Lactobacillus fermentum hf19 having an ability to alleviate symptoms of chronic ulcerative colitis and use thereof

Abstract

The application discloses a lactobacillus fermentum HF19 with the ability of relieving symptoms of chronic ulcerative colitis and application thereof. Lactobacillus fermentum ​ ), which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO.M20252279; and a 16S rDNA sequence is shown as SEQ ID:1. The HF19 has the potential of inhibiting inflammatory reactions and protecting an intestinal epithelial barrier; further evaluation of acid resistance, bile salt resistance, in-vitro adhesion and sensitivity to commonly used antibiotics shows that the HF19 still has certain growth ability under the conditions of pH 3.0 acid and 0.1% bile salt, has the potential of resisting gastrointestinal environment, has detectable intestinal epithelial cell adhesion capacity, and does not show extensive drug resistance to most commonly used antibiotics, and has a safe basis for further development and application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a fermenting lactobacillus, specifically a fermenting lactobacillus that can relieve symptoms of chronic ulcerative colitis. Lactobacillus fermentum HF19 and its applications. Background Technology

[0002] In recent years, with the Westernization of dietary structure, changes in lifestyle, and environmental factors, the incidence of ulcerative colitis (UC) has been increasing year by year. Ulcerative colitis is a chronic, relapsing inflammatory bowel disease, mainly characterized by abdominal pain, diarrhea, mucus and bloody stools, and weight loss. The disease course is protracted and prone to relapse, severely impacting patients' quality of life. Currently, clinical interventions for ulcerative colitis mainly include aminosalicylic acid drugs, glucocorticoids, immunosuppressants, and biologics. Some severely ill patients or those with poor drug control require surgical treatment. However, long-term use of existing drugs may be accompanied by increased risk of infection, liver and kidney damage, immunosuppression, and secondary loss of response; surgery may also bring risks such as disease recurrence, anastomotic leakage, intestinal adhesions, intestinal obstruction, and short bowel syndrome. Therefore, developing novel probiotics that are safe, low-cost, suitable for long-term intake, and can help relieve the symptoms of ulcerative colitis has significant application value.

[0003] Lactic acid bacteria are a class of beneficial microorganisms widely found in traditional fermented foods and the human gut, playing roles in regulating gut microbiota, reducing inflammatory responses, enhancing intestinal barrier function, and maintaining gut microecological homeostasis. Studies have shown that some lactic acid bacteria can alleviate ulcerative colitis-related intestinal damage by reducing the release of pro-inflammatory factors, increasing tight junction protein expression, and promoting the recovery of goblet cells and the intestinal mucus barrier. Therefore, screening for lactic acid bacteria strains with good gastrointestinal tolerance, safety, and anti-inflammatory activity is an important direction for developing probiotic products to improve the symptoms of ulcerative colitis.

[0004] Traditional fermented dairy products from the Qinghai-Tibet Plateau undergo natural fermentation under unique geographical conditions, including low temperatures, low oxygen levels, and a high-fat milk matrix, resulting in abundant and regionally distinctive lactic acid bacteria resources. Lactic acid bacteria derived from these products possess potential advantages in areas such as gastrointestinal tolerance, intestinal adhesion, immune regulation, and barrier protection. Therefore, screening lactic acid bacteria strains with anti-inflammatory activity, intestinal barrier protection capabilities, and good safety profiles from traditionally fermented yak milk products from the Qinghai-Tibet Plateau is of great significance for developing probiotic preparations, functional foods, or pharmaceutical compositions that can help alleviate symptoms of ulcerative colitis. Summary of the Invention

[0005] To address the aforementioned problems in the background art, this invention provides a fermenting lactobacillus HF19 with the ability to alleviate symptoms of chronic ulcerative colitis (UC) and its applications. This invention isolates and purifies lactic acid bacteria from traditional fermented dairy products from the Qinghai-Tibet Plateau, screening for strains that reduce UC symptoms. Based on the strain's ability to inhibit NO production in RAW264.7 inflammatory cells and protect the monolayer membrane of Caco-2 cells in vitro, and considering its probiotic characteristics such as acid and bile salt tolerance and antibiotic resistance, a lactic acid bacterium with good overall properties and the ability to alleviate UC symptoms was screened. Its function was further verified through animal experiments.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A type of Lactobacillus fermentum HF19 with the ability to relieve symptoms of chronic ulcerative colitis, is Lactobacillus fermentum ( Lactobacillus fermentum The 16S rDNA sequence was deposited on October 21, 2025, at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, China (abbreviated as CCTCC); accession number: CCTCC NO.M20252279; SEQ ID:1.

[0008] A method for preparing the above-mentioned Lactobacillus fermentum HF19 includes the following steps:

[0009] Step (1) uses dilution coating and streak plate method to isolate and purify a single lactic acid bacteria;

[0010] Step (2) By comparing the ability of different isolated strains to alleviate UC symptoms through in vitro experiments, lactic acid bacteria with the function of inhibiting UC symptoms were screened out;

[0011] Step (3) By conducting experiments on the acid and bile salt resistance, adhesion characteristics and antibiotic resistance of the isolated strains, strains with good probiotic characteristics are obtained.

[0012] The application of the above-mentioned Lactobacillus fermentum HF19 in the relief of chronic ulcerative colitis.

[0013] The use of the above-mentioned Lactobacillus fermentum HF19 in the preparation of a composition having the function of relieving symptoms of chronic ulcerative colitis.

[0014] The application of the above-mentioned Lactobacillus HF19 in the preparation of a food product that relieves the symptoms of chronic ulcerative colitis, wherein the food product is a fermented dairy product.

[0015] The application of the above-mentioned Lactobacillus fermentum HF19 in the preparation of a live bacterial preparation with the function of relieving symptoms of chronic ulcerative colitis.

[0016] A composition comprising the aforementioned Lactobacillus fermentum HF19.

[0017] A food product containing the aforementioned Lactobacillus fermentum HF19.

[0018] A dairy product comprising the aforementioned Lactobacillus fermentum HF19.

