Lactobacillus acidophilus hc1356 for relieving gastrointestinal mucosal injury caused by chemotherapy, probiotic preparation and application

Abstract

The present application relates to the technical field of probiotics, in particular to a lactobacillus acidophilus HC1356 for relieving gastrointestinal mucosal injury caused by chemotherapy, a probiotic preparation and application. Lactobacillus acidophilus The lactobacillus acidophilus (HC1356) has been preserved in the China General Microbiological Culture Collection Center on March 23, 2023, the address is No. 1, Xibahe Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 26888. The lactobacillus acidophilus HC1356 provided by the present application has very excellent gastrointestinal colonization ability and adhesion ability. It is verified by animal experiments that the lactobacillus acidophilus HC1356 has a significant improvement effect on various related indexes of male and female rats in a cisplatin-induced gastrointestinal mucositis model, and the improvement trend of the indexes of male and female rats is basically consistent. The strain has no toxic effect and no hepatointestinal translocation phenomenon in the whole test process.

Description

Technical Field

[0001] This invention relates to the field of probiotic technology, specifically to a strain of Lactobacillus acidophilus HC1356 that alleviates chemotherapy-induced gastrointestinal mucosal damage, probiotic preparations, and applications. Background Technology

[0002] Chemotherapy is one of the mainstream methods of cancer treatment at present. While using chemical drugs to inhibit the growth, reproduction and spread of cancer cells, it inevitably damages normal cells, leading to a series of side effects, including chemotherapy-induced gastroenteritis.

[0003] Chemotherapy-induced gastrointestinal mucositis can have multifaceted impacts on cancer patients. Symptoms such as nausea and vomiting can lead to adjustments or suspension of chemotherapy dosages, weakening the anti-cancer effect, increasing the risk of recurrence and metastasis, and prolonging the treatment cycle. Furthermore, it can damage the integrity of the gastrointestinal mucosa, reduce the intestinal immune barrier function, allowing bacteria and toxins to enter the bloodstream and cause systemic infections, increasing the difficulty of treatment and even endangering life. It can also lead to decreased appetite and malabsorption, inducing malnutrition, thereby reducing chemotherapy tolerance and delaying the patient's recovery period. Simultaneously, persistent physical discomfort severely affects daily life, exacerbates psychological stress, and induces anxiety, depression, and other problems, significantly reducing quality of life. Therefore, effective intervention for gastrointestinal mucositis is of great significance for improving the effectiveness of cancer treatment.

[0004] Cisplatin, as a broad-spectrum anti-tumor drug, induces gastrointestinal mucositis through a combination of direct toxic damage and indirect inflammatory response to the gastrointestinal mucosa. After reaching the gastrointestinal mucosa via the bloodstream, its platinum ions cross-link with the DNA of mucosal epithelial cells, forming a DNA-platinum complex. This complex interferes with DNA replication and transcription, directly causing cytotoxicity to rapidly proliferating gastrointestinal mucosal epithelial cells, leading to apoptosis or necrosis. Simultaneously, it induces the production of large amounts of reactive oxygen species, triggering oxidative stress, damaging cell membrane integrity and mitochondrial function, further disrupting cellular energy metabolism, and exacerbating mucosal inflammation and barrier function. Furthermore, cisplatin can activate inflammatory signaling pathways such as NF-κB, promoting the release of pro-inflammatory factors such as tumor necrosis factor-α and interleukin-6, exacerbating mucosal congestion and edema. The inflammatory response and mechanical barrier damage form a vicious cycle, ultimately manifesting as symptoms such as diarrhea and abdominal pain. At the same time, cisplatin can damage the enteric nervous system, leading to gastrointestinal motility disorders. Coupled with autonomic nervous system dysfunction, this may cause functional dyspepsia or even intestinal obstruction. In addition, the combined use of broad-spectrum antibiotics during chemotherapy can easily disrupt the intestinal microecological balance with cisplatin, leading to the overgrowth of pathogenic bacteria such as Clostridium difficile, further aggravating mucosal inflammation and diarrhea symptoms.

[0005] Currently, there are various treatment methods for chemotherapy-induced gastrointestinal mucositis, but each has its limitations. Nutritional support therapy can improve the patient's nutritional status, enhance immunity, and promote mucosal repair by providing high-calorie, easily digestible foods or enteral nutrition preparations. However, the difficulty in eating after chemotherapy makes this difficult, with some patients requiring nasogastric feeding or intravenous nutrition, and the related costs are high, resulting in a significant economic burden. In drug therapy, antacids, antiemetics, and mucosal protectants can quickly relieve symptoms such as nausea and vomiting, but some drugs may cause adverse reactions, and long-term use may affect the recovery of gastrointestinal function. Traditional Chinese medicine treatment includes herbal medicine and acupuncture. Herbal medicine can regulate spleen and stomach function with few side effects and high safety, but the efficacy varies greatly from person to person, has a slow onset of action, and the quality of the herbs and processing methods may affect the efficacy. Acupuncture regulates gastrointestinal function by stimulating acupoints, but its efficacy depends on the operator's skill, the effect is short-lived, and multiple treatments are required. Intravenous infusion therapy can quickly correct dehydration and electrolyte imbalances, and is suitable for patients with severe vomiting and inability to eat. However, it usually requires hospitalization or frequent medical visits, increasing time and financial costs. Furthermore, intravenous puncture may cause complications, leading to additional pain and health risks. Probiotic therapy can regulate the balance of the intestinal microecology, enhance intestinal barrier function, reduce inflammatory responses, and improve gastrointestinal symptoms. It has a relatively high safety profile, but different strains have different effects, and it is necessary to find suitable strains.

[0006] Currently, although some research exists on the treatment of enteritis with probiotics, the phenotypic symptoms of enteritis vary significantly due to different causes. For example, CN118620805A discloses the efficacy and application of a probiotic composition of Lactobacillus acidophilus and Bifidobacterium longum subsp. longum in treating lipopolysaccharide-induced intestinal inflammation. This type of intestinal inflammation is an infection-related or endotoxin-mediated common intestinal inflammation, with damage concentrated in the intestinal mucosa and without the direct toxic effects of chemotherapy drugs on mucosal cells. The probiotic composition mainly works by reducing pro-inflammatory factors TNF-α and IL-6, increasing the anti-inflammatory factor IL-10, and increasing intestinal tight junction proteins, but it does not have the effects of repairing mucosal epithelial cells or restoring mucosal barrier function.

[0007] Therefore, it is necessary to provide a probiotic strain suitable for treating gastrointestinal mucosal inflammation induced by the chemotherapy drug cisplatin. Summary of the Invention

[0008] To address the current lack of probiotic products that can alleviate chemotherapy-induced gastrointestinal mucosal damage, this invention provides a strain of Lactobacillus acidophilus HC1356 that can alleviate chemotherapy-induced gastrointestinal mucosal damage, a probiotic preparation, and its application.

[0009] The technical solution of this invention is as follows:

[0010] In a first aspect, the present invention provides a strain of Lactobacillus acidophilus HC1356 that alleviates chemotherapy-induced gastrointestinal mucosal damage. Lactobacillus acidophilus HC1356 was deposited on March 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26888, and classified as *Lactobacillus acidophilus*. Lactobacillus acidophilus .

[0011] Secondly, the present invention provides the application of Lactobacillus acidophilus HC1356 in the preparation of a product for alleviating chemotherapy-induced gastrointestinal mucosal damage, specifically, the product is a pharmaceutical product.

[0012] Thirdly, the present invention provides a probiotic preparation comprising raw powder of Lactobacillus acidophilus HC1356, wherein the preparation method of raw powder of Lactobacillus acidophilus HC1356 includes the following steps:

[0013] Fermentation broth of Lactobacillus acidophilus HC1356 was centrifuged and the bacterial sludge was collected. The bacterial sludge was then coated and freeze-dried under vacuum to obtain freeze-dried blocks. The freeze-dried blocks were then crushed to obtain raw powder of Lactobacillus acidophilus HC1356.

