluid only kills a small number of cells, while the majority survive. This indicates that strain MPA01 has strong tolerance to both acid and pepsin, allowing it to survive well in the simulated gastric fluid environment. After 2 hours of incubation in the simulated gastric fluid environment, strain MPA01 enters the intestinal fluid environment, where it has a survival rate of 97.6% after 1 hour of incubation. This indicates that after strain MPA01 enters the intestinal fluid environment, the high bile salt concentration in the intestinal fluid only kills a small number of cells, while the majority survive. This indicates that strain MPA01 can tolerate the high bile salt environment in the intestine and is resistant to trypsin, allowing it to survive well in the simulated intestinal fluid environment.

[0071] In summary, the strain Lactobacillus curvatum MPA01 of the present invention thrived after a series of digestions in simulated gastric and intestinal fluids, and was able to survive well in the intestines. Since the ability of a strain to enter and colonize the intestines is a prerequisite for its effectiveness, the strain MPA01 of the present invention has high intestinal application value.

[0072] Example 5 Determination of the ability of strain MPA01 to degrade uric acid 1. Determination of xanthine oxidase inhibition activity The strain MPA01 was activated to the second generation, cultured at 37°C for 24 h, and then centrifuged at 12000 rpm / min to discard the supernatant. 500 μL of PBS buffer (pH 7.0) was added to resuspend the bacteria as the sample solution to be tested.

[0073] The reaction was carried out in a PBS buffer system with a pH of 7.0. The amount of reagent added for each test item is shown in Table 5. Taking the addition process of test item A as an example: 125 μL of the sample solution to be tested, 100 μL of a 0.1 U / mL xanthine oxidase solution, and 575 μL of PBS buffer were added to the test tube. The mixture was shaken for 30 seconds and reacted at 37°C for 10 minutes. Then, 200 μL of a 2 mmol / L xanthine solution was added. The mixture was shaken for 30 seconds and reacted at 37°C for 10 minutes. The supernatant was centrifuged and the absorbance at 295 nm was measured. This was repeated three times. The xanthine oxidase inhibition rate was calculated. The test results are shown in Table 6. The oxidase inhibition rate is shown in formula (5): Xanthine oxidase inhibition rate (%) = [1-(AB) / (CD)] × 100% (5) Table 5 Xanthine oxidase inhibition activity assay system configuration

[0074] Table 6 MPA01 inhibition of xanthine oxidase activity

[0075] According to formula (5) and Table 6, the inhibition rate of strain MPA01 on xanthine oxidase was (82.94±0.17)%, which had a very significant inhibitory effect on xanthine oxidase.

[0076] 2. Determination of uric acid degradation ability The strain MPA01 was activated to the second generation and inoculated into MRS liquid culture medium supplemented with 700 μmol / L uric acid at a 3% (v / v) inoculum. A blank control group without the strain was set up and cultured at 37°C for 24 h. After centrifugation at 12,000 rpm / min, the supernatant was taken as the test solution. The reaction reagents were added according to the operating system instructions of the Nanjing Jiancheng Uric Acid Detection Kit in Table 7, mixed, and incubated at 37°C for 10 min. The absorbance A was read at a wavelength of 510 nm to determine the initial uric acid content and the residual uric acid content, as shown in formula (6). The concentration of the C calibrator was 400 μmol / L. The uric acid degradation rate was calculated as shown in formula (7). The test results are shown in Table 8.

[0077] UA (μmol / L) = [(A 测定 -A 空白 ) / (A 标准 -A 空白 )] C 校准品 (6) Uric acid degradation rate (%) = (UA 初始- UA 剩余 ) / UA 初始 100%(7) Table 7 Operating System Instructions

[0078] Table 8 Determination of uric acid degradation ability of MPA01

[0079] According to formulas (6) and (7), Table 8 shows that the degradation rate of uric acid by strain MPA01 is 80.54%, which shows that it has a significant degradation effect on uric acid.

