A fermented Lactobacillus AK223-12 and its application in preparing a product for treating amyotrophic lateral sclerosis

Abstract

The present invention belongs to the field of probiotic technology, and specifically relates to a fermented Lactobacillus AK223-12 and its application in the preparation of a product for treating amyotrophic lateral sclerosis. The strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 8, 2024, with the deposit number GDMCC No: 64839. The fermented Lactobacillus ( Limosilactobacillus leaven AK223‑12 is a probiotic strain approved for use by the National Health Commission and has shown significant improvement in the treatment of ALS. Daily administration of 45 billion cells of this strain and 300 mg of its metabolites can sustainably provide essential endogenous amounts of quercetin, vitamin B6, vitamin D, and potassium during intestinal growth and reproduction. This has broad potential for preventing, alleviating, and treating ALS and maintaining a healthy nervous system.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and particularly relates to a Lactobacillus fermentum AK223-12 and an application thereof in preparing a product for treating amyotrophic lateral sclerosis. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] ALS, also known as amyotrophic lateral sclerosis (ALS), is a neurological disease of unknown etiology involving motor neurons, often accompanied by degeneration of related neurons. This condition can lead to progressive paralysis, decreased speech, swallowing, and motor function, and ultimately respiratory insufficiency and death. Nowadays, the intestinal flora is inseparable from human health. When the intestinal flora undergoes pathological changes, its metabolites also change, causing corresponding changes in the normal physiological functions of the human body through immune and circulatory mechanisms, thereby affecting the development of the disease. Conversely, the disease can also affect the intestines, leading to changes in the intestinal microbiome.

[0004] The inventors have found in the prior art (Research Progress on Amyotrophic Lateral Sclerosis and Gut Microbiota, Gong Zhenxiang et al.; Potential roles of gut microbiome & metabolites in modulation of murine ALS, Nature, 2019, 572, 474-480, etc.) that the relationship between amyotrophic lateral sclerosis and gut microbiota is inseparable, as described below:

[0005] 1. Intestinal barrier damage caused by intestinal flora imbalance in patients with amyotrophic lateral sclerosis

[0006] Gut microbiota and their metabolites, such as short-chain fatty acids, help protect the integrity of the intestinal epithelial barrier. Studies have shown that pathological changes in the composition of the intestinal microbiota can lead to intestinal epithelial barrier disruption and increased mucosal permeability. In addition to inducing intestinal motility dysfunction, this disruption of barrier integrity can also lead to gut-brain axis dysregulation via the ascending tract of the gut-brain pathway, triggering central nervous system neuroinflammation and neurodegenerative diseases such as amyotrophic lateral sclerosis, multiple sclerosis, and Parkinson's disease.

[0007] Gut Microbiota and Genetics in Patients with ALS

[0008] Interactions between the host genome and the gut microbiota influence normal signaling along the gut-brain axis. Genetic variation can modulate the gene-editing system of the native gut microbiota, affecting the composition of enterobacteria. Genetic variation activates bacteriophages that naturally coexist with the gut microbial host. Phages can cause immune disturbances by recognizing foreign genetic elements and destroying their DNA fragments. Chronic neuroinflammation caused by immune disturbances triggers the accumulation of misfolded proteins in nervous system cells and peripheral nerves, further leading to neuronal death. Disruption of glial fibrillary acidic protein in the enteric nervous system suggests that the gut microbiota influences intestinal neuromuscular structure and function, and an imbalance in the gut microbiota may be a potential trigger for degenerative diseases. Furthermore, γ-glutamylamino acids may be inversely associated with the risk of developing amyotrophic lateral sclerosis, while γ-glutamylphenylalanine is a specific risk factor for the disease. Two of its metabolites, 1-arachidonoyl-gpi and 3-methyl-2-oxobutyric acid, have been found to increase the risk of ALS, while increased levels of 4-acetylaminobutyric acid may reduce the risk of ALS.

[0009] 3. Gut flora dysbiosis and autophagy in patients with amyotrophic lateral sclerosis

[0010] Metabolites from the gut microbiota regulate intestinal inflammation through the autophagy pathway. Furthermore, autophagy, in turn, degrades invading pathogens, regulates pathogens, triggers the release of proinflammatory cytokines, and participates in antigen presentation and lymphocyte development. Dysregulated autophagy, mediated by dysbiosis, compromises intestinal barrier integrity and increases intestinal permeability, thereby transferring gut microbes, their metabolites, and microbe-associated molecular patterns to mesenteric lymphoid tissue. The resulting neuroinflammatory response contributes to the development and progression of neurological diseases.

[0011] 4. Gut flora dysbiosis and immunity in patients with amyotrophic lateral sclerosis

[0012] The gut microbiota and the intestinal immune system interact through the gut-brain axis to maintain immune tolerance and shape immune responses during inflammation. The maturation and function of central nervous system macrophages and microglia are controlled by the gut microbiota, and changes in gut bacteria indirectly affect factors such as the macrophage-to-microglia ratio.

[0013] Studies have shown that the composition and products of the intestinal flora have a strong influence on the immune response, and the central nervous system is also affected by the immune system. Short-chain fatty acids, as important metabolites of the intestinal flora, can regulate the immune response and serve as important communication signals for the brain-gut axis. By using probiotics, the number of microorganisms that produce short-chain fatty acids in the intestine can be increased. In addition to the potential to restore microbial homeostasis, probiotics can also be considered as carriers of neuroactive compounds, which play an important role in the brain-gut axis. However, the current exploration of functional probiotics and their active metabolites for the prevention and treatment of amyotrophic lateral sclerosis is still limited, and the effective ingredients and mechanisms of action need to be elucidated. Summary of the Invention

[0014] In response to the shortcomings of the prior art, the present invention aims to provide a Lactobacillus fermentum AK223-12 strain and its use in the preparation of a product for treating ALS. This strain, isolated from rose cakes, was identified as Lactobacillus fermentum by 16S rRNA sequencing (16S ribosomal RNA gene sequencing). It has the potential to regulate intestinal flora balance, promote digestion and absorption, and effectively treat ALS.

