Lactobacillus paracasei cfcfc018, bacterial agent and application thereof

Abstract

The application discloses a lactobacillus paracasei CFCFC018, a bacterial agent and application thereof, and belongs to the technical field of microorganisms. Lacticaseibacillus paracasei The lactobacillus paracasei CFCFC018 is isolated from the intestinal contents of a field cat in a semi-agricultural and semi-pastoral area in Inner Mongolia, and the strain can highly express amino acids and neurotransmitters without genetic engineering modification, can significantly reduce the damage of mammary gland alveolar structure in a mouse with mastitis induced by staphylococcus aureus, avoid the thickening of the interalveolar matrix layer of the mammary gland, reduce the number of neutrophil infiltrates, and reduce the damage degree of the mammary gland tissue, and has a certain protective effect on the mammary gland tissue. Researches also show that the strain can reduce the abundance of harmful bacteria in feces, improve intestinal inflammation, and reduce the submucosal edema of the intestinal tract, which provides a new idea for preventing and treating mastitis by regulating the intestinal flora.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus paracasei CFCFC018, its bacterial agent, and its application. Background Technology

[0002] Mastitis is a common disease in dairy cows, not only reducing the quality of dairy products but also having serious adverse effects on animal health. Studies have found that more than 20% of bovine mastitis is caused by Staphylococcus aureus, which is characterized by its difficulty in eradication, low antibiotic cure rate, and persistent transmission, and can even cause chronic, subclinical, and hyperacute gangrenous mastitis. Antibiotics are commonly used drugs for the clinical prevention and treatment of Staphylococcus aureus infections; however, drug residues and resistance in dairy cows can affect the safety and quality of milk and dairy products. Antibiotic-resistant Staphylococcus aureus isolated from bovine mastitis has become one of the main causes of mastitis recurrence. Therefore, finding safe and effective drugs for the treatment of mastitis is crucial and necessary.

[0003] The gut microbiota has been proven to be a complex ecosystem that plays a positive role in host metabolism, immune regulation, and overall health. Microorganisms colonizing the gut can also influence tissues or organs outside the gastrointestinal tract, thereby participating in or regulating the occurrence and development of diseases. Recent research indicates that the gut microbiota plays a crucial role in the development of mastitis in dairy cows, and that it influences inflammatory diseases. This provides a new approach to preventing and treating mastitis by modulating the gut microbiota.

[0004] Lactobacillus paracasei ( Lactobacillus paracasei *Lactobacillus paracasei* is a facultative anaerobic, non-motile, non-spore-forming lactic acid bacterium. It possesses not only strong antibacterial activity but also various immune response regulatory functions, exhibiting beneficial effects on human health. It can be obtained from various animal intestines, oral cavities, silage, milk, and other environments. Currently, 34 species of *Lactobacillus paracasei* have been isolated, 8 of which were isolated from the digestive system flora of humans or other animals. Given current taxonomic techniques, all are classified as *Lactobacillus paracasei* species, but their properties vary significantly depending on the isolation and screening sources. Therefore, further research into the probiotic characteristics and antibacterial effects of *Lactobacillus paracasei* from different isolation sources is of great significance for applications in various fields. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a strain of Lactobacillus paracasei CFCFC018, a bacterial agent, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CFCFC018, the Lactobacillus paracasei ( Lacticaseibacillus paracasei CFCFC018 was deposited on July 15, 2025 at the China Center for Type Culture Collection (No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province), with accession number CCTCC M 20251610.

[0008] In a second aspect, the present invention provides a microbial agent containing the aforementioned *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei )CFCFC018.

[0009] The bacterial agent contains Lactobacillus paracasei ( Lacticaseibacillus paracasei CFCFC018 exists in the form of live bacterial suspension or fermentation broth.

[0010] In a third aspect, the present invention provides the aforementioned *Lactobacillus paracasei* (… Lacticaseibacillus paracasei The application of CFCFC018 or the above-mentioned microbial agents in the following (1) or (2):

[0011] (1) Production of amino acids and their derivatives;

[0012] (2) Production of neurotransmitters and their precursors.

[0013] The amino acids and their derivatives include: glutamic acid, arginine, alanine, leucine, isoleucine, or valine.

[0014] The neurotransmitters and their precursors include tyramine, dopa (DOPA), norepinephrine, and kynurenine.

[0015] Amino acids are fundamental molecules in protein synthesis and metabolic regulation. Glutamic acid, which can participate in metabolism as an amino acid and also act as a neurotransmitter, is commonly used in the preparation of seasonings, anti-anxiety foods, and pharmaceuticals. Arginine can improve vascular function and is used in the preparation of cardiovascular health products. Leucine, isoleucine, and valine can promote muscle synthesis. Neurotransmitters are key molecules regulating the function of the nervous system and can be applied to the intervention of brain diseases and the regulation of the gut-brain axis. Natural strains do not synthesize large amounts of amino acids themselves, but mainly obtain amino acids by breaking down milk proteins through proteolytic systems (such as cell membrane proteases PrtP and peptidases PepO / PepX). The *Lactobacillus paracasei* provided in this invention (… Lacticaseibacillus paracasei The differences between CFCFC018 and existing Lactobacillus paracasei include: the strain does not require genetic engineering and can express amino acids (and their derivatives) and neurotransmitters (and their precursors) at high levels.