[0019] A live bacteria preparation comprising the aforementioned Lactobacillus fermentum HF19.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention provides a strain of *Lactobacillus fermentum* HF19 with the effect of alleviating intestinal inflammation. Screening experiments using in vitro anti-inflammatory and intestinal barrier protection methods revealed that HF19 has the potential to inhibit inflammatory responses and protect the intestinal epithelial barrier. Further evaluation using acid and bile salt tolerance, in vitro adhesion, and sensitivity to commonly used antibiotics showed that HF19 still has a certain growth capacity under acidic conditions of pH 3.0 and 0.1% bile salts, demonstrating its potential for tolerance to the gastrointestinal environment. This strain exhibits detectable intestinal epithelial cell adhesion ability and has not shown extensive resistance to most commonly used antibiotics, providing a safe basis for further development and application. In vivo validation was performed using a DSS-induced mouse model of chronic ulcerative colitis. Results showed that HF19 intervention significantly reduced the disease activity index (DAI) (p<0.05), improved model-induced colonic shortening, restoring colonic length to near-normal levels, and could regulate spleen and liver indices, alleviating abnormal immune organ responses and liver metabolic burden under inflammatory conditions, while having no significant adverse effects on kidney indices. ELISA results showed that HF19 significantly reduced the level of the pro-inflammatory cytokine IL-1β in colonic tissue. Specifically, HF19 group D also significantly reduced IL-6 levels and increased the level of the anti-inflammatory cytokine IL-10, suggesting that it can alleviate intestinal inflammation by regulating the balance of pro-inflammatory / anti-inflammatory factors. Combined with H&E and AB-PAS staining results of colonic tissue, HF19 can improve colonic mucosal structural damage, reduce inflammatory cell infiltration, and promote the recovery of crypt structure and goblet cell mucus secretion function, thereby maintaining the integrity of the intestinal mucosal barrier. In conclusion, *Lactobacillus fermentum* (… Lactobacillus fermentum HF19 has acid and bile salt resistance, anti-inflammatory regulation and intestinal barrier protection effects. It can be used as a probiotic starter or functional food ingredient to prepare products that relieve symptoms related to inflammatory bowel disease. Attached Figure Description

[0022] Figure 1 The secretion levels of the inflammatory effector molecule NO in the RAW264.7 inflammatory cell model by strain HF19 were: a) the amount of secretion of pro-inflammatory factors TNF-α and IL-1β, and the amount of secretion of the anti-inflammatory factor IL-10.

[0023] Figure 2 To demonstrate the protective effect of strain HF19 on the Caco-2 monolayer cell membrane barrier, NC: normal group; MC: model group; HF19 F group: HF19 live bacteria fermentation supernatant group; HF19 A: HF19 live bacteria fermentation broth; HF19 D: HF19 heat-inactivated fermentation broth;

[0024] Figure 3 The changes in absorbance of strain HF19 at 600 nm wavelength over 12 hours were measured under different pH and bile salt concentrations.

[0025] Figure 4 To investigate the effects of strain HF19 on the disease activity index (DAI) and perianal condition in a mouse model of chronic ulcerative colitis, a) DAI index of mice in each group; b) representative perianal morphological photographs of mice in each group.

[0026] Figure 5 The effects of strain HF19 on colon length and organ indices in a mouse model of chronic ulcerative colitis were investigated. a) Colon length; b) Spleen index; c) Liver index; d) Kidney index.

[0027] Figure 6 The results of AB-PAS staining and H&E staining of colonic pathological sections from mice with chronic ulcerative colitis model caused by strain HF19 are shown. a) AB-PAS staining results; b) HE staining results.

[0028] Figure 7 The effects of strain HF19 on the levels of inflammatory factors in the colonic tissue of mice with chronic ulcerative colitis were investigated, including: a) IL-1β levels; b) IL-6 levels; and c) IL-10 levels. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0030] Example 1

[0031] In this embodiment, lactic acid bacteria were isolated from the collected samples using the traditional plate separation method. The strains with anti-inflammatory and intestinal barrier protection properties were screened in vitro. By simulating different pH conditions and bile salt concentrations, strains with acid and bile salt tolerance were screened. The anti-inflammatory and intestinal barrier effects were studied in UC model mice to explore the role of the strains and their fermentation products in alleviating UC symptoms in vivo.

[0032] The relevant culture medium formulations are as follows:

[0033] (1) MRS liquid culture medium: 10.0 g peptone, 8.0 g beef meal, 4.0 g yeast powder, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, final pH 5.7±0.2.

[0034] (2) MRS solid culture medium: 10.0 g peptone, 8.0 g beef meal, 4.0 g yeast powder, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, 15.0 g agar, final pH 6.2±0.2.

[0035] 1. Isolation of lactic acid bacteria

[0036] Samples of yak milk, cheese, fermented milk, and fermented cream were collected from Qinghai, Gansu, Sichuan, and other places. Single lactic acid bacteria were isolated using dilution coating and streak plate methods.

[0037] 2. Screening for strains with in vitro anti-inflammatory capabilities.

[0038] With a density of 5×10 5 RAW264.7 cells / well were cultured in DMEM containing 10% fetal bovine serum and a 1% penicillin / streptomycin / gentamicin mixture for 24 h at 37 °C and 5% CO2. Before inoculating the cells with lactic acid bacteria, the old medium in the 24-well plates was discarded and replaced with cell culture medium free of antibiotics and fetal bovine serum. The strains were washed twice with sterile PBS, collected by centrifugation (8000 g, 4 min), and then resuspended in DMEM medium. Based on the results of co-culturing lactic acid bacteria with inflammatory cells and under different gradient concentrations of lactic acid bacteria, the optimal concentration of the lactic acid bacteria to be screened was determined to be 1 × 10⁻⁶. 7 CFU / mL, so this concentration was used for subsequent experiments. The strain was then prepared at a concentration of 1×10⁻⁶ CFU / mL. 7 CFU / mL was seeded into each well and co-cultured with cells for 2 h. After 2 h, lipopolysaccharide (final concentration 1 μg / mL) was added, and the cells were cultured for 16 h. The cell supernatant was collected for subsequent experiments. Further investigation was conducted on the concentrations of pro-inflammatory factors TNF-α and IL-10 in the cell supernatant after co-culturing the strain with inflammatory cells, and strains that reduced the production of pro-inflammatory factors and promoted the secretion of anti-inflammatory factors were screened. The in vitro anti-inflammatory capacity results are as follows: Figure 1 As shown.

