Lactobacillus paracasei Lpc-14 strain with functions of resisting oxidation and promoting gastrointestinal motility and preparation method of lactobacillus paracasei Lpc-14 strain

Abstract

The invention belongs to the technical field of microorganisms, and relates to a lactobacillus paracasei Lpc-14 strain with functions of resisting oxidation and promoting gastrointestinal motility and a preparation method of the lactobacillus paracasei Lpc-14, the lactobacillus paracasei is classified and named as Lactobacillus paracasei, the preservation number of the lactobacillus paracasei is CGMCC No. 29666, the preservation time is January 18, 2024, and the preservation number of the lactobacillus paracasei Lpc-14 is CGMCC No. 29666. The preservation unit is China General Microbiological Culture Collection Center, and the address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain is separated and purified from naturally-fermented Chinese sauerkraut fermentation liquor, has the capacity of resisting oxidation and promoting gastrointestinal motility of mice with constipation and improving secretion of gastrointestinal regulating peptides, further has the capacity of resisting acid and alkali and resisting artificial simulated gastrointestinal fluid and bile, and has the characteristics of probiotics.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Lactobacillus paracasei Lpc-14 with antioxidant and gastrointestinal motility-promoting functions and its preparation method. Background Technology

[0002] The number of people suffering from constipation is increasing due to irregular eating habits, mental stress, and other factors. Constipation sufferers commonly experience slow intestinal motility and gut microbiota imbalance. Studies have shown that probiotics can increase the secretion of regulatory peptides in the gastrointestinal tract, promote normal intestinal motility and mucus secretion, and are significantly effective in improving constipation symptoms, and are currently widely used in constipation treatment. However, probiotics have the following problems in improving constipation: some probiotics lack the ability to synthesize active electron chains, affecting their survival in aerobic environments; and the presence of excessive free radicals in the intestines affects intestinal health. Therefore, it is necessary to screen for probiotics that possess both antioxidant properties and the ability to improve intestinal motility, so that probiotics can better relieve constipation.

[0003] Free radicals are constantly generated during bodily metabolism. Excessive accumulation disrupts the redox balance, leading to DNA hydroxylation, protein denaturation, and lipid peroxidation. This, in turn, can trigger apoptosis, cause bodily damage, and affect intestinal function, exacerbating intestinal problems in constipated patients. Lactic acid bacteria, by regulating the levels of antioxidant enzymes and scavenging free radicals, reduce free radical accumulation and minimize damage to the host gut. They play a crucial role in improving slow peristalsis caused by intestinal oxidative damage in constipated patients. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Lactobacillus paracasei Lpc-14 with antioxidant and gastrointestinal motility-promoting functions and its preparation method, so as to solve the above-mentioned problems.

[0005] This invention provides a strain of Lactobacillus paracasei Lpc-14 with antioxidant and gastrointestinal motility-promoting functions. The strain is Lactobacillus paracasei Lpc-14, classified as Lactobacillus paracasei, with accession number CGMCC No. 29666, deposited on January 18, 2024, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Furthermore, the Lactobacillus paracasei Lpc-14 strain was isolated and purified from the fermentation broth of naturally fermented sauerkraut.

[0007] The *Lactobacillus paracasei* Lpc-14 strain with antioxidant and gastrointestinal motility-promoting functions provided by this invention is prepared by the following method:

[0008] Chop the cabbage and bok choy and put them into a sterile container. Add salt water to submerge the vegetables, seal the container, and ferment in a cool, shady place indoors. During fermentation, open the lid to release the air. After 4 months of pickling, you will get naturally fermented sauerkraut.

[0009] The fermentation broth of naturally fermented sauerkraut was diluted with physiological saline and spread onto MRS agar medium, and incubated at 37°C for 48 hours.

[0010] Single colonies of different shapes and sizes were picked from the culture dish and purified by streaking multiple times on MRS agar medium until they were identified as single colonies. The single colonies were then examined under a microscope, and physiological, biochemical and molecular biological identifications were performed. The isolated strains were then preserved in cryogenic glycerol.