[0016] In a fourth aspect, the present invention provides the aforementioned *Lactobacillus paracasei* (… Lacticaseibacillus paracaseiCFCFC018 or the above-mentioned microbial agent in the preparation of products for preventing and treating mastitis and intestinal inflammation.

[0017] The prevention and treatment of mastitis specifically manifests as: significantly reducing the damage to the mammary gland alveolar structure, avoiding the thickening of the interalveolar matrix layer, reducing the number of neutrophil infiltrates, and reducing the degree of damage to the mammary gland tissue.

[0018] The prevention and treatment of intestinal inflammation specifically manifests as: reducing the submucosal edema of the intestinal tract.

[0019] In a fifth aspect, the present application provides a probiotic preparation for preventing and treating mastitis, wherein the probiotic preparation is Lactobacillus paracasei (CFCFC018) Lacticaseibacillus paracasei CFCFC018 is the active ingredient.

[0020] The beneficial effects of the present application are:

[0021] The present application isolates a strain of Lactobacillus paracasei (CFCFC018) from the intestinal contents of a semi-agricultural and semi-pastoral area in Inner Mongolia. Lacticaseibacillus paracasei CFCFC018, which does not need to be genetically engineered, can express amino acids and neurotransmitters itself, can significantly reduce the damage to the mammary gland alveolar structure in the mammary gland tissue of mice induced by Staphylococcus aureus, avoid the thickening of the interalveolar matrix layer, reduce the number of neutrophil infiltrates, and reduce the degree of damage to the mammary gland tissue, and has a certain protective effect on the mammary gland tissue. Studies have also shown that the strain can reduce the abundance of harmful bacteria in feces, improve intestinal inflammation, and reduce submucosal edema of the intestinal tract, which provides a new idea for preventing and treating mastitis by regulating intestinal microbial communities. BRIEF DESCRIPTION OF DRAWINGS

[0022] Lacticaseibacillus paracasei Figure 1 is the morphological characteristics of strain CFCFC018; wherein, the colony morphology of strain CFCFC018 (left), the optical microscope photograph (middle), and the scanning electron microscope observation photograph (right).

[0023] Figure 1 Figure 2 is the growth characteristic curve of the strain; wherein, Figure 2 A is the growth curve of strain CFCFC018, Figure 2 B is the acid production curve of strain CFCFC018.

[0024] Figure 2 Figure 3 is the morphology of CFCFC018 strain on a sheep blood plate.

[0025] Figure 3 Figure 4 is a mouse abdominal dissection diagram; wherein, Figure 4 A is the abdominal dissection diagram of the control group of mice, Figure 4 B is the abdominal dissection diagram of the CFCFC018 strain treatment group of mice.

[0026] Figure 4 Inhibition of S. aureus by P. parapsilvii in vitro.

[0027] Figure 5 H&E staining to detect the pathological changes of mouse mammary tissue induced by S. aureus; wherein, Figure 6 a is the pathological changes of the mammary tissue in the negative control group, Figure 6 b is the pathological changes of the mammary tissue in the P. parapsilvii pretreatment group, Figure 6 c is the damage score of the mammary tissue.

[0028] Figure 6 ELISA to detect the contents of inflammatory factors and MPO in the mammary tissue and serum; wherein, Figure 7 a is the contents of IL-1β, IL-6, and TNF-α in the mammary tissue, Figure 7 b is the MPO activity in the mammary tissue, Figure 7 c is the contents of IL-1β, IL-6, and TNF-α in the serum, Figure 7 d is the MPO activity in the serum.

[0029] Figure 7 Changes in the diversity of fecal bacterial community in mice with mastitis; wherein, Figure 8 a-d are the Goods_coverage value, chao1, shannon, and simpson index of the fecal bacterial community in mice before S. aureus infection (P>0.05); Figure 8 e-h are the Goods_coverage value, chao1, shannon, and simpson index of the intestinal bacterial community in mice after S. aureus infection (P>0.05); PS and PL represent the negative control group and the P. parapsilvii pretreatment group before infection, respectively, and S and L represent the negative control group and the P. parapsilvii pretreatment group after infection, respectively.

[0030] Figure 8 Changes in the distribution of intestinal microbial community in mice with mastitis (unweighted UniFrac distance); wherein, Figure 9 a is the distribution of intestinal bacterial community in mice before S. aureus infection, Figure 9 b is the distribution of intestinal bacterial community in mice after S. aureus infection; PS and PL represent the negative control group and the P. parapsilvii pretreatment group before infection, respectively, and S and L represent the negative control group and the P. parapsilvii pretreatment group after infection, respectively.

[0031] Figure 9 Changes in the composition of fecal microbial community in mice with mastitis; wherein, Figure 10In Figure A, the changes in the phylum level of the intestinal flora in mice after Staphylococcus aureus infection are shown. Figure 10 In the middle B, the changes in the genus level of the intestinal flora of mice after Staphylococcus aureus infection are shown; S and L represent the negative control group and the Lactobacillus paracasei pretreatment group after infection, respectively.