[0039] The RAW264.7 cell line is a mouse-derived macrophage line that can effectively mimic the functions of macrophages in vivo, such as phagocytosis, antigen processing, antigen presentation, and the production of inflammatory mediators. Lipopolysaccharide-induced polarization of RAW264.7 cells leads to the production of large amounts of inflammatory inducing substances, such as NO and TNF-α. Simultaneously, RAW264.7 cells also produce the anti-inflammatory cytokine IL-10. The IL-10 / TNF-α ratio directly reflects the cellular inflammatory homeostasis, and the ratio shows a certain negative correlation with the cellular inflammatory state. Figure 1 As shown, compared with the blank control group, the levels of NO, TNF-α, and IL-1β in the model group RAW264.7 cells were significantly increased (p<0.05), indicating that lipopolysaccharide stimulation successfully induced a significant inflammatory response in the cells. Specifically, NO secretion increased from 1.222±0.101 μM / L to 5.717±0.653 μM / L, TNF-α level increased from 112.235±13.421 pg / mgprot to 254.467±54.015 pg / mgprot, and IL-1β level increased from 15.622±6.381 pg / mgprot to 52.794±6.898 pg / mgprot. After intervention with live HF19 bacteria, the levels of NO, TNF-α, and IL-1β in RAW264.7 cells were significantly reduced (p<0.05). Specifically, NO secretion decreased to 1.925±1.189 μM / L, TNF-α level decreased to 159.968±32.468 pg / mg prot, and IL-1β level decreased to 10.184±4.421 pg / mg prot. Significance analysis showed no significant difference in TNF-α and IL-1β levels between the HF19 intervention group and the blank control group (p>0.05), indicating that live HF19 bacteria can effectively inhibit the abnormal release of pro-inflammatory factors induced by lipopolysaccharide, bringing the cellular inflammatory response closer to normal levels. Furthermore, the IL-10 level in the model group decreased from 32.955±3.945 pg / mg prot in the blank control group to 25.291±3.917 pg / mg prot. After HF19 intervention, the IL-10 level rebounded to 27.929±4.025 pg / mg prot, and the difference between the groups was not statistically significant (p>0.05). After HF19 intervention, the IL-10 / TNF-α ratio increased from 0.099 in the model group to 0.175, suggesting that HF19 live bacteria, while inhibiting the release of pro-inflammatory mediators such as TNF-α, IL-1β, and NO, helps to improve the balance of pro-inflammatory / anti-inflammatory factors in RAW264.7 cells. These results indicate that HF19 live bacteria can effectively alleviate the lipopolysaccharide-induced inflammatory state of RAW264.7 macrophages, reduce the risk of excessive inflammatory activation and its cascade reaction, and has good anti-inflammatory regulatory potential.

[0040] 3. Screening for strains capable of protecting the Caco-2 monolayer cell membrane in vitro.

[0041] Caco-2 cells obtained from the China Center for Type Culture Collection, Wuhan University, were seeded in culture medium (90% DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin-gentamicin) and cultured at 37°C in a CO2 environment with a volume fraction of 5%. The culture medium was changed every 2-3 days. When the Caco-2 cells adhered to approximately 90% of the bottom area of ​​the culture flask, they were digested with trypsin and then passaged or cryopreserved.

[0042] Caco-2 cells were loaded at 2.5 × 10⁻⁶. 5 pcs / cm 2 Inoculated into Transwell plates (pore size 0.4 μm, diameter 12 mm, surface area 1.12 cm²). 2 Add 0.5 mL of cell suspension and 1.5 mL of cell culture medium to each well of the upper and lower chambers of a 12-well cell culture plate, respectively. Change the culture medium after 24 h of culture. Wash the inside (AP) and outside (BL) of the cell culture chamber with Hanks buffer during the replacement. Change the culture medium every other day for the first 7 days, and then daily thereafter until 21 days of culture, at which point the Caco-2 cells have fully differentiated into a dense monolayer structure.

[0043] The transepthelial electrical resistance (TEER) of Caco-2 cell monolayers was measured using a resistance meter. The TEER of Caco-2 cell monolayers gradually increased with increasing culture time; a higher TEER indicated better monolayer compactness. Specifically, a TEER value ≥ 500 Ω·cm was considered acceptable. 2 Caco-2 cell monolayers can be used to simulate small intestinal epithelial cell transport experiments.

[0044] Alkaline phosphatase (AKP) is a marker enzyme of the brush border of intestinal epithelial cells, reflecting the degree of differentiation and functional status of these cells. A ratio of AP to BL greater than 1.5 indicates successful monolayer cell differentiation. Culture media from AP and BL were collected at 3, 14, and 21 days, and AKP activity was measured using an alkaline phosphatase kit. After measurement, the media were washed and replaced.

[0045] The concentration of the cultured probiotic solution was adjusted to 5×10⁻⁶. 6A live bacterial suspension was prepared by resuspending the bacteria in physiological saline solution at CFU / ml. A portion of the live bacterial suspension was then autoclaved at 121°C for 15 min and allowed to cool to room temperature to obtain an inactivated bacterial solution. The bacterial solution was centrifuged at 8000 g for 4 min at 4°C to obtain the supernatant. After vacuum freezing concentration, the supernatant was resuspended in physiological saline solution at 20% of its original volume, and the pH was adjusted to 7. The supernatant was then filtered through a 0.22 μm filter membrane to obtain a sterile supernatant.

[0046] After successful establishment of the Caco-2 cell monolayer, 50 μL of live bacterial suspension, inactivated bacterial solution, supernatant, or physiological saline was added to the AP side. To the BL side, TNF-α stimulation at a final concentration of 10 ng / mL or the same volume of physiological saline was added, and the cells were incubated for 48 h. The resistance values ​​at 0 h and 48 h were recorded, and the TEER increase at 48 h was calculated.

[0047] Calculate the TEER growth value using the following formula:

[0048]

[0049] In the formula: TEER48 h is the resistance value measured at 48 h; TEER0 h is the resistance value measured at 0 h.

[0050] Caco-2 cells are human colon cancer epithelial cells with strong proliferative capacity. Under prolonged culture conditions, they can differentiate into polarized, tightly connected intestinal epithelial-like cells with microvilli-like structures, effectively mimicking the physical state and barrier function of the intestinal epithelium. After 21 days of continuous culture, the ratio of AKP on the AP side to the BL side increased to 1.896 ± 0.125, and the TEER value stabilized at 567.84–650.72, marking the successful establishment of the intestinal epithelial cell simulation model. Figure 2As shown, after TNF-α treatment, the TEER value of Caco-2 monolayer cells in the Model group decreased from 206.333±2.082 in the Control group to 148.667±12.662, and there was a significant difference compared with the Control group (p<0.05). This indicates that the density of Caco-2 monolayer cell membrane decreased significantly and the cell barrier permeability increased significantly after TNF-α destruction. After intervention with different components of HF19, both the HF19-F group and the HF19-A group were able to increase the TEER value of Caco-2 monolayer cells under TNF-α injury conditions. The TEER value of the HF19-F group recovered to 206.333±17.502, which was not significantly different from the Control group (p>0.05) and was 38.79% higher than the Model group, indicating that the HF19 fermentation supernatant can significantly improve TNF-α-induced intestinal epithelial barrier damage. The TEER value of the HF19-A group increased to 185.667±19.425, which was 24.89% higher than the Model group and was significantly different from the Model group (p<0.05), indicating that the HF19 live bacterial suspension can also enhance the integrity of the Caco-2 monolayer cell membrane barrier. In contrast, the TEER value of the HF19-D group was 145.667±13.650, which was not significantly different from that of the Model group (p>0.05), suggesting that it had no significant effect on improving TNF-α-induced Caco-2 cell barrier damage. Overall, both the HF19 fermentation supernatant and the live bacterial suspension could alleviate the decline in Caco-2 monolayer cell barrier function caused by TNF-α to some extent, with the HF19 fermentation supernatant showing a more significant protective effect. This indicates that HF19 may enhance the density of the intestinal epithelial barrier through its active metabolites or cell-related functional components, thus possessing a potential protective effect on the intestinal barrier.