[0080] Example 6 Preparation of a bacterial agent using the present invention's Lactobacillus curvatum MPA01 Lactobacillus curvatum MPA01 was inoculated at 3% (v / v) of the culture medium mass into MRS liquid medium sterilized at 121°C for 15 minutes. 12% enzymatically hydrolyzed skim milk, 0.8% glucose, 1.5% soy peptone, 0.6% yeast extract, and the remainder water were added to the total mass of the culture medium, and the pH was adjusted to 6.8. Then, after culturing at 37°C for 48 hours, the culture was centrifuged at 6000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the culture was washed 2-4 times with pH = 7.2 phosphate buffer to obtain bacterial sludge, which was then resuspended in a protective agent to a concentration of 10 11 CFU / mL. The protective agent contained 150 g / L skim milk, 40 mL / L glycerol, 20 g / L sorbitol, 150 g / L trehalose, and 10 g / L sodium L-glutamate. Finally, the suspension was pre-incubated at 37°C for 60 minutes, then pre-frozen at -40°C for 2 hours and freeze-dried. The survival rate of MPA01 cells was calculated after freeze-drying.

[0081] The calculation formula for bacterial survival rate is: Bacterial survival rate (%) = (N / N0) × 100% (8) In formula (8), N is the number of viable bacteria after reconstitution of freeze-dried bacterial powder, CFU / mL; N0 is the number of viable bacteria in the bacterial suspension before freeze-drying, CFU / mL.

[0082] The data obtained from the above experiment were obtained by formula (8), and the cell survival rate of the freeze-dried strain MPA01 of the present invention was 93.5%, which well maintained the activity of the strain.

[0083] Example 7 Preparation of probiotic products using Lactobacillus curvatum MPA01 of the present invention The probiotic product is a composition of Lactobacillus curvatus and other ingredients; in the product, the number of viable Lactobacillus curvatus is not less than 1×10 11 CFU / mL or 1×10 11 CFU / g.

[0084] The ingredients may include one or more of prebiotics, fillers, acidulants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, and filter aids.

[0085] The dosage form of the probiotic product can be compressed candy, solid beverage, beverage, granule, capsule, tablet, pill or oral liquid, etc.

[0086] 7.1 Lactobacillus curvatum MPA01 Compressed Candy In this embodiment, a Lactobacillus curvatum MPA01 tablet candy is provided, which is prepared according to the following steps: Preparation of active bacterial powder: culturing the Lactobacillus curvatum MPA01 strain in a liquid culture medium, collecting and washing the bacterial cells, adding auxiliary materials, and drying to prepare active bacterial powder; Take 4 parts of bacterial powder, 60 parts of sorbitol, 15 parts of whole milk powder, 6 parts of fermented kiwi powder, 2 parts of isomaltooligosaccharide, 2 parts of magnesium stearate, 0.3 parts of vitamin C, 1 part of galacto-oligosaccharide, 1 part of fructo-oligosaccharide, and 1 part of polydextrose, mix well to obtain a pre-made powder; The prefabricated powder is tableted and packaged using a candy tableting device to obtain a Lactobacillus curvatus tablet candy. The number of viable Lactobacillus curvatus in the tablet candy is not less than 1×10 11 CFU / g.

[0087] 7.2 Lactobacillus curvatum MPA01 Probiotic Solid Beverage In this embodiment, a probiotic solid beverage of Lactobacillus curvatum MPA01 is also provided, which is prepared according to the following steps: Preparation of active bacterial powder: activating the Lactobacillus curvatum MPA01 and culturing it in MRS liquid medium, collecting and washing the bacterial cells, adding auxiliary materials, and drying to obtain active bacterial powder; 20% active bacteria powder, 15% bonito peptide, 10% erythritol, 15% skim milk powder, 15% fermented kiwi fruit powder, 10% maltodextrin, 5% fructooligosaccharide, 5% galacto-oligosaccharide and 5% isomaltooligosaccharide are taken by weight, mixed evenly, and packaged to obtain a Lactobacillus curvatum MPA01 probiotic solid beverage; The viable count of Lactobacillus curvatum MPA01 is not less than 1×10 10 CFU / g.