[0032] Figure 10 Differences in the relative abundance of fecal microbial communities in mastitis mice (genus level); among them, Figure 11 In Figure 'a', the difference in the relative abundance of fecal microorganisms in mice before Staphylococcus aureus infection is represented. Figure 11 In the figure, b represents the difference in the relative abundance of fecal microorganisms in mice after Staphylococcus aureus infection; PS and PL represent the pre-infection negative control group and the Lactobacillus paracasei pretreatment group, respectively, and S and L represent the post-infection negative control group and the Lactobacillus paracasei pretreatment group, respectively.

[0033] Figure 11 The effect of Lactobacillus paracasei on fecal matter in mice with DSS-induced colitis.

[0034] Figure 12 Colonic intestinal sections for DSS-induced colitis test using Lactobacillus paracasei. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0037] Example 1: Isolation, purification and identification of strains

[0038] 1. Isolation and purification of strains

[0039] We collected 10g of rectal feces from semi-free-range cats in pastoral areas using the rectal feces collection method. The feces were placed in MRS liquid culture medium and quickly brought back to the laboratory for bacterial isolation.

[0040] In a sterile operating table, the enriched bacterial solution was subjected to 10 [units of treatment]. -1 ~10 -6Gradient dilution (100 μL of culture broth was added to 900 μL of physiological saline), 200 μL of the diluted bacterial solution was spread on MRS solid plate, and placed in a constant temperature incubator at 37 °C. After the colonies grew, different morphological colonies were picked on the plate using a loop in a clean bench. After the colonies grew again, the streaking operation was repeated until single colonies formed on the plate, and the strain purification operation was completed. The purified strain was inoculated into MRS slant medium for further culture for staining and microscopic examination.

[0041] 2. Strain identification

[0042] (1) Colony morphology and fungal staining microscopy

[0043] After the bacterial solution was diluted to the appropriate concentration, it was spread on the plate and cultured for two days. The colony morphology was observed, and the bacterial cell morphology was observed by Gram staining microscopy. Figure 13 It can be seen that the colony is milky white, the surface is smooth, and the growth state on the plate is good. The colony is convex, the texture is thick, and the microscopic observation is Gram-positive, blunt round at both ends, and rod-shaped bacteria.

[0044] (2) Bacterial physiological and biochemical identification

[0045] According to the "Berger's Bacterial Identification Manual" (8th edition), the physiological and biochemical tests of carbon source oxidation and carbon source utilization of the strain were carried out.

[0046] Table 1: Physiological and biochemical characteristics of strain CFCFC018 - enzyme activity

[0047]

[0048] +: positive reaction; - : negative reaction

[0049] Table 2: Physiological and biochemical characteristics of strain CFCFC018 - acid production using carbon sources

[0050]

[0051] +: positive reaction; - : negative reaction

[0052] (3) Bacterial 16S rRNA sequence homology analysis

[0053] Using 27f, 1492r lactic acid bacteria universal primer for sequence detection, Shanghai Shengong Biotechnology Co., Ltd. was used for sequencing, the 16S rRNA gene sequence of the strain was added to the NCBI database for comparison, Bacillus subtilis IAM12118 (AB042061) was used as the outgroup to construct the Neighbor-Joining phylogenetic tree, and the strain was identified to belong to Paracaseolyticus by phylogenetic analysis Figure 1The strain is biologically preserved, and the preservation information is as follows:

[0054] Strain name: Paracasei CFCFC018;

[0055] Classification name: Lacticaseibacillus paracasei ;

[0056] Preservation agency: China Center for Type Culture Collection;

[0057] Address: Wuhan University, Wuhan, Hubei, China;

[0058] Preservation date: July 15, 2025;

[0059] Preservation number: CCTCC M 20251610.

[0060] Example 2: Evaluation of probiotic properties and safety of Paracasei CFCFC018

[0061] Test strain: The test strain Paracasei CFCFC018 was isolated by the laboratory, and Staphylococcus aureus (ATCC25923), Salmonella typhimurium (ATCC14028) and Escherichia coli (ATCC25922) were purchased from domestic microorganism strain preservation agencies.

[0062] 1. Growth characteristics and acid production performance of Paracasei CFCFC018

[0063] The strain isolated in Example 1 was activated and cultured, then 100 uL of the bacterial solution after 18~24 h of activation was inoculated into MRS liquid medium at a 2% inoculation amount. The culture was carried out at 28 ℃ with constant temperature oscillation at 160 rpm, and during the culture, the absorbance of the culture solution at 600 nm (OD 600 ) was measured every 1 h for the first 5 h using a spectrophotometer, and the absorbance of the culture solution at 600 nm (OD 600 ) was measured every 2 h after 6 h using a spectrophotometer. At the same time, the pH of the culture solution was measured every 2 h using a handheld pH meter. The acid production curve was plotted with time as the X axis and the pH value of the culture solution as the Y axis.

[0064] From the growth curve of strain A in Lacticaseibacillus paracasei , it can be seen that within 0~2 h after subculture, the growth rate of the strain was slow, and it was in the lag phase. After 2 h, the growth rate of the strain increased significantly, and it entered the logarithmic growth phase; after 14 h of subculture, the growth curve tended to be flat, and it entered the stationary phase. Figure 2The acid production curve of the C. parapsilosis CFCFC018 strain showed that the pH value decreased slowly within 2 h after inoculation, and then decreased rapidly after 2 h. The pH value decreased to below 4.0 after 8 h.