[0014] Furthermore, Lactobacillus acidophilus HC1356 was transferred from the refrigerated environment to room temperature and then inoculated into primary liquid culture medium and cultured for 16-20 h to prepare primary seed; it was then transferred to secondary seed tank and cultured for 12-15 h to obtain secondary seed; subsequently, it was transferred to tertiary fermenter for fermentation to obtain fermentation broth of Lactobacillus acidophilus HC1356.

[0015] Furthermore, the primary liquid culture medium consists of peptone 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, and Tween 80 1.0 g / L;

[0016] The culture medium used in the secondary seed tank consists of 10.0 g / L peptone, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.0 g / L edible oil.

[0017] The culture medium used in the tertiary seed tank consists of whey powder 13 g / L, glucose 30 g / L, yeast powder 20 g / L, concentrated carrot juice 5 g / L, concentrated tomato juice 5 g / L, soybean peptone 10 g / L, manganese sulfate 0.2 g / L, magnesium sulfate 0.1 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 1 g / L, sodium acetate 2 g / L, Tween 80 1.0 g / L, and edible oil 1.0 g / L.

[0018] Furthermore, the coating method for the bacterial sludge is as follows: by mass percentage, 10% of sterilized skim milk powder, 8% of trehalose, 3% of lactose, 1% of soy protein isolate, 0.5% of L-glutamate, 0.1% of ascorbic acid, and 77.4% of distilled water are mixed evenly and then homogenized with the Lactobacillus acidophilus HC1356 bacterial sludge obtained by centrifugation at a mass ratio of 1:1-2. The pH value is adjusted to 6.5-7.0 to obtain the bacterial sludge coating solution.

[0019] Furthermore, based on the number of Lactobacillus acidophilus HC1356, the specifications of probiotic preparations are selected from ≥10 billion CFU / g, ≥20 billion CFU / g, ≥50 billion CFU / g, ≥100 billion CFU / g, or ≥200 billion CFU / g.

[0020] Furthermore, probiotic preparations also contain fructooligosaccharides, galactooligosaccharides, inulin, lactulose, pumpkin powder, and banana powder.

[0021] Furthermore, the mass ratio of fructooligosaccharides, galactooligosaccharides, inulin, lactulose, pumpkin powder, and banana powder is 4:2:1:0.5:0.4:0.3.

[0022] Fourthly, the present invention also provides the application of a probiotic preparation in the preparation of a product for alleviating chemotherapy-induced gastrointestinal mucosal damage, specifically, the product is a pharmaceutical product.

[0023] Furthermore, chemotherapy-induced gastrointestinal mucosal damage refers to the toxic effects of cisplatin on gastrointestinal mucosal tissues, as well as damage to the heart, liver, spleen, kidneys, thymus, appetite suppression, inflammatory damage, and oxidative damage.

[0024] The beneficial effects of this invention are as follows:

[0025] The *Lactobacillus acidophilus* HC1356 provided by this invention possesses excellent gastrointestinal colonization and adhesion abilities. Animal experiments have verified that *Lactobacillus acidophilus* HC1356 significantly improves various relevant indicators in male and female rats with cisplatin-induced gastrointestinal mucositis, and the improvement trends in these indicators are generally consistent between male and female rats. Throughout the experiment, this strain showed no toxic effects and no hepato-intestinal translocation.

[0026] The *Lactobacillus acidophilus* HC1356 provided by this invention can alleviate cisplatin-induced weight loss and appetite suppression by regulating the intestinal microecology and improving intestinal barrier function. It can also reduce cisplatin-induced inflammatory damage by lowering the expression levels of pro-inflammatory factors IL-6, IL-1β, IL-18, and TNF-α, and increasing the expression level of anti-inflammatory factor IL-10. Furthermore, it can reduce cisplatin-induced oxidative damage by lowering serum ROS and MDA levels and increasing SOD and GSH-Px activities, thereby regulating intestinal flora or activating antioxidant pathways. It can also reduce cisplatin-induced liver and kidney damage by lowering serum ALT and AST activities and BUN and Cr levels. Additionally, it can reduce NF-κB levels in the rat gastric antrum and colon and alleviate gastrointestinal mucosal inflammation by reducing the toxic effects of cisplatin on the gastrointestinal mucosa. Finally, it can improve cisplatin-induced damage to the heart, liver, spleen, kidneys, and thymus, and help maintain or protect the function of immune organs, thus alleviating cisplatin-induced gastrointestinal mucosal inflammation. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a line graph showing the weight changes of the male rats in Example 5.

[0029] Figure 2 This is a line graph showing the weight changes of the female mice in Example 5.

[0030] Figure 3 This is a line graph showing the changes in food intake of male rats in Example 5.

[0031] Figure 4 This is a line graph showing the changes in food intake of the female mice in Example 5.

[0032] Figure 5 This is a bar chart showing the IL-6 content in the serum cells of rats in each group in Example 5; Figure 5 In the figure, (A) is a bar chart of serum IL-6 content in male mice, and (B) is a bar chart of serum IL-6 content in female mice.

[0033] Figure 6 This is a bar chart showing the IL-10 content in the serum cells of rats in each group in Example 5; Figure 6 In the figure, (A) is a bar chart of serum IL-10 content in male mice, and (B) is a bar chart of serum IL-10 content in female mice.

[0034] Figure 7This is a bar chart showing the IL-1β content in the serum cells of rats in each group in Example 5; Figure 7 In the figure, (A) is a bar chart of serum IL-1β content in male mice, and (B) is a bar chart of serum IL-1β content in female mice.

[0035] Figure 8 This is a bar chart showing the IL-18 content in the serum cells of rats in each group in Example 5; Figure 8 In the figure, (A) is a bar chart of serum IL-18 content in male mice, and (B) is a bar chart of serum IL-18 content in female mice.

[0036] Figure 9 This is a bar chart showing the TNF-α content in the serum cells of rats in each group in Example 5; Figure 9 In the figure, (A) is a bar chart of serum TNF-α content in male mice, and (B) is a bar chart of serum TNF-α content in female mice.

[0037] Figure 10 This is a bar chart showing the serum ROS levels of rats in each group in Example 5; Figure 10 In the figure, (A) is a bar chart of serum ROS content in male mice, and (B) is a bar chart of serum ROS content in female mice.

[0038] Figure 11 This is a bar chart showing the SOD content in the serum of rats in each group in Example 5; Figure 11 In the figure, (A) is a bar chart of serum SOD content in male mice, and (B) is a bar chart of serum SOD content in female mice.

[0039] Figure 12 This is a bar chart showing the serum MDA content of rats in each group in Example 5; Figure 12 In the figure, (A) is a bar chart of serum MDA content in male mice, and (B) is a bar chart of serum MDA content in female mice.

[0040] Figure 13 This is a bar chart showing the serum GSH-Px content of rats in each group in Example 5; Figure 13 In the figure, (A) is a bar chart of serum GSH-Px content in male mice, and (B) is a bar chart of serum GSH-Px content in female mice.

[0041] Figure 14 This is a bar chart showing the serum ALT levels in each group of rats in Example 5; Figure 14 In the figure, (A) is a bar chart of serum ALT content in male mice, and (B) is a bar chart of serum ALT content in female mice.

[0042] Figure 15 This is a bar chart showing the serum AST levels of rats in each group in Example 5; Figure 15 In the figure, (A) is a bar chart of serum AST content in male mice, and (B) is a bar chart of serum AST content in female mice.

[0043] Figure 16 This is a bar chart showing the serum BUN content of rats in each group in Example 5; Figure 16 In the figure, (A) is a bar chart of serum BUN content in male mice, and (B) is a bar chart of serum BUN content in female mice.

[0044] Figure 17 This is a bar chart showing the serum Cr content of rats in each group in Example 5; Figure 17 In the figure, (A) is a bar chart of serum Cr content in male mice, and (B) is a bar chart of serum Cr content in female mice.