[0011] The present invention has the following technical effects:

[0012] 1. The Lactobacillus paracasei Lpc-14 of the present invention has both antioxidant function and the function of promoting gastrointestinal motility.

[0013] 2. The Lactobacillus paracasei Lpc-14 of this invention exhibits good resistance to acid and alkali, as well as tolerance to simulated gastrointestinal fluids and bile. When orally administered to the human body, it maintains a high survival rate in the gastrointestinal tract.

[0014] 3. The Lactobacillus paracasei Lpc-14 of this invention has strong antioxidant capacity, with scavenging abilities of 94.42±4.67%, 99.31±2.75%, 85.68±6.22%, and 95.39±5.68% for DPPH free radicals, hydroxyl free radicals, ABTS+ free radicals, and superoxide anion free radicals, respectively, and a total reducing power of 2.57±0.29.

[0015] 4. The Lactobacillus paracasei Lpc-14 of this invention can regulate the secretion of constipation-related gastrointestinal regulatory peptides (MLT, SP, VIP) in mouse serum, including promoting the secretion of excitatory neurotransmitters (MLT, SP) and reducing the secretion of inhibitory neurotransmitters (VIP). Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The colony morphology and Gram staining results of Lactobacillus paracasei Lpc-14 in culture dishes;

[0018] Figure 2The acid resistance of Lactobacillus paracasei Lpc-14;

[0019] Figure 3 The alkali resistance of Lactobacillus paracasei Lpc-14;

[0020] Figure 4 The ability of Lactobacillus paracasei Lpc-14 to tolerate artificially simulated gastrointestinal fluid and bile;

[0021] Figure 5 The results show the determination of the free radical scavenging rate of Lactobacillus paracasei Lpc-14 bacterial culture;

[0022] Figure 6 A comparison chart of MTL levels in the serum of mice in each group;

[0023] Figure 7 A comparison chart of SP levels in the serum of mice in each group;

[0024] Figure 8 This is a comparison chart of VIP levels in the serum of mice in each group. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] The present invention isolates Lactobacillus paracasei Lpc-14 from naturally fermented sauerkraut. It has strong antioxidant capacity, which can reduce the inhibition of peristalsis caused by intestinal oxidation. At the same time, it can regulate the secretion of gastrointestinal regulatory peptides and increase gastrointestinal motility. It can be used as a probiotic to relieve or treat constipation.

[0028] Example 1: Screening and identification of Lactobacillus paracasei Lpc-14

[0029] 1. Strains Isolation and Purification

[0030] Chop the cabbage and bok choy and place them in a sterile container. Add salt water to cover the vegetables, seal the container, and place it in a cool, dark place to ferment. During fermentation, open the lid to release the gas, and the mixture will become sauerkraut. Take 1.0 mL of sauerkraut juice, dilute it with physiological saline, and then take 100 μL of the diluted solution at 10:10 concentrations. -2 ~10 -7The fermentation broth was evenly spread onto MRS medium and incubated at 37°C for 48 hours. Single colonies of different morphologies and sizes were picked from the culture dishes and streaked multiple times on MRS agar medium for purification until a pure strain was confirmed.

[0031] 2. Microscopic examination

[0032] Single colonies were picked and inoculated into MRS broth medium. After culturing for 24 hours, Gram staining was performed. The bacterial culture was smeared, Gram-stained, and the cell morphology and arrangement were observed under a light microscope. The observations were recorded and photographed for preservation. Finally, Gram-positive cultures were identified as suspected lactic acid bacteria, and 1.0 mL of pure culture was transferred to a storage tube containing 50% glycerol for later use.

[0033] 3. Physiological and biochemical tests for identification

[0034] Physiological and biochemical tests, including hydrogen peroxide test, indole test, and carbohydrate utilization test, were used to identify and screen bacterial strains. The isolated and purified strain was white, with a raised center, a smooth and moist surface, and a diameter of 0.83±0.12 mm; the strain was Gram-positive, and the cells were short rod-shaped and arranged in pairs. Figure 1 Its physicochemical characteristics include a negative hydrogen peroxide test; a negative indole reaction; a positive aescin hydrolysis test; and the ability to ferment cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose.