[0065] 2. Study on the bacteriostatic ability of C. parapsilosis CFCFC018

[0066] To determine the growth inhibition effect of the C. parapsilosis CFCFC018 strain on three common intestinal pathogenic bacteria, i.e. Staphylococcus aureus (ATCC25923), Salmonella typhimurium (ATCC14028), and Escherichia coli (ATCC25922). The Oxford cup agar diffusion method was used to study the antibacterial activity of the CFCFC018 strain.

[0067] The CFCFC018 bacterial solution activated for 24 h was centrifuged at 4°C and 6000 r / min for 10 min in a high-speed centrifuge, and was ready for use. 200 uL of the E. coli, Salmonella, and S. aureus bacterial solution was uniformly coated on the LB solid culture medium with a coating rod. When the bacterial solution was completely absorbed, two Oxford cups were placed on the LB plate, and 200 uL of the supernatant and 200 uL of the original bacterial solution were added, respectively. The plate was cultured in a 37°C constant temperature incubator for 24 h. Whether an inhibition zone was formed was observed, and the diameter of the inhibition zone was accurately measured with a vernier caliper. When the diameter of the inhibition zone was equal to 7-8 mm, it was defined as insensitive; when it was 10-20 mm, it was defined as moderately sensitive; and when it was 21-30 mm or the diameter of the inhibition zone was greater than 30 mm, it was defined as highly sensitive.

[0068] The results showed that the CFCFC018 fermentation solution had a moderate inhibitory effect on E. coli, Salmonella, and S. aureus, and the supernatant had a moderate inhibitory effect on Salmonella (Table 3).

[0069] Table 3 Diameter of the inhibition zone of the CFCFC018 fermentation supernatant and the original solution on common pathogenic bacteria

[0070]

[0071] 3. Hemolytic determination of C. parapsilosis CFCFC018

[0072] Bacterial hemolysis can be divided into α-hemolysis, β-hemolysis, and γ-hemolysis. Among them, α-hemolysis (grass green appears around the colony) and γ-hemolysis (no grass green and transparent circle appears around the colony) are not pathogenic, and only β-hemolysis (transparent circle appears around the colony) is associated with pathogenicity.

[0073] Take the activated culture 18~24 h of bacteria, with inoculation ring dipped in the plate streak, each streak from the end of the last streak, try not to repeat, streak 3~4 can. Placed in 37℃ constant temperature anaerobic culture 24 h, observation whether produce hemolytic ring.

[0074] From Figure 2 It can be seen that Lactobacillus paracasei CFCFC018 is gray in color after 24 h culture on sheep blood plate, no hemolytic ring around the colony, determined as gamma hemolysis, that is, non-hemolytic type, which is very safe strain.

[0075] 4. Safety evaluation of Lactobacillus paracasei CFCFC018

[0076] (1) Experimental method

[0077] SPF C57BL / 6 mice provided by Sbi Bio-Technology Co., Ltd. (Beijing) were 12, half male and half female, 5 weeks old, body weight (22±2) g. 12 h day and night alternation, temperature 22~26℃, humidity 50%~70%. 12 mice were randomly divided into 2 groups, control group and experimental group, 6 in each group, and were fed in separate cages. After 3 d of adaptive feeding, the test began, and the mice were free to drink and eat during feeding.

[0078] Methods refer to "National food safety standard acute oral toxicity test" (GB15193.3-2014). The control group was given 200 uL / only physiological saline per day, and the test group was given 200 uL / only bacteria solution per day, with 1.0×10 9 cfu / mL in the process, without limiting the food intake, free to drink water, and gavage for 21 days. During the experiment, the food intake, mental state, body weight change and death of the mice were recorded in detail.

[0079] (2) Experimental results

[0080] During the observation period, no abnormalities were found in the mice of the control group and the experimental group, and no deaths occurred. After autopsy, no ocular lesions were observed in the mouse organs, and no bacterial growth was observed in tissue culture. It is proved that Lactobacillus paracasei CFCFC018 has no pathogenicity to mice. As shown in Table 4, compared with the control group, the body weight of the bacteria solution treatment group decreased, the daily weight gain decreased, and other indicators had no obvious change.

[0081] Table 4 Safety evaluation index

[0082]

[0083] As shown in Table 5, compared with the control group, gavage of CFCFC018 bacteria solution had no significant effect on the blood routine index of mice.

[0084] Table 5 Blood routine index determination

[0085]

[0086] As shown in Table 5, the blood routine index determination results of the mice in the blank control group, the model control group, and the Lactobacillus paracasei CFCFC018 group were as follows: the white blood cell count, the red blood cell count, the hemoglobin content, the hematocrit, and the platelet count were normal, and the results were within the normal range. The results showed that the Lactobacillus paracasei CFCFC018 strain did not cause adverse effects on the blood routine index of the mice. Figure 3

[0087] Example 3: Amino acid and neurotransmitter quantitative determination of Lactobacillus paracasei

[0088] Instrument: AB Sciex QTRAP 6500+ mass spectrometer, AB Sciex ExionLC™ AD liquid chromatograph.