[0088] It should be noted that the above concept is to prepare a probiotic product by combining Lactobacillus curvatus and other ingredients, and the selection of ingredients and the ratio of the ingredient components can be adaptively adjusted, including but not limited to the embodiments.

[0089] Example 8 Fermentation product of Lactobacillus curvatum MPA01 Preparation of active bacterial powder: activating the Lactobacillus curvatum MPA01 and culturing it in MRS liquid medium, collecting and washing the bacterial cells, adding auxiliary materials, and drying to obtain active bacterial powder; Mix 2.0 kg of water, 0.5 kg of concentrated blueberry juice, and 0.6 kg of concentrated pineapple juice, inoculate 63 g of Lactobacillus curvatus MPA01 powder, and ferment at 32°C for 48 hours. The pH at the fermentation end point is 3.62 to obtain a Lactobacillus curvatus MPA01 fermentation product. The number of viable Lactobacillus curvatus MPA01 in the fermentation product is ≥ 1 × 10 11 CFU / g or 1×10 11 CFU / mL.

[0090] It should be noted that the above concept is to add active bacterial powder of Lactobacillus curvatum MPA01 or other forms of Lactobacillus curvatum MPA01 to the fermentation system to obtain a fermentation product. The selection and ratio of the various components of the fermentation culture medium in the fermentation system can be adaptively adjusted, including but not limited to concentrated blueberry juice, concentrated pineapple juice, etc. in the embodiment scheme.

[0091] Example 9: Probiotic preparation of Lactobacillus curvatum MPA01 The present invention provides a microecological preparation, the components of which include active bacterial powder of Lactobacillus curvatum MPA01 and / or fermentation liquid of Lactobacillus curvatum MPA01; The active bacterial powder of Lactobacillus curvatus MPA01 is vacuum freeze-dried bacterial powder and / or spray-dried bacterial powder, and the number of viable bacteria is ≥1×10 11 CFU / g; The Lactobacillus curvatum MPA01 fermentation broth includes any one or a combination of multiple of a fermentation supernatant, a cell-free extract, and bacterial cell metabolites.

[0092] Example 10: Microplastic Adsorbent The present invention provides a microplastic adsorbent, the components of which include Lactobacillus curvatum MPA01 or a composite bacteria including Lactobacillus curvatum MPA01 and Lactobacillus plantarum MPs-68.

[0093] 10.1 Lactobacillus curvatum MPA01 Microplastic Adsorbent In this embodiment, a microplastic adsorbent of Lactobacillus curvatum MPA01 is provided, which is prepared according to the following steps: A single colony of Lactobacillus curvatus MPA01 was selected and inoculated into MRS liquid medium and cultured at 37°C for 24 h to obtain an activated solution. The activated solution was inoculated into MRS liquid medium at a rate of 3% (v / v) and cultured at 37°C for 20 h to obtain the first-level seed solution. The first-level seed solution was inoculated into MRS liquid medium at a rate of 3% (v / v) and cultured at 37°C for 20 h to obtain the second-level seed solution; The secondary seed liquid was inoculated into MRS liquid medium at a rate of 3% (v / v) and cultured at 37°C for 20 h to obtain bacterial liquid; The bacterial solution was centrifuged at 4°C and 6000 rpm for 10 min to collect the precipitate; The precipitate was washed twice with sterile PBS buffer (pH = 7.4), centrifuged again at 4°C and 6000 rpm for 10 min, the supernatant was discarded, and the bacteria were collected; A protective agent solution containing 100 g / L skim milk powder, 30 g / L crystalline trehalose, 20 g / L fructooligosaccharide and 10 g / L sodium glutamate was emulsified and mixed with the Lactobacillus curvatum MPA01 bacteria in a weight ratio of 1:1, and freeze-dried to obtain a Lactobacillus curvatum MPA01 microplastic adsorbent; Among them, the number of viable Lactobacillus curvatum MPA01 was ≥1×10 11 CFU / g.

