Lactobacillus kefiranofaciens KM15, microbial preparation, composition and application thereof in alzheimer's disease

Abstract

The application provides a lactobacillus kefiri KM15, a microbial preparation, a composition and application thereof in Alzheimer's disease, and belongs to the technical field of microorganisms. The preservation number of the lactobacillus kefiri KM15 is CGMCC No. 38244. The strain can improve cognitive dysfunction, anxiety-like behavior and learning and memory ability of AD model mice, enhance antioxidant capacity, improve SOD, CAT, GSH-Px and T-AOC activity, reduce MDA level, regulate inflammation, reduce pro-inflammatory factors IL-6, IL-1beta and TNF-alpha, increase anti-inflammatory factor IL-10, repair colon mucosa damage, and enhance intestinal barrier. The strain can be used for preparing a medicine for preventing or assisting in improving Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Lactobacillus equina KM15, microbial preparations, compositions, and their application in Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment, primarily affecting the elderly and pre-aging populations. Its typical pathological features include senile plaques formed by the aggregation of β-amyloid protein (Aβ), neurofibrillary tangles caused by hyperphosphorylation of tau protein, and the resulting neuronal apoptosis, synapse loss, brain atrophy, and neuroinflammatory responses. With the accelerating aging of the global population, AD has become a major public health problem. However, its exact pathogenesis is not yet fully understood, and there are currently no effective drugs for treating AD. Therefore, seeking safe and effective prevention and adjunctive interventions for AD has significant scientific and social value.

[0003] In recent years, numerous studies have confirmed that the gut microbiota communicates bidirectionally with the central nervous system through the "microbiota-gut-brain axis." This axis, through neural signals, immune signals, and gut microbiota metabolites, independently or synergistically regulates brain function and the pathological process of Alzheimer's disease (AD). This theory provides a novel research approach for targeting the gut microbiota to intervene in the AD process and improve cognitive function. Therefore, developing probiotic preparations that can exert neuroprotective effects by regulating the balance of the gut microbiota has become a potentially effective strategy for preventing or delaying the development of AD.

[0004] Lactobacillus hummus ( Lactobacillus kefiranofaciens *Lactobacillus hummi* is a Gram-positive, facultative anaerobic lactic acid bacterium belonging to the family Lactobacillusceae and the genus *Lactobacillus*. It was first isolated from kefir, a traditional fermented dairy product. Current research indicates that this bacterium possesses various probiotic functions: it exhibits excellent survival ability in the acidic gastrointestinal environment; it can scavenge free radicals and alleviate oxidative stress; it inhibits the growth and colonization of various pathogenic bacteria through acid production and the synthesis of antibacterial substances; in terms of immune regulation, it can reduce inflammatory responses such as airway inflammation and colitis; simultaneously, it also shows regulatory effects on metabolic and neuropsychiatric disorders such as obesity, diabetes, and depressive behaviors. Furthermore, *Lactobacillus hummi* can reshape the intestinal flora structure, increase the abundance of beneficial bacteria, and show positive effects in maintaining intestinal microecological balance.

[0005] Although existing studies have suggested that *Lactobacillus hummi* possesses various probiotic functions, to date, there have been no systematic reports on whether it can improve Alzheimer's disease-related cognitive impairment through the gut-microbiota-brain axis pathway, or on its specific mechanisms of action and dose-response relationships. Therefore, in-depth exploration of the potential application value of *Lactobacillus hummi* in the prevention or adjuvant improvement of Alzheimer's disease, and elucidating its mechanism of action, is of significant theoretical importance and application prospect for developing new AD intervention strategies based on gut microbiota regulation. Summary of the Invention

[0006] One of the objectives of this invention is to provide a *Lactobacillus hummilaceus* (Horse milk wine-like bacteria). Lactobacillus kefiranofaciens KM15 was deposited on April 14, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.38244.

[0007] The second objective of this invention is to provide a microbial preparation containing Lactobacillus hummilaceus KM15.

[0008] Preferably, the microbial preparation is a solid or liquid preparation.

[0009] Preferably, the solid formulation includes lyophilized powder, tablets, capsules, and granules; the liquid formulation includes solutions, suspensions, and emulsions.

[0010] Preferably, the microbial preparation also contains pharmaceutically acceptable excipients.

