Lactobacillus paracasei CFCFC018, microbial inoculum and application of lactobacillus paracasei CFCFC018

Abstract

The invention discloses lactobacillus paracasei CFCFC018, a microbial agent and application of the lactobacillus paracasei CFCFC018, and belongs to the technical field of microorganisms. The lactobacillus paracasei CFCFC018 is separated from intestinal contents of rural cats in half-farming and half-pastoral areas in Inner Mongolia, does not need genetic engineering modification, can highly express amino acids and neurotransmitters, can remarkably relieve mammary gland alveolar structure damage in mammary gland tissues of rats suffering from mastitis induced by staphylococcus aureus, and has the advantages of being high in safety and good in stability. The thickening of a mammary gland interacellular matrix layer is avoided, the infiltration number of neutrophils is reduced, the damage degree of mammary gland tissues is reduced, and a certain protection effect on the mammary gland tissues is achieved. The research also shows that the strain can reduce the abundance of harmful bacteria in excrement, improve intestinal inflammation and relieve edema of intestinal submucosa, which provides a new thought for preventing and treating mastitis by regulating intestinal microbiota.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus paracasei CFCFC018, a bacterial agent and applications thereof. Background Art

[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 over 20% of mastitis in dairy cows is caused by Staphylococcus aureus, a bacterium that is difficult to eradicate, has a low cure rate with antibiotics, and continues to spread. It can even cause chronic, subclinical, and hyperacute gangrenous mastitis. Antibiotics are commonly used in the clinical prevention and treatment of S. aureus infections. However, drug residues and resistance in dairy cows can affect the safety and quality of milk and dairy products. Antibiotic-resistant S. aureus isolated from bovine mastitis has become a major cause of recurrent mastitis. Therefore, the search for safe and effective treatments for mastitis is crucial and essential.

[0003] The intestinal microbiome has been shown to be a complex ecosystem that plays an active role in host metabolism, immune regulation, and overall health. Microorganisms that colonize the intestine can also influence tissues and organs beyond the gastrointestinal tract, thereby participating in or regulating the development and progression of diseases. Recent research indicates that the intestinal microbiome plays a key role in the development of mastitis in dairy cows and that the intestinal microbiome can influence inflammatory diseases. This provides a new approach to preventing and treating mastitis by modulating the intestinal microbiome.

[0004] Lactobacillus paracasei ( Lactobacillus paracasei ) is a facultative anaerobic, non-motile, non-spore-forming lactic acid bacterium that not only has strong antibacterial activity but also has multiple immune response regulatory functions. It has a good probiotic effect on human health and can be obtained from a variety of animal environments such as the intestines, mouths, silage, and milk. Currently, 34 species of Lactobacillus paracasei have been isolated, 8 of which were isolated from the digestive system flora of humans or other animals. Based on current taxonomic technology, all are classified as Lactobacillus paracasei, but their properties vary greatly due to different isolation and screening sources. Therefore, expanding research on the probiotic properties and antibacterial effects of Lactobacillus paracasei from different isolation sources is of great significance for applications in different fields. Summary of the Invention

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

[0006] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei) CFCFC018, the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) CFCFC018 was deposited in the China Center for Type Culture Collection (No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on July 15, 2025, and its deposit number is CCTCC M 20251610.

[0007] The second aspect of the present invention provides a bacterial agent, which contains the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei )CFCFC018.

[0008] The bacterial agent contains Lactobacillus paracasei ( Lacticaseibacillus paracasei ) CFCFC018 exists in the form of live bacterial suspension or fermentation broth.

[0009] The third aspect of the present invention provides the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of CFCFC018 or the above-mentioned microbial agents in the following (1) or (2): (1) Production of amino acids and their derivatives; (2) Produce neurotransmitters and their precursors.

[0010] The amino acids and their derivatives include: glutamic acid, arginine, alanine, leucine, isoleucine or valine.

[0011] The neurotransmitters and their precursors include: tyramine, dopa DOPA, norepinephrine and kynurenine.

[0012] Amino acids are the basic molecules for protein synthesis and metabolic regulation. Glutamic acid is an amino acid that can participate in metabolism as an amino acid and can also play a role in signal transmission as a neurotransmitter. It is often used in the preparation of condiments or anti-anxiety foods and medicines; arginine can improve vascular function and is used to prepare cardiovascular health products; leucine, isoleucine and valine can promote muscle synthesis. Neurotransmitters are key molecules that regulate the function of the nervous system and can be used to intervene in brain diseases and regulate the gut-brain axis. Natural strains do not synthesize amino acids in large quantities by themselves, but mainly obtain amino acids by decomposing milk proteins through protein hydrolysis systems (such as cell envelope protease PrtP, peptidase PepO / PepX). The Lactobacillus paracasei provided by the present invention ( Lacticaseibacillus paracasei ) The differences between CFCFC018 and existing Lactobacillus paracasei include: the strain does not require genetic engineering and can highly express amino acids (and their derivatives) and neurotransmitters (and their precursors) on its own.