[0051] 4. Determination of the probiotic properties of lactic acid bacteria

[0052] 4.1 Determination of acid and bile salt tolerance of lactic acid bacteria

[0053] The experimental method used by Ramos et al. to determine the acid and bile salt resistance of bacterial strains was improved. Lactic acid bacteria were inoculated at a 2% inoculum into modified pH-MRS (MRS medium, pH adjusted to 1.5, 2, and 3) and BAs-MRS (MRS medium, bile salt concentrations of 0.1%, 0.3%, and 0.5%). The absorbance of the culture at 600 nm was measured at 0 h and 8 h, and then every 4 h at 600 nm to assess the growth capacity of the probiotics under different conditions.

[0054] After entering the host, lactic acid bacteria must successively experience the complex physiological environments of the oral cavity, stomach, and upper digestive tract, and tolerate adverse factors such as gastric acid, pepsin, bile salts, and pancreatic enzymes to maintain their activity and further exert their probiotic effects. Therefore, acid resistance and bile salt tolerance are important indicators for evaluating the gastrointestinal adaptability and in vivo survival potential of probiotics. This study investigated the growth changes of *Lactobacillus fermentum* HF19 under different acidic conditions (pH 1.5, 2.0, 3.0) and different bile salt concentrations (0.1%, 0.3%, 0.5%) for 12 hours, and measured the absorbance values ​​every 4 hours. The results are as follows: Figure 3 As shown.

[0055] The pH of human gastric juice typically ranges from 1.5 to 4.5, fluctuating depending on the type of food, the state of ingestion, and the gastric emptying process. Experimental results showed that HF19 exhibited a slight increase in absorbance after 12 hours of treatment at pH 3.0, indicating that this strain can adapt to a certain acidic environment and maintain a certain proliferative capacity after adaptation, suggesting good survival potential under relatively mild gastric acid conditions. After passing through the stomach, the strain needs to further tolerate adverse factors such as bile salts in the small intestine. Bile salts have surface activity and can affect bacterial cell membrane integrity and cell metabolism; therefore, bile salt tolerance is an important indicator for evaluating the intestinal adaptability of probiotics. The results showed that HF19's absorbance increased to 1.145 ± 0.003 after 12 hours of treatment with 0.1% bile salts, indicating that it can adapt well to a low-bile-salt environment. In conclusion, *Lactobacillus fermentum* HF19 possesses certain acid and bile salt tolerance capabilities, providing a potential basis for maintaining activity and exerting probiotic functions in the gastrointestinal environment.

[0056] 4.2 Adhesion ability of lactic acid bacteria

[0057] Using LGG as a positive control, the bacterial culture was centrifuged (8000 g, 4 min) to obtain bacterial cells, washed three times with sterile physiological saline, and resuspended to obtain a concentration of 5 × 10⁻⁶. 8 CFU / mL bacterial suspension. Add 10 μL of 10 mM CFDASE fluorescent dye to 1 mL of bacterial suspension, mix, and incubate at 37 °C for 30 minutes. After incubation, wash the bacterial cells three times with sterile physiological saline to remove CFDASE that has not bound to the lactic acid bacteria.

[0058] Caco-2 cells were used at a rate of 5 × 10 5Caco-2 cells were seeded at a concentration of [number] cells / mL in 24-well plates and cultured for 48 hours until dense cell membranes formed. Two hours prior to this, the culture medium was discarded and the cells were washed with sterile PBS, then replaced with antibiotic-free medium (90% DMEM + 10% fetal bovine serum). 100 μL of CFDASE-labeled lactic acid bacteria was added to each well, and the bacteria and cells were co-cultured at 37°C with 5% CO2 for 2 hours. After 2 hours, the culture medium was discarded, and the cell membranes were washed three times with sterile PBS to remove any lactic acid bacteria not adhering to the Caco-2 cell membranes. The cells were digested with 0.25% trypsin for 5 minutes, and after thorough regurgitation, 100 μL was transferred to a black 96-well plate. The absorbance (A1) was recorded using a fluorescence microplate reader. The fluorescent dye was used in the dark throughout the process to prevent fluorescence quenching. Three replicates were performed for each group, and the absorbance values ​​were measured at an excitation wavelength of 485 nm and an emission wavelength of 515 nm.

[0059] Calculate the adhesion rate using the following formula:

[0060]

[0061] In the formula, The absorbance of the sample; Add the same volume of trypsin as described above to the CFDASE-labeled bacterial suspension; The absorbance values ​​are for equal ratios of PBS and trypsin.

[0062] The ability of probiotics to adhere to the gastrointestinal tract allows them to colonize the tract and exert their health-promoting effects, while also preventing pathogens from adhering to the intestine. Using the LGG strain as a positive control, the in vitro adhesion ability of the HF19 strain was evaluated. The results showed that the adhesion rates in the LGG group, measured in three replicates, were 3.31%, 3.34%, and 3.51%, with an average adhesion rate of 3.39%; while the adhesion rates in the HF19 group, measured in three replicates, were 0.32%, 0.31%, and 0.33%, with an average adhesion rate of 0.32%. Compared to LGG, the adhesion rate of the HF19 strain was lower, indicating that HF19 has some adhesion ability to intestinal epithelial cells, but its in vitro adhesion ability is weaker than that of the LGG positive control strain. Therefore, HF19 is not a highly adhesive strain, but it still exhibits detectable intestinal epithelial cell adhesion ability, providing a basis for its brief stay in the intestine and the exertion of its probiotic functions.

[0063] 4.3 Antibiotic resistance assay of probiotics

[0064] Lactic acid bacteria solution was evenly spread onto the surface of sterile MRS solid culture medium, divided into zones and numbered. Antibiotic discs (dried) were then affixed to the spread medium surface, and the culture was incubated upside down at 37 °C for 24 h. The diameter of the inhibition zone was measured using a ruler with an accuracy of 1 mm and recorded. Each sample was measured in triplicate. The selected antibiotics included rifampin, amikacin, neomycin, gentamicin, kanamycin, chloramphenicol, erythromycin, norfloxacin, levofloxacin, lincomycin, penicillin, chlorcacillin, tetracycline, doxycycline, vancomycin, ceftriaxone, cefotaxime, ciprofloxacin, and polymyxin B. Antibiotic resistance of the strains was determined according to the CLSI 2017 standard.