[0015] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0016] The first aspect of the present invention provides a strain of Lactobacillus fermentum (Limosilactobacillus fermentum) AK223-12, which was deposited in the Guangdong Provincial Microbial Culture Collection Center (address: 5th Floor, Laboratory Building, Guangdong Provincial Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou) on July 8, 2024, with a deposit number of GDMCC No: 64839.

[0017] The second aspect of the present invention provides a method for culturing the above-mentioned Lactobacillus fermentum AK223-12, which comprises: expansion culture, primary seed culture, seed tank culture and fermentation culture.

[0018] Preferably, the culture medium for the expanded culture contains, per 1000 mL of water, 15-25 g of trehalose, 5-7 g of bovine colostrum powder, 5-7 g of beef extract powder, 0.5-0.8 g of sodium acetate, 0.3-0.7 mL of Tween-80, and 1.0-1.8 g of potassium dihydrogen phosphate. The pH value of the culture medium for the expanded culture is adjusted to 7.0 (alkaline solution adjustment). Preferably, the culture conditions for the expanded culture are: culturing at 36-38° C. for 10-20 hours.

[0019] Further preferably, the culture medium for the expanded culture contains, per 1000 mL of water, 20 g of trehalose, 6 g of bovine colostrum powder, 6 g of beef extract powder, 0.7 g of sodium acetate, 0.5 mL of Tween-80, and 1.5 g of potassium dihydrogen phosphate. The pH value of the culture medium for the expanded culture is adjusted to 7.0 (alkaline solution adjustment). Preferably, the culture conditions for the expanded culture are: culturing at 37° C. for 16 hours.

[0020] Preferably, the culture medium for the primary seed culture contains, per 1000 mL of water, 8-12 g of trehalose, 5-7 g of peptone, 3-5 g of beef extract powder, 1.5-2.0 g of metasilicic acid, 0.5-1.2 g of sodium acetate, and 0.5-1.2 mL of Tween-80. The pH value of the culture medium for the primary seed culture is natural. Preferably, the culture conditions for the primary seed culture are: culture at 36-38° C. for 20-30 hours; the inoculum size of the primary seed culture is 0.5%-0.6% by volume.

[0021] Further preferably, the culture medium for the primary seed culture contains the following: 10g trehalose, 6g peptone, 4g beef extract powder, 1.8g metasilicic acid, 1g sodium acetate, and 1mL Tween-80 per 1000mL of water, and the pH value of the culture medium for the primary seed culture is natural. Preferably, the culture conditions for the primary seed culture are: culture at 37°C for 24h; the inoculum size of the primary seed culture is 0.5% by volume.

[0022] Preferably, the culture medium for the seed tank culture comprises, by weight percentage, 2-5% trehalose, 0.5-1.2% casein peptone, 0.1-0.3% dipotassium hydrogen phosphate, 1-5% corn peptide, 0.1-0.5% sodium acetate, 0.05-0.15% Tween-80, 0.05-0.15% polyether defoamer, and 90-92% tap water. The pH value of the culture medium for the seed tank culture is natural. Preferably, the culture conditions for the seed tank culture are: a tank pressure of 0.04-0.06 MPa, culture at 36-38°C for 10-20 hours; the inoculum size for the seed tank culture is 1%-3%, by volume.

[0023] Further preferably, the culture medium for the seed tank culture comprises, by weight percentage, 4% trehalose, 1% casein peptone, 0.2% dipotassium hydrogen phosphate, 3% corn peptide, 0.3% sodium acetate, 0.1% Tween-80, 0.1% polyether defoamer, and 91.3% tap water, and the pH value of the culture medium for the seed tank culture is natural. Further preferably, the culture conditions for the seed tank culture are: a tank pressure of 0.05 MPa, culturing at 37°C for 15 hours; and the inoculum size for the seed tank culture is 2% by volume.

[0024] Preferably, the fermentation culture medium comprises, by weight percentage, 2-3% trehalose, 0.5-1.2% casein peptone, 0.8-1.2% L-arabinose, 1-1.8% isomaltooligosaccharide, 2-5% yeast extract, 0.8-2.0% sodium acetate, 0.1-0.3% Tween-80, 0.5-1.0% arginine, 0.5-1.5% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 85-88% tap water. The pH value of the fermentation culture medium is adjusted to 6.5. Preferably, the fermentation culture conditions are: a tank pressure of 0.04-0.06 MPa, culturing at 36-38°C for 25-40 hours; the inoculum size of the fermentation culture is 4%-6%, by volume.

[0025] Further preferably, the fermentation culture medium comprises, by weight percentage, 2.5% trehalose, 1% casein peptone, 1% L-arabinose, 1.5% isomaltooligosaccharide, 3% yeast extract, 1.5% sodium acetate, 0.2% Tween-80, 0.8% arginine, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 87.4% tap water. The pH of the fermentation culture medium is adjusted to 6.5. Further preferably, the fermentation culture conditions are: a tank pressure of 0.05 MPa, culturing at 37°C for 36 hours, and an inoculum size of 5% by volume.