[0011] Preferably, the pharmaceutically acceptable excipient is selected from one or more of excipients, binders, disintegrants, lubricants, preservatives, antioxidants, or flavoring agents.

[0012] A third objective of this invention is to provide the use of Lactobacillus equina milkiformis KM15 or a microbial preparation in the preparation of a drug for the prevention and / or adjuvant improvement of Alzheimer's disease.

[0013] Preferably, the drug is used to improve cognitive impairment, anxiety-like behavior, oxidative stress, or gut microbiota dysbiosis associated with Alzheimer's disease.

[0014] The fourth objective of this invention is to provide a composition for the prevention and / or adjuvant improvement of Alzheimer's disease, comprising Lactobacillus equinasum KM15 and a pharmaceutically acceptable carrier.

[0015] Preferably, the pharmaceutically acceptable carrier is selected from one or more of lactose, starch, microcrystalline cellulose, mannitol, and magnesium stearate.

[0016] Preferably, the composition for the prevention and / or adjunctive improvement of Alzheimer's disease is a probiotic product.

[0017] Preferably, the composition for the prevention and / or adjunctive improvement of Alzheimer's disease is a pharmaceutical composition.

[0018] Preferably, the probiotic product is used to improve Alzheimer's disease-related cognitive impairment, anxiety-like behavior, oxidative stress, or gut microbiota dysbiosis.

[0019] The beneficial effects of this invention are: The *Lactobacillus hummiflorum* KM15 provided by this invention has been verified in animal experiments to have the following effects: (1) Improve cognitive dysfunction in AD model mice: Through behavioral experiments such as open field test, Y maze, and new object recognition, it was confirmed that Lactobacillus equina-KM15 can significantly increase the time mice spend in the central area of ​​the open field, improve the spontaneous alternation rate and new object recognition index, indicating that it can effectively improve the spatial learning ability, short-term memory ability and anxiety-like behavior of AD mice.

[0020] (2) Enhance the body's antioxidant capacity: After intervention with Lactobacillus equina KM15, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) in the serum of AD mice increased, while the content of malondialdehyde (MDA) decreased, indicating that this strain can effectively alleviate oxidative stress damage in AD mice.

[0021] (3) Regulation of inflammatory response: Lactobacillus equina KM15 can reduce the levels of pro-inflammatory factors IL-6, IL-1β and TNF-α in the serum of AD mice, while increasing the expression of anti-inflammatory factor IL-10, showing good anti-inflammatory activity.

[0022] (4) Repairing intestinal tissue damage: H&E staining results of colon tissue showed that Lactobacillus equinae KM15 could repair the damaged colonic mucosal structure of AD mice, restore the integrity of crypt structure, increase the number of goblet cells, reduce inflammatory cell infiltration, thereby enhancing the intestinal mucosal barrier function.

[0023] (5) Regulation of gut microbiota homeostasis: 16S rDNA sequencing analysis showed that *Lactobacillus equinasum* KM15 could increase the α- and β-diversity of gut microbiota in AD mice, increase the Firmicutes / Bacteroidetes (F / B) ratio, and increase the number of Lactobacillus families ( Lactobacillaceae ), family Trichophyceae ( Lachnospiraceae Akkermansiaceae ( Akkermansiaceae The abundance of beneficial bacteria such as Staphylococcus ( ) and the reduction of Staphylococcus ( Staphylococcaceae The abundance of pathogenic bacteria such as α and β can effectively correct AD-induced intestinal flora imbalance.

[0024] In summary, the Lactobacillus equina-KM15 provided by this invention exerts its preventive and adjunctive effects on Alzheimer's disease through the gut-brain axis pathway, improving cognitive behavior, anti-oxidation, anti-inflammation, repairing the intestinal barrier, and regulating gut microbiota. It has the advantages of high safety, clear efficacy, and clear mechanism, and can be used to prepare drug formulations for the prevention or adjunctive improvement of Alzheimer's disease. Attached Figure Description