[0013] A fourth aspect of the present invention provides the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of CFCFC018 or the above-mentioned bacterial agent in the preparation of products for preventing and treating mastitis and enteritis.

[0014] The prevention and treatment of mastitis is specifically manifested in: significantly reducing damage to the mammary gland alveolar structure, avoiding thickening of the mammary gland inter-alveolar matrix layer, reducing the number of neutrophil infiltration, and alleviating the degree of mammary gland tissue damage.

[0015] The specific performance of the enteritis prevention and treatment is: alleviating edema of the intestinal submucosa.

[0016] In a fifth aspect, the present invention provides a probiotic preparation for preventing and treating mastitis, wherein the probiotic preparation is prepared with the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei ) CFCFC018 is the active ingredient.

[0017] Beneficial effects of the present invention: The present invention isolated a strain of Lactobacillus paracasei (Lactobacillus paracasei) from the intestinal contents of farm cats in the semi-agricultural and semi-pastoral areas of Inner Mongolia. Lacticaseibacillus paracasei ) CFCFC018, a strain that requires no genetic engineering and naturally produces high levels of amino acids and neurotransmitters, significantly reduces structural damage to mammary alveoli in mice with Staphylococcus aureus-induced mastitis, prevents thickening of the interacinar matrix, reduces neutrophil infiltration, and alleviates the severity of mammary tissue damage, demonstrating a protective effect. Studies have also shown that this strain can reduce the abundance of harmful bacteria in feces, improve intestinal inflammation, and reduce intestinal submucosal edema, offering a new approach for preventing and treating mastitis by modulating the intestinal microbiome. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The morphological characteristics of strain CFCFC018; including the colony morphology of strain CFCFC018 (left), optical microscope examination photo (middle) and scanning electron microscope observation photo (right).

[0019] Figure 2 is the strain growth characteristic curve; Figure 2 A in the middle is the growth curve of strain CFCFC018, Figure 2 Middle B is the acid production curve of strain CFCFC018.

[0020] Figure 3 The figure shows the morphology of strain CFCFC018 on sheep blood agar plate.

[0021] Figure 4 This is the mouse abdominal cavity anatomy diagram; Figure 4 A in the middle is the abdominal anatomical diagram of the mice in the control group. Figure 4 Middle B is the abdominal anatomical diagram of mice in the CFCFC018 strain treatment group.

[0022] Figure 5 The in vitro inhibitory effect of Lactobacillus paracasei on Staphylococcus aureus.

[0023] Figure 6 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. parnassius pretreatment group, Figure 6 c is the mammary tissue damage score.

[0024] Figure 7 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.

[0025] Figure 8 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 mouse fecal bacterial community before S. aureus infection (P>0.05); Figure 8 e-h are the Goods_coverage value, chao1, shannon, and simpson index of the mouse intestinal bacterial community after S. aureus infection (P>0.05); PS and PL represent the negative control group and the P. parnassius pretreatment group before infection, respectively, and S and L represent the negative control group and the P. parnassius pretreatment group after infection, respectively.

[0026] Figure 9 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. parnassius pretreatment group before infection, respectively, and S and L represent the negative control group and the P. parnassius pretreatment group after infection, respectively.

[0027] Figure 10 Changes in the composition of fecal microbial community in mice with mastitis; wherein, Figure 10 A is the change in phylum level of intestinal bacterial community in mice after S. aureus infection, Figure 10 B is the change in genus level of intestinal bacterial community in mice after S. aureus infection; S and L represent the negative control group and the P. parnassius pretreatment group after infection, respectively.

[0028] Figure 11 Differences in relative abundance of fecal microbiota of mice with mastitis (genus level); wherein, Figure 11 a is the difference in relative abundance of fecal microorganisms of mice before S. aureus infection, Figure 11 b is the difference in relative abundance of fecal microorganisms of mice after S. aureus infection; PS and PL represent the negative control group before infection and the P. casei pretreatment group, respectively, and S and L represent the negative control group and the P. casei pretreatment group after infection, respectively.

[0029] Figure 12 Effect of P. casei on feces of DSS-induced colitis mice.

[0030] Figure 13 Test colon intestinal tract section of P. casei on DSS-induced colitis mice. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed description is illustrative only, and is intended to provide further description of the application. Unless otherwise defined, 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 belongs.