[0026] The third aspect of the present invention provides a microbial agent comprising an active ingredient, wherein the active ingredient comprises the Lactobacillus fermentum or its fermentation liquid according to the first aspect.

[0027] Preferably, the microbial agent comprises inactivated Lactobacillus fermentum and its fermentation liquid.

[0028] A fourth aspect of the present invention provides use of the aforementioned Lactobacillus fermentum AK223-12 or the aforementioned microbial agent in preparing food.

[0029] A fifth aspect of the present invention provides use of the aforementioned Lactobacillus fermentum AK223-12 or the aforementioned microbial agent in the preparation of a health food that helps regulate intestinal flora or aids digestion.

[0030] A sixth aspect of the present invention provides the use of the above-mentioned Lactobacillus fermentum AK223-12 or the above-mentioned microbial agent in the preparation of a drug for treating ALS.

[0031] Preferably, the product is in the form of freeze-dried bacterial powder.

[0032] The seventh aspect of the present invention provides a method for preparing freeze-dried bacterial powder for treating ALS, comprising: inactivating and centrifuging the fermentation bacterial liquid obtained after fermentation and cultivation in the second aspect to obtain inactivated wet bacterial mud metabolites, and freeze-drying the inactivated wet bacterial mud metabolites, and then mixing them with auxiliary materials to obtain a suspension, and freeze-drying the suspension to obtain the product.

[0033] Preferably, the inactivation refers to maintaining the fermentation broth at 100-140° C. for 5-8 seconds for UHT inactivation treatment; further preferably, the inactivation refers to maintaining the fermentation broth at 120° C. for 6 seconds for UHT inactivation treatment.

[0034] Preferably, the centrifugal speed is 8000 r / min to 12000 r / min, specifically, the centrifugal speed is 3 to 5 kg / min.

[0035] Preferably, the drying conditions for freeze drying are: vacuum degree 8-12 Pa, temperature 25-28° C., and drying time 8-12 h; further preferably, the drying conditions for freeze drying are: vacuum degree 10 Pa, temperature 25° C., and drying time 10 h.

[0036] Preferably, the moisture content of the freeze-dried bacterial powder is ≤4% by mass.

[0037] Preferably, the excipients include whey peptides, pullulan, arginine, and choline; further preferably, the mass addition ratio of the inactivated wet bacterial mud metabolites to the excipients is 1:2-3.5; further preferably, the mass addition ratio of the inactivated wet bacterial mud metabolites to the excipients is 1:2.95.

[0038] Beneficial technical effects of one or more of the above technical solutions:

[0039] (1) The fermented Lactobacillus AK223-12 provided by the present invention is a probiotic and a major component of the normal intestinal flora of the human body. It has the characteristics of being non-toxic, effective, safe, and green, and can play an important role in human health.

[0040] (2) Studies have shown that the fermented Lactobacillus AK223-12 provided by the present invention has a significant improvement effect on the treatment of ALS. Daily administration of 45 billion strains of the present invention and 300 mg of its metabolites can sustainably provide the necessary endogenous amounts of quercetin, vitamin B6, vitamin D, and potassium during intestinal reproduction and growth, and has broad application prospects in preventing, alleviating, and treating the occurrence of ALS and maintaining a healthy nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without any creative work.

[0042] Figure 1 The changes in the weight of mice and the trend of delaying the development of the disease in Experimental Example 1 of the present invention, wherein (a) is the weight change of mice; (b) is the morbidity test;

[0043] Figure 2 The test results are as follows: (a) the residence time, hanging residence time, and hind limb clasping degree score test of mice in Experiment 1 of the present invention, wherein (b) is the hanging time test; (c) is the hind limb clasping degree score test;

[0044] Figure 3 The SOD1-positive staining area in mouse brain tissue sections, the SOD1 level in mouse spinal cord lysate, and the OC and A11-positive prefibrillar oligomer levels in the spinal cord lysate in Experimental Example 1 of the present invention are tested, wherein (a) is the SOD1-positive staining area test in mouse brain tissue sections; (b) is the SOD1 level test in mouse spinal cord lysate, and (c) is the OC and A11-positive prefibrillar oligomer level test in the spinal cord lysate;

[0045] Figure 4 The test results are as follows: (a) the area occupied by motor neurons and the number of motor neurons in the spinal cord and brainstem of mice in Experimental Example 1 of the present invention; (b) the number of motor neurons;

[0046] Figure 5 The number of apoptotic motor neurons in the spinal cord and brainstem of mice, the number of activated microglia in the spinal cord and brainstem of mice, and the number of activated astrocytes in the spinal cord and brainstem of mice in Experimental Example 1 of the present invention are tested, wherein (a) is a test for the number of apoptotic motor neurons; (b) is a test for activated microglia; and (c) is a test for activated astrocytes.