[0094] 10.2 Composite Microplastic Adsorbent of Lactobacillus curvatum MPA01 and Lactobacillus plantarum MPs-68 In this embodiment, a composite microplastic adsorbent of Lactobacillus curvatum MPA01 and Lactobacillus plantarum MPs-68 is also provided, which is prepared according to the following steps: Single colonies of Lactobacillus curvatus MPA01 and Lactobacillus plantarum MPs-68 were selected and inoculated into MRS liquid culture medium, and cultured at 37°C for 24 h to obtain activation solution. The activated solution was inoculated into MRS liquid medium at a rate of 3% (v / v) and cultured at 37°C for 20 h to obtain the primary seed solution. The first-level seed solution was inoculated into MRS liquid medium at a 3% (v / v) inoculum and cultured at 37°C for 20 h to obtain the second-level seed solution. The secondary seed solution was inoculated into the same MRS liquid medium at a 3% (v / v) inoculation rate and cultured at 37°C for 20 h to obtain the composite bacterial solution. The composite bacterial solution was centrifuged at 4°C and 6000 rpm for 10 min to collect the precipitate; The precipitate was washed twice with sterile PBS buffer (pH = 7.4), centrifuged again at 4 °C and 6000 rpm for 10 min, the supernatant was discarded, and the composite bacterial cells were collected; A protective agent solution containing 100 g / L skim milk powder, 30 g / L crystalline trehalose, 20 g / L fructooligosaccharide and 10 g / L sodium glutamate was emulsified and mixed with the composite bacteria in a weight ratio of 1:1, and then freeze-dried to obtain a composite microplastic adsorbent of Lactobacillus curvatum MPA01 and Lactobacillus plantarum MPs-68. Among them, the number of viable bacteria of the composite bacteria is ≥1×10 11 CFU / g.

[0095] It should be noted that in this embodiment, the number of viable bacteria of Lactobacillus plantarum MPs-68: the number of viable bacteria of Lactobacillus curvatus MPA01 in the composite bacteria is 1:1. Those skilled in the art can adaptively adjust the ratio according to the functional effects of Lactobacillus curvatus MPA01 and Lactobacillus plantarum MPs-68 provided by the present invention to meet corresponding needs.

[0096] In summary: The Lactobacillus curvatus of the present invention is a natural strain of marine origin and has high food safety. It has a relatively obvious adsorption effect on microplastics polystyrene (PS) and polyethylene (PE), and has significant antioxidant capacity, is resistant to gastric acid and bile salts, and has good intestinal colonization ability. It also has a good ability to degrade uric acid. Therefore, it has important application value in the fields of food or health products such as auxiliary adsorption of microplastics, anti-oxidation, and uric acid reduction. For example: (1) Lactobacillus curvatum is used as a raw material component of the bacterial agent to prepare a bacterial agent with the above functions. (2) obtaining a fermentation product having the above-mentioned function by fermenting Lactobacillus curvatum; (3) Lactobacillus curvatus is used as a raw material component of a probiotic preparation to prepare a probiotic preparation having the above-mentioned efficacy. The Lactobacillus curvatus may be present in the probiotic preparation in the form of fermentation liquid and / or bacterial powder of Lactobacillus curvatus MPA01.

[0097] Similarly, since Lactobacillus curvatum MPA01 has the properties of adsorbing microplastics polystyrene and polyethylene, anti-oxidation, acid and bile resistance, and degrading uric acid, it can be used in functional products including probiotic products, functional foods that assist in the adsorption of microplastics, antioxidant foods, acid and bile resistant foods, uric acid-lowering functional foods, etc.

[0098] The Lactobacillus curvatum MPA01 provided by the present invention has the following beneficial effects: The Lactobacillus curvatum MPA01 provided by the present invention is isolated and screened from oyster samples aseptically collected by marine fishermen in Xiamen, Fujian Province. It is a natural marine strain with high food safety. The adsorption rate of Lactobacillus curvatus MPA01 on microplastics PS was 42.80%, and the adsorption rate on PE was 22.13%, while the adsorption rate of the composite bacteria of MPA01 and MPs-68 on microplastics PS was 66.25% ( p<0.05 ), the adsorption rate for PE is 52.70% ( p <0.05). Compared with the adsorption rate of Lactobacillus curvatus MPA01 alone on microplastics PS and PE, the adsorption rate of the composite bacteria of MPA01 and MPs-68 on microplastics PS and PE was significantly improved, showing a synergistic effect, especially the synergistic effect on PE adsorption was more significant. It can be applied to the production of fermentation products and other products that adsorb microplastics.