[0035] 4. Molecular biological identification

[0036]

[0037] Example 2: Tolerance test of Lactobacillus paracasei Lpc-14

[0038] 1. Results of acid resistance test

[0039] After two generations of activation in MRS medium at 37℃, bacterial suspensions were prepared and resuspended in MRS medium with pH adjusted to 5.0, 4.0, 3.0, 2.0, and 1.0 using 4.0 mol / L HCl. The suspensions were then incubated at 37℃ for 24 h, and the bacterial suspensions were collected for OD measurement. 600 The nm value was calculated in triplicate, with the OD value of MRS medium (pH 6.0) as a control. The survival rate was then calculated. That is, survival rate = N. t / N0

[0040] Where: N t —OD values ​​of MRS culture media with pH values ​​ranging from 1.0 to 5.0;

[0041] N0—OD value of MRS medium at pH 6.0.

[0042] In healthy individuals, the pH of gastric juice on an empty stomach ranges from 0.9 to 1.8. After ingestion, the gastric juice pH changes to approximately 1.8 to 5.0. Therefore, this experiment selected a pH range of 1 to 5 to test the bacterial strain's tolerance to acid. The results are as follows... Figure 2 As shown, by Figure 2 It can be seen that *Lactobacillus paracasei* Lpc-14 exhibits poor tolerance at pH 1.0, with a survival rate of only 7.08% after 24 hours of treatment; however, it demonstrates strong tolerance at pH 4.0, with a survival rate of 51.85% after 24 hours of treatment. This indicates that the bacterium has a strong ability to tolerate acidic conditions.

[0043] 2. Alkali resistance test results

[0044] After two generations of activation in MRS medium at 37℃, bacterial suspensions were prepared and resuspended in MRS medium at pH values ​​of 11.0, 10.0, 9.0, 8.0, and 7.0. The suspensions were then incubated at 37℃ for 24 hours before the bacterial culture was collected; OD was measured. 600 The nm value was calculated in triplicate, with the OD value of MRS medium (pH 6.0) as a control. The survival rate was then calculated. That is, survival rate = N. t / N0, where: N t —OD values ​​of MRS culture media with pH values ​​ranging from 7.0 to 11.0;

[0045] N0—OD value of MRS medium at pH 6.0.

[0046] This experiment used alkaline conditions (pH 7-11) to test the strain's tolerance to alkali. The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that the survival rate of Lactobacillus paracasei Lpc-14 reached over 98% at pH 6-8. As the pH increased further, the survival rate decreased, but after 24 hours of cultivation at pH 11, the survival rate was still 67.43%. This indicates that this strain has a strong ability to tolerate alkaline conditions.

[0047] 3. Results of simulated gastrointestinal fluid tolerance test

[0048] Lactobacillus paracasei Lpc-14 strain was activated and passaged twice in MRS medium at 37℃. A bacterial suspension was prepared, resuspended in simulated gastric fluid, and treated at 37℃ for 3 hours. After centrifugation and discarding the supernatant, the bacterial sludge was collected and washed three times with sterile physiological saline. The sludge precipitate was collected using aseptic techniques, and physiological saline was added at a ratio of bacterial sludge to physiological saline of 1:5. The mixture was thoroughly diluted serially, and then colony counting was performed.

[0049] Take 1.0g of bacterial sludge after 3 hours of treatment with the gastric fluid, add it to simulated bile, and treat at 37℃ for 20 minutes. Centrifuge and discard the supernatant, collect the bacterial sludge, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of 1:5 to bacterial sludge, mix thoroughly, perform serial dilution, and count the colonies.

[0050] Take 1.0g of bacterial sludge after treating the bile for 20 minutes, add it to simulated intestinal fluid, and treat at 37℃ for 4 hours. Centrifuge and discard the supernatant, collect the bacterial sludge, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of bacterial sludge to physiological saline of 1:5, mix well, perform serial dilution, and count the colonies.

[0051] Survival rate of artificial gastrointestinal fluid and bile = N t / N0

[0052] Where: N t —The number of live bacteria after artificially simulating the treatment of gastric juice, bile, and intestinal juice;

[0053] N0—Number of viable bacteria before treatment.