[0047] Figure 6 The present invention is to measure the inflammatory factors (IL-1β, IL-6, TNF-α) in the tissue extract in Test Example 1;

[0048] Figure 7 The determination of IKKβ, p65, and p-p65 in the tissue extract in Experimental Example 1 of the present invention;

[0049] Figure 8 This is the LC-MS mass spectrum raw data information diagram obtained by measuring the main metabolite information of the present invention;

[0050] Figure 9 The effects of the control group, model group and experimental groups 1 to 5 on the MDA and SOD levels in cells induced by H2O2 in Experimental Example 2 of the present invention;

[0051] Figure 10 The cytotoxic effects of the control group, LSP group, and experimental groups 1 to 5 in Experimental Example 2 of the present invention on cells;

[0052] Figure 11 The effects of the control group, LSP group and experimental groups 1 to 5 on cell NO secretion in Experimental Example 2 of the present invention;

[0053] Figure 12 The effects of the control group, LSP group and experimental groups 1 to 5 on cell secretion of TNF-α in Experimental Example 2 of the present invention;

[0054] Figure 13 The effects of the control group, LSP group and experimental groups 1 to 5 on the secretion of IL-1β, IL-6 and IL-10 by cells in Experimental Example 2 of the present invention are shown;

[0055] Figure 14 This is the effect of the control group, model group and experimental groups 1 to 5 on the rotarod residence time and cage cover hanging time in Experimental Example 2 of the present invention, where (a) is the rotarod residence time test; (b) is the cage cover hanging time test. DETAILED DESCRIPTION

[0056] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0057] It should be noted that the present invention does not impose any restrictions on the detection methods for quercetin, vitamin B6, vitamin D, and potassium content. As long as the content can be determined, those skilled in the art can select an appropriate detection method based on actual conditions. For example, quercetin content can be determined by high performance liquid chromatography; vitamin B6 content can be determined by liquid chromatography-tandem mass spectrometry; vitamin D content can be determined by liquid chromatography-tandem mass spectrometry; and potassium content can be determined by inductively coupled plasma mass spectrometry.

[0058] Equipment used in production: culture incubator, clean bench, constant temperature incubator, various test tubes, Erlenmeyer flasks, seed tanks, fermentation tanks, vacuum freeze dryer, three-dimensional blender, low-temperature crusher, vacuum packaging machine.

[0059] Considering that maintaining intestinal microbial balance is helpful for alleviating the symptoms of ALS patients, the present invention proposes a strain of Lactobacillus fermentum and its application in preparing a product for treating ALS.

[0060] Specifically, in the first typical embodiment of the present invention, a strain of Lactobacillus fermentum AK223-12 is provided, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center (address: 5th Floor, Laboratory Building, Guangdong Provincial Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou) on July 8, 2024, with a deposit number of GDMCC No: 64839.

[0061] In a second typical embodiment of the present invention, a culture method of the above-mentioned Lactobacillus fermentum AK223-12 is provided, which comprises: expansion culture, primary seed culture, seed tank culture and fermentation culture.

[0062] In some embodiments, the culture medium for the expanded culture contains, per 1000 mL of water, 15-25 g of trehalose, 5-7 g of bovine colostrum powder, 5-7 g of beef extract powder, 0.5-0.8 g of sodium acetate, 0.3-0.7 mL of Tween-80, and 1.0-1.8 g of potassium dihydrogen phosphate. The pH of the culture medium for the expanded culture is adjusted to 7.0 (alkaline solution adjustment). In some embodiments, the culture conditions for the expanded culture are: culturing at 36-38° C. for 10-20 hours.

[0063] In some embodiments, the expanded culture medium contains, per 1000 mL of water, 20 g of trehalose, 6 g of bovine colostrum powder, 6 g of beef extract powder, 0.7 g of sodium acetate, 0.5 mL of Tween-80, and 1.5 g of potassium dihydrogen phosphate. The pH of the expanded culture medium is adjusted to 7.0 (alkaline solution). In some embodiments, the expanded culture is cultured at 37° C. for 16 hours.

[0064] In some embodiments, the culture medium for the primary seed culture contains, per 1000 mL of water, 8-12 g of trehalose, 5-7 g of peptone, 3-5 g of beef extract powder, 1.5-2.0 g of metasilicic acid, 0.5-1.2 g of sodium acetate, and 0.5-1.2 mL of Tween-80. The pH value of the culture medium for the primary seed culture is natural. In some embodiments, the culture conditions for the primary seed culture are: incubation at 36-38° C. for 20-30 hours; and the inoculum size of the primary seed culture is 0.5% to 0.6% by volume.

[0065] In some embodiments, the culture medium for the primary seed culture comprises, per 1000 mL of water, 10 g of trehalose, 6 g of peptone, 4 g of beef extract powder, 1.8 g of metasilicic acid, 1 g of sodium acetate, and 1 mL of Tween-80. The pH of the culture medium for the primary seed culture is natural. In some embodiments, the culture conditions for the primary seed culture are: incubation at 37°C for 24 hours; and an inoculum size of 0.5% by volume.

[0066] In some embodiments, the culture medium for the seed tank culture comprises, by weight percentage, 2-5% trehalose, 0.5-1.2% casein peptone, 0.1-0.3% dipotassium hydrogen phosphate, 1-5% corn peptide, 0.1-0.5% sodium acetate, 0.05-0.15% Tween-80, 0.05-0.15% polyether defoamer, and 90-92% tap water. The pH value of the culture medium for the seed tank culture is natural. In some embodiments, the culture conditions for the seed tank culture are: a tank pressure of 0.04-0.06 MPa, culturing at 36-38°C for 10-20 hours; and the inoculum size for the seed tank culture is 1%-3%, by volume.

[0067] In some embodiments, the culture medium for the seed tank culture comprises, by weight percentage, 4% trehalose, 1% casein peptone, 0.2% dipotassium hydrogen phosphate, 3% corn peptide, 0.3% sodium acetate, 0.1% Tween-80, 0.1% polyether defoamer, and 91.3% tap water. The pH of the culture medium for the seed tank culture is natural. In some embodiments, the culture conditions for the seed tank culture are: a tank pressure of 0.05 MPa, culturing at 37°C for 15 hours; and the inoculum size for the seed tank culture is 2% by volume.