[0032] The specific embodiments of the present application will be further described in conjunction with the following examples. The following detailed description is illustrative only, and is not intended to limit the scope of the present application.

[0033] Example 1: Isolation and identification of strains 1. Isolation and purification of strains Semi-free-range state of the pastoral cat, by rectal method, take 10 g of rectal feces, placed in MRS liquid medium, quickly taken back to the laboratory, and the strain isolation was carried out.

[0034] In a sterile operation table, 10 -1 ~ 10 -6 Gradient dilution (100 µL of culture medium was added to 900 µL of physiological saline), 200 µL of diluted bacterial solution was spread on MRS solid plate, and incubated at 37 ℃. After the colonies grew, the different morphological colonies were picked up on the plate with 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.

[0035] 2. Identification of strains (1) Colony morphology and fungal staining microscopy After diluting the bacterial solution to an appropriate concentration, spread it on a plate and culture it for two days to observe the colony morphology; observe the bacterial cell morphology through Gram staining microscopy. Figure 1 It can be seen that the colonies are milky white, with a smooth surface, and are in good growth condition on the plate. The colonies have protrusions and a thick texture. Microscopic observation shows that they are Gram-positive, rod-shaped bacteria with blunt ends.

[0036] (2) Bacterial physiological and biochemical identification Physiological and biochemical tests of carbon source oxidation and carbon source utilization were performed on the strains according to Bergey's Manual of Bacterial Identification (8th edition).

[0037] Table 1: Physiological and biochemical characteristics of strain CFCFC018 – enzyme activity +: positive reaction; -: negative reaction; Table 2: Physiological and biochemical characteristics of strain CFCFC018 – Acid production using carbon sources +: positive reaction; -: negative reaction; (3) Bacterial 16S rRNA sequence homology analysis Sequence detection was performed using universal primers for lactic acid bacteria, 27f and 1492r, and the strain was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The 16S rRNA gene sequence of the strain was added to the NCBI database for comparison. A Neighbor-Joining phylogenetic tree was constructed with Bacillus subtilis IAM12118 (AB042061) as an outbranch. Phylogenetic analysis showed that the strain belonged to Lactobacillus paracasei. Lacticaseibacillus paracasei The strain was biologically deposited, and the deposit information is as follows: Bacteria name: Lactobacillus paracasei CFCFC018; Category naming: Lacticaseibacillus paracasei ; Depository: China Center for Type Culture Collection; Address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province; Deposit date: July 15, 2025; Accession number: CCTCC M 20251610.

[0038] Example 2: Probiotic properties and safety evaluation of Lactobacillus paracasei CFCFC018 Test strains: The experimental strain Lactobacillus paracasei CFCFC018 was isolated by our laboratory, and Staphylococcus aureus (ATCC25923), Salmonella typhimurium (ATCC14028) and Escherichia coli (ATCC25922) were purchased from domestic microbial culture collection institutions.

[0039] 1. Growth characteristics and acid production performance of Lactobacillus paracasei CFCFC018 The strain isolated in Example 1 was activated and cultured. Subsequently, 100 μL of the bacterial solution after 18 to 24 hours of activation culture was evenly inoculated into MRS liquid culture medium at a 2% inoculum size. The culture was carried out under constant temperature and shaking conditions of 28°C and 160 rpm. During the culture process, regular sampling was adopted. The absorbance of the culture solution at 600 nm (OD) was accurately measured every hour for the first 5 hours using a spectrophotometer. 600 After 6 h, the absorbance of the culture medium at 600 nm (OD 600 ) value. Simultaneously, sample the appropriate culture medium every 2 hours and measure the pH using a handheld pH meter. Plot an acid production curve with time on the X-axis and the pH of the culture medium on the Y-axis.

[0040] from Figure 2 The growth curve of strain A shows that within 0-2 hours after passage, the strain grew slowly, entering the growth retardation phase. After 2 hours, the growth rate increased significantly, entering the logarithmic growth phase. After 14 hours of passage, the growth curve flattened and entered the stable phase. Figure 2 From the acid production curve of strain B, it can be seen that the pH value of strain CFCFC018 decreases slowly within 2 hours of inoculation, then decreases rapidly after 2 hours, and drops below 4.0 after 8 hours.

[0041] 2. Study on the antibacterial ability of Lactobacillus paracasei CFCFC018 To determine the growth inhibition of Lactobacillus paracasei CFCFC018 against three common enteric pathogens, including Staphylococcus aureus (ATCC25923), Salmonella typhimurium (ATCC14028), and Escherichia coli (ATCC25922), the antibacterial activity of strain CFCFC018 was studied using the Oxford cup agar diffusion method.