[0065] The susceptibility of Lactobacillus fermentum HF19 to 19 common antibiotics was evaluated using the disk susceptibility testing method. The results showed that HF19 exhibited differential tolerance characteristics to different classes of antibiotics. Among the tested antibiotics, HF19 was sensitive to rifampin, chloramphenicol, erythromycin, penicillin, ampicillin, tetracycline, doxycycline, vancomycin, ceftriaxone, cefotaxime, and neomycin, with inhibition zone diameters of 24.3±1.5 mm, 31.0±1.0 mm, 28.0±1.0 mm, 34.7±1.5 mm, 35.7±0.6 mm, 20.0±0.0 mm, 27.0±1.7 mm, 18.3±0.6 mm, 28.0±1.0 mm, 29.7±0.6 mm, and 16.3±1.5 mm, respectively, indicating that these antibiotics effectively inhibited the growth of HF19. Among them, HF19 is relatively sensitive to penicillin antibiotics. The inhibition zones of penicillin and ampicillin are relatively large, reaching 34.7±1.5 mm and 35.7±0.6 mm respectively, indicating that HF19 has high sensitivity to β-lactam antibiotics.

[0066] Meanwhile, HF19 showed intermediate sensitivity to gentamicin and lincomycin, with inhibition zone diameters of 13.7±0.6 mm and 27.3±1.2 mm, respectively, indicating that the strain's sensitivity to these antibiotics was at an intermediate level. HF19 exhibited resistance to amikacin, kanamycin, norfloxacin, levofloxacin, ciprofloxacin, and polymyxin B. The inhibition zone diameters for norfloxacin, ciprofloxacin, and polymyxin B were all 0.0±0.0 mm, indicating strong resistance to some quinolone and peptide antibiotics. The inhibition zone diameters for amikacin and kanamycin were 9.7±0.6 mm and 7.7±0.6 mm, respectively, indicating that HF19 also showed some resistance to some aminoglycoside antibiotics.

[0067] Overall, HF19 was sensitive to 11 of the 19 common antibiotics tested, intermediately resistant to 2, and resistant to 6. This strain was relatively sensitive to penicillins, tetracyclines, cephalosporins, and some macrolides, but showed resistance to some aminoglycosides, quinolones, and polymyxin B. These results indicate that HF19 does not exhibit widespread resistance to most commonly used antibiotics, and therefore has the potential for further development and application.

[0068] Table 1

[0069]

[0070] 5. Alleviating effect of strain HF19 on UC model mice

[0071] 5.1 Laboratory Animals and Housing Conditions

[0072] Thirty 6-week-old SPF-grade male C57BL / 6J mice, weighing 19±2g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The mice were placed in an environment with alternating light and dark conditions for 12 hours, a temperature of 22±1℃, and a humidity of 55±5%. During the experiment, the mice had free access to water and food. The experiment began after 1 week of acclimatization feeding.

[0073] (1) Preparation of HF19 live bacterial suspension: The bacteria were activated and passaged, and each passage was carried out using MRS medium for the same duration of 18 h. The bacterial suspension was centrifuged at 8000 g for 4 min, the supernatant was removed, the bacterial cells were collected, and washed twice with sterile PBS. The bacterial concentration was adjusted to 1.0 × 10⁻⁶. 9 CFU / mL.

[0074] (2) Heat-inactivated HF19 cells: Centrifuge the bacterial solution at 8000 g for 4 min, discard the supernatant, collect the cells, and wash twice with sterile PBS. Adjust the cell concentration to 1.0 × 10⁻⁶. 9 CFU / mL. After resuspending, the bacterial solution was heated at 121 °C for 15 min, and then cooled to obtain the heat-inactivated bacterial preparation.

[0075] After the adaptation period, male C57BL / 6 mice were randomly divided into 12 groups (n=6). A modeling cycle lasting two weeks was observed, with a total of three cycles. During the first week of each cycle, mice were fed sterile water containing 2% DSS (w / v), and in the second week, they were fed standard drinking water. The control group did not participate in the modeling process and consumed standard drinking water throughout the entire feeding cycle. Starting on day 18, the experimental group mice were administered a probiotic preparation via gavage, with each gavage administration containing 200 μL of live or dead bacteria (1×10⁻⁶). 9 The concentration of CFU / mL was reduced, and the control and modeling groups were replaced with the same volume of PBS. Specific groupings are shown in Table 2.

[0076] Table 2 Grouping and Feeding Methods of Laboratory Animals

[0077]

[0078] 5.2 Collection of mouse tissue samples

[0079] Starting 18 days after the acclimatization period, mice were administered gavage daily at 2 PM according to their grouping requirements, once a day, until the end of the experiment. Throughout the experimental period, the mice's growth status, weight, fecal morphology, and degree of occult blood were recorded. One day before the end of the experiment, feces from each group of mice were collected using metabolic cages and quickly stored at -80 °C for subsequent experiments. After the experiment, all mice were fasted but allowed free access to water for 12 hours. They were then anesthetized by an injection of ketamine (100 mg / kg bw) into the abdomen, and blood was collected from the orbital venous plexus of each group of mice. Serum was prepared by centrifuging the blood samples at 4 °C and 3000 r / min for 15 min and quickly stored at -80 °C. After blood collection, the mice were euthanized by cervical dislocation. The length of the colon was measured, and the liver, kidneys and spleen were photographed and weighed. Then, a portion of the tissue was fixed with 10% formalin, and the other portion was wrapped in aluminum foil and quickly frozen in liquid nitrogen. Finally, it was stored in a -80°C freezer.

[0080] 5.3 Disease Activity Index Score and Mouse Phenotypic Assessment

[0081] The severity of ulcerative colitis (UC) could be assessed using the Disease Activity Index (DAI) and mouse phenotype. During the experimental period, weekly changes in fecal occult blood, fecal morphology, and body weight were recorded to calculate the DAI score, and perianal conditions were photographed. The DAI score was calculated using weight loss score, fecal morphology score, and fecal occult blood score. The formula for calculating the DAI score is as follows: DAI = (Weight Loss Score + Fecal Morphology Score + Fecal Occult Blood Score) / 3. Evaluation indicators are shown in Table 3.