[0099] The Lactobacillus curvatum MPA01 of the present invention has a DPPH free radical scavenging rate of (93.12±2.2)%, exhibits significant antioxidant capacity, and has good tolerance to gastric acid and bile salts, as well as the ability to colonize the intestine, thereby improving the structure of the intestinal flora and promoting intestinal motility. It also has a significant inhibitory effect on xanthine oxidase, with an inhibition rate of (82.94±0.17)%, and has a significant ability to degrade uric acid, with a degradation rate of 80.54%. It can be used in the production of uric acid-lowering fermented products or in probiotic products, and has high commercial value.

[0100] Although terms such as fermentation, microplastics, and functional foods are frequently used herein, the possibility of using other terms is not excluded. These terms are used only to more conveniently describe and explain the essence of the present invention; interpreting them as any additional limitations is contrary to the spirit of the present invention.

[0101] The Lactobacillus curvatum MPA01 provided by the present invention can be used as a raw material component in probiotic products, functional foods, and fermented products. Its benefits include, but are not limited to, microplastic adsorption, uric acid reduction, and antioxidant activities, all of which have beneficial effects on the human body. Product forms include solid preparations such as powders and tablets, or liquid preparations suitable for oral administration.

[0102] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Lactobacillus curvatus MPA01 capable of absorbing microplastics and degrading uric acid, characterized in that: Its deposit number is CGMCC No.31977.

2. A probiotic product of Lactobacillus curvatus MPA01, characterized in that: Its components include the Lactobacillus curvatus MPA01 with the ability to adsorb microplastics and degrade uric acid as described in claim 1.

3. A functional food of Lactobacillus curvatum MPA01, characterized in that: Its components include the Lactobacillus curvatus MPA01 with the ability to adsorb microplastics and degrade uric acid as described in claim 1.

4. The functional food of Lactobacillus curvatus MPA01 according to claim 3, characterized in that: The functional foods include one or more combinations of functional foods that assist in the adsorption of microplastics accumulated in the human body, antioxidant foods, acid-resistant and bile-resistant foods, and functional foods that degrade uric acid.

5. A fermentation product of Lactobacillus curvatus MPA01, characterized in that: Its components include the Lactobacillus curvatus MPA01 with the ability to adsorb microplastics and degrade uric acid as described in claim 1.

6. A bacterial agent of Lactobacillus curvatus MPA01, characterized in that: Its components include the Lactobacillus curvatus MPA01 with the ability to adsorb microplastics and degrade uric acid as described in claim 1.

7. The Lactobacillus curvatus MPA01 bacterial agent according to claim 6, characterized in that: The viable bacterial count of Lactobacillus curvatus MPA01 is ≥ 1×10 11 CFU / g or ≥1×10 11 CFU / mL.

8. A microecological preparation, characterized in that: Its components include the Lactobacillus curvatus MPA01 with the ability to adsorb microplastics and degrade uric acid as described in claim 1.

9. A microplastic adsorbent, characterized in that: Its components include Lactobacillus curvatus MPA01 or a composite bacteria including Lactobacillus curvatus MPA01 and Lactobacillus plantarum MPs-68; The viable bacterial count of Lactobacillus curvatus MPA01 is ≥ 1×10 9 CFU / mL or ≥1×10 9 CFU / g; The viable count of the composite bacteria is ≥ 1×10 9 CFU / mL or ≥1×10 9 CFU / g; The Lactobacillus curvatum MPA01 is the Lactobacillus curvatum MPA01 with the ability to adsorb microplastics and degrade uric acid as described in claim 1; the preservation number of the Lactobacillus plantarum MPs-68 is CGMCC No.31976.

10. Use of the microplastic adsorbent as claimed in claim 9 in adsorbing microplastic polystyrene and / or polyethylene.

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