[0025] Figure 1 This is a curve showing the change in mouse body weight; Figure 2 The percentage change in mouse body weight; Figure 3 This represents the change in body weight of mice at week 8. Figure 4 The activity trajectory of mice in an open field experiment; Figure 5 This represents the total distance traveled by the mouse in the open field experiment. Figure 6 The average velocity of mouse activity in an open field experiment; Figure 7 The time that mice spend in the central region during open field experiments; Figure 8 The activity trajectory of mice in the elevated cross maze experiment; Figure 9 The time spent exploring the elevated cruciate maze in mice within the open arm; Figure 10 The percentage of time spent exploring the open arm in the elevated cruciate maze experiment in mice; Figure 11 The activity trajectory of mice in the new object recognition experiment; Figure 12 The time the mouse spends with the new object; Figure 13 Recognition index for novel object recognition experiments in mice; Figure 14 The trajectory of the mouse in the Y-maze experiment; Figure 15 The spontaneous alternation rate in the mouse Y-maze experiment; Figure 16 The pain time was measured for mice by tail flicking. Figure 17 The level of total antioxidant capacity in mouse serum; Figure 18 The level of malondialdehyde in mouse serum; Figure 19 The level of superoxide dismutase activity in mouse serum; Figure 20 The level of catalase activity in mouse serum; Figure 21 The level of glutathione peroxidase activity in mouse serum; Figure 22 The level of IL-10 in mouse serum; Figure 23 The level of IL-6 in mouse serum; Figure 24 This refers to the level of IL-1β in mouse serum; Figure 25 The level of TNF-α in mouse serum; Figure 26 H&E staining of mouse colon tissue; Figure 27 Venn plot showing the distribution of OTUs in the gut microbiota of mice in each group; Figure 28 The Shannon index of mouse gut microbiota; Figure 29 The Simpson index of mouse gut microbiota; Figure 30 The Chao1 index of mouse gut bacteria; Figure 31 ACE index of mouse gut bacteria; Figure 32 PCoA analysis of β-diversity in mouse gut microbiota; Figure 33 Species composition analysis at the phylum level of mouse gut microbiota; Figure 34 The ratio of Firmicutes to Bacteroidetes in the mouse gut; Figure 35 Analysis of species composition at the family level in the mouse gut microbiota; Figure 36 Species composition analysis at the genus level of mouse gut microbiota; Figure 37 Microscopic image of Lactobacillus hummiflorum KM15; The Lactobacillus malariae provided by this invention ( Lactobacillus kefiranofaciens KM15 was deposited on April 14, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.38244. Detailed Implementation

[0026] The present application will be further described in detail below with reference to the embodiments. The preparation method of *Lactobacillus equina* KM15, the formulation of the microbial preparation, the construction of animal experimental models, and the evaluation of effects of the present invention can all be implemented with reference to the following embodiments. However, it should be understood that the following embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention. All equivalent substitutions, simple modifications, or conventional optimizations based on the technical concept of the present invention are within the scope of protection of the present invention. Those skilled in the art can make several adjustments and improvements to the raw materials, strain culture conditions, animal model parameters, dosage, and combination forms used without departing from the principle of the present invention, and these adjustments and improvements should also be considered within the scope of protection of the present invention.

[0027] Example 1 Preparation of Lactobacillus hummilaceus KM15 bacterial suspension Lactobacillus hummiflorum KM15 was isolated from kefir grains collected in Gaolan County, Lanzhou City, Gansu Province in June 2025. The kefir grain samples were inoculated into raw milk and anaerobically cultured at 37℃ for 72 hours. One mL of the fermented yogurt was then serially diluted 10⁻⁶ times. 4 ~10 6 After dilution, the culture medium was spread onto modified MRS solid medium and incubated anaerobically at 37°C for 72 hours. White, moist, and round colonies were selected by colony morphology, purified three times using the three-zone streak method, and finally obtained as Lactobacillus maltophilus KM15 by Gram staining and microscopic observation.

[0028] The modified MRS medium consisted of: 20.0 g / L glucose, 45.0 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 0.2 g / L manganese sulfate, 0.15 g / L sodium chloride, 15 g / L sodium acetate, and 1.0 g / L Tween 80, with the pH adjusted to 6.2.

[0029] Preparation of modified MRS solid medium: Before sterilization, add 20 g / L of agar powder to the above modified MRS medium, heat to completely dissolve the agar powder, and then autoclave at 115℃ for 20 min to obtain modified MRS solid medium.

[0030] Lactobacillus equina-like bacteria KM15 was inoculated into modified MRS solid medium using an inoculation loop and cultured at 37°C under anaerobic conditions for 72 h to activate the bacteria. After Gram staining and microscopic observation, a single colony was picked and inoculated into 3 mL of modified MRS liquid medium and cultured statically at 37°C under anaerobic conditions for 48 h to prepare a seed culture.