[0089] Reagents: 23 kinds of amino acid standard and 2 kinds of stable isotopes (L-Alanine-d4 (deuterated alanine-d4), deuterated phenylalanine-d8 (Phenylalanine-d8)) labeled standard were purchased from Sigma-Aldrich; ammonium acetate (AR, Sigma-Aldrich, USA), methanol, acetonitrile, formic acid (LC-MS, Thermo-Fisher, USA), ultrapure water Mill-Q (Millipore, USA). 23 kinds of neurotransmitter standard and 2 kinds of stable isotope labeled standard were purchased from Shanghai Zhenjun Biological Technology Co., Ltd.

[0090] Table 6 Amino acid and its derivative information

[0091]

[0092] Amino acid standard preparation: 23 kinds of amino acid standards were accurately weighed, and a mixed standard linear mother liquor with a concentration of 5 mg / mL was prepared. The linear mother liquor was diluted with methanol to obtain a series of concentrations as shown in Table 8. A solution of L-Alanine-d4 and Phenylanine-d2 with a certain concentration was prepared, and the internal standard solution (IS) was obtained by mixing. The linear, internal standard, and quality control mother liquor and working solution were stored in a-20℃ refrigerator.

[0093] Neurotransmitter standard preparation: 23 kinds of neurotransmitter standards were accurately weighed, and a mixed standard linear mother liquor was prepared. The linear mother liquor was diluted with methanol to obtain a series of concentrations of working solution. A solution of L-Tyrosine-d4, Alanine-d4, and IAA-d4 with a certain concentration was prepared to obtain an internal standard solution (IS). The linear, internal standard, and quality control mother liquor and working solution were stored in a-20℃ refrigerator.

[0094] Table 7 Neurotransmitter and its precursor information

[0095] ​

[0096] Amino acid metabolite extraction: Take sample into mass spectrometry water, vortex mix, as dilution sample; take 50 μL of dilution sample, add 200 μL of precipitant containing mixed internal standard (acetonitrile:methanol=1:1), vortex mix, stand on ice for 30 min, centrifuge at 12000 rpm at 4°C for 10 min, take all supernatant for LC-MS analysis.

[0097] Neurotransmitter metabolite extraction: Take sample into mass spectrometry water, vortex mix, as dilution sample; take 100 μL of dilution sample, add 400 μL of precipitant containing mixed internal standard (acetonitrile:water=8:2), vortex mix, stand on ice for 30 min, centrifuge at 12000 rpm at 4°C for 10 min, take all supernatant for LC-MS analysis.

[0098] Amino acid metabolite chromatography, mass spectrometry method:

[0099] Chromatographic column: ACQUITY UPLC BEH Amide (2.1x100mm, 1.7μm);

[0100] Mobile phase: A phase: 5mM ammonium acetate aqueous solution containing 0.1% formic acid B phase: acetonitrile containing 0.1% formic acid;

[0101] Column temperature: 50°C;

[0102] Injection volume: 1 μL;

[0103] Flow rate: 0.3 mL / min.

[0104] Amino acid chromatography concentration gradient is shown in the following table:

[0105] Table 8 Amino acid chromatography concentration gradient

[0106]

[0107] Mass spectrometry conditions: Electrospray ionization source (ESI), positive ionization mode. Ion source temperature 550°C, ion source voltage 5500V, curtain gas 35 psi, atomization gas 50 psi, auxiliary gas 60 psi. Scanning is carried out by multiple reaction monitoring (MRM).

[0108] Neurotransmitter chromatography, mass spectrometry method:

[0109] Chromatographic column: Waters XSelect HSS T3 (2.1x150mm, 2.5μm);

[0110] Mobile phase: Phase A: 5 mM ammonium acetate in water with 0.1% formic acid Phase B: acetonitrile with 0.1% formic acid;

[0111] Column temperature: 45 °C;

[0112] Injection volume: 2 μL;

[0113] Flow rate: 0.3 mL / min.

[0114] The chromatographic concentration gradient of neurotransmitters is shown in Table 9 below:

[0115] Table 9 Chromatographic concentration gradient of neurotransmitters

[0116]

[0117] Mass spectrometry conditions: electrospray ionization source (ESI), negative ionization mode. Ion source temperature 550 °C, ion source voltage -4500 V, gas curtain gas 35 psi, nebulization gas 60 psi, auxiliary gas 60 psi. Scanning was performed using multiple reaction monitoring (MRM).

[0118] (1) Quantitative detection of amino acids synthesized by Lactobacillus paracasei

[0119] As shown in Table 10, the amino acids highly expressed by Lactobacillus paracasei were glutamic acid, arginine, alanine, and branched-chain amino acids (leucine, isoleucine, valine, etc.).

[0120] Table 10 Quantitative detection of amino acids and their derivatives in Lactobacillus paracasei

[0121]

[0122] (2) Quantitative detection of neurotransmitters in Lactobacillus paracasei

[0123] As shown in Table 11, the neurotransmitters highly expressed by Lactobacillus paracasei were tyramine, dopamine DOPA, norepinephrine, and kynurenine.