[0045] Figure 18 This is a bar chart showing the expression levels of NF-κB in the gastric antrum of rats in each group in Example 5; Figure 18 In the figure, (A) is a bar chart of NF-κB content in the gastric antrum of male rats, and (B) is a bar chart of NF-κB content in the gastric antrum of female rats.

[0046] Figure 19 This is a bar chart showing the expression levels of NF-κB in the colon of rats in each group in Example 5; Figure 19 In the figure, (A) is a bar chart of NF-κB content in the colon of male mice, and (B) is a bar chart of NF-κB content in the colon of female mice.

[0047] Figure 20 This is a bar chart of the gastrointestinal mucosal data of each group of rats in Example 5; Figure 20 In the figure, (A) is a bar chart of gastric mucosal thickness of rats in each group, (B) is a bar chart of ileal villus thickness of rats in each group, (C) is a bar chart of ileal villus height of rats in each group, (D) is a bar chart of ileal villus width of rats in each group, and (E) is a bar chart of colonic mucosal thickness of rats in each group.

[0048] Figure 21 This is a bar chart of organ indices for each group of rats in Example 5; Figure 21 In the figure, (A) is a bar chart of the heart index of each group of rats, (B) is a bar chart of the liver index of each group of rats, (C) is a bar chart of the spleen index of each group of rats, (D) is a bar chart of the kidney index of each group of rats, (E) is a bar chart of the lung index of each group of rats, and (F) is a bar chart of the thymus index of each group of rats.

[0049] Figures 5 to 21 In this context, the same lowercase letter indicates no significant difference between groups, while different lowercase letters indicate significant differences. Detailed Implementation

[0050] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0051] Example 1

[0052] On January 15, 2023, fermented goat milk made using traditional methods by local herders in Hulunbuir City, Inner Mongolia Autonomous Region, and showing no signs of spoilage, was collected in sterile sampling bottles. 5-10 mL samples were collected under aseptic conditions and quickly transported to the laboratory in a 4℃ refrigerator.

[0053] During the isolation process, the collected yogurt samples were first serially diluted with sterile physiological saline. 0.1 mL of each dilution was then spread onto MRS agar plates and anaerobically cultured at 37°C for 48-72 h. After single colonies appeared on the plates, they were picked based on their morphology. The isolated single colonies were purified three times using a three-zone streak plating technique. Each single colony was then inoculated into fresh MRS liquid medium for enrichment and culture for 24 h. The enriched bacterial solution was then spread onto MRS agar plates again. This process of picking single colonies and inoculating was repeated 2-3 times. The purified bacterial strain was finally obtained through Gram staining microscopy and physiological and biochemical characterization.

[0054] One of the purified strains was named HC1356, and 16S rDNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd., using 27F and 1492R primers.

[0055] The sequencing results (sequence 1) are as follows:

[0056]

[0057] The sequences and 16S rDNA genes were compared in the NCBI database, and a phylogenetic tree was constructed using MEGA 5.0 software. BLAST analysis showed that strain HC1356 is very similar to *Lactobacillus acidophilus*. Lactobacillus acidophilus (99% related), so the strain was named Lactobacillus acidophilus HC1356.

[0058] The identified Lactobacillus acidophilus HC1356 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 23, 2023, with accession number CGMCC No. 26888, and classified as Lactobacillus acidophilus. Lactobacillus acidophilus .

[0059] Example 2: Determination of the colonization ability of Lactobacillus acidophilus HC1356

[0060] The key to probiotics' ability to fully exert their health-promoting effects lies in their adhesion and colonization capabilities in the gastrointestinal tract. Beneficial bacteria such as lactic acid bacteria can prolong their residence time by adhering to the intestinal mucosa, thereby regulating the intestinal microecological balance or secreting beneficial metabolites. It is worth noting that gastric acid and bile salts in the gastrointestinal tract have a certain inhibitory effect on bacteria. Therefore, assessing the tolerance of bacterial strains to simulated gastric juice and bile salts has become an important indicator for screening high-quality probiotic strains.

[0061] I. Preparation of simulated gastrointestinal fluid solution

[0062] (1) Gastric fluid buffer: Accurately weigh 0.5144 g KCl, 0.1225 g KH2PO4, 2.75085 g NaCl, 2.1002 g NaHCO3, 0.0203 g MgCl2·6H2O, 0.0480 g (NH4)2CO3, and 0.0083 g CaCl2, and make up to 1000 mL.

[0063] (2) Simulated gastric juice (pH 3.0): 3 g of pepsin was dissolved in 1000 mL of gastric juice buffer, the pH was adjusted to 3.0 with 1 mol / L HCl, and sterilized by filtration through a 0.22 μm filter membrane. It was prepared fresh for use.

[0064] (3) Intestinal fluid buffer: Accurately weigh 0.5069 g KCl, 0.1089 g KH2PO4, 2.2442 g NaCl, 7.1408 g NaHCO3, 0.0067 g MgCl2·6H2O, and 0.0333 g CaCl2, and make up to 1000 mL.

[0065] (4) Simulated intestinal fluid (0.1% bile salt): 1000 mL of intestinal fluid buffer, add 1 g of trypsin and 1 g of bile salt to dissolve, adjust the pH of the simulated intestinal fluid to 8.0 with 1 mol / L NaOH, filter with a 0.22 μm filter membrane for sterilization, and prepare fresh for use.

[0066] II. Determination of the colonization ability of bacterial strains under simulated gastrointestinal fluid digestion conditions

[0067] After activation, the strain was inoculated into liquid culture medium at a 1% inoculum and incubated aerobically at 37°C for 24 h. Under aseptic conditions, the culture was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the bacterial sludge was collected. After washing three times with sterile PBS buffer, the enriched bacterial cells were obtained for later use.

[0068] (1) Gastric juice simulates digestion:

[0069] Take 1 mL of enriched bacterial cells and add 9 mL of simulated gastric fluid. Set up 3 replicates, incubate at 37℃ in a constant temperature water bath, and shake at 100 r / min for 0 h, 1 h, 2 h, 3 h, and 4 h. Calculate the viable count using the dilution plating method. Calculate the colonization survival rate of Lactobacillus acidophilus according to the following formula:

[0070] Gastric juice colonization survival rate = number of colonized and surviving bacteria / initial number of bacteria × 100%.

[0071] The results are shown in Table 1 below.

[0072] Table 1. Survival rate of strains at different times during simulated gastric digestion.

[0073]

[0074] (2) Intestinal fluid simulates digestion:

[0075] Take 1 mL of enriched bacterial cells and add 9 mL of simulated intestinal fluid. Set up 3 replicates, incubate at 37℃ in a constant temperature water bath, and shake at 100 r / min for 0 h, 12 h, 24 h, 36 h, and 48 h. Calculate the viable count using the dilution plating method. Calculate the colonization survival rate of Lactobacillus acidophilus according to the following formula:

[0076] Intestinal colonization survival rate = number of colonized and surviving bacteria / initial number of bacteria × 100%.

[0077] The results are shown in Table 2 below.

[0078] Table 2 Survival rates of strains at different times during simulated intestinal digestion

[0079]

[0080] Based on the strain survival data, although the survival rate of this strain gradually decreased with the extension of digestion time, its overall performance was excellent. During continuous digestion in a simulated gastric juice environment for 4 hours, the overall survival rate of the strain remained above 85%, demonstrating strong acid resistance and good tolerance to the acidic environment of the stomach. Furthermore, during a digestion process in simulated intestinal juice lasting up to 48 hours, its overall survival rate also exceeded 80%, indicating that this strain has good tolerance to components such as bile salts and trypsin in intestinal juice.