[0031] Before subculturing, the Lactobacillus malathiformes KM15 in the seed culture needs to be Gram-stained and examined under a microscope. The microscopic image is shown below. Figure 37As shown, it was identified as purified Lactobacillus maltophilus by morphological analysis. Subsequently, the seed culture was inoculated into 5 mL of modified MRS liquid medium at an inoculum volume of 3%, and activated by static incubation at 37°C under anaerobic conditions for 48 h, thus obtaining Lactobacillus maltophilus KM15 bacterial culture.

[0032] Example 2 Establishment and grouping intervention of AD mouse model Six-month-old SPF-grade male C57BL / 6 mice, weighing 20±10.0 g, were used in the experiment. The Alzheimer's disease model was established by intraperitoneal injection of D-galactose (120 mg / kg, 0.2 mL) combined with gavage administration of aluminum chloride (20 mg / kg, 0.2 mL). Mice were randomly divided into a blank control group, a model group, a positive drug group, and a KM15 treatment group, with 10 mice in each group. From week 2, the blank control group received daily gavage administration of the same volume of physiological saline as the injection; the model group, positive drug group, and KM15 treatment group continuously received D-galactose and AlCl3 combined for modeling. Simultaneously, in addition to continuous modeling, the positive drug group received daily gavage administration of 0.2 mL (1 mg / kg) donepezil hydrochloride, and the KM15 treatment group received daily gavage administration of 0.2 mL (1×10⁻⁶) donepezil hydrochloride (1 mg / kg). 8 CFU / kg) of Lactobacillus equina KM15 bacterial suspension was administered, with the intervention lasting for 8 weeks.

[0033] Example 3 Effects of Lactobacillus flavus on body weight and organ index in AD mice During the experiment, mouse weight was measured and recorded at fixed times each week to observe their general growth. After the intervention ended and the mice were euthanized, major organs such as the heart, liver, spleen, lungs, and kidneys were quickly separated. Surface-attached fat and connective tissue were removed, and the organs were rinsed thoroughly with pre-cooled 0.9% physiological saline. After blotting with filter paper, the weight of each organ was accurately measured using an electronic analytical balance. The organ index was calculated as the ratio of organ weight to the mouse's final body weight. Organ index (mg / g) = organ mass (mg) / mouse final body mass (g).

[0034] Sample collection and processing: After all behavioral experiments were completed, mice were fasted for 12 hours but allowed free access to water, and then anesthetized by intraperitoneal injection of 5% tribromoethanol hydrate. Whole blood was collected by ocular sampling, allowed to stand at room temperature for 20 minutes, and then centrifuged at 4°C and 3500 rpm for 15 minutes. The supernatant serum was separated, aliquoted, and stored at -80°C for subsequent biochemical analysis. Following blood collection, the mice were rapidly dissected, and the whole brain was isolated and rinsed thoroughly with pre-cooled 0.9% saline. The brain was symmetrically divided into left and right halves along the sagittal line: the left brain tissue was fixed in 10% fixative for HE staining; the right brain tissue was rapidly separated from the hippocampus and cerebral cortex on ice, aliquoted, and quickly immersed in liquid nitrogen, then transferred to a -80°C cryogenic freezer for subsequent gene and protein level analysis. Simultaneously, colon tissue was isolated, and some colon samples were fixed in 10% fixative for intestinal histopathological observation. Cecal contents and periodically collected fecal samples were collected, rapidly flash-frozen in liquid nitrogen, and stored at -80°C for gut microbiota analysis. In addition, organs such as the heart, liver, spleen, lungs, and kidneys are separated, fatty tissue is removed, and the organs are weighed and recorded after being dried with filter paper for the purpose of calculating organ indices.

[0035] During the intervention with *Lactobacillus hummi*, the mice exhibited normal morphology, diet, and water intake, with no abnormal reactions or deaths. For example... Figure 1 , Figure 2 and Figure 3 As shown, over time, the control group exhibited natural weight gain, while the model group experienced a slow decline in body weight. The positive control group showed a significant rebound in weight gain in the later stages, indicating that the intervention had a delayed effect. The Lactobacillus hummibens KM15 group showed the smallest change throughout the period, indicating that this strain had no significant toxic side effects on mouse body weight. Statistical analysis results showed that during the experiment, except for the model group, the body weight of mice in other groups steadily increased, and at week eight, the body weight of the model group was significantly lower than that of the control group.