[0124] Table 11 Quantitative detection of neurotransmitters and their precursors in Lactobacillus paracasei

[0125]

[0126] The results of the quantitative analysis of amino acid composition and neurotransmitters showed that the strain highly expressed glutamic acid (801.09 U / g), arginine (858.16 U / g), alanine (401.30 U / g), and branched-chain amino acids (leucine 272.59, isoleucine 252.58, valine 216.03 U / g).

[0127] The neurotransmitter data confirmed that the strain highly expressed tyramine (2584.72 ng / mL), dopamine DOPA (1991.08 ng / mL), norepinephrine (560.83 ng / mL), and kynurenine (421.15 ng / mL).

[0128] Example 4: Protective effect of Paracaseicoccus caseicola CFCFC018 on Staphylococcus aureus-induced mastitis in mice

[0129] Test animals: 8-week-old SPF Kunming mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. (Animal Production License No. SCXK (Jing) 2019-0010). The feeding and management of experimental animals and experimental operations were in accordance with the Experimental Animal Welfare Ethical Review Guidelines (GB / T 35892-2018).

[0130] 1. Detection of the protective effect of Paracaseicoccus caseicola CFCFC018 on the mammary gland

[0131] (1) Construction of a mouse mastitis model

[0132] On the 8th day after delivery, the mice and their pups were housed separately, and the fourth pair of mammary glands of the mice were used for modeling. 50 μL of Staphylococcus aureus bacterial suspension was injected into the fourth pair of mammary glands of the mice to establish a mouse model of mastitis.

[0133] The 14 successfully modeled mice were divided into two groups: a negative control group (group S) and a Paracaseicoccus caseicola prophylactic treatment group (group L), with 7 mice in each group. The mice in group L were injected with 50 μL of Paracaseicoccus caseicola bacterial suspension into each mammary gland; the mice in group S were injected with an equal amount of sterile normal saline into each mammary gland. After 24 hours, the mice were sacrificed by decapitation, and sampling was performed. All experimental mice were fasted for 12 hours before sample collection.

[0134] (2) Histopathological detection of the mammary gland

[0135] The fourth pair of mammary gland tissues of the mice were removed, and one side was placed in a 4% paraformaldehyde solution for overnight fixation, followed by H&E staining and sectioning. Histological scores ranged from 1 to 5, with higher scores indicating more severe tissue damage. A score of 1 indicated no histopathological features (i.e., no necrosis, no neutrophil and lymphocyte infiltration), a score of 2 indicated minimal histopathological features (i.e., single neutrophil infiltration), a score of 3 indicated mild histopathological features (i.e., a small amount of neutrophil infiltration), a score of 4 indicated moderate histopathological features (i.e., more neutrophil infiltration, with slight damage to the glandular structure), and a score of 5 indicated severe histological features (i.e., a large number of neutrophil infiltration, with severe damage to the glandular structure).

[0136] As Figure 4As shown, S. aureus caused obvious pathological damage, the number of neutrophils in mammary gland alveoli increased, and the interalveolar basement layer thickened Figure 6 a). Compared with S group, P. paracasei pretreatment significantly reduced the damage of mammary gland alveolar structure, reduced the number of neutrophil infiltration, and the alveolar state was good Figure 6 b). After semi-quantitative scoring of mammary gland tissue, it was found that P. paracasei pretreatment could significantly reduce the degree of mammary gland tissue damage (P<0.05) Figure 6 c).

[0137] (3) Detection of inflammatory cytokines and MPO activity in mammary gland tissue and serum

[0138] The contents of IL-1β, IL-6, TNF-α and MPO in the mammary gland tissue of mice were detected by ELISA kit. The blood was collected by enucleation of the eyeball, and the blood was collected in a coagulation blood collection tube. After centrifugation at 4 ℃ and 4000 r / min for 10 min, the supernatant was collected to detect the contents of IL-1β, IL-6, TNF-α and MPO in the serum of mice. The test operation and sample detection were strictly carried out according to the requirements of the kit instruction manual.

[0139] The test results showed that Figure 6 ), the content of IL-1β in the mammary gland tissue of L group increased slightly, the contents of IL-6 and TNF-α decreased slightly, and the differences were not significant (P>0.05); the MPO activity in the mammary gland tissue of the two groups was not significantly different (P>0.05). The contents of IL-1β, IL-6 and TNF-α in the serum of L group were slightly low, but the difference was not significant compared with S group (P>0.05); P. paracasei had no effect on the MPO activity in the serum (P>0.05).

[0140] (4) 16S rRNA gene sequencing and analysis of fecal bacterial community

[0141] Fresh feces were collected from mice in each group before and after the establishment of the mastitis model and placed in sterile cryovials. The samples were then flash-frozen in liquid nitrogen and transported to Beijing Novogene Biotechnology Co., Ltd. under dry ice conditions. The paired-end region of the V3-V4 region of the 16S rRNA gene in the fecal samples from 14 mice was sequenced using the Illumina NovaSeq platform. The amplification primers were 341F (SEQ ID No. 1: 5'-CCTAYGGGRBGCASCAG-3') and 806R (SEQ ID No. 2: 5'-GGACTACNNGGGTATCTAAT-3'; Note: N represents "any natural nucleotide", specifically any one of adenine (A), cytosine (C), guanine (G), or thymine (T). The WIPO ST.26 sequence listing uses "A" to represent this. Denoising of amplicon sequence variants (ASVs) was performed using DADA2 to obtain valid data, followed by species annotation and abundance analysis. Alpha diversity was analyzed using the T-test, and Beta diversity was analyzed using principal component analysis (PCA). At the phylum and genus levels, the species abundance and composition of the samples were statistically analyzed, and the T-test was used to analyze species with significant differences between groups at the genus level.