[0068] In some embodiments, the fermentation culture medium comprises, by weight percentage, 2-3% trehalose, 0.5-1.2% casein peptone, 0.8-1.2% L-arabinose, 1-1.8% isomaltooligosaccharide, 2-5% yeast extract, 0.8-2.0% sodium acetate, 0.1-0.3% Tween-80, 0.5-1.0% arginine, 0.5-1.5% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 85-88% tap water. The pH of the fermentation culture medium is adjusted to 6.5. In some embodiments, the fermentation culture conditions are: a tank pressure of 0.04-0.06 MPa, culturing at 36-38°C for 25-40 hours, and an inoculum size of 4%-6% by volume.

[0069] In some embodiments, the fermentation culture medium comprises, by weight percentage, 2.5% trehalose, 1% casein peptone, 1% L-arabinose, 1.5% isomaltooligosaccharide, 3% yeast extract, 1.5% sodium acetate, 0.2% Tween-80, 0.8% arginine, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 87.4% tap water. The pH of the fermentation culture medium is adjusted to 6.5. In some embodiments, the fermentation culture conditions are: a tank pressure of 0.05 MPa, culturing at 37°C for 36 hours, and an inoculum size of 5% by volume.

[0070] In a third typical embodiment of the present invention, a microbial agent is provided, comprising an active ingredient, wherein the active ingredient comprises the Lactobacillus fermentum or a fermentation broth thereof according to the first aspect.

[0071] In some embodiments, the microbial agent comprises inactivated Lactobacillus fermentum and its fermentation broth.

[0072] In a fourth typical embodiment of the present invention, there is provided use of the above-mentioned Lactobacillus fermentum AK223-12 or the above-mentioned bacterial agent in preparing food.

[0073] In a fifth typical embodiment of the present invention, there is provided a use of the above-mentioned Lactobacillus fermentum AK223-12 or the above-mentioned bacterial agent in the preparation of a health food that helps regulate intestinal flora or aids digestion.

[0074] In a sixth typical embodiment of the present invention, there is provided a use of the above-mentioned Lactobacillus fermentum AK223-12 or the above-mentioned bacterial agent in the preparation of a drug for treating ALS.

[0075] In some embodiments, the product is in the form of freeze-dried bacterial powder.

[0076] The seventh aspect of the present invention provides a method for preparing freeze-dried bacterial powder for treating ALS, comprising: inactivating and centrifuging the fermentation bacterial liquid obtained after fermentation and cultivation in the second aspect to obtain inactivated wet bacterial mud metabolites, and freeze-drying the inactivated wet bacterial mud metabolites, and then mixing them with auxiliary materials to obtain a suspension, and freeze-drying the suspension to obtain the product.

[0077] In some embodiments, the inactivation refers to maintaining the fermentation broth at 100-140° C. for 5-8 seconds for UHT inactivation treatment.

[0078] In some embodiments, the inactivation refers to maintaining the fermentation broth at 120° C. for 6 seconds for UHT inactivation treatment.

[0079] In some embodiments, the centrifugal speed is 8000 r / min to 12000 r / min, specifically, the centrifuge is performed at a feed rate of 3 to 5 kg / min.

[0080] In some embodiments, the freeze-drying conditions are: vacuum degree 8-12 Pa, temperature 25-28° C., and drying time 8-12 h.

[0081] In some embodiments, the freeze-drying conditions are: vacuum degree 10 Pa, temperature 25° C., and drying time 10 h.

[0082] In some embodiments, the moisture content of the freeze-dried bacterial powder is ≤4% by mass.

[0083] In some embodiments, the excipients include whey peptides, pullulan, arginine, and choline.

[0084] In some embodiments, the mass addition ratio of the inactivated wet bacterial mud metabolites to the auxiliary materials is 1:2 to 3.5.

[0085] In some embodiments, the mass addition ratio of the inactivated wet bacterial mud metabolites to the auxiliary materials is 1:2.95.

[0086] The present invention is further illustrated below by way of examples, but the present invention is not limited to the scope of the embodiments described. Based on the embodiments in the present invention, those skilled in the art, without making any creative premise, any variation of the present invention all fall within the scope of protection of the present invention. Meanwhile, in the examples of the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0087] Example 1: Strain Identification

[0088] A strain of Lactobacillus fermentum was isolated from rose cake and identified as Lactobacillus fermentum AK223-12 by 16S rRNA method. The 16S rDNA sequence of the strain is shown in SEQ ID NO: 1.

[0089] The fermented Lactobacillus (Limosilactobacillus fermentum) AK223-12 screened above was deposited in the Guangdong Provincial Microbial Culture Collection Center (address: 5th Floor, Laboratory Building, Guangdong Provincial Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou) on July 8, 2024, with the deposit number GDMCC No: 64839.

[0090] Example 2: Cultivation of strains

[0091] 1. Expand bacterial culture:

[0092] The fermented Lactobacillus AK223-12 obtained in Example 1 and stored in a -80°C refrigerator was expanded and cultured. The culture medium was prepared as follows: 20 g of trehalose, 6 g of bovine colostrum powder, 6 g of beef extract powder, 0.7 g of sodium acetate, 0.5 mL of Tween-80, 1.5 g of potassium dihydrogen phosphate, and 1000 mL of tap water. The pH was adjusted to 7.0 (adjusted with alkali solution). The trehalose and other raw materials in the above formula were added to 1000 mL of water in the formula without any difference in order, stirred for 10 minutes to dissolve, and then dispensed into test tubes, each containing 5 mL. The tubes were sealed with kraft paper stoppers and sterilized in a medical sterilizer at 0.1-0.12 MPa for 20 minutes. The culture medium was then prepared.