[0054] Gastric juice contains mucus, gastric acid, pepsin, etc. For probiotics to exert their beneficial effects, they must survive in the stomach, meaning they need to be able to tolerate the acidic environment and resist pepsin. Food typically stays in the stomach for 3 hours; therefore, this invention uses simulated gastric juice to treat Lactobacillus paracasei Lpc-14 bacterial culture for 3 hours. After digestion in the stomach, food enters the intestines. The small intestine environment is weakly alkaline and contains trypsin and bile. Probiotics can achieve a viable count of 10⁻⁶ in the intestines. 6Probiotics need to have a concentration of at least CFU / mL to exert their probiotic function; therefore, they need to be able to tolerate the weakly alkaline environment of the intestine and resist trypsin and bile. Food typically stays in the small intestine for 3-8 hours. Therefore, this experiment used simulated intestinal fluid to treat *Lactobacillus paracasei* Lpc-14 bacterial culture for 4 hours. The results are as follows... Figure 4 As shown. By Figure 4 It can be seen that after 3 hours of treatment with gastric juice, the survival rate of *Lactobacillus paracasei* Lpc-14 was 82.77%, with a viable count of 15.162 ± 0.004 lg (cfu / g). After 20 minutes of treatment with bile, the survival rate was 81.59%, with a viable count of 14.946 ± 0.021 lg (cfu / g). After 4 hours of treatment with intestinal juice, the survival rate was still 71.29%, with a viable count of 13.059 ± 0.018 lg (cfu / g), indicating that *Lactobacillus paracasei* Lpc-14 has a strong ability to tolerate simulated gastrointestinal fluid.

[0055] Example 3: In vitro antioxidant capacity test of Lactobacillus paracasei Lpc-14

[0056] Table 1 lists the probiotic strains with antioxidant activity disclosed in existing research.

[0057] Table 1. Probiotic strains with antioxidant activity disclosed in existing studies.

[0058] Strain name Antioxidant degradation rate Reference Lactobacillus paracaseisubsp. Tolerans RQ1, Lactobacillus paracaseisubsp. Tolerans RQ10 The average DPPH free radical scavenging rates of RQ1 and RQ10 were 91.66% and 90.91%, respectively. Zhang J, Li L, Ren Q, et al. Study on the probiotic properties and antioxidant activity of lactic acid bacteria in Inner Mongolia cheese [J]. China Condiment, 2025, 50(4): 36-43. Lactobacillus XBMU436-2 The DPPH free radical, hydroxyl radical, and superoxide anion radical scavenging rates were 57.55±0.54%, 80.64±0.39%, and 83.83±0.20%, respectively. Shi S, Guo X, Ma J, et al. Optimization of GABA-producing lactic acid bacteria process and antioxidant activity of the product [J]. Food Industry, 2023, 44(05): 86-91. Lactobacillus pentosus AC9 The DPPH free radical, hydroxyl radical scavenging rates, and total reducing power were 94.85±0.20%, 44.03±0.41%, and 2.233±0.003, respectively. Wang J, Wu X, Liu X. Screening and identification of antioxidant lactic acid bacteria in fermented meat products [J]. Cereals and Oils, 2023, 36(11): 149-153. Sake Lactobacillus XM-LS01 The DPPH free radical, hydroxyl radical, and superoxide anion radical scavenging rates were 89.73%, 35.28%, and 88.95%, respectively. Wang X, Yan S, Gao Y, et al. Screening and identification of human-derived lactic acid bacteria and study on their antioxidant activity [J]. China Dairy Industry, 2022, 50(06): 16-21+64. L. plantarum subsp. Plantarum CICC 20279, L. casei CICC 23184, L. acidophilus ATCC 4356 The DPPH free radical of L. plantarum subsp. plantarum CICC 20279 was more than 95%, the hydroxyl radical scavenging rate of L. casei CICC 23184 was the highest at 40.7%, and the reducing power of L. acidophilus ATCC 4356 was about 0.4. Xu C, Zhang S, Meng J, et al. Preliminary study on antioxidant effect and cholesterol degradation capacity of different lactic acid bacteria [J]. Food and Fermentation Industries, 2023, 49(02): 152-158+165. Lactobacillus plantarum Y3 The DPPH free radical, hydroxyl radical, and superoxide anion radical scavenging rates were more than 90%, 51.26±0.62%, and 20.16±0.51%, respectively. Lin Y, Zhang Y, Li K, et al. Isolation and screening of lactic acid bacteria from traditional sour porridge and analysis of their antioxidant capacity in vitro. Food Sci Technol, 2023, 44(20): 126-134.