[0142] Depend on Figure 7 It can be seen that the Goods-coverage values ​​of all samples are distributed between 0.996 and 0.999, indicating that the sequencing depth of mouse fecal microbiota is above 99%, which can comprehensively reflect the types and structure of mouse fecal microbiota. Figure 8 a, Figure 8 (e). This study found no significant differences in the chao1, Shannon, and Simpson indices among all mice before modeling. Figure 8 b, Figure 8 The result showed that (p>0.05) indicated that there was no significant difference in fecal bacterial community diversity and abundance between the two groups of mice before the experiment. After modeling, the chao1, Shannon, and Simpson indices increased in the L group (p>0.05). Figure 8 f, Figure 8 g, Figure 8 The result showed that intraductal perfusion of Lactobacillus paracasei for 24 h (P>0.05) indicated that the fecal bacterial community diversity and abundance increased slightly after 24 h of perfusion of the lactiferous ducts, but the difference was not statistically significant.

[0143] PCA is used to reflect differences in the composition and structure of fecal microbiota in mice. PCA analysis based on unweighted UniFrac distance showed that the composition of fecal bacterial communities was relatively similar across groups before Staphylococcus aureus infection. Figure 8 (a) After infection, the diversity of gut bacterial communities in mice in each group increased, and the inter-individual differences increased.Figure 9 (b)

[0144] At the level of the door ( Figure 9 In the middle group (A), Bacteroidetes was the main bacteria in group S (48.8%) and group L (61.9%). At the level of other phyla, group S consisted of Firmicutes (33.9%), Verrucous Microbes (11.4%), Dethiobacterium (1.8%), and Campylobacter (1.1%); group L consisted of Firmicutes (28.7%), Proteobacterium (3.0%), Dethiobacterium (2.4%), and Verrucous Microbes (1.8%). This indicates that Lactobacillus paracasei altered the phylum-level structure of the fecal flora in mastitis mice.

[0145] Further analysis of fecal microbiota composition in mastitis mice at the genus level ( Figure 10 In the middle B group and the S group, the species were composed of Bacteroides (11.5%), Akkermania (11.4%), Lactobacillus (6.6%) and Dubosiella (4.5%). In the L group, the species of Bacteroides (23.1%) increased relatively, while the species of Lactobacillus (4.8%) and Dubosiella (2.5%) decreased relatively, indicating that Lactobacillus paracasei altered the genus-level structure of the fecal flora in mastitis mice.

[0146] The levels of Prevotellaceae_NK3B31_group (associated with liver injury and autoimmune inflammation) and unidentified_Gastranaerophilales (increased abundance is often accompanied by inflammation and decreased immunity) were significantly reduced (P<0.05).

[0147] In summary, perfusion with Lactobacillus paracasei can reduce neutrophil infiltration in mammary tissue of mice with Staphylococcus aureus-induced mastitis and decrease the abundance of harmful bacteria in feces, thus providing some protection for mammary tissue. However, it has no significant effect on the content of related inflammatory factors.

[0148] Example 5: Protective effect of Lactobacillus paracasei against DSS-induced colitis in mice.

[0149] 1. Establishment of a mouse enteritis model

[0150] After 7 days of acclimatization feeding, the mice were randomly divided into three groups: a blank control group, an enteritis model group, and a CFCFC018 *Lactobacillus paracasei* treatment group, with 8 mice in each group. A 3% DSS solution was prepared in sterile physiological saline. Except for the blank control group, the enteritis model group and the CFCFC018 *Lactobacillus paracasei* treatment group were administered 200 μL of DSS solution by gavage daily for 7 consecutive days to establish a mouse ulcerative colitis model. Specific grouping and treatment methods are shown in Table 12.

[0151] Table 12 Mouse grouping and treatment

[0152]

[0153] During modeling, the state of the mice was observed and recorded in detail every day, including the daily weight gain of the mice, eating, stool characteristics, and blood in the stool, and the DAI score was given according to the scoring standard (Table 13). Disease activity index (DAI) = body weight loss rate score + stool characteristics score + occult blood score.

[0154] Table 13 DAI scoring standard

[0155]

[0156] Comprehensive evaluation of body weight, stool consistency, and fecal occult blood showed that the body weight of the blank group mice did not change, and the stool consistency was normal, and the fecal occult blood was negative; the body weight of the DSS group mice decreased significantly, the stool consistency was not clear, and there was visible blood stool; the DSS+CFCFC018 group mice significantly reduced the body weight loss, the stool was mucous, and the stool was blue (Table 14). Collect mouse feces, fresh feces of DSS group mice is visible blood stool, stool is soft. After drying, it is red, black or green. As can be seen from the figure, compared with the DSS group, the feces of the mice in the bacterial solution CFCFC018 group improved, and the blood stool phenomenon decreased significantly (Fig. 2). Figure 10

[0157] Table 14 DAI score

[0158]

[0159] (2) Effect on the pathological changes of colon tissue of intestinal inflammation mice

[0160] After the 4% paraformaldehyde fixed colon tissue was successively dehydrated, transparent, immersed in wax, and embedded, hematoxylin-eosin (H&E) staining was performed, and the H&E staining pictures of the colon tissue were scored according to the scoring standard in Table 15.