[0093] Inoculation: Under sterile conditions (clean bench), open the -80℃ low-temperature freezer to store the frozen tube of bacteria, and dissolve it at room temperature. Then take one sterilized test tube culture medium, add 0.5mL of the dissolved bacterial solution to the test tube, seal the tube mouth with a rubber stopper and kraft paper, shake it gently by hand, and place it in a constant temperature incubator at 37℃ for 16 hours. This is called expansion culture medium.

[0094] 2. Primary seed cultivation

[0095] Primary seed culture formula: 10g trehalose, 6g peptone, 4g beef extract powder, 1.8g metasilicic acid, 1g sodium acetate, 1mL Tween-80, 1000mL tap water, pH natural. Procedure: Add all ingredients, without any prior mixing, to 1000mL of tap water. Start a blender and stir for 10 minutes until completely dissolved. This is called the primary seed culture medium.

[0096] Add the stirred and dissolved first-level seed culture solution into the triangular flask according to the dosage, then wrap the bottle mouth with 6 layers of gauze and a layer of kraft paper, and put it into the sterilizer for sterilization. Before sterilization, first open the exhaust valve on the sterilizer and wait until a small amount of steam is discharged. Close it. When the pressure on the sterilizer reaches 0.05MPa, open the exhaust valve on the sterilizer to exhaust for 6-8 minutes, then close the exhaust valve and continue heating. Start timing when the steam pressure in the sterilizer reaches 0.1-0.12MPa. The sterilization time is 20 minutes. After completion, leave the heat source and let it cool down naturally. When the pressure gauge on the sterilizer returns to zero, open the sterilizer, take out the sterilized culture medium and place it in the clean workbench to cool naturally. When the temperature drops to 37°C, transfer the above cultured expanded bacterial liquid to the sterilized culture medium in the triangular flask. The inoculation amount is 0.5%, that is, 100mL of the first-level seed culture medium and 0.5mL of the expanded bacterial liquid. After completion, seal the bottle mouth with the original gauze and kraft paper, shake it slightly by hand to mix the accessed bacteria and culture medium evenly, and then place it in a constant temperature box and culture it at 37°C for 24 hours. This is called the first-level bacterial liquid.

[0097] 3. Seed tank cultivation

[0098] Sterilization of fermentation equipment, pipelines and sterile filtration systems: First, open the valves of all inlet and outlet pipelines and sterile air pipelines, pass 0.12-0.14MPa steam, allow the steam to connect with the pipeline valves and discharge a small amount of steam, ventilate for 40 minutes, then close the valves of all inlet and outlet pipelines and set aside.

[0099] Sterilization of empty seed tanks and fermentation tanks: Close all valves, open the drain valve at the bottom of the tank and the drain valve in the tank interlayer, open the direct steam valve to introduce 0.12-0.14MPa steam into the pipe, start timing when the temperature in the tank reaches 121℃, the sterilization time is 40min, then close the drain valve at the bottom of the tank and the drain valve in the interlayer, and the temperature in the tank will naturally drop to 37℃ for standby use.

[0100] Seed tank culture formula: trehalose 4%, casein peptone 1%, dipotassium hydrogen phosphate 0.2%, corn peptide 3%, sodium acetate 0.3%, Tween-80 0.1%, polyether defoamer 0.1%, tap water 91.3%, natural pH value. Specific operation: First, add the tap water used in the formula into the sterilized seed tank that has been empty, start the mixer, and add the required raw materials in the formula respectively (the order of addition does not matter). Then, stir continuously and pass steam to heat. First, use the steam in the tank interlayer to heat to 95°C, and then switch to direct steam heating. When the temperature in the tank reaches 121°C, maintain it for 20 minutes to achieve the sterilization effect. Then turn off the steam and start cooling through the tap water in the tank interlayer. When the temperature in the tank reaches 37°C, it is ready for use. This is called seed culture medium. Then, the cultured first-level bacterial culture liquid is inoculated into the seed tank under sterile conditions. The inoculation amount is 2% of the culture medium in the tank. 100 kg of seed tank culture medium can be inoculated with 2 kg of first-level bacterial liquid. Then, cover the inoculation cap on the tank and start stirring and culturing. The culture conditions are: temperature 37°C, natural pH value, stirring speed 140r / min, tank pressure 0.05MPa, and culture time 15h to meet the requirements. If the tank body is lowered, sterile air can be introduced to maintain the tank pressure. If the tank pressure is too high, the air release valve on the tank top can be used to adjust it. This is called seed tank culture.

[0101] 4. Fermentation

[0102] Fermentation formula: 2.5% trehalose, 1% casein peptone, 1% L-arabinose, 1.5% isomaltooligosaccharide, 3% yeast extract powder, 1.5% sodium acetate, 0.2% Tween-80, 0.8% arginine, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, 87.4% tap water. Specific steps: First, add the tap water used in the formula to an empty, sterilized fermentation tank. Start the mixer and add the ingredients in the formula (the order of addition is irrelevant). Then, apply steam for heating. First, use interlayer steam to heat to 95℃, then switch to direct steam heating. Start timing when the temperature in the tank reaches 121℃, and maintain it for 30 minutes to achieve the sterilization effect. Then cool it down with tap water in the interlayer. When the temperature in the tank drops to 37℃, start to inoculate the bacteria. Before inoculation, first increase the tank pressure of the seed tank to 0.1MPa, and maintain the tank pressure of the fermentation tank at 0.05MPa. Then open the inoculation pipeline valve, and transport the seeds cultured in the seed tank to the fermentation tank through pressure difference, and then close the inoculation channel valve.