[0059] 1. The ability of the strain in this embodiment to scavenge DPPH free radicals.

[0060] Prepare a 0.1 mmol / L DPPH solution with anhydrous ethanol, store protected from light, and adjust the OD with anhydrous ethanol before use. 517 The absorbance at 517 nm is 1.25 ± 0.05. Take 2.0 mL of bacterial culture into a 10 mL test tube, add 2.0 mL of DPPH solution, and incubate at room temperature in the dark for 20 min. Measure the absorbance at 517 nm and record it as A1. Record the absorbance of ethanol instead of DPPH as A2. Record the absorbance of ethanol instead of the sample as A0. Calculate the DPPH free radical scavenging rate according to the formula.

[0061] ;

[0062] The results are as follows Figure 5 As shown, the scavenging rate of DPPH free radicals by Lactobacillus paracasei Lpc-14 bacterial culture was 96.58±7.55%.

[0063] 2. Ability to scavenge hydroxyl radicals

[0064] Take 1.0 mL of bacterial culture and place it in a test tube. Then, add 1.0 mL of 9 mmol / L ferrous sulfate solution, 1.0 mL of 9 mmol / L salicylic acid ethanol solution, and 1.0 mL of 8.8 mmol / L H₂O₂ solution sequentially. Shake well and incubate the test tube in a 37°C water bath for 30 min. After cooling to room temperature, measure the absorbance at 510 nm, which is A1. The absorbance of H₂O instead of H₂O₂ is A2. The absorbance of H₂O instead of the sample is A0. Calculate the hydroxyl radical scavenging rate according to the formula.

[0065] ;

[0066] The results are as follows Figure 5 As shown, the scavenging rate of Lactobacillus paracasei Lpc-14 bacterial culture for hydroxyl radicals was 99.31±2.75%.

[0067] 3. Regarding ABTS + Free radical scavenging ability

[0068] First, prepare a 7.0 mmol / L ABTS solution and a 2.45 mmol / L K₂S₂O₈ solution, mix them in equal proportions, and react at 4°C in the dark for 12–16 hours. Then, dilute with anhydrous ethanol to an OD₂ concentration before use. 734 The nm value was 0.7 ± 0.02. 0.5 mL of bacterial culture was placed in a test tube, and 4.5 mL of diluted ABTS stock solution was added. The mixture was reacted at 30°C for 10 min, and the OD value was measured. 734 nm is A1. H2O replaces the OD of ABTS. 734 nm is A2. H2O replaces the OD of the sample. 734 nm is A0. Calculate ABTS according to the formula. + Free radical scavenging rate.

[0069] ;

[0070] The results are as follows Figure 5 As shown, the scavenging rate of ABTS+ free radicals by Lactobacillus paracasei Lpc-14 bacterial culture was 85.68±6.22%.

[0071] 4. Ability to scavenge superoxide anion free radicals

[0072] Take 1.0 mL of bacterial culture into a test tube, then add 1.0 mL of 10 mmol / L pyrogallol solution and 5.0 mL of 50 mmol / L Tris-HCl (pH 8.2). React at 25 °C for 4 min, then add 1.0 mL of 8 mol / L HCl to terminate the reaction. Measure the OD.320 nm represents Al, and H2O replaces the OD of pyrogallol. 320 nm is A2, and H2O replaces the OD of the sample. 320 nm is A0. Calculate the superoxide anion radical scavenging rate according to the formula.

[0073] ;

[0074] The results are as follows Figure 5 As shown, the scavenging rate of superoxide anion free radicals by Lactobacillus paracasei Lpc-14 bacterial culture was 95.39±5.68%.