[0081] (3) Gastric juice-intestinal juice two-step method to simulate digestion:

[0082] Take 1 mL of enriched bacterial cells, add 9 mL of simulated gastric fluid, set up 3 replicates, incubate at 37℃ in a constant temperature water bath, rotate at 100 r / min for 3 h, then remove and quickly centrifuge. Add another 9 mL of simulated intestinal fluid, and culture under the same conditions for 24 h. Finally, count the bacteria using the dilution plating method. Calculate the colonization survival rate of Lactobacillus acidophilus according to the following formula:

[0083] Intestinal fluid colonization survival rate = number of colonized and surviving bacteria / initial number of bacteria × 100%.

[0084] Calculations showed that the survival rate of the strain after simulated gastric-intestinal two-step digestion remained at 81.31% ± 1.87%, a result that more closely reflects its actual survival status in the human digestive tract. These experimental data indicate that *Lactobacillus acidophilus* HC1356 is an excellent strain with outstanding acid and bile salt tolerance, capable of successfully colonizing the gastrointestinal tract.

[0085] Example 3: Determination of the adhesion ability of Lactobacillus acidophilus HC1356

[0086] I. Determination of adhesion ability to mucin

[0087] (1) Establishment of the mucin model: Weigh 10 mg of mucin and prepare a mucin solution with a concentration of 1 mg / mL using sterile PBS buffer, and store at -20℃. Take 500 µL of mucin solution and fix it in a 24-well cell culture plate for 1 h, incubate overnight at 4℃, then add an equal volume of mucin and continue incubation at 37℃ for 2 h to fill the blank sites, and then wash twice with sterile PBS buffer.

[0088] (2) Adhesion ability test: After overnight culture, the bacterial cells were collected and resuspended in sterile PBS buffer, and the bacterial count was adjusted to 10. 8CFU / mL, counted and recorded using a plating method. Add 500 µL of bacterial suspension to the mucin model, incubate at 37℃ for 1 h, wash 5 times with sterile citrate buffer to remove unbound bacteria, add 1 mL of 0.5% Tween 80 (v / v) to collect adherent bacteria, and finally count using the dilution plating method. The experiment was conducted in triplicate. *Lactobacillus rhamnosus* LGG was used as a control. The adhesion rate of *Lactobacillus acidophilus* to mucin was calculated using the following formula:

[0089] Mucin adhesion rate = number of bacteria adhered / initial number of bacteria × 100%.

[0090] The results are shown in Table 3 below.

[0091] II. Determination of adhesion ability to HT-29 cells (intestinal epithelial cell model)

[0092] (1) Culture of HT-29 cells: HT-29 cells were inoculated into DMEM complete medium containing 10% bovine serum + 1% penicillin + 1% streptomycin and incubated at 37°C in a carbon dioxide incubator with 5% CO2 and 95% air. The culture medium was changed daily and the cells were digested and passaged every week with 0.25% trypsin-EDTA digestion solution.

[0093] (2) Adhesion ability test: After the strain was activated, it was inoculated into liquid culture medium at a 1% inoculum and incubated aerobically at 37°C for 24 h. Under aseptic conditions, it was centrifuged at 6000 rpm for 10 min, the supernatant was discarded and the bacterial sludge was collected. After washing three times with sterile PBS buffer, 1×10⁻⁶ bacteria were prepared. 6 Prepare a bacterial suspension of CFU / mL for later use. Adjust the subcultured HT-29 cells to a concentration of 5 × 10⁶. 6 Cells were inoculated at a density of 1 mL / mL into 12-well cell culture plates. Once the cells reached a monolayer, the culture medium was discarded, and the plates were washed three times with sterile PBS buffer. 1 mL of the prepared bacterial suspension was added to each well, and the plates were gently shaken to mix. 20 μL of the bacterial suspension was then aspirated and counted to determine the initial cell count. The cell culture plates were then placed in a 5% CO2, 95% air incubator at 37°C for 2 h. The culture medium was discarded, and the plates were washed three times with sterile PBS buffer. 0.7 mL of trypsin-EDTA (0.25%) was added to each well to digest the cells for 10 min. After complete cell detachment, 0.3 mL of DMEM complete medium was added to terminate the digestion. 20 μL of the bacterial suspension was aspirated and counted to determine the number of adherent cells. The experiment was conducted in triplicate. *Lactobacillus rhamnosus* LGG was used as a control. The adhesion rate of *Lactobacillus acidophilus* to HT-29 cells was calculated using the following formula:

[0094] HT-29 cell adhesion rate = number of adhered bacteria / initial number of bacteria × 100%.

[0095] The results are shown in Table 3 below.

[0096] Table 3 Adhesion Ability Test Results

[0097]

[0098] As shown in Table 3, *Lactobacillus acidophilus* HC1356 exhibits superior adhesion performance to mucin and HT-29 cells compared to *Lactobacillus rhamnosus* LGG. Regarding mucin, the adhesion rate of *Lactobacillus acidophilus* HC1356 reached 46.97% ± 0.62%, significantly higher than that of *Lactobacillus rhamnosus* LGG (36.47% ± 1.53%). This indicates that *Lactobacillus acidophilus* HC1356 more readily binds to intestinal mucosal mucin, facilitating colonization in the intestinal mucosal layer and constructing a biological barrier to resist harmful bacteria. In terms of adhesion rate to HT-29 cells, *Lactobacillus acidophilus* HC1356 achieved 19.32% ± 1.47%, far exceeding that of *Lactobacillus rhamnosus* LGG (12.04% ± 1.68%). This demonstrates its superior ability to adhere to intestinal epithelial cells, regulating the intestinal microecology through competitive site occupancy and secretion of beneficial metabolites. Furthermore, it can participate in immune regulation, exhibiting greater application potential in improving intestinal health and addressing intestinal inflammatory responses.

[0099] Example 4: Preparation of probiotic formulation

[0100] (1) Preparation of culture medium: Prepare the culture medium used in the expansion process according to the following ratios.

[0101] The 3.5 L liquid culture medium consists of peptone 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, and Tween 80 1.0 g / L.

[0102] The culture medium used in the 100 L seed tank consists of 10.0 g / L peptone, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.0 g / L edible oil.

[0103] The culture medium used in the 1000 L fermenter consists of whey powder 13 g / L, glucose 30 g / L, yeast powder 20 g / L, concentrated carrot juice 5 g / L, concentrated tomato juice 5 g / L, soybean peptone 10 g / L, manganese sulfate 0.2 g / L, magnesium sulfate 0.1 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 1 g / L, sodium acetate 2 g / L, Tween 80 1.0 g / L, and edible oil 1.0 g / L.

[0104] (2) Propagation of strain: The refrigerated Lactobacillus acidophilus HC1356 strain was warmed to room temperature for 1 h, inoculated into 3.5L liquid culture medium and cultured for 16-20 h to make primary seed, and then transferred to 100 L seed tank and cultured for 12-15 h to obtain secondary seed. Subsequently, 10% of the inoculum was transferred to 1000 L fermenter for fermentation to obtain fermentation broth of Lactobacillus acidophilus HC1356.

[0105] (3) Preparation of raw powder: The fermentation broth of Lactobacillus acidophilus HC1356 was centrifuged to obtain bacterial sludge; 10% skim milk powder, 8% trehalose, 3% lactose, 1% soy protein isolate, 0.5% sodium L-glutamate, 0.1% ascorbic acid, and 77.4% distilled water were sterilized in batches by high pressure steam and then homogenized with the Lactobacillus acidophilus HC1356 bacterial sludge obtained by centrifugation at a mass ratio of 1:1-2. The pH value was adjusted to 6.5-7.0 to obtain bacterial sludge coating liquid. After vacuum freeze-drying, the freeze-dried block was taken out and crushed to obtain raw powder of Lactobacillus acidophilus HC1356.