[0103] The inoculation amount is 5 kg of bacteria cultured in the seed tank for every 100 kg of culture medium in the fermentation tank. The culture conditions are: culture temperature 37°C, stirring speed 180r / min, tank pressure maintained at 0.05MPa, and the acid and alkali automatic control system is used to maintain the pH value at 6.5 during the fermentation process. If the tank pressure drops, sterile air can be introduced to maintain the tank pressure until the fermentation is completed. The total fermentation time is 36 hours, and then the temperature is lowered to 20°C. This is the fermentation liquid.

[0104] 5. Inactivation and freeze-drying of Lactobacillus fermentation broth

[0105] 1. The fermentation broth obtained by fermentation culture was subjected to UHT inactivation treatment at 120°C for 6 seconds.

[0106] 2. Pump the fermentation liquid into a storage tank for centrifugation. Operation: Start the centrifuge, adjust the speed to 10,000 r / min, and centrifuge at a feed rate of 4 kg / min. After centrifugation, collect the inactivated wet bacterial sludge metabolites and freeze-dry the collected inactivated wet bacterial sludge metabolites.

[0107] 3. Freeze-drying of inactivated bacterial sludge metabolites

[0108] Preparation of suspension: Take 1000 g of inactivated wet bacterial sludge metabolites, add 4000 mL of sterile saline, 800 g of whey peptide, 2000 g of pullulan, 30 g of arginine, and 120 g of choline.

[0109] Operation: First, take 2500mL of normal saline in the formula, add whey peptide and stir to dissolve, then add 1000g of inactivated wet bacterial mud metabolites, and then add the remaining normal saline and pullulan and excipients in the formula and stir thoroughly to obtain a suspension. The suspension is freeze-dried (place the suspension in a freeze-drying tray, start the freeze dryer, cool to -50°C in 120 minutes, maintain for 1 hour, turn on the vacuum pump, heat to 25°C at 4°C / h, maintain for 10 hours, vacuum degree reaches 10 Pa, moisture 3%, water activity 0.18), and finally control the moisture content to 4%, which becomes freeze-dried bacterial powder of inactivated bacterial metabolites.

[0110] The freeze-dried bacterial powder can be used in food or medicine; the food includes baked food, solid beverage, liquid beverage, and compressed candy.

[0111] After testing, the freeze-dried powder obtained in Example 2 had an extracellular polysaccharide content of 32%, a polypeptide content of 6%, an amino acid content of 26%, and an inactivated bacterial cell content of 150 billion / g.

[0112] Test Example 1: Animal Experimental Study

[0113] 1. Experiment on regulating the function of mouse intestinal flora:

[0114] Mice were divided into a control group and an experimental group, with 10 mice in each group. The control group received a normal diet, while the experimental group received a probiotic product containing inactivated bacterial cells and metabolites. Mouse feces were sterilely collected, weighed, and diluted 10-fold with a sterile diluent to create a uniform suspension. The suspension was then diluted in 10-fold increments, and the appropriate dilution was selected and inoculated onto various selective media. The bacterial count per gram of wet feces was calculated, and the logarithm was taken for statistical analysis. Twenty-four hours after the last administration of the probiotic product (freeze-dried powder), rectal feces were collected, and the intestinal flora was analyzed as shown in Table 1.

[0115] Table 1 Suitable culture conditions and detection of intestinal flora (x - ±SD)

[0116]

[0117] As shown in Table 1, probiotic products can increase the number of beneficial bacteria (Bifidobacteria and Lactobacilli), while the number of harmful bacteria (Enterobacter and Enterococcus) did not increase but showed a slight downward trend, indicating that probiotics can regulate the function of the intestinal flora in mice.

[0118] 2. Mouse Gastrointestinal Motility Experiment

[0119] Mice were divided into a control group, a model group, and an experimental group, with 10 mice in each group. The control and model groups were given distilled water by gavage, while the experimental group was given a probiotic product containing inactivated bacteria and metabolites. After 15 days of gavage, all mice were fasted for 16 hours. The model and experimental groups were then given difenococcal compound by gavage, while the control group was given distilled water by gavage. Thirty minutes later, all mice were given gum arabic ink by gavage. Thirty minutes later, the mice were killed by cervical dissection. Following cervical dissection, the abdomen was rapidly incised along the midline to dissect the mesentery. The intestinal loop was cut from the pylorus to the ileocecal region, and the small intestine was gently pulled into a straight line. The intestinal length was measured as the "total small intestinal length," and the distance from the pylorus to the ink front was the "ink propagation length." The ink propagation rate was calculated according to the following formula. The ink propagation rate for the small intestine is shown in Table 2.

[0120]

[0121] Table 2 Small intestinal ink propulsion rate (x - ±SD)

[0122]

[0123]

[0124] As shown in Table 2, the intestinal motility inhibition model was established in mice after administration of compound difenocool. Compared with the model group, the small intestinal ink propulsion rate increased in the experimental group, indicating that probiotics can promote small intestinal digestion.

[0125] 3. Animal model experiments

[0126] (1) Animal behavior experimental plan: 11-week-old mice were divided into WT mice and SOD1-G93A transgenic mice. WT mice served as the control group, and SOD1-G93A transgenic mice served as the model group and experimental group. The control group and the model group were fed normally. The experimental group was fed with a probiotic product containing 4.5 billion inactivated bacteria and 30 mg of metabolites (i.e., the freeze-dried powder prepared in Example 2). The food was fed once a day for 5 weeks. The test items were as follows:

[0127] ①During the feeding period, the weight of mice was measured and recorded once a week. The results are shown in Figure 1 .