[0075] 5. Overall Restoration Capacity

[0076] Add 0.5 mL of sample, 0.5 mL of potassium ferricyanide (1% by mass), and 0.5 mL of sterile phosphate buffer (0.2 M, pH 6.6), and mix well. Incubate at 50 °C for 20 min. Rapidly cool, add 0.5 mL of trichloroacetic acid (TCA, 10% by mass), and centrifuge at 4500 × g for 10 min at 4 °C. Collect the supernatant. Take 1.0 mL of the supernatant, add 1.0 mL of ferric chloride (0.1% by mass), and 1.0 mL of sterile ultrapure water, and vortex to mix. Let stand for 10 min, and measure the absorbance at 700 nm. Perform three replicates for each sample.

[0077] Total restoration capacity = A1 - A0

[0078] Where: A1—the absorbance measured on the sample;

[0079] A0—The absorbance measured using 0.5 mL of ultrapure distilled water instead of 0.5 mL of sample.

[0080] Table 2 Total reducing capacity of Lactobacillus paracasei Lpc-14

[0081] Treatment method OD700nm Total reducing capacity [A0] 0.06±0.001 / [A1] 2.63±0.287 2.57±0.286

[0082] The results are shown in Table 2. The total reducing power of Lactobacillus paracasei Lpc-14 bacterial culture was 2.57 ± 0.29.

[0083] Example 4: Lactobacillus paracasei Lpc-14 promotes gastrointestinal motility in constipated mice:

[0084] Table 3 lists the probiotic strains that have been publicly disclosed in existing research as having the ability to regulate the secretion of gastrointestinal regulatory peptides.

[0085] Table 3. Probiotic strains with the ability to regulate the secretion of gastrointestinal regulatory peptides as reported in existing studies.

[0086] Strain name Regulation of gastrointestinal regulatory peptide secretion capacity Reference Bifidobacterium animalissubsp. lactisTG11 MTL < 300 pg / mL; SP < 60 pg / mL. Ma W, Zhao Y, Liu Y et al. Bifidobacterium animalis subsp. lactisTG11 ameliorates loperamide-induced constipation in mice by modulating gut microbiota[J]. Frontiers in Microbiology, 2025, 16. L. rhamnosusCCFM1068 MTL < 150 pg / mL. Wang G, Yang S, Sun S et al. Lactobacillus rhamnosus StrainsRelieve Loperamide-Induced Constipation via Different PathwaysIndependent of Short-Chain Fatty Acids[J]. 2020, Volume 10 - 2020. L. plantarum AHQ-14, L. plantarum HM-22 MTL < 240 pg / mL; VIP > 300 pg / mL. Heilongjiang Feihe Dairy Co., Ltd., Dalian University of Technology. A synbiotic composition for improving chronic constipation symptoms and its preparation method and application: CN 116803300 A, 2023-09-26 [P]. L. casei MG551225 MTL was 57.55±4.40 pg / mL; SP was 50.25±4.72 pg / mL. Zhu YQ, Li S, Jiang Y, et al. Effects of Lactobacillus casei on the levels of gastrointestinal regulatory peptides and intestinal flora in constipated mice[J / OL]. Food Sci Technol, 2025, 1-12. L. paracasei LPC-F SP < 130 pg / mL. Wang L, Yang S, Wang J, et al. Lactobacillus paracasei LPC-F Relieves Constipation by Promoting Interstitial Cells of Cajal Proliferation[J]. Food and Fermentation Industries, 2022, 48(4): 1-9. B. animalis subsp. lactis CCFM 625 MTL was 195.5±3.33 pg / mL; SP was 85.11±4.7 pg / mL. Wang L, Wang G, Zhang H, et al. Effects of Bifidobacterium animalis subsp. lactis with adhesion properties on the levels of gastrointestinal regulatory peptides in serum of constipated mice[J]. Chinese Journal of Food Science, 2019, 19(06): 13-20.

[0087] 1. Animal treatment and experimental grouping of the bacterial strains in this embodiment

[0088] Forty mice (BALB / c mice, 6-8 weeks old) were acclimatized for 7 days and then randomly divided into four groups: blank group, model group, positive control group, and Lactobacillus paracasei Lpc-14 intervention group, with 10 mice in each group.