[0036] The organ index refers to the ratio of the weight of an animal's internal organs to its total weight. Under normal physiological conditions, the ratio of the mass of each organ to its body weight remains relatively constant. When the body is in a pathological state, this ratio usually changes. The organ index reflects the functional state of an animal's internal organs and is a comprehensive representation of the body's nutritional metabolism and health status. As shown in Table 1, there were no significant differences in the organ indices of the heart, liver, spleen, lungs, and kidneys between the control group and the model group mice, indicating that the organ indices of the model group mice did not develop pathological changes after modeling. Compared with the control group or the model group, the intervention of *Lactobacillus hummi* KM15 had no significant effect on the important tissues and organs (heart, liver, spleen, lungs, and kidneys) of AD mice, preliminarily indicating that gavage administration of *Lactobacillus hummi* KM15 had no significant toxic side effects on the normal growth of mice in each group.

[0037] Table 1. Changes in organ indices of mice in each group.

[0038] Example 4 Effects of Lactobacillus flavus on cognitive impairment in AD mice 1. Effects of Lactobacillus equinae on anxiety-like behavior in AD mice - open field test Open field tests (OFTs), also known as spontaneous activity tests, are widely used to assess the voluntary movement abilities of laboratory animals and reflect their anxiety-like behavioral characteristics. Examples include trajectory tracking. Figure 4 The results showed that the activity trajectories of the control group mice were intricate and interwoven throughout the various areas of the open field box, demonstrating their strong desire to explore and their good mental state. Compared with the control group, the model group mice tended to move along the edge of the open field box, with most of their movement trajectories overlapping and no activity traces appearing in the central area of ​​the open field box, demonstrating their weaker desire to learn about unfamiliar environments and their anxiety in a confined environment. Compared with the model group, after intervention with positive drugs and Lactobacillus equina KM15, it could be observed that the mice's activity trajectories did not completely circle around the box, but showed traces of moving towards the central area, demonstrating their curiosity and exploration of the new environment.

[0039] like Figure 5 As shown, compared with the blank group, the total distance traveled by mice in the model group was significantly reduced; compared with the model group, the total distance traveled by mice in the KM15 treatment group was significantly increased.

[0040] like Figure 6 As shown, compared with the blank group, the average speed of the mice in the model group was significantly reduced; compared with the model group, the average speed of the KM15 treatment group was significantly increased.

[0041] like Figure 7 As shown, compared with the blank group, the model group mice spent significantly less time in the central region; compared with the model group, the KM15 treatment group spent significantly more time in the central region.

[0042] Studies have shown that in spontaneous activity tests, lower average speed, shorter total distance traveled, and less time spent in the central area indicate more pronounced anxiety-like behavior in mice. Therefore, *Lactobacillus equinasum* KM15 can, to some extent, improve D-galactose / AlCl3-induced anxiety-like behavior in AD mice.

[0043] 2. Elevated cross maze - assessing anxiety levels and spatial exploration behavior in mice. The Elevated Plus Maze (EPM) is a behavioral paradigm based on the conflict between innate fear and exploratory drive in rodents. It is a classic, efficient, and well-validated experimental model for assessing anxiety-like behaviors.

[0044] like Figure 8 , Figure 9 and Figure 10 As shown, compared with the blank group, the model group mice had significantly less time spent exploring the open arm and a significantly lower percentage of time spent exploring the open arm; compared with the model group, the KM15 treatment group had significantly more time spent exploring the open arm and a significantly higher percentage of time spent exploring the open arm.

[0045] Studies have shown that in spontaneous exploration tests, the model group exhibited significantly reduced time spent exploring the open arm and a significantly reduced proportion of time spent exploring the open arm, indicating more pronounced anxiety-like behavior and lower spatial exploration ability in the mice. Therefore, *Lactobacillus equinasum* KM15 can, to some extent, improve D-galactose / AlCl3-induced AD mice's anxiety-like behavior and spatial exploration ability.

[0046] 3. Novel object recognition - assessing the learning and memory abilities of mice Novel object recognition is a non-operational cognitive behavioral paradigm based on the spontaneous exploratory behavior of rodents. It is specifically designed to assess non-spatial declarative memory, especially its core component—recognition memory. Its core theoretical basis lies in the natural tendency of rodents to spontaneously explore novel stimuli, i.e., "novelty preference."