[0082] Table 3 DAI Scoring Criteria

[0083]

[0084] DSS (sodium dextran sulfate), with its strong negative charge, successfully established a UC model by chemically dissecting the colonic epithelial barrier in mice. This barrier damage rapidly triggers a strong immune response and intestinal inflammation, leading to symptoms such as decreased appetite, rapid weight loss, and severe diarrhea in mice, often resulting in death due to complications such as dehydration, electrolyte imbalance, and hemorrhagic anemia. The destructive effect of DSS on the mouse intestine can be visually represented by the DAI score and the cleanliness of the perianal area, as shown in the results. Figure 4 As shown.

[0085] like Figure 4 As shown, the Disease Activity Index (DAI) score and perianal condition observation of mice were used to evaluate the effect of HF19 strain intervention on the improvement of symptoms in experimental enteritis model mice. The results showed that the DAI score of the normal control group (NC group) was 0.00±0.00, and the perianal area was clean, with no obvious redness, swelling, bleeding, or fecal contamination, indicating that mice under normal feeding conditions did not exhibit obvious intestinal inflammation-related symptoms. Compared with the NC group, the DAI score of the model group (MC group) was significantly increased to 3.67±0.33 (p<0.05), and the perianal area of ​​the mice showed obvious redness, swelling, bloodstains, and contamination, suggesting that the modeling treatment induced a significant intestinal inflammatory response and disease activity, indicating that the experimental model was successfully established.

[0086] After intervention with the positive control drug, the DAI score of mice decreased to 2.00±0.33, a reduction of 45.50% compared with the MC group, and the difference was significant (p<0.05), indicating that mesalazine treatment could effectively alleviate the enteritis symptoms induced by the model. The DAI scores of mice in the HF19 A group and HF19 D group were 2.22±0.51 and 2.11±0.38, respectively, a reduction of 39.51% and 42.51% compared with the MC group, and both were significantly different from the MC group (p<0.05).

[0087] The observation of perianal phenotypes in mice showed that the cleanliness of the perianal area was significantly reduced in the MC group, accompanied by redness, swelling, bloodstains, and filth. In contrast, after intervention with the PC group, live HF19 bacteria, and heat-inactivated HF19, the redness, swelling, bleeding, and filth around the perianal area were significantly reduced, and the perianal condition was significantly improved compared to the MC group. These results indicate that both live HF19 bacteria and heat-inactivated HF19 can significantly reduce the disease activity index in experimental enteritis model mice, improve perianal inflammation and appearance symptoms induced by the model, and alleviate intestinal inflammation-related damage, demonstrating a potential role in alleviating enteritis symptoms and protecting intestinal health.

[0088] 5.4. Mouse colon length and organ index

[0089] After dissecting the mice, the length of the colon was measured, and the liver, spleen, and kidneys were weighed. The organ index was calculated using the formula below.

[0090]

[0091] In the formula, The weight of the organ is (g). The final weight of the mouse (g).

[0092] In patients with ulcerative colitis (UC), the colon is in a prolonged state of inflammation, resulting in continuous damage and repair of the intestinal mucosa. Over time, this leads to localized fibrosis in the large intestine, decreased intestinal elasticity, and shortened colon length. Therefore, the degree of colon length shortening is positively correlated with the severity of chronic ulcerative colitis. Statistical analysis was performed on the colon length in mice. Organ indices are preliminary indicators for evaluating the systemic inflammatory response in mice, reflecting to some extent the structure and function of organs. Abnormal changes in organ indices reflect the severity of the systemic inflammatory response after successful model establishment in mice. The results of colon length statistics and various organ indices are as follows: Figure 5 As shown.

[0093] Depend on Figure 5 a) It was found that the colon length of mice in the NC group was 6.92 cm, while the colon length in the MC group decreased to 5.80 cm, significantly lower than that in the NC group (p<0.05), indicating that the modeling treatment caused significant colon shortening in mice, and the intestinal inflammation model was successfully established. The colon length in the PC group increased to 7.21 cm, significantly higher than that in the MC group (p<0.05). After HF19 intervention, the colon length of mice in the HF19 A group and HF19 D group increased to 7.43 cm and 7.59 cm, respectively, both significantly higher than that in the MC group (p<0.05), and there was no significant difference compared with the NC and PC groups (p>0.05), indicating that different HF19 treatments could effectively alleviate the model-induced colon shortening and restore the colon length to near-normal levels.

[0094] Depend on Figure 5 (b) It was found that the spleen index in the NC group was 0.21%, while it significantly increased to 0.76% in the MC group (p<0.05), indicating that the model induced a significant enhancement of the spleen's immune response. The spleen index in the PC group decreased to 0.46%, which was significantly lower than that in the MC group (p<0.05). The spleen indices in the HF19 A group and the HF19 D group were 0.43% and 0.48%, respectively, both significantly lower than those in the MC group (p<0.05), and there was no significant difference from the PC group (p>0.05), suggesting that HF19 can effectively alleviate the model-induced increase in the spleen index and reduce the abnormal immune organ response in enteritis.

[0095] Depend on Figure 5c) It was found that the liver index in the NC group was 3.74%, while it significantly increased to 4.91% in the MC group (p<0.05), suggesting that DSS induction may increase the metabolic burden on the liver or cause liver-related stress responses. The liver index in the PC group decreased to 4.25%, which was significantly lower than that in the MC group (p<0.05). The liver indices in the HF19 A group and HF19 D group were 4.41% and 4.50%, respectively, both significantly lower than those in the MC group (p<0.05), and there was no significant difference from the PC group (p>0.05). This indicates that HF19 intervention can reduce the model-induced increase in liver index to some extent and alleviate liver-related stress changes under inflammatory conditions.

[0096] Depend on Figure 5 d) It was found that the kidney indices of the NC group, MC group, PC group, HF19 A group, and HF19 D group were 1.10%, 1.19%, 1.10%, 1.09%, and 1.16%, respectively, with no significant differences among the groups (p>0.05). These results indicate that under the experimental conditions, neither 2% DSS treatment nor HF19 intervention caused significant abnormal changes in the kidney indices of mice, suggesting that HF19 has no adverse effect on kidney tissue indices.

[0097] In summary, both HF19 group A and HF19 group significantly improved colonic shortening in experimental enteritis model mice, reduced model-induced increases in liver and spleen indices, and had no significant effect on kidney indices. This indicates that HF19 can exert a potential protective effect on intestinal health by alleviating intestinal inflammatory damage, reducing stress responses in related organs, and regulating abnormal changes in immune organs.