[0161] Table 15 Histopathological scoring of colon tissue H&E staining pictures

[0162]

[0163] The results showed that the submucosal layer of the DSS model group was edematous, the tight connection with the muscle layer was damaged, the number of neutrophils increased, and the number of goblet cells did not change significantly. The bacterial solution treatment group significantly reduced the submucosal edema phenomenon. Figure 12 Figure 13

[0164] ​​(3) Effect on serum immunological indexes of intestinal inflammation mice

[0165] The detection of serum immunological indexes was performed according to the kit instructions, and the procedure was as follows:

[0166] (1) Equilibrium: take out the microplate in the kit and equilibrate at room temperature for 30 min;

[0167] (2) Sample addition: no sample was added to the blank hole, 50 μL of sample solution was added to the sample hole, and 50 μL of standard solution of different concentrations was added to the standard hole;

[0168] (3) Incubation: incubate at 37℃ for 30 min;

[0169] (4) Washing: wash with washing solution for 5 times, and shake dry;

[0170] (5) Incubation: add 50 μL of enzyme-labeled reagent, and incubate at 37℃ for 30 min;

[0171] (6) Washing: wash with washing solution for 5 times, and shake dry;

[0172] (7) Color development: sequentially add 50 μL of color developing agent A and 50 μL of color developing agent B, and incubate at 37℃ in the dark for 30 min;

[0173] (8) Determination: add 50 μL of stop solution to terminate the reaction, and determine the absorbance at 450 nm by using a microplate ultraviolet-visible spectrophotometer;

[0174] (9) Calculation: draw a standard curve, substitute the absorbance of the sample into the standard curve, and calculate the content of the detected cytokine.

[0175] The detection results (Table 16) showed that in the DSS-induced colon inflammation test of Paracasei, the levels of proinflammatory factors TNF-α and IL-1β in the serum of mice were reduced after treatment with CFCFC018 bacterial solution, but the statistical difference was not significant (P>0.05). P Compared with the blank group, the IL-2 level of the DSS group and the CFCFC018 bacterial solution treatment group was increased, but the statistical difference was not significant (P>0.05). P Compared with the blank control group, the levels of anti-inflammatory cytokines in the serum of the group containing DSS were lower than those of the blank control group.

[0176] Table 16 Serum immunological indexes in DSS intestinal inflammation test

[0177]

[0178] Note: TNF-α, IL-1β, and IL-2 are common proinflammatory factors; IL-4 and IL-10 are anti-inflammatory cytokines.

[0179] (4) Effect on serum biochemical indicators of intestinal inflammation mice

[0180] The serum biochemical indicators were determined by using a laboratory automatic biochemical analyzer. The detection items and determination methods are shown in Table 17.

[0181] Table 17 Serum biochemical detection items and determination methods

[0182]

[0183] The detection results (Table 18) showed that in the DSS test, the addition of L. paracasei CFCFC018 could increase the alkaline phosphatase level in the serum of mice, and there was no significant difference compared with the DSS group. Compared with the blank control group, the ALP content of the two treatment groups decreased significantly (P < 0.05). P The cholesterol content of the DSS group was higher than that of the blank group, and although it decreased after treatment with the bacterial solution, there was no significant difference (P > 0.05). The high-density lipoprotein content of the two treatment groups increased significantly compared with the blank group (P < 0.05). P P

[0184] Table 18 Serum biochemical indicators of DSS intestinal inflammation test

[0185]

[0186] In summary, L. paracasei CFCFC018 plays an important role in improving intestinal inflammation and is expected to become a new type of food additive and health food.

[0187] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications

[0188] within the spirit and principles of the present application, such as equivalent replacements, improvements, etc., shall be included in the protection scope of the present application.​​

Claims

1. A Lactobacillus paracasei (L. paracasei) strain (CFCFC018), characterized by, Lacticaseibacillus paracasei ) the nucleotide sequence of the 16S rRNA gene of which is represented in SEQ ID NO: 1 and which has the following characteristics: The Lactobacillus paracasei (Lactobacillus paracasei) Lacticaseibacillus paracasei CFCFC018 was deposited with China Center for Type Culture Collection on July 15, 2025, and the deposit number is CCTCC M 20251610.

2. The Lactobacillus paracasei (Lactobacillus paracasei) of claim 1 Lacticaseibacillus paracasei ) CFCFC018 for use in (1) or (2) as follows: (1) producing amino acids; (2) producing neurotransmitters; The amino acids are glutamic acid, arginine, alanine, leucine, isoleucine or valine. The neurotransmitters are tyramine, dopamine DOPA, norepinephrine and kynurenine.

3. Lactobacillus paracasei (CNCM® I-2081) for use according to claim 1. Lacticaseibacillus paracasei ) CFCFC018 for use in the preparation of a medicament for the prevention and treatment of mastitis and for the prevention and treatment of enteritis.

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