[0047] like Figure 11 , Figure 12 and Figure 13 As shown, compared with the control group, the model group mice spent significantly less time on new objects; compared with the model group, the KM15 treatment group spent significantly more time on new objects. The model group mice had a low Recognition Index (RI), indicating poor memory and no difference in exploration of new and old objects. This study shows that *Lactobacillus hummilaceus* KM15 can significantly improve the learning and memory abilities of D-galactose / AlCl3-induced AD mice. 4. Y-maze - Assessing spatial learning and memory abilities in mice The Y-maze spontaneous alternation behavior test is a behavioral experiment based on the endogenous exploratory drive of rodents, primarily used to assess their spatial working memory and prefrontal-hippocampal dependent executive function. This experiment does not rely on exogenous rewards or punishments; it reflects short-term spatial memory updates and behavioral flexibility by quantifying the animal's tendency to avoid repeatedly entering the same arm in successive choices. The method involves sequentially recording the order in which mice enter each arm and calculating the spontaneous alternation rate.

[0048] In healthy mice, the spontaneous alternation rate is approximately 60-80%, such as Figure 14 and Figure 15The model group had a lower spontaneous alternation rate than the control group, indicating that the model group mice had impaired spatial working memory. After intervention with Lactobacillus equina KM15, the spontaneous alternation rate of the mice was close to that of the control group, reflecting that the mice had good spatial working memory ability.

[0049] 5. Tail-flick pain test The basic principle of the mouse tail-flick pain tester is to shine a beam of light on the mouse tail to generate a heat-collecting effect, causing the local temperature of the mouse tail to rise and produce pain. When the pain exceeds the animal's pain threshold, the animal will flick its tail to escape. This method is used to determine the level and changes of the animal's pain threshold.

[0050] like Figure 16 As shown, there were no significant differences in pain perception among the blank group, model group, positive drug group, and Lactobacillus equina KM15 group in AD mice, indicating that the different behavioral differences among the groups of mice were not due to pain.

[0051] Example 5 Effects of Lactobacillus hummilaceus on oxidative stress in AD mice Oxidative stress is the cell damage and dysfunction caused by the production of reactive oxygen species (ROS) and free radicals in the body exceeding the capacity of the antioxidant defense system. As the largest digestive organ, the intestines inevitably come into contact with foreign substances and reactive oxygen species.

[0052] This study measured serum oxidative stress indicators in mice of each group, and the results are as follows: Figure 17 and Figure 18 As shown in the figure, compared with the control group, the total antioxidant capacity (T-AOC) activity in the serum of the model group mice was significantly reduced, and the malondialdehyde (MDA) content was significantly increased, indicating that AD mice experienced oxidative stress. Compared with the model group, the MDA content in the serum of the KM15-treated group mice was significantly reduced, and the T-AOC activity was significantly increased, indicating that both exerted antioxidant effects in mice.

[0053] like Figure 19 , Figure 20 and Figure 21 As shown, compared with the control group, the serum activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) in the model group mice were significantly reduced, indicating oxidative damage in AD mice. Compared with the model group, the serum activities of SOD, CAT, and GSH-PX in the Lactobacillus equina KM15 group mice were significantly increased, indicating that all of them exert antioxidant effects in mice.

[0054] Example 6 Effects of Lactobacillus hummilaceus on serum inflammation in AD mice IL-1β, TNF-α, IL-6, and IL-10 are key inflammatory factors, and their levels are closely related to the inflammatory response. By detecting these indicators, a comprehensive understanding of the inflammatory status in an AD mouse model can be obtained.

[0055] like Figure 22 Compared with the control group, the serum IL-10 level in the model group mice was significantly reduced, indicating that the intestinal barrier was damaged and an inflammatory response occurred, thus successfully establishing the AD model. Compared with the model group, the KM15 treatment group increased the expression of the anti-inflammatory factor IL-10.

[0056] like Figure 23 , Figure 24 and Figure 25 As shown, compared with the blank group, the serum levels of IL-6, IL-1β, and TNF-α in the model group mice were significantly increased. Compared with the model group, the serum levels of IL-6, IL-1β, and TNF-α in the KM15 treatment group mice were significantly decreased, indicating that it has a certain anti-inflammatory effect, can reduce the release of pro-inflammatory factors, and promote the expression of anti-inflammatory factors, thereby inhibiting the intestinal inflammatory response.