[0089] A constipation model was established by continuous gavage for 14 days. The specific grouping, drug treatment, and experimental bacterial strain treatment are as follows:

[0090] The blank control group received 0.2 mL of normal saline via gavage; the model group received 0.2 mL of loperamide hydrochloride (1 mg / mL, based on a modeling standard of 10 mg / kg b.w) via gavage + 0.2 mL of normal saline; the positive control group received 0.2 mL of loperamide hydrochloride + 0.2 mL of 1.0 × 10⁹ CFU / mL Bifidobacterium triple live bacteria capsules (Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd.) via gavage; the intervention group received 0.2 mL of loperamide hydrochloride + 0.2 mL of 1.0 × 10⁹ CFU / mL Bifidobacterium triple live bacteria capsules via gavage. 9 cfu / mL Lactobacillus paracasei Lpc-14 bacterial suspension.

[0091] The experimental mice were housed at the SPF-grade experimental animal center of Harbin University of Commerce. Environmental control parameters were strictly set at a temperature of 20±2℃ and a relative humidity of 50±5%, and an automatic light control system was installed to achieve a 12-hour day / night cycle. Strict animal welfare standards were followed during the experiment, with cage cleaning and bedding changes performed twice weekly. The animal experimental procedures involved in this invention strictly followed the relevant requirements of the Experimental Animal Ethics Committee of Heilongjiang University (Experimental Animal Ethics Review Acceptance Number: 20220523006). After a 2-week intervention, the experimental animals were fasted for 12 hours, anesthetized, and blood samples were collected by enucleation for subsequent experimental measurements.

[0092] 2. Results of the mouse motilin (MTL) content assay

[0093] After anesthetizing mice, blood was collected by enucleation. The blood samples were centrifuged at 3500×g for 10 min at 4°C, and serum was collected. The concentration of MTL in the serum was determined using an ELISA kit. Each measurement was repeated three times, and the average value was taken.

[0094] Constipated mice exhibit weakened intestinal function, and there is a very close correlation between intestinal neurotransmitters and intestinal muscle motility. Serum gastrointestinal regulatory peptides are highly sensitive to changes in gastrointestinal function; they can improve constipation by regulating muscle contraction and mucus secretion. MTL is one of the main hormones promoting gastrointestinal motility; MTL can stimulate bile secretion and accelerate intestinal peristalsis. Figure 6 It was evident that the constipation model group showed a significant difference compared to the control group (p<0.05), with a significant decrease in the concentration of excitatory neurotransmitters, at 193.56±7.87 pg / mL and 397.44±6.94 pg / mL, respectively. In contrast, the concentrations of excitatory neurotransmitters in the serum of mice treated with Bifidobacterium triple live bacteria and Lpc-14 via gavage increased, at 308±7.64 pg / mL and 331.33±7.64 pg / mL, respectively, indicating that Lactobacillus paracasei Lpc-14 promotes the production of excitatory neurotransmitters. Furthermore, the MTL concentration in the serum of mice in the Lpc-14 intervention group was significantly higher than (p<0.05) in the Bifidobacterium triple live bacteria positive control group, suggesting that Lpc-14 is superior to commercially available Bifidobacterium triple live bacteria in accelerating intestinal peristalsis. Moreover, the experiment used a single strain, and its MTL regulation level was higher than that of commercially available triple live bacteria.

[0095] 3. Results of the test for determination of substance P (SP) content

[0096] After anesthetizing mice, blood was collected by enucleation. The blood samples were centrifuged at 3500×g for 10 min at 4°C, and serum was collected. The concentration of SP in the serum was determined using an ELISA kit. Each measurement was repeated three times, and the average value was taken.