[0042] After 24 hours of activation culture, the CFCFC018 bacterial suspension was centrifuged at 6000 rpm at 4°C for 10 minutes and set aside. 200 μL of each of the E. coli, Salmonella, and Staphylococcus aureus bacterial suspensions were evenly spread onto LB solid medium using a spreading rod. Once the suspensions were completely absorbed, two Oxford cups were placed on the LB plate and 200 μL of the supernatant and 200 μL of the original bacterial suspension were added, respectively. The plates were incubated at 37°C for 24 hours. The formation of inhibition zones was observed and the diameter of the inhibition zones was accurately measured using a vernier caliper. An inhibition zone diameter of 7–8 mm was defined as insensitive; 10–20 mm was moderately sensitive; and 21–30 mm and inhibition zone diameters >30 mm were considered highly sensitive.

[0043] The results showed that the fermentation broth of CFCFC018 had moderate inhibitory effects on Escherichia coli, Salmonella, and Staphylococcus aureus, and the supernatant had a moderate inhibitory effect on Salmonella (Table 3).

[0044] Table 3 Inhibition zone diameters of the fermentation supernatant and stock solution of strain CFCFC018 against common pathogens 3. Hemolytic activity assay of Lactobacillus paracasei CFCFC018 Bacterial hemolysis can be divided into α-hemolysis, β-hemolysis and γ-hemolysis. Among them, α-hemolysis (grass green appears around the colonies) and γ-hemolysis (no grass green and no transparent circle around the colonies) are not pathogenic. Only β-hemolysis (transparent circle appears around the colonies) is related to pathogenicity.

[0045] Take the activated culture solution from 18–24 hours and streak it onto the plate with an inoculating loop. Each streak begins at the end of the previous streak, avoiding duplication. Three to four streaks are sufficient. Incubate the plate at 37°C for 24 hours under constant temperature and anaerobically observe for the formation of a hemolytic zone.

[0046] from Figure 3 It can be seen that after culturing Lactobacillus paracasei CFCFC018 on the sheep blood agar plate for 24 hours, the colony color is grayish white with no hemolytic ring around it, and it is determined to be γ-type hemolytic, that is, non-hemolytic, and is a very safe strain.

[0047] 4. Safety Evaluation of Lactobacillus paracasei CFCFC018 (1) Experimental methods Twelve SPF-grade C57BL / 6 mice, half male and half female, 5 weeks old, weighing (22 ± 2) g, were provided by Spefoc Biotechnology (Beijing) Co., Ltd. They were housed in separate cages under a 12-hour day / night cycle, with a temperature of 22–26°C and a humidity of 50%–70%. The mice were randomly divided into two groups, a control group and an experimental group, with six mice in each group. The experiment began after a 3-day adaptive feeding period, with free access to water and food.

[0048] The method was based on the National Food Safety Standard Acute Oral Toxicity Test (GB15193.3-2014). The control group was given 200 μL of normal saline per day, and the experimental group was given 200 μL of bacterial solution per day. The viable bacterial count was 1.0 × 10 9 cfu / mL. During this process, mice were given free access to food and water for 21 days. During the experiment, detailed records were kept of their food intake, mental state, weight changes, and mortality.

[0049] (2) Experimental results During the observation period, no abnormalities were observed in either the control or experimental group of mice, and no deaths occurred. Post-mortem examination of the mice's organs revealed no gross lesions, and tissue culture revealed no bacterial growth. This suggests that Lactobacillus paracasei CFCFC018 is not pathogenic to mice. As shown in Table 4, compared with the control group, the group treated with the bacterial solution experienced decreased body weight and daily weight gain, while other parameters remained unchanged.

[0050] Table 4 Safety evaluation indicators As shown in Table 5, compared with the control group, oral administration of CFCFC018 bacterial solution had no significant effect on the routine blood indicators of mice.

[0051] Table 5 Blood routine index determination like Figure 4 As shown, after the experiment, the mice were dissected and examined, revealing no bleeding in any organs, and no abnormalities in the liver or spleen. These results indicate that the Lactobacillus paracasei CFCFC018 strain had no adverse effects on the mice's internal organs.

[0052] Example 3: Quantitative determination of amino acids and neurotransmitters of Lactobacillus paracasei Instruments: AB Sciex QTRAP 6500+ mass spectrometer, AB SciexExionLCTMAD liquid chromatograph.