[0057] Example 7 Effects of Lactobacillus hummilaceus on small intestinal tissue damage in AD mice Colon histopathological section results as follows Figure 26 As shown in the figure, the colonic tissue of mice in the blank control group had intact mucosa, submucosa, muscularis, and serosa, with clear crypt structures. The cells and glands in the upper colonic mucosa were neatly arranged, containing a large number of goblet cells, and there was no inflammatory cell infiltration in the submucosa. In the model group, the mucosal structure was severely damaged, with disordered shedding of upper mucosal cells, destroyed crypt structures, a significantly reduced number of goblet cells, and a large number of inflammatory cells infiltrating the submucosa. In the positive drug group and the KM15 treatment group, the colonic structure was intact, with complete crypt structures, clear mucosal structures, increased goblet cells, and a significant reduction in inflammatory cells. This indicates that the intervention of *Lactobacillus equinatum* KM15 improved colonic tissue damage in AD mice to some extent, maintained the colonic mucosal structure, and strengthened the intestinal mucosal barrier.

[0058] Example 8 The gut microbiota is an interactive micro-ecosystem, and its richness and diversity play an important role in maintaining gut homeostasis and health.

[0059] An operational taxonomic unit (OTU) is a concept in ecology and microbiology used to classify and describe microbial communities. Specifically, an OTU is a taxonomic unit defined based on gene sequence similarity, typically used to describe microbial groups with similarity exceeding a certain threshold. Its main function is to group microorganisms according to similar characteristics, thereby facilitating their study and comparison.

[0060] like Figure 27 The Venn diagram showed that the number of common OTUs in the four groups was 714, while the number of OTUs specific to the control group was 1530. The model group exhibited significantly reduced gut microbiota diversity, with only 616 specific OTUs remaining. In contrast, the KM15 treatment group had 932 OTUs. This indicates that modeling disrupts gut microbiota diversity in mice, and that *Lactobacillus hummiflorum* can positively regulate gut microbiota homeostasis.

[0061] Example 9 Alpha diversity analysis Alpha diversity of gut microbiota refers to the degree of diversity of microbial species within a sample. High alpha diversity in the gut microbiota generally indicates a healthy and functionally diverse gut microbiota, while low alpha diversity may be associated with some diseases or health problems. Commonly used indicators include the Chao1 index, ACE index, Shannon index, and Simpson index; the Chao1 and ACE indices measure species richness, while the Shannon and Simpson indices measure species diversity.

[0062] Depend on Figure 28 , Figure 29 , Figure 30 and Figure 31 It can be seen that, compared with the blank group, the gut microbiota diversity index (Shannon, Simpson) and richness index (Chao1, ACE) of the model group mice were significantly decreased; this indicates that the original gut microbiota structure was destroyed, resulting in a decrease in richness and diversity; while after intervention with Lactobacillus equina KM15, all indices were significantly improved.

[0063] Example 10 Beta diversity analysis Beta diversity refers to the difference in microbial community composition between different samples and is an important indicator for assessing differences between sample groups. Principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) are commonly used to study the beta diversity of microbial communities in different samples. The closer two samples are, the more similar their composition is. Unlike PCoA analysis, NMDS reduces the dependence on actual distance values ​​and places more emphasis on the ranking of values, thus downplaying the absolute degree of difference between values.

[0064] The results are as follows Figure 32 As shown, there were significant differences in the gut microbiota structure between the control group and the model group, with the two groups exhibiting complete separation in their microbiota distribution. This indicates that the gut microbiota composition changed after modeling. The gut microbiota distribution in the KM15-treated group partially overlapped with that in the control group. This demonstrates that *Lactobacillus hummiflorum* KM15 can resist gut microbiota dysbiosis.

[0065] Example 11 Species composition analysis – phylum, family, and genus levels At the phylum level, the dominant phylum in each group was Bacteroidetes (…). Bacteroidetes Firmicutes ( Firmicutes Proteobacteria ( Proteobacteria ) and Actinobacteria ( Actinobacteria After induction, the model group mice... Firmicutes Relative abundance decreased Bacteroidota The abundance increased, and after intervention with Lactobacillus hummilaceus KM15, Firmicutes The relative abundance increased. Bacteroidota Abundance decreased.