[0128] according to Figure 1 It can be seen that mice lost significant weight at 13-15 weeks. By feeding the mice with probiotic products, it was found that probiotic products can significantly slow down the weight loss of mice and have a tendency to delay the development of the disease.

[0129] ② Use the rotarod device to evaluate the motor coordination of mice. The rotarod experiment was carried out for 3 consecutive days, and each day consisted of a training phase and a test phase. In the training phase, the rotarod speed was 4r / min, and the mice trained on the rotarod for 5 minutes, after which they were put back into the cage and entered the test phase 1 hour later. In the test phase, the rotarod speed was uniformly accelerated from 0r / min to 40r / min within 5 minutes. The observer recorded the duration of the mouse on the rotarod, and 300s was recorded as long as it did not fall. The test was conducted 3 times a day, with an interval of 30 minutes between each test. The experimental results of the third day were selected for statistical analysis, and the results are shown in the table. Figure 2 .

[0130] according to Figure 2 As shown in (a), the SOD1-G93A transgenic model group mice stayed on the rotarod the shortest time, and the stay time was prolonged after being fed with probiotic products, indicating that probiotic products can improve the motor coordination of mice.

[0131] ③ Place the mouse on the cage lid. Turn the lid over and place it 50 cm away from the cage. Observe for 60 seconds and record the time the mouse remains suspended on the cage lid. Any time the mouse remains suspended for more than 60 seconds is counted as 60 seconds.

[0132] according to Figure 2 As shown in (b), the mice in the SOD1-G93A transgenic model group had the shortest hanging time on the cage cover, and the time was prolonged after being fed with probiotic products, indicating that probiotic products can improve the motor coordination of mice.

[0133] ④ Suspend the mouse head downward in the air and observe for 15 seconds. The degree of hind limb clasping is graded based on the mouse's performance. The scale is as follows: Grade 0 = both hind limbs extended outward and away from the abdomen; Grade 1 = one hind limb retracted toward the abdomen for at least half of the observation period; Grade 2 = both hind limbs partially retracted toward the abdomen for at least half of the observation period; Grade 3 = both hind limbs fully retracted toward the abdomen for at least half of the observation period. Performance between the two scores is scored as 0.5, 0.5, or 2.5.

[0134] according to Figure 2 As shown in (c), the hind limb clasping score of mice in the SOD1-G93A transgenic model group was the highest, indicating that the motor coordination of this model mice was the worst. After feeding the probiotic product, the hind limb clasping score decreased, indicating that the probiotic product can improve the motor coordination of mice.

[0135] (2) After the animal behavior experiment, the mice were anesthetized (250 μL 2% sodium pentobarbital was injected intraperitoneally); PBS (containing 10 U / mL heparin) pre-cooled at 4°C was perfused into the hearts of the mice; the mouse brain was taken and the brain tissue was divided into two parts. At the same time, the lumbar spinal cord was taken. Half of the brain and spinal cord were fixed with 4% paraformaldehyde solution at 4°C overnight, and then dehydrated with sucrose (concentrations of 10%, 20%, and 30%) in a gradient manner. After the end, they were embedded in OCT and then frozen and sectioned. The sections were stored at -80°C. The remaining tissue was lysed with RIPA lysis buffer with protease inhibitors added, centrifuged in a centrifuge (4°C, 12000r, 30min), the supernatant was collected, and stored at -80°C after aliquoting. The test items are as follows:

[0136] ① Rewarm the tissue sections at -80℃ for 30min, perform TUNEL and NISSL staining, and analyze the results. Figures 3 to 5 The SOD1 level in mouse spinal cord lysate was detected by Western blotting. Figure 3 As shown in (b). The level of SOD1 oligomers in tissues was detected by dot blot. The results are shown in Figure 2 As shown in (c).

[0137] There are a large number of mutant SOD1 aggregates deposited in the brainstem and spinal cord of ALS patients and transgenic mice, and mutant SOD1 is prone to misfolding, oligomerization and formation of fibrous deposits, leading to motor neuron degeneration. Figure 2 As shown in Figures (a) to (c), the levels of SOD1 aggregates and oligomers in the brainstem and spinal cord of mice fed the probiotic product were significantly reduced, indicating that the probiotic product can reduce the fibrillar deposition of mutant SOD1 aggregates. Loss of motor neurons in the spinal cord is a key pathological feature of ALS.

[0138] according to Figure 4As shown in Figures (a) and (b), feeding the probiotic product significantly increased the area and number of motor neurons in the spinal cord and brainstem of mice, significantly improving neuronal survival. Mutated SOD1 is prone to forming toxic oligomers, leading to proteasome damage and endoplasmic reticulum stress in neurons, ultimately activating apoptosis pathways.

[0139] according to Figure 5 As shown in (a) to (c), after feeding the probiotic product, the number of apoptotic neurons in the spinal cord and brainstem of mice was significantly reduced.

[0140] ② Determination of IL-1β, IL-6, and TNF-α in tissue extracts. The determination of the three factors was performed using the ELISA kit method. The results are shown in Figure 6 The levels of IKKβ, p65 and phosphorylated p65 (p-p65) in mouse spinal cord lysates were determined by immunoblotting. Figure 7 .

[0141] The release of inflammatory factors can cause the pathology and death of motor neurons. Figure 6 It can be seen that by feeding probiotic products, the levels of IL-1β, IL-6, and TNF-α are significantly reduced. NF-kB signaling is one of the main pathways for inflammation, and NF-kB upregulation occurs in ALS patients and transgenic mice. Figure 7 The