[0053] Reagents: 23 amino acid standards and two stable isotope-labeled standards (L-Alanine-d4 (deuterated alanine-d4) and deuterated phenylalanine-d8 (deuterated phenylalanine-d8)) were purchased from Sigma-Aldrich. Ammonium acetate (AR, Sigma-Aldrich, USA), methanol, acetonitrile, and formic acid (LC-MS, Thermo-Fisher, USA), and ultrapure water Mill-Q (Millipore, USA) were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. Table 6 Information on amino acids and their derivatives Amino acid standard preparation: Accurately weigh 23 amino acid standards to prepare a 5 mg / mL mixed standard stock solution. Dilute the stock solution with methanol to obtain a concentration series as shown in Table 8. Prepare L-Alanine-d4 and Phenylanine-d2 solutions at specific concentrations and mix thoroughly to obtain the internal standard solution (IS). Store the stock solutions and working solutions for the linearity, internal standard, and quality control assays at -20°C.

[0054] Preparation of neurotransmitter standards: Accurately weigh 23 neurotransmitter standards to prepare mixed linearization stock solutions. Dilute the linearization stock solutions with methanol to create a series of working solutions. Prepare internal standard solutions (IS) with specific concentrations of L-Tyrosine-d4, Alanine-d4, and IAA-d4. Store the linearization, internal standard, and quality control stock solutions and working solutions at -20°C.

[0055] Table 7 Neurotransmitters and their precursors Extraction of amino acid metabolites: Take the sample and add it to mass spectrometry water, vortex mix to prepare the diluted sample; take 50 μL of the diluted sample, add 200 μL of precipitant containing mixed internal standards (acetonitrile: methanol = 1:1), vortex mix, let it stand on ice for 30 min, centrifuge at 12000 rpm at 4℃ for 10 min, and take the entire supernatant for LC-MS analysis.

[0056] Extraction of neurotransmitter metabolites: Take the sample and add it to mass spectrometry water, vortex mix to prepare the diluted sample; take 100 μL of the diluted sample and add 400 μL of precipitant containing mixed internal standards (acetonitrile: water = 8:2), vortex mix, let it stand on ice for 30 min, centrifuge at 12000 rpm at 4℃ for 10 min, and take the entire supernatant for LC-MS analysis.

[0057] Amino acid metabolite chromatography and mass spectrometry methods: Column: ACQUITY UPLC BEH Amide (2.1 × 100 mm, 1.7 μm); Mobile phase: Phase A: 5 mM ammonium acetate aqueous solution containing 0.1% formic acid; Phase B: acetonitrile containing 0.1% formic acid; Column temperature: 50 °C; Injection volume: 1 μL; Flow rate: 0.3 mL / min.

[0058] The amino acid chromatographic concentration gradient is shown in the following table: Table 8 Amino acid chromatographic concentration gradient Mass spectrometry conditions: electrospray ionization (ESI) in positive ionization mode. Source temperature: 550°C, source voltage: 5500 V, curtain gas: 35 psi, nebulizer gas: 50 psi, auxiliary gas: 60 psi. Multiple reaction monitoring (MRM) scanning was used.

[0059] Neurotransmitter chromatography and mass spectrometry methods: Column: Waters XSelect HSS T3 (2.1 × 150 mm, 2.5 μm); Mobile phase: Phase A: 5 mM ammonium acetate aqueous solution containing 0.1% formic acid; Phase B: acetonitrile containing 0.1% formic acid; Column temperature: 45 °C; Injection volume: 2 μL; Flow rate: 0.3 mL / min.

[0060] The neurotransmitter chromatographic concentration gradient is shown in Table 9 below: Table 9 Neurotransmitter chromatographic concentration gradient Mass spectrometry conditions: electrospray ionization (ESI) in negative ionization mode. Source temperature: 550°C, source voltage: -4500 V, curtain gas: 35 psi, nebulizer gas: 60 psi, auxiliary gas: 60 psi. Multiple reaction monitoring (MRM) scanning was used.

[0061] (1) Quantitative detection of amino acids synthesized by Lactobacillus paracasei As shown in Table 10, the amino acids most expressed by Lactobacillus paracasei were glutamic acid and arginine, followed by alanine and branched-chain amino acids (leucine, isoleucine, valine, etc.).

[0062] Table 10 Quantitative detection of amino acids and derivatives thereof of Lactobacillus paracasei (2) Quantitative detection of neurotransmitters of Lactobacillus paracasei As shown in Table 11, the neurotransmitters highly expressed by Lactobacillus paracasei are tyramine, DOPA, norepinephrine and kynurenine.

[0063] Table 11 Quantitative detection of Lactobacillus paracasei neurotransmitters and their precursors The results of integrated amino acid composition and neurotransmitter quantitative analysis showed that the strain highly expressed glutamate (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).

[0064] Neurotransmitter data confirmed that the strain highly expressed tyramine (2584.72 ng / mL), DOPA (1991.08 ng / mL), norepinephrine (560.83 ng / mL), and kynurenine (421.15 ng / mL).