[0106] (4) Raw material mixing: The raw powder of Lactobacillus acidophilus HC1356 was thoroughly mixed and diluted with fructooligosaccharide (purchased from Baolingbao Biotechnology Co., Ltd.), galactooligosaccharide (purchased from Shandong Bailong Chuangyuan Biotechnology Co., Ltd.), inulin (purchased from Cosucra, Belgium), lactulose (purchased from Jiangsu Jiujia Biotechnology Co., Ltd., product purity 98%), pumpkin powder (purchased from Jiangsu Runhui Food Co., Ltd.), and banana powder (purchased from Shaanxi Xintianyu Biotechnology Co., Ltd.) to obtain probiotic preparations with Lactobacillus acidophilus counts of ≥10 billion CFU / g, ≥20 billion CFU / g, ≥50 billion CFU / g, ≥100 billion CFU / g, and ≥200 billion CFU / g, respectively. When mixing, the mass ratio of fructooligosaccharide, galactooligosaccharide, inulin, lactulose, pumpkin powder, and banana powder was controlled to be 4:2:1:0.5:0.4:0.3.

[0107] Example 5: Animal model experiment of chemotherapy-induced gastric mucosal loss

[0108] I. Establishment of a rat model of cisplatin-induced gastrointestinal mucositis

[0109] (1) Animal experiment grouping: 90 eight-week-old SPF-grade Wistar rats, half male and half female, with an initial weight of 250 g ± 10 g for males and 180 g ± 10 g for females. They were fed rat-specific feed, with free access to water and food. The bedding was changed regularly. After one week of acclimatization, abnormal rats were removed, and only 40 male and 40 female rats were selected for use. The 40 male rats and 40 female rats were randomly divided into 5 groups: blank control group (CK group), cisplatin model group (MOD group), low-dose probiotic group (LPG group), medium-dose probiotic group (MPG group), and high-dose probiotic group (HPG group).

[0110] (2) Animal modeling: The total experimental period was 8 days. All rats were fed a basic diet throughout the experimental period. After the start of the experiment, the low-dose probiotic group received a low-dose probiotic (1×10⁻⁶) group. 7 CFU / 100 g body weight / dose) was administered via gavage as an adjunct to feeding. The medium-dose probiotic group received a medium-dose probiotic (5×10) 7 CFU / 100 g body weight / dose) was administered via gavage as an adjunct to feeding; the high-dose probiotic group received a high dose of probiotics (1×10). 8 The rats were administered a supplementary feeding dose of CFU / 100 g body weight per dose via gavage. The blank control group and the cisplatin model group were administered an additional feeding dose of sterile saline via gavage. The gavage dose was calculated based on changes in rat body weight throughout the experimental period, and the gavage times were fixed at 8:00 and 20:00.

[0111] The probiotics administered by gavage to the low-dose, medium-dose, and high-dose probiotic groups was Lactobacillus acidophilus HC1356. First, the Lactobacillus acidophilus HC1356 bacterial count was prepared according to the method described in Example 4, with a count of 5 × 10⁶. 9 A probiotic preparation of CFU / g was prepared, and then diluted with sterile physiological saline 500 times, 100 times, and 50 times to obtain concentrations of 1×10⁻⁶ CFU / g. 7 CFU / mL, 5×10 7 CFU / mL, 1×10 8 CFU / mL probiotic solution. At each gavage, 1 mL of the corresponding concentration of probiotic solution was administered to rats in the low-dose, medium-dose, and high-dose probiotic groups.

[0112] On day 4 of the experimental period, the cisplatin model group, low-dose probiotic group, medium-dose probiotic group and high-dose probiotic group were injected intraperitoneally with cisplatin 6 mg / kg body weight, while the blank control group was injected intraperitoneally with an equal volume of sterile saline.

[0113] II. Indicator Collection and Measurement

[0114] (1) Changes in rat body weight: The rats were weighed at fixed times every day, and the changes in rat body weight were observed. The results are as follows: Figure 1 , Figure 2 As shown.

[0115] Throughout the experimental period, comparing data from male and female mice, in the blank control group, the initial weight of male mice (approximately 270 g) was higher than that of female mice (approximately 205 g), and the weight gain of male mice (approximately 40 g) was greater than that of female mice (approximately 20 g), consistent with sex differences in growth and development. In the cisplatin model group, the weight of both male and female mice showed a decreasing trend, with the weight loss in male mice (approximately 10 g) being slightly greater than that in female mice (approximately 5 g), which may be related to differences in physiological metabolism and cisplatin sensitivity between males and females. Although the weight ranges of male and female mice differed within their respective age groups, the trends in weight change were basically consistent, allowing for simultaneous analysis.

[0116] After grouping rats of the same sex, their body weights showed different trends, with significant differences between groups. Regardless of sex, the weight gain in the blank control group was relatively stable, conforming to the normal weight range for rats of that age, providing a healthy physiological benchmark for subsequent experiments. The weight changes in the cisplatin model group exhibited a clear phased pattern. From days 1 to 3, there was no significant difference in weight changes between this group and other groups. However, after intraperitoneal injection of cisplatin on day 4, the weight showed a significant and continuous decreasing trend. This is because cisplatin, as a chemotherapy drug, is toxic and adversely affects the rats, inhibiting growth and causing weight loss. These results confirm the successful establishment of the cisplatin toxicity model, demonstrating that its toxicity can alter the body's metabolism and physiological state. In the first 3 days of the experiment, the weight changes in the low-dose probiotic group, medium-dose probiotic group, and high-dose probiotic group were basically the same as those in the blank control group and the cisplatin model group, indicating that the administration of different doses of probiotics by gavage at this stage did not significantly interfere with the weight of the rats. However, after intraperitoneal injection of cisplatin on the 4th day, the weight loss in the low-dose probiotic group, medium-dose probiotic group, and high-dose probiotic group was more moderate than that in the cisplatin model group, and there was a difference between the groups: the high-dose probiotic group maintained the best weight, followed by the medium-dose probiotic group, and the low-dose probiotic group had the most significant weight loss among the three groups, but overall all three groups were better than the cisplatin model group.

[0117] The above results indicate that gavage administration of Lactobacillus acidophilus HC1356 has a certain alleviating effect on cisplatin-induced weight loss, and the effect of high-dose Lactobacillus acidophilus HC1356 is better than that of low and medium doses. This alleviating effect is universally applicable to both male and female mice.

[0118] In summary, cisplatin can cause a significant decrease in body weight in rats. This embodiment successfully constructed a cisplatin toxicity injury model, which can simulate the adverse effects of chemotherapy drugs on the body's growth status. Gavage administration of Lactobacillus acidophilus HC1356 can alleviate the weight loss in rats caused by cisplatin-induced gastrointestinal mucositis, reduce the toxic side effects of cisplatin, and maintain the body's growth status. The alleviating effect of high-dose probiotics is relatively better.

[0119] (2) Food intake of rats: The weight of food consumed by rats was measured at a fixed time each day, and the changes in daily food intake were observed. The results are as follows: Figure 3 , Figure 4 As shown.

[0120] Throughout the experimental period, the changes in food intake of both male and female rats showed clear and regular trends. Furthermore, the trends in food intake in female rats closely correlated with those in male rats, allowing for comprehensive analysis. In the first three days of the experiment, there were no significant differences in food intake among the control group, cisplatin model group, low-dose probiotic group, medium-dose probiotic group, and high-dose probiotic group. This indicates that the gavage intervention in the first three days and the initial basal feeding did not affect the rats' feeding behavior. It wasn't until day four, after intraperitoneal injection of cisplatin in the cisplatin model group, low-dose probiotic group, medium-dose probiotic group, and high-dose probiotic group, that food intake in these groups began to show varying degrees of decrease compared to the control group. The cisplatin model group showed the most significant decrease in food intake, which remained at a low level throughout the subsequent experimental phases. In contrast, the low-dose, medium-dose, and high-dose probiotic groups showed relatively moderate decreases in food intake, with the high-dose probiotic group exhibiting the best intake, followed by the medium-dose group, and the low-dose group the worst, but overall still outperforming the cisplatin model group.