[0098] 5.5 Histopathological Analysis of Mouse Tissue

[0099] To further evaluate the effect of HF19 on improving colonic tissue damage and mucus barrier in mice with experimental enteritis, H&E staining and AB-PAS staining were performed on colonic tissues from each group of mice. Mouse colonic tissues fixed in 10% formaldehyde solution were dehydrated using an ethanol gradient, embedded in paraffin, sectioned, and stained with H&E. Pathological changes in the tissues were observed under a microscope. AB-PAS staining was used to observe the improvement of colonic damage and intestinal mucus distribution caused by the bacterial intervention. Colonic tissue section staining provides a direct view of the physiological state of mice in different experimental groups. H&E staining reveals the pathological condition of the tissue through the different affinities of different dyes for substances of different properties, making it a powerful tool for pathological analysis. AB-PAS staining marks acidic mucus substances as blue and neutral mucopolysaccharides and glycoproteins as purple-red, thus directly displaying the distribution of goblet cells and changes in mucus type. Results are as follows: Figure 6 As shown.

[0100] AB-PAS staining results showed ( Figure 6a) In the NC group, the colonic mucosa of mice showed a higher number of goblet cells with continuous and relatively uniform blue positive staining, indicating that their mucus secretion function and mucosal barrier structure were normal. In the MC group, the blue positive area in the colonic tissue of mice was significantly reduced, the staining intensity was weakened, and the number of goblet cells decreased, indicating that DSS treatment led to reduced intestinal mucus secretion, impaired goblet cell function, and disruption of the mucus barrier integrity. Compared with the MC group, the blue positive staining area and goblet cell distribution in the PC group were somewhat restored. In the HF19 A and HF19 D groups, the blue positive area in the colonic tissue of mice was significantly increased compared to the MC group, the staining intensity was enhanced, and the distribution of goblet cells and mucus layer structure were improved, suggesting that HF19 can promote or maintain colonic mucus secretion function and improve DSS-induced mucus barrier damage.

[0101] H&E staining results showed ( Figure 6 (b) In the NC group, the colonic tissue structure of mice was intact, with continuous mucosal epithelium, neat and regular crypt arrangement, and relatively uniform distribution of goblet cells. No obvious epithelial shedding, tissue destruction, or large-scale inflammatory cell infiltration was observed. The colonic tissue of mice in the MC group showed obvious pathological damage, manifested as mucosal structure destruction, epithelial cell shedding, disordered crypt arrangement, and even local loss, accompanied by increased inflammatory cell infiltration, suggesting significant inflammatory damage to the colon after DSS induction. Compared with the MC group, the degree of colonic tissue damage in the PC group was reduced, and the mucosal structure and crypt morphology were somewhat restored. After HF19 intervention, the colonic tissue structure of mice in the HF19 A and HF19 D groups was significantly improved compared with the MC group, showing enhanced mucosal epithelial continuity, more regular crypt arrangement, reduced tissue destruction, and decreased inflammatory cell infiltration, indicating that different forms of HF19 treatment can alleviate DSS-induced pathological damage to colonic tissue.

[0102] In summary, the results of AB-PAS staining and H&E staining together indicate that both HF19 group A and HF19 group D can improve DSS-induced colonic mucosal structural damage, reduce inflammatory cell infiltration, and promote the recovery of crypt structure and goblet cell mucus secretion function, thereby helping to maintain the integrity of the intestinal mucosal barrier and reduce the degree of intestinal inflammatory damage. This suggests that HF19 has potential intestinal barrier protection and enteritis relief effects.

[0103] 5.6 Determination of biochemical indicators in mice

[0104] Colon tissue samples stored at -80 ℃ were removed and slowly thawed at 4 ℃ to maximize protein activity. After thawing, an appropriate amount of colon tissue was accurately weighed and added to pre-cooled phosphate buffer at a weight-to-volume ratio. The tissue was then thoroughly homogenized using a tissue homogenizer under ice bath conditions to prepare a tissue homogenate. Subsequently, the homogenate was centrifuged at 12000 rpm for 15 minutes at 4 ℃, and the supernatant was carefully collected and aliquoted. Following the instructions of the enzyme-linked immunosorbent assay (ELISA) kit, the concentrations of inflammatory factors IL-6, IL-1β, and IL-10 in the supernatant were determined using a double-antibody sandwich method. Standard wells and sample replicates were provided during the experiment. After incubation, washing, and color development, the absorbance of each well was measured at 450 nm using a microplate reader. Finally, the specific content of each factor was calculated based on the standard curve. The inflammatory factor content of each group of mice is shown below. Figure 7 As shown.

[0105] like Figure 7 As shown in a), the IL-1β level in the colon tissue of mice in the NC group was 48.23 pg / mg prot, while that in the MC group was significantly increased to 141.44 pg / mg prot, an increase of 193.25% compared to the NC group (p<0.05), indicating that the modeling treatment induced a significant pro-inflammatory response. The IL-1β level in the PC group decreased to 41.00 pg / mg prot, a decrease of 71.01% compared to the MC group, and the difference was significant (p<0.05). After HF19 intervention, the IL-1β levels in the HF19 A group and HF19 D group decreased to 41.96 pg / mg prot and 34.11 pg / mg prot, respectively, a decrease of 70.33% and 75.88% compared to the MC group, and both were significantly lower than those in the MC group (p<0.05). The significance analysis results showed that there was no significant difference between HF19 A group and HF19 D group and NC group and PC group (p>0.05), indicating that different treatments of HF19 can effectively inhibit the abnormal increase of IL-1β induced by the model and restore it to near normal level.

[0106] like Figure 7As shown in b), the IL-10 level in the colon tissue of mice in the NC group was 34.43 pg / mg prot, while that in the MC group decreased to 14.96 pg / mg prot, a decrease of 56.54% compared to the NC group, and the difference was significant (p<0.05), indicating that the modeling treatment inhibited the expression of the anti-inflammatory factor IL-10. The IL-10 level in the PC group rebounded to 19.92 pg / mg prot, an increase of 33.12% compared to the MC group. The IL-10 level in the HF19 A group was 16.22 pg / mg prot, with no significant difference from the MC group (p>0.05), suggesting that HF19 A had no significant effect on promoting IL-10 secretion; the IL-10 level in the HF19 D group increased to 26.29 pg / mg prot, an increase of 75.68% compared to the MC group, and significantly higher than the MC group (p<0.05), but with no significant difference from the PC group (p>0.05). The above results indicate that HF19 D can significantly promote the recovery of the level of the anti-inflammatory factor IL-10, which helps to enhance the local anti-inflammatory regulatory capacity of the intestine.