[0065] Example 4: Protective effect of Lactobacillus paracasei CFCFC018 on mastitis in mice induced by Staphylococcus aureus Experimental animals: 8-week-old SPF Kunming mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., Animal Production License No.: SCXK (Beijing) 2019-0010. The husbandry and experimental procedures of experimental animals were in accordance with the Guidelines for the Ethical Review of Laboratory Animals—Welfare (GB / T 35892-2018).

[0066] 1. Detection of the protective effect of Lactobacillus paracasei CFCFC018 on mammary glands (1) Construction of mouse mastitis model On the 8th day after birth, the mice and their pups were housed in separate cages. The fourth pair of mammary glands of the mice were taken for modeling. 50 μL of Staphylococcus aureus suspension was injected into the fourth pair of mammary glands of the mice to establish a mouse model of mastitis.

[0067] Fourteen mice successfully modeled were divided into two groups: a negative control group (S group) and a Lactobacillus paracasei preventive treatment group (L group), with seven mice in each group. Mice in group L received a 50 μL suspension of Lactobacillus paracasei injected into each mammary gland; mice in group S received an equal volume of sterile saline injected into each mammary gland. Twenty-four hours later, the mice were sacrificed by cervical dislocation for sampling. All mice were fasted for 12 hours before sampling.

[0068] (2) Breast tissue pathology examination The fourth pair of mammary glands of mice were removed, and one side was fixed overnight in 4% paraformaldehyde solution. Hematoxylin and eosin (H&E) staining and sectioning were performed. Histological scores range from 1 to 5, with higher scores indicating more severe tissue damage. A score of 1 indicates no histopathological features (i.e., no necrosis and no neutrophil or lymphocyte infiltration), 2 indicates minimal histopathological features (i.e., a single neutrophil infiltration), 3 indicates mild histopathological features (i.e., a small number of neutrophil infiltration), 4 indicates moderate histopathological features (i.e., a large number of neutrophil infiltration and mild damage to the glandular structure), and 5 indicates severe histological features (i.e., a large number of neutrophil infiltration and severe damage to the glandular structure).

[0069] like Figure 6 As shown in Figure 2, S. aureus caused significant pathological damage, with an increase in the number of neutrophils in the mammary alveoli and thickening of the inter-alveolar matrix layer ( Figure 6 Compared with the S group, pretreatment with Lactobacillus paracasei significantly alleviated the damage to the mammary gland alveolar structure and reduced the number of neutrophil infiltration, and the alveolar state was good ( Figure 6 Semi-quantitative scoring of breast tissue revealed that pretreatment with Lactobacillus paracasei significantly reduced the degree of breast tissue damage (P<0.05) ( Figure 6 Middle c).

[0070] (3) Detection of inflammatory cytokines and MPO activity in breast tissue and serum ELISA kits were used to detect the levels of IL-1β, IL-6, TNF-α, and MPO in mouse mammary tissue. Eyeballs were removed and blood was collected in procoagulant blood collection tubes. The blood was centrifuged at 4°C, 4000 rpm for 10 min, and the supernatant was aspirated to detect the levels of IL-1β, IL-6, TNF-α, and MPO in mouse serum. The experimental operation and sample testing were carried out strictly in accordance with the requirements of the kit instructions.

[0071] The test results show that ( Figure 7 In group L, the IL-1β level in breast tissue was slightly increased, while the IL-6 and TNF-α levels were slightly decreased, with no significant differences (P>0.05). There was no significant difference in MPO activity in breast tissue between the two groups (P>0.05). The serum IL-1β, IL-6, and TNF-α levels in group L were slightly lower, but not significantly different from those in group S (P>0.05). Lactobacillus paracasei did not affect serum MPO activity (P>0.05).

[0072] (4) 16SrRNA gene sequencing and analysis of fecal bacterial communities Fresh feces were collected from each group of mice before and after the mastitis model was established, placed in sterile cryovials, snap-frozen in liquid nitrogen, and shipped on dry ice to Beijing Novogene Biotechnology Co., Ltd. Paired-end sequencing of the V3-V4 region of the 16S rRNA gene was performed using the Illumina NovaSeq platform to analyze bacterial community DNA fragments from fecal samples from 14 mice. 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 adenine (A), cytosine (C), guanine (G), or thymine (T), and is represented by "A" in the WIPO ST.26 sequence listing. DADA2 was used to denoise amplicon sequence variants (ASVs), and species annotation and abundance analysis were performed after obtaining valid data. Alpha diversity was analyzed by 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 T-test was used to analyze species with significant differences between groups at the genus level.