[0121] This phenomenon indicates that cisplatin significantly inhibits food intake in rats, while gavage administration of Lactobacillus acidophilus HC1356 has a certain alleviating effect on cisplatin-induced food intake inhibition, with the alleviating effect being more pronounced at higher doses of probiotics. It is speculated that Lactobacillus acidophilus HC1356 may alleviate the toxic stress caused by cisplatin-induced gastrointestinal mucositis by regulating the intestinal microecology and improving intestinal barrier function, thereby reducing the inhibitory effect of cisplatin on feeding behavior.

[0122] (3) Blood collection and related index determination of rats: On the 8th day of the experimental cycle, all groups of rats were fasted but allowed to drink water for 12 h. After the fasting period, sodium pentobarbital was injected intraperitoneally for anesthesia. After the anesthesia took effect, blood was collected from the heart. After standing at 4℃ for 3 h, the blood was frozen and centrifuged at 3000 rpm for 16 min at 4℃. The serum was separated and aliquoted and stored at -80℃ for later use, avoiding repeated freeze-thaw cycles.

[0123] Serum IL-6, IL-10, IL-1β, IL-18, and TNF-α are all cytokines that play crucial roles in physiological and pathological processes such as immune regulation and inflammatory responses. IL-6 is an important pro-inflammatory cytokine, participating in immune activation and inflammation amplification; IL-10 is a key anti-inflammatory cytokine that can inhibit the release of pro-inflammatory cytokines and maintain immune homeostasis; IL-1β is a core factor in the initiation of inflammation, triggering an inflammatory cascade and inducing fever; IL-18 can enhance cellular immunity, synergistically promote inflammation, and participate in metabolic regulation; TNF-α is a potent pro-inflammatory cytokine that mediates inflammatory responses and immune cell regulation, and its overexpression can lead to tissue damage and is also associated with tumor apoptosis regulation. The results of serum IL-6, IL-10, IL-1β, IL-18, and TNF-α levels are as follows: Figures 5 to 9 As shown.

[0124] In this experiment, compared with the blank control group, the expression levels of pro-inflammatory factors IL-6, IL-1β, IL-18, and TNF-α in the serum of both male and female mice in the cisplatin model group were significantly increased, while the expression level of anti-inflammatory factor IL-10 was significantly decreased, indicating that the cisplatin-induced systemic inflammation model was successfully established. After intervention with different doses of probiotics in the low-dose, medium-dose, and high-dose probiotic groups, the levels of pro-inflammatory factors IL-6, IL-1β, IL-18, and TNF-α showed a decreasing trend compared with the cisplatin model group, while the anti-inflammatory factor IL-10 showed an increasing trend. This indicates that the inflammatory damage induced by cisplatin can be regulated by probiotic intervention to regulate the balance of inflammatory factors, and the relief effect of the medium-dose and high-dose probiotic groups was better than that of the low-dose probiotic group. Regarding sex differences, the absolute values ​​of pro-inflammatory factors in male mice in the cisplatin model group were generally higher than those in female mice after cisplatin induction, indicating that male mice are more sensitive to cisplatin-induced inflammation, which may be related to the influence of male hormones and metabolic characteristics. In terms of basic anti-inflammatory capacity, the expression level of IL-10 in female mice in the blank control group was higher than that in male mice. After probiotic intervention, the increase in serum IL-10 expression level in female mice and the response to dosage were also better than those in male mice. This reflects that the anti-inflammatory pathway in male mice responds weakly to probiotics than that in female mice.

[0125] In summary, cisplatin can induce an imbalance of inflammatory factors, and the response to this imbalance differs between genders. The use of Lactobacillus acidophilus HC1356 can alleviate inflammatory damage and regulate the balance of inflammatory factors, with medium- and high-dose probiotics showing better relief.

[0126] The results of serum reactive oxygen species (ROS) content measurement are as follows: Figure 10As shown in the figure. In this experiment, compared with the blank control group, the serum ROS levels in both female and male rats in the cisplatin model group were significantly increased, indicating that cisplatin can induce rats to produce a large amount of reactive oxygen species, causing oxidative stress damage, and the model construction was effective. Compared with the cisplatin model group, the serum ROS levels in the low-dose probiotic group, medium-dose probiotic group, and high-dose probiotic group all showed a decreasing trend after probiotic intervention. The medium-dose and high-dose probiotic groups showed better effects than the low-dose probiotic group. It can be inferred that the intervention of Lactobacillus acidophilus HC1356 may reduce ROS and alleviate oxidative damage by directly secreting antioxidant metabolites or indirectly regulating intestinal flora and activating antioxidant pathways.

[0127] Superoxide dismutase (SOD), as a core antioxidant enzyme, can scavenge harmful superoxide anion free radicals, reflecting the body's antioxidant and anti-aging capabilities. The results of serum superoxide dismutase activity assays are as follows: Figure 11 As shown in the figure. In this experiment, regardless of whether the mice were female or male, the SOD activity in the cisplatin model group was significantly lower than that in the blank control group. This indicates that cisplatin damages mitochondria, inhibits the synthesis of antioxidant enzymes, and reduces SOD activity, verifying the cisplatin-induced oxidative stress damage, which is consistent with the increase in serum ROS. Compared with the cisplatin model group, the low-dose probiotic group, medium-dose probiotic group, and high-dose probiotic group all showed an increasing trend in serum SOD activity, and the medium-dose and high-dose probiotic groups were more effective than the low-dose probiotic group. Therefore, it is speculated that Lactobacillus acidophilus HC1356 can increase SOD activity by directly secreting SOD-containing metabolites or indirectly affecting the pathway of SOD synthesis activation by intestinal flora.

[0128] Malondialdehyde (MDA), a marker product of lipid peroxidation, reflects the degree of oxidative damage in the body. Serum MDA activity assay results are as follows... Figure 12 As shown in the figure. In this experiment, the serum MDA content in the cisplatin model group was significantly higher than that in the blank control group in both female and male mice. Combined with previous experimental results, cisplatin promotes ROS generation, triggers a lipid peroxidation chain reaction leading to MDA accumulation, thus verifying the cisplatin-induced oxidative stress damage. The serum MDA content in the low-dose probiotic group, medium-dose probiotic group, and high-dose probiotic group was lower than that in the cisplatin model group, and the medium-dose and high-dose probiotic groups were more effective than the low-dose probiotic group. This indicates that Lactobacillus acidophilus HC1356 can reduce lipid peroxidation substrates by scavenging ROS, increase the activity of antioxidant enzymes such as SOD to block the oxidation chain reaction, reduce MDA generation, and thus alleviate oxidative damage.

[0129] Glutathione peroxidase (GSH-Px) is an important antioxidant enzyme whose core function is to remove harmful peroxides (such as hydrogen peroxide and lipid peroxides) from the body, protecting cells from oxidative damage. Serum GSH-Px activity assay results are as follows... Figure 13 As shown in the figure. In this experiment, the serum GSH-Px activity in the cisplatin model group was significantly lower than that in the blank control group in both female and male mice. This indicates that cisplatin can induce oxidative stress, consume GSH-Px or inhibit its synthesis, and weaken the body's antioxidant capacity, which is consistent with the previous results of increased ROS and MDA accumulation. The serum GSH-Px activities in the low-dose probiotic group, medium-dose probiotic group, and high-dose probiotic group were all higher than those in the cisplatin model group, and the medium-dose and high-dose probiotic groups were more effective than the low-dose probiotic group. This indicates that Lactobacillus acidophilus HC1356 can indirectly enhance the enzyme activity of GSH-Px by promoting GSH-Px synthesis or supplementing reduced glutathione substrate.