[0107] like Figure 7 As shown in c), the IL-6 level in the colon tissue of mice in the NC group was 38.40 pg / mg prot, while that in the MC group it increased to 56.19 pg / mg prot, a 46.31% increase compared to the NC group, and the difference was significant (p<0.05), suggesting that the modeling treatment induced an enhanced IL-6-mediated inflammatory response. The IL-6 levels in the PC group and HF19 A group were 45.90 pg / mg prot and 46.19 pg / mg prot, respectively, which were lower than those in the MC group, but there was no significant difference between them and the MC group (p>0.05). The IL-6 level in the HF19 D group decreased to 36.76 pg / mg prot, a 34.57% decrease compared to the MC group, and significantly lower than that in the MC group (p<0.05), but there was no significant difference compared to the NC group (p>0.05), indicating that HF19 D can effectively inhibit the model-induced increase in IL-6, restoring it to near-normal levels.

[0108] In summary, both HF19 A and HF19 D significantly reduced IL-1β levels, with HF19 D also significantly reducing IL-6 levels and increasing IL-10 levels, suggesting that HF19 D plays a more significant role in regulating the balance of pro-inflammatory / anti-inflammatory factors. These results indicate that HF19 can alleviate model-induced colonic inflammation by inhibiting the release of pro-inflammatory factors and regulating anti-inflammatory factor levels, thus possessing a potential role in alleviating intestinal inflammation.

[0109] 6. Identification of lactic acid bacteria

[0110] 16S rDNA sequence identification of strain

[0111] Bacterial genomic DNA was extracted and used as a template for PCR amplification using universal 16S rDNA primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-CTACGGCTACCTTGTTACGA-3'). The PCR reaction system consisted of: 2.0 μL of 10×Ex Taq buffer, 0.2 μL of 5u Ex Taq, 1.6 μL of 2.5 mM dNTP Mix, 1.1 μL of 5p Primer, 1.1 μL of 5p Primer, 0.5 μL of DNA, and 13.7 μL of ddH2O. The PCR conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 25 cycles; and 72℃ extension for 10 min. The PCR products were detected by agarose gel electrophoresis. The amplified fragment length was approximately 1.6 bp. Figure 6 .

[0112] The sequencing was performed by Shanghai Paisennong Biotechnology Co., Ltd., and the sequencing results were submitted to NCBI for online BLAST comparison. The results showed that the strain of this invention is 100% Lactobacillus fermentum. Lactobacillus fermentum Lactobacillus fermentum ( ) Lactobacillus fermentum HF19, deposited at the China Center for Type Culture Collection (CCTCC) on October 28, 2025; accession number: CCTCC NO.M 20252279, 16S rDNA sequence is shown below:

[0113] ctcgaaagca tgggtagcga acaggattag ataccctggt agtccatgcc gtaaacgatg780

[0114] agtgctaggt gttggagggt ttccgccctt cagtgccgga gctaacgcat taagcactcc840

[0115] gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc900

[0116] ggtggagcat gtggtttaat tcgaagctac gcgaagaacc ttaccaggtc ttgacatctt960

[0117] gcgccaaccc tagagatagg gcgtttcctt cgggaacgca atgacaggtg gtgcatggtc1020

[0118] gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtta1080

[0119] ctagttgcca gcattaagtt gggcactcta gtgagactgc cggtgacaaa ccggaggaag1140

[0120] gtggggacga cgtcagatca tcatgcccct tatgacctgg gctacacacg tgctacaatg1200

[0121] gacggtacaa cgagtcgcga actcgcgagg gcaagcaaat ctcttaaaac cgttctcagt1260

[0122] tcggactgca ggctgcaact cgcctgcacg aagtcggaat cgctagtaat cgcggatcag1320

[0123] catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgagagtt1380

[0124] tgtaacaccc aaagtcggtg gggtaacctt ttaggagcca gccgcctaag gtgggacaga1440

[0125] Example 2

[0126] This embodiment provides a method for producing fermented milk containing strain HF19. The method includes the following steps: preheating the raw milk to 40°C, adding 7% sucrose and stirring until dissolved, homogenizing under a pressure of 20 MPa and a temperature of 55°C; sterilizing by heat treatment at 95°C for 5 minutes, cooling to a fermentation temperature of 37°C, inoculating with Lactobacillus fermentum HF19 at a quantity of 2%–3%; fermenting at 37°C until the acidity reaches 70°T, breaking the emulsion, and ripening at 4°C for 16 hours to obtain the fermented milk product.

[0127] Example 3

[0128] This embodiment provides a method for preparing a powdered product of Lactobacillus fermentum strain HF19. The method includes the following steps: Activated Lactobacillus fermentum HF19 is inoculated into MRS broth medium at a 3% inoculum size, cultured at 37°C for 24 hours, centrifuged at 10,000 rpm for 10 minutes, and the supernatant is discarded to obtain Lactobacillus fermentum HF19 bacterial sludge. The bacterial sludge is mixed with skim milk at a weight ratio of 1:10 and then freeze-dried under vacuum to obtain bacterial powder. The bacterial powder can be combined with milk powder, oligosaccharides, and different flavoring agents to formulate products for daily use.

Claims

1. A type of Lactobacillus fermentum HF19 with the ability to relieve symptoms of chronic ulcerative colitis, characterized in that... The HF19 is Lactobacillus fermentum ( Lactobacillus fermentum (), deposited at the China Center for Type Culture Collection, accession number CCTCC NO.M20252279.

2. The *Lactobacillus fermentum* HF19 with the ability to relieve symptoms of chronic ulcerative colitis according to claim 1, characterized in that... The 16S rDNA sequence of HF19 is shown in SEQ ID:

1.

3. The use of Lactobacillus fermentum HF19 as described in any one of claims 1-2 in the relief of chronic ulcerative colitis.

4. The use of the Lactobacillus fermentum HF19 according to any one of claims 1-2 in the preparation of a composition having the function of relieving symptoms of chronic ulcerative colitis.

5. The use of the Lactobacillus HF19 of any one of claims 1-2 in the preparation of a food product having the function of relieving symptoms of chronic ulcerative colitis.

6. The use of Lactobacillus fermentum HF19 as described in any one of claims 1-2 in the preparation of a live bacterial preparation having the function of relieving symptoms of chronic ulcerative colitis.

7. A composition, characterized in that... The composition comprises Lactobacillus fermentum HF19 as described in any one of claims 1-2.

8. A food product, characterized in that... The food contains Lactobacillus fermentum HF19 as described in any one of claims 1-2.

9. A dairy product, characterized in that... The dairy product comprises Lactobacillus fermentum HF19 as described in any one of claims 1-2.

10. A live bacteria preparation, characterized in that... The live bacteria preparation comprises Lactobacillus fermentum HF19 as described in any one of claims 1-2.

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