[0066] Studies have shown that a healthy gut microbiota typically has a balanced Firmicutes to Bacteroidetes ratio (F / B), such as Figure 33 and Figure 34 As shown, the F / B ratio significantly increased after D-galactose / AlCl3 induction, indicating gut microbiota dysbiosis in mice. However, the F / B ratio was significantly regulated to normal by Lactobacillus hummibens KM15.

[0067] like Figure 35 As shown, at the family level, compared with the control group, the model group mice had a significantly increased number of Staphylococcus aureus (Staphylococcus family). Staphylococcaceae The abundance of Lactobacillus ( ) decreased, reducing the abundance of Lactobacillus family ( Lactobacillaceae ), family Trichophyceae ( Lachnospiraceae Akkermansiaceae ( Akkermansiaceae The abundance of Staphylococcus species was significantly reduced in the KM15 treatment group compared to the model group, while the abundance of Lactobacillus species was increased. Lactobacillaceae ), family Trichophyceae ( Lachnospiraceae Akkermansiaceae ( AkkermansiaceaeThe abundance of ) indicates an increase in harmful bacteria and a decrease in beneficial bacteria in the mouse gut microbiota. Studies have shown that Staphylococcus families can exacerbate the condition by disrupting the balance of gut microbiota and inducing immune responses. For example, Staphylococcus aureus can produce toxins and pro-inflammatory factors, exacerbating intestinal inflammation and increasing the risk of bacterial translocation by disrupting the intestinal barrier function; Lactobacillus families can participate in the breakdown of complex carbohydrates to produce short-chain fatty acids, playing an important role in maintaining the diversity and stability of the gut microbiome; in addition, Trichophyton families ( Lachnospiraceae Belonging to the Firmicutes phylum, these bacteria are typically found in the gut of healthy organisms, cooperating in the metabolism of nutrients and contributing to the stability of the gut microbiota. This indicates that AD mice exhibit an increase in harmful bacteria and a decrease in beneficial bacteria in their gut microbiota. Under the protective effect of *Lactobacillus hummilaceus* KM15, the colonization of pathogenic bacteria can be reduced, while the abundance of beneficial bacteria that produce short-chain fatty acids can be increased.

[0068] like Figure 36 As shown, at the genus level, the gut microbiota composition of each group of mice was mainly composed of Staphylococcus aureus (Staphylococcus family). Staphylococcaceae Lactobacillus family ( Lactobacillaceae ), family Trichophyceae ( Lachnospiraceae Akkermansiaceae ( Akkermansiaceae ), Duborella ( Dubosiella Bacteroides ( Bacteroides The dominant bacteria were *Staphylococcus*. Compared with the control group, AD mice showed an increase in the relative abundance of *Lactobacillus*, *Ackermania*, *Trichophyton*, *Duborella*, and *Bacteroides*. Compared with the model group, *Lactobacillus hummi* KM15 increased the relative abundance of *Lactobacillus*, *Ackermania*, *Trichophyton*, *Duborella*, and *Bacteroides*, and decreased the relative abundance of *Staphylococcus*. This indicates that the protective effect of *Lactobacillus hummi* KM15 can reduce the colonization of pathogenic bacteria and increase the abundance of beneficial bacteria such as short-chain fatty acids.

[0069] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A strain of Lactobacillus malathiformis ( Lactobacillus kefiranofaciens KM15, characterized in that, The Lactobacillus equina-like bacteria KM15 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 14, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.38244.

2. A microbial preparation, characterized in that, It contains Lactobacillus equinae KM15 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation is either a solid or liquid preparation.

4. The microbial preparation according to claim 2 or 3, characterized in that, The microbial preparation also contains pharmaceutically acceptable excipients.

5. The use of the Lactobacillus equinae KM15 of claim 1 or the microbial preparation of any one of claims 2-4 in the preparation of a medicament for the prevention and / or adjuvant improvement of Alzheimer's disease.

6. The application according to claim 5, characterized in that, The drug is used to improve cognitive impairment, anxiety-like behavior, oxidative stress, or gut microbiota dysbiosis associated with Alzheimer's disease.

7. A composition for the prevention and / or adjunctive improvement of Alzheimer's disease, characterized in that, The composition is a pharmaceutical composition comprising Lactobacillus equinasum KM15 as described in claim 1 and a pharmaceutically acceptable carrier.

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