[0130] Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) can reflect liver damage, while blood urea nitrogen (BUN) and serum creatinine (Cr) can reflect kidney function. The results of serum ALT, AST, BUN, and Cr measurements are as follows: Figures 14 to 17 As shown in the figure. In this experiment, in both female and male mice, the cisplatin model group showed significantly higher serum ALT and AST activities, as well as significantly higher BUN and Cr levels compared to the blank control group. This indicates that cisplatin directly damages hepatocytes, increasing the release of transaminases, and also damages glomeruli and renal tubules, leading to impaired excretion of urea nitrogen and creatinine. This verifies the hepatotoxicity and nephrotoxicity induced by cisplatin, which is consistent with the previous data on oxidative damage. Serum ALT and AST activities in the low-dose, medium-dose, and high-dose probiotic groups were all lower than those in the cisplatin model group, and BUN and Cr levels were also lower in the cisplatin model group. Furthermore, the medium-dose and high-dose probiotic groups showed better alleviating effects on this damage than the low-dose probiotic group. This indicates that Lactobacillus acidophilus HC1356 can reduce hepatocyte oxidative damage and inhibit mitochondrial apoptosis by secreting metabolites such as short-chain fatty acids, reduce intestinal-derived liver damage by regulating gut microbiota, reduce intestinal urease activity to decrease BUN production or promote creatinine metabolism, and reduce the impact of endotoxemia on renal blood flow by regulating gut microbiota.

[0131] Based on the above serum data, cisplatin-induced oxidative stress has a toxic effect on the body. It can increase serum ROS, accumulate MDA, and decrease the activity of antioxidant enzymes such as SOD and GSH-Px, thereby triggering a series of oxidative stress responses. It can also induce an increase in serum pro-inflammatory factors IL-6, IL-1β, IL-18, and TNF-α, and a decrease in anti-inflammatory factor IL-10, causing certain inflammatory damage. Furthermore, it can lead to an increase in serum ALT, AST, BUN, and Cr levels, thereby causing liver and kidney damage. Intervention with medium or high doses of Lactobacillus acidophilus HC1356 can alleviate cisplatin-induced oxidative stress, inflammatory responses, and liver and kidney damage by regulating the oxidative-anti-inflammatory balance, thereby achieving the effect of relieving cisplatin-induced gastrointestinal mucositis.

[0132] (4) Determination of relevant indicators in rat gastrointestinal tissues: In the regulation of inflammation and apoptosis, NF-κB, as a core transcription factor, has an expression level that is a key indicator for assessing the inflammatory status of the gastrointestinal tract. Rats were euthanized by cervical dislocation after blood collection from the heart on day 8 of the experimental period. Tissues from the gastric antrum (pylorus) and colon (fixed site) were rapidly collected, rinsed with pre-cooled PBS, and then flash-frozen in liquid nitrogen. The entire process was performed at low temperatures. The expression levels of NF-κB in the gastric antrum and colon were measured using a rat nuclear factor κB subunit p65 (NF-κB p65) affinity peptide ELISA kit. The results are as follows: Figure 18 , Figure 19 As shown.

[0133] In this experiment, the trends in NF-κB levels were highly consistent between female and male mice, showing no significant sex differences. This indicates the sex-specific universality of cisplatin's inflammatory toxicity and the anti-inflammatory effects of probiotics. Furthermore, in both female and male mice, the NF-κB levels in the gastric antrum and colon of the blank control group remained at low baseline levels, reflecting the inflammatory homeostasis of the rat gastrointestinal tract under normal physiological conditions. The NF-κB levels in the gastric antrum and colon of the cisplatin model group were significantly elevated, indicating that cisplatin induces acute inflammatory damage in the gastrointestinal tract by damaging the gastrointestinal mucosal epithelium, triggering the recruitment of immune cells and the release of inflammatory mediators, ultimately activating the NF-κB signaling pathway. The experimental results of low-dose, medium-dose, and high-dose probiotic groups showed that the NF-κB levels in the gastric antrum and colon of rats gradually decreased with increasing doses of Lactobacillus acidophilus HC1356. Among them, the high-dose probiotic group showed the most relief effect, which was closest to the normal level of the blank control group. It is speculated that this effect is due to the triple action of probiotics: first, regulating the intestinal flora structure, inhibiting the abundance of pro-inflammatory bacteria and reducing endotoxin release; second, strengthening the intestinal barrier function and reducing the stimulation of the mucosa by pathogens and toxins; and third, directly regulating the NF-κB phosphorylation process of immune cells and blocking the amplification of the inflammatory cascade.

[0134] Based on the above results, cisplatin can induce gastrointestinal inflammation by activating the NF-κB signaling pathway, while the oral administration of Lactobacillus acidophilus HC1356 in this invention can alleviate gastrointestinal mucosal inflammation caused by this damage process, with high-dose probiotics showing relatively better alleviating effects.

[0135] (5) Evaluation of gastric mucosal damage index: The whole stomach of the euthanized rats was removed and dissected. After being rinsed with sterile PBS buffer, the stomach was spread out on a coded sterile plate and observed with the naked eye. The gastric mucosal damage index was evaluated according to the GUTH method. The length of each lesion in the whole stomach was scored according to the GUTH standard and then summed to obtain the damage index. The scoring criteria are shown in Table 4 below.

[0136] Table 4. Gastric Mucosal Injury Index Scoring Criteria

[0137]

[0138] Table 5 shows the gastric mucosal damage index for different groups. In Table 5, the same lowercase letter indicates no significant difference between groups, while different lowercase letters indicate significant differences.

[0139] Table 5. Gastric Mucosal Injury Index (UI) in Different Groups

[0140]

[0141] Table 5 shows the gastric mucosal injury index for different groups. The UI in the blank control group was 0.00±0.00, indicating almost no damage to the gastric mucosa in normal rats. The UI in the cisplatin model group surged to 6.43±0.58, showing a highly significant difference from the CK group, indicating that cisplatin strongly induced gastric mucosal injury in rats, and the model was successfully established. The UI in the low-dose probiotic group was 5.40±0.46, significantly lower than the cisplatin model group, indicating that low-dose probiotics could alleviate cisplatin-induced gastric mucosal injury to some extent, but the improvement was limited. The UI in the medium-dose probiotic group was 4.30±0.55, and the UI in the high-dose probiotic group was 3.99±0.52, both showing highly significant differences from the cisplatin model group, indicating that medium and high-dose probiotics had a more prominent alleviating effect on gastric mucosal injury, and the degree of injury was closer to normal. Within the experimentally determined dosage range, higher probiotic doses may have a stronger protective effect on the gastric mucosa. However, the UI difference between the medium-dose probiotic group and the high-dose probiotic group was relatively smaller, suggesting that the marginal benefit of the protective effect may weaken after the dosage reaches a certain level.

[0142] It is evident that Lactobacillus acidophilus HC1356 has a clear protective effect against cisplatin-induced gastric mucosal damage in rats.

[0143] (6) Measurement of gastrointestinal mucosal data: Cisplatin, as a commonly used chemotherapy drug, can cause significant toxic damage to gastrointestinal tissues, leading to problems such as destruction of the gastrointestinal mucosal structure. During rat dissection, 1 cm × 1 cm tissue samples were precisely and quickly taken from the gastric body (avoiding the antrum and cardia), and 3 cm samples were taken from above and below the ileocecal junction. 3 cm specimens of the ileum and colon were taken for histopathological examination. Pathological specimens were fixed with 4% formaldehyde solution, routinely dehydrated, and embedded in paraffin, with the tissue blocks embedded perpendicularly. HE staining was used to observe the pathological changes in rat gastrointestinal tissues and to measure gastric mucosal thickness, ileal villus thickness, ileal villus height, ileal villus width, and colonic mucosal thickness. The results are as follows: Figure 20 As shown.

[0144] Compared with the blank control group, the cisplatin model group showed significantly lower values ​​for gastric mucosal thickness, ileal villus thickness, ileal villus height, ileal villus width, and colonic mucosal thickness, directly demonstrating the toxic effects of cisplatin on rat gastrointestinal tissues, leading to a substantial decrease in gastrointestinal mucosal structure-related indicators. In contrast, the low-dose, medium-dose, and high-dose probiotic groups all showed varying degrees of increase in these gastrointestinal tissue indicators compared to the cisplatin model group. Furthermore, as the probiotic dose gradually increased from low to medium to high, the indicator values ​​showed a gradual upward trend, with the high-dose probiotic group's values ​​being closer to those of the blank control group.