[0097] SP is the most potent excitatory substance for gastrointestinal smooth muscle in the body. It is a neurotransmitter that regulates intestinal motility; increased SP concentration can accelerate gastrointestinal contractions and motility. Figure 7 It was evident that the concentration of SP in the constipation model group was significantly lower than that in the control group (p<0.05), at 136.95±3.93 pg / mL and 216.75±6.06 pg / mL, respectively. Meanwhile, the concentrations of excitatory neurotransmitters in the serum of mice administered via gavage with a combination of Bifidobacterium triple live bacteria and Lactobacillus paracasei Lpc-14 were both increased, at 166.82±3.27 pg / mL and 166.16±3.06 pg / mL, respectively, indicating that Lactobacillus paracasei Lpc-14 promotes the production of excitatory neurotransmitters. Figure 7 As can be seen, the SP concentration levels in the Lactobacillus paracasei Lpc-14 intervention group and the positive control group of Bifidobacterium triple live bacteria were comparable, with no significant difference (p>0.05). Furthermore, the experiment used a single strain, indicating that a single strain can achieve the SP regulation level of commercially available Bifidobacterium triple live bacteria.

[0098] 4. Results of the assay for the content of vasoactive intestinal peptide (VIP) in mice

[0099] After anesthetizing mice, blood was collected by enucleation. The blood samples were centrifuged at 3500×g for 10 min at 4°C, and the serum was collected. The concentration of VIP in the serum was determined using an ELISA kit. Each measurement was repeated three times, and the average value was taken.

[0100] Elevated VIP levels can weaken segmental colonic motility while enhancing propulsive motility. VIP may regulate intestinal fluid metabolism through the intestinal cAMP-PKA signaling pathway and aquaporin expression, and can also relieve constipation by promoting the proliferation of beneficial bacteria, thus restoring the gut microbiota to a normal state. Figure 8 As can be seen, the constipation model group showed a significant difference compared with the control group (p<0.05), with a significant increase in the concentration of inhibitory neurotransmitters, reaching 131.83±2.60 pg / mL and 94.61±2.68 pg / mL, respectively. In contrast, the concentrations of inhibitory neurotransmitters in the serum of mice administered with Bifidobacterium triple live bacteria and Lactobacillus paracasei Lpc-14 decreased, reaching 100.17±5.83 pg / mL and 105.03±3.78 pg / mL, respectively. This indicates that Lactobacillus paracasei Lpc-14 has an inhibitory effect on the production of inhibitory neurotransmitters. The VIP concentration levels in the serum of mice administered with Lactobacillus paracasei Lpc-14 and the positive control group of Bifidobacterium triple live bacteria were comparable, with no significant difference (p>0.05). The VIP concentration levels in the experimental strain intervention group and the positive control group of Bifidobacterium triple live bacteria were comparable, with no significant difference (p>0.05). Furthermore, the experiment used a single strain, indicating that a single strain can achieve the same level of regulation of VIP by commercially available Bifidobacterium triple live bacteria. The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of *Lactobacillus paracasei* Lpc-14 with antioxidant and gastrointestinal motility-promoting functions, characterized in that... The strain is Lactobacillus paracasei Lpc-14, with the cultural name Lactobacillus paracasei, accession number CGMCC No. 29666, accession date January 18, 2024, and depositary institution China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The *Lactobacillus paracasei* Lpc-14 strain with antioxidant and gastrointestinal motility-promoting functions according to claim 1, characterized in that... The Lactobacillus paracasei Lpc-14 strain was isolated and purified from the fermentation broth of naturally fermented sauerkraut.

3. The *Lactobacillus paracasei* Lpc-14 strain with antioxidant and gastrointestinal motility-promoting functions according to claim 1 or 2, characterized in that... The Lactobacillus paracasei Lpc-14 strain was prepared by the following method: Chop the cabbage and bok choy and put them into a sterile container. Add salt water to submerge the vegetables, seal the container, and ferment in a cool, shady place indoors. During fermentation, open the lid to release the air. After 4 months of pickling, you will get naturally fermented sauerkraut. The fermentation broth of naturally fermented sauerkraut was diluted with physiological saline and spread onto MRS agar medium, and incubated at 37°C for 48 hours. Single colonies of different shapes and sizes were picked from the culture dish and purified by streaking multiple times on MRS agar medium until they were identified as single colonies. The single colonies were then examined under a microscope, and physiological, biochemical and molecular biological identifications were performed. The isolated strains were then preserved in cryogenic glycerol.

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