[0073] Depend on Figure 8 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 microbiome is above 99%, which can more comprehensively reflect the types and structures of mouse fecal microbial communities ( Figure 8 Middle a, Figure 8 This study found that there was no significant difference in chao1, Shannon and Simpson indexes among all mice before modeling ( Figure 8 Middle b, Figure 8 d) (P>0.05), indicating that there was no significant difference in the diversity and abundance of fecal bacterial communities between the two groups of mice before the experiment. After modeling, the chao1, Shannon and Simpson indices of the L group increased ( Figure 8 Middle f, Figure 8 Middle g, Figure 8 The results showed that intraductal instillation of Lactobacillus paracasei for 24 h could slightly increase the diversity and abundance of fecal bacterial communities, but there was no significant difference.

[0074] PCA was used to reflect the differences in the composition and structure of the mouse fecal microbiome. PCA analysis based on unweighted UniFrac distance showed that the composition of the mouse fecal bacterial community in each group was similar before Staphylococcus aureus infection ( Figure 9 Middle (a), after infection, the diversity of intestinal bacterial communities in mice increased within each group, and the differences between individuals increased ( Figure 9 Middle b).

[0075] At door level ( Figure 10 Figure (A) shows that Bacteroidetes was the main bacteria in group S (48.8%) and group L (61.9%). At the level of other phyla, group S was composed of Firmicutes (33.9%), Verrucomicrobia (11.4%), Desulfobacteria (1.8%), and Campylobacter (1.1%); group L was composed of Firmicutes (28.7%), Proteobacteria (3.0%), Desulfobacteria (2.4%), and Verrucomicrobia (1.8%), indicating that Lactobacillus paracasei changed the phylum-level structure of the fecal flora in mice with mastitis.

[0076] The fecal microbiota composition of mastitis mice was further analyzed at the genus level ( Figure 10 In the middle (B), the S group was composed of Bacteroides (11.5%), Akkermansia (11.4%), Lactobacillus (6.6%), and Dubosiella (4.5%). In the L group, the Bacteroides (23.1%) increased relatively, while the Lactobacillus (4.8%) and Dubosiella (2.5%) decreased relatively, indicating that Lactobacillus paracasei changed the genus-level structure of the fecal flora in mice with mastitis.

[0077] The levels of Prevotellaceae_NK3B31_group (associated with liver damage and autoimmune inflammation) and unidentified_Gastranaerophilales (increased abundance is often accompanied by inflammation and decreased immunity) were significantly decreased (P<0.05).

[0078] In summary, infusion of Lactobacillus paracasei can reduce the infiltration of neutrophils in the mammary tissue of mice with mastitis induced by Staphylococcus aureus, reduce the abundance of harmful bacteria in feces, and have a certain protective effect on mammary tissue, but has no significant effect on the content of related inflammatory factors.

[0079] Example 5: Protective effect of Lactobacillus paracasei on dextran sulfate sodium (DSS)-induced colitis in mice 1. Establishment of Mouse Enteritis Model After 7 days of adaptive 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 eight mice in each group. A 3% DSS solution was prepared in sterile saline. Except for the blank group, the enteritis model group and the CFCFC018 Lactobacillus paracasei treatment group were gavaged daily with 200 μL of DSS solution for seven consecutive days to establish a mouse ulcerative enteritis model. Specific grouping and treatment methods are shown in Table 12.

[0080] Table 12 Grouping and treatment of mice During the modeling period, the mice's condition was observed and recorded daily, including daily weight gain, food intake, stool characteristics, and blood in stool. The DAI score was calculated according to the scoring criteria (Table 13). The disease activity index (DAI) = weight loss rate score + stool characteristics score + occult blood score.

[0081] Table 13 DAI scoring criteria Comprehensive evaluation of body weight, stool viscosity and fecal occult blood showed that the weight of mice in the blank group did not change, and the stool viscosity was normal, and the fecal occult blood was negative; the weight of mice in the DSS group decreased significantly, the stool viscosity was still unclear, and there was blood in the stool visible to the naked eye; the weight loss of mice in the DSS+CFCFC018 group was significantly reduced, and the stool was mucous and blue (Table 14). The mouse feces were collected. The fresh feces of mice in the DSS group were bloody stools visible to the naked eye, and the feces were soft and loose. After drying, they were red, black or green. As can be seen from the figure, compared with the DSS group, the fecal condition of mice in the bacterial liquid CFCFC018 group was improved, and the blood in the stool was significantly reduced ( Figure 12 ).

[0082] Table 14 DAI score (2) Effects on pathological changes in colon tissue of enteritis mice The colon tissue fixed with 4% paraformaldehyde was dehydrated, transparentized, wax-impregnated, and embedded in sequence, and then stained with hematoxylin-eosin (H&E). The histopathological scores of the H&E-stained images of the colon tissue were performed according to the scoring criteria in Table 15.