[0145] This shows that cisplatin causes significant toxic damage to the gastrointestinal tract tissues of rats, significantly reducing gastric mucosal thickness, ileal villi, and other related indicators. However, Lactobacillus acidophilus HC1356 has a protective effect against cisplatin-induced gastrointestinal tissue damage in rats. Within a certain range, the protective effect of Lactobacillus acidophilus HC1356 on gastrointestinal tissues may be stronger with increasing dose, and it can improve the gastrointestinal mucosal structural damage caused by cisplatin to some extent.

[0146] (7) Determination of organ indices in rats: The stability of organ indices in rats directly reflects their immune level and health status. During rat dissection, the heart, liver, spleen, kidneys, lungs, and thymus of the rat are accurately and quickly removed, weighed, and the data are recorded. The organ indices of the rat are calculated according to the following formula:

[0147] Cardiac index = Heart weight (g) / Body weight (g) × 100%;

[0148] Liver index = Liver weight (g) / Body weight (g) × 100%;

[0149] Spleen index = Spleen weight (g) / Body weight (g) × 100%;

[0150] Kidney index = Kidney weight (g) / Body weight (g) × 100%;

[0151] Lung Index = Lung weight (g) / Body weight (g) × 100%;

[0152] Thymus index = thymus weight (g) / body weight (g) × 100%.

[0153] The measurement results are as follows Figure 21 As shown in the figure. In this experiment, the study of organ indices (heart, liver, spleen, kidney, lung, thymus) in female and male rats revealed relatively significant differences between different treatment groups. Furthermore, the trends in organ indices were highly synchronized between male and female rats, with no significant sex differences observed. This indicates that the organ toxicity of cisplatin and the protective effect of probiotics have similar modes of action in male and female individuals. Comparing the bar charts of organ indices, the cisplatin model groups in both female and male rats showed a decreasing trend in heart and thymus indices compared to the control group. However, the liver, spleen, and kidney indices in the cisplatin model groups showed an increasing trend compared to the control group. These inter-group differences strongly confirm that intraperitoneal injection of cisplatin can induce multi-organ damage in rats, interfering with organ development and functional maintenance. In the low-dose, medium-dose, and high-dose probiotic groups after probiotic intervention, the heart, liver, spleen, kidney, and thymus indices of both male and female mice showed varying degrees of restorative recovery compared to the cisplatin model group. The high-dose probiotic group showed a more significant recovery in organ indices, approaching the levels of the blank control group, followed by the medium-dose group, while the low-dose group showed the weakest effect. Notably, in both male and female mice, the lung index in the cisplatin model group did not show a significant increase compared to other treatment groups. This may be because the lungs possess multiple protective mechanisms and are the primary excretion pathway for non-cisplatin metabolites. Probiotics may alleviate cisplatin-induced organ damage by regulating the gut microbiota, enhancing immune responses, reducing oxidative stress, or directly antagonizing cisplatin toxicity, thus achieving multi-organ protection.

[0154] In summary, cisplatin toxicity can cause varying degrees of damage to various organs. In this invention, oral administration of Lactobacillus acidophilus HC1356 can improve cisplatin-induced damage to the heart, liver, spleen, kidneys, and thymus, and help maintain or protect the function of immune organs, thereby alleviating cisplatin-induced gastrointestinal mucositis. Among these methods, the protective effect of high-dose probiotics is relatively superior.

[0155] (8) Liver probiotic detection: After weighing the liver, cut off 1 g of liver and add it to sterile PBS buffer for suspension and homogenization. Take 1 mL of the solution and spread it on MRS plate. Incubate at 37℃ for 48 h in both aerobic and anaerobic conditions. Detect whether bacteria grow on the plate.

[0156] In this experiment, no colonies were observed in rat liver tissue after 48 h of aerobic and anaerobic culture on MRS plates. This indicates that *Lactobacillus acidophilus* HC1356 does not undergo hepato-gut translocation in animals.

[0157] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A type of Lactobacillus acidophilus ( Lactobacillus acidophilus The application of HC1356 in the preparation of products that alleviate chemotherapy-induced gastrointestinal mucosal damage, characterized in that... Lactobacillus acidophilus HC1356 was deposited on March 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26888, and classified as Lactobacillus acidophilus. Lactobacillus acidophilus ; Chemotherapy-induced gastrointestinal mucosal damage refers to the toxic effects of cisplatin on gastrointestinal mucosal tissues.

2. The application of a probiotic preparation in the preparation of a product to alleviate chemotherapy-induced gastrointestinal mucosal damage, characterized in that, The probiotic preparation contains raw powder of Lactobacillus acidophilus HC1356, which was deposited on March 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26888 and classified as Lactobacillus acidophilus. Lactobacillus acidophilus The preparation method of Lactobacillus acidophilus HC1356 raw powder includes the following steps: The fermentation broth of Lactobacillus acidophilus HC1356 was centrifuged and the bacterial sludge was collected. The bacterial sludge was coated and then freeze-dried under vacuum to obtain freeze-dried blocks. The freeze-dried blocks were crushed to obtain the raw powder of Lactobacillus acidophilus HC1356. Chemotherapy-induced gastrointestinal mucosal damage refers to the toxic effects of cisplatin on gastrointestinal mucosal tissues.

3. The application as described in claim 2, characterized in that, Lactobacillus acidophilus HC1356 was transferred from the refrigerated environment to room temperature and then inoculated into primary liquid culture medium and cultured for 16-20 h to prepare primary seed; it was then transferred to secondary seed tank and cultured for 12-15 h to obtain secondary seed; subsequently, it was transferred to tertiary fermenter for fermentation to obtain fermentation broth of Lactobacillus acidophilus HC1356.

4. The application as described in claim 3, characterized in that, The primary liquid culture medium consists of peptone 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, and Tween 80 1.0 g / L. The culture medium used in the secondary seed tank consists of 10.0 g / L peptone, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.0 g / L edible oil. The culture medium used in the tertiary seed tank consists of whey powder 13 g / L, glucose 30 g / L, yeast powder 20 g / L, concentrated carrot juice 5 g / L, concentrated tomato juice 5 g / L, soybean peptone 10 g / L, manganese sulfate 0.2 g / L, magnesium sulfate 0.1 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 1 g / L, sodium acetate 2 g / L, Tween 80 1.0 g / L, and edible oil 1.0 g / L.

5. The application as described in claim 2, characterized in that, The coating method for the bacterial sludge is as follows: by mass percentage, 10% of sterilized skim milk powder, 8% of trehalose, 3% of lactose, 1% of soy protein isolate, 0.5% of L-glutamate, 0.1% of ascorbic acid, and 77.4% of distilled water are mixed evenly and then homogenized with the Lactobacillus acidophilus HC1356 bacterial sludge obtained by centrifugation at a mass ratio of 1:1-2. The pH value is adjusted to 6.5-7.0 to obtain the bacterial sludge coating solution.

6. The application as described in claim 2, characterized in that, Based on the number of Lactobacillus acidophilus HC1356, the specifications of probiotic preparations are selected from ≥10 billion CFU / g, ≥20 billion CFU / g, ≥50 billion CFU / g, ≥100 billion CFU / g, or ≥200 billion CFU / g.

7. The application as described in any one of claims 2-6, characterized in that, Probiotic preparations also contain fructooligosaccharides, galactooligosaccharides, inulin, lactulose, pumpkin powder, and banana powder.

8. The application as described in claim 7, characterized in that, The mass ratio of fructooligosaccharides, galactooligosaccharides, inulin, lactulose, pumpkin powder, and banana powder is 4:2:1:0.5:0.4:0.3.

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