[0083] Table 15 Histopathological scoring of H&E stained images of colon tissue The results showed that the DSS model group had edema in the submucosal layer, the tight junction with the muscularis 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 edema in the submucosal layer ( Figure 13 ).

[0084] (3) Effects on serum immunological indicators in enteritis mice Serum immunological index detection was performed according to the kit instructions. The steps are as follows: (1) Equilibration: Take out the microplate from the test kit and equilibrate it at room temperature for 30 min; (2) Sample addition: No sample is added to the blank wells, 50 μL of sample solution is added to the sample wells, and 50 μL of standard solutions of different concentrations are added to the standard wells; (3) Incubation: incubate at 37°C for 30 min; (4) Washing: Wash 5 times with detergent and spin dry; (5) Incubation: Add 50 μL of enzyme-labeled reagent and incubate at 37°C for 30 min; (6) Washing: Wash 5 times with detergent and spin dry; (7) Color development: Add 50 μL of color developer A and 50 μL of color developer B in sequence and incubate at 37°C in the dark for 30 min; (8) Determination: Add 50 μL of stop solution to terminate the reaction, and measure the absorbance at 450 nm using a microplate UV-visible spectrophotometer; (9) Calculation: Draw a standard curve, substitute the sample absorbance into the standard curve, and calculate the cytokine content.

[0085] The test results showed (Table 16) that in the DSS-induced colitis test of Lactobacillus paracasei, the levels of inflammatory factors TNF-α and IL-1β in mouse serum were reduced after treatment with CFCFC018 bacterial solution, but the difference was not statistically significant ( P >0.05). Compared with the blank group, the IL-2 levels in the DSS group and the CFCFC018 bacterial solution treatment group increased, but the difference was not statistically significant. P >0.05). Compared with the blank control group, the levels of anti-inflammatory cytokines in the serum of the DSS-containing group were lower than those of the blank control group.

[0086] Table 16 Serum immune indicators of DSS enteritis test Note: TNF-α, IL-1β, and IL-2 are common inflammatory factors; IL-4 and IL-10 are anti-inflammatory cytokines.

[0087] (4) Effects on serum biochemical indicators in mice with enteritis Serum biochemical indicators were measured using a laboratory automatic biochemical analyzer. The test items and measurement methods are shown in Table 17.

[0088] Table 17 Serum biochemical test items and determination methods The test results showed (Table 18) that in the DSS test, the addition of Lactobacillus paracasei CFCFC018 increased the level of alkaline phosphatase in the serum of mice. 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); the cholesterol content in the DSS group was higher than that in the blank group. Although it was reduced after treatment with bacterial solution, there was no significant difference ( P >0.05), the high-density lipoprotein levels in the two treatment groups increased significantly compared with the blank group ( P <0.05).

[0089] Table 18 Serum biochemical indicators of DSS enteritis test In summary, Lactobacillus paracasei CFCFC018 plays an important role in improving intestinal inflammation and is expected to become a new type of food additive and health food.

[0090] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principles of the present application shall be Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei ) CFCFC018, characterized in that, The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) CFCFC018 was deposited in the China Center for Type Culture Collection on July 15, 2025, with the deposit number CCTCC M 20251610.

2. A bacterial agent, characterized in that The bacterial agent contains the Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei )CFCFC018.

3. The microbial agent according to claim 2, characterized in that The bacterial agent contains Lactobacillus paracasei ( Lacticaseibacillus paracasei ) CFCFC018 exists in the form of live bacterial suspension or fermentation broth.

4. Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei ) Use of CFCFC018 or the bacterial agent described in claim 2 in the following (1) or (2): (1) Production of amino acids and their derivatives; (2) Produce neurotransmitters and their precursors.

5. The use according to claim 4, characterized in that The amino acids and their derivatives include: glutamic acid, arginine, alanine, leucine, isoleucine or valine.

6. The use according to claim 4, characterized in that The neurotransmitters and their precursors include: tyramine, dopa DOPA, norepinephrine and kynurenine.

7. Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei ) Use of CFCFC018 or the bacterial agent described in claim 2 in the preparation of products for preventing and treating mastitis and enteritis.

8. The use according to claim 7, characterized in that The prevention and treatment of mastitis is specifically manifested in: significantly reducing damage to the mammary gland alveolar structure, avoiding thickening of the mammary gland inter-alveolar matrix layer, reducing the number of neutrophil infiltration, and alleviating the degree of mammary gland tissue damage.

9. The use according to claim 7, characterized in that The specific performance of the enteritis prevention and treatment is: alleviating edema of the intestinal submucosa.

10. A probiotic preparation for preventing and treating mastitis, characterized in that: The probiotic preparation is prepared with the Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei ) CFCFC018 is the active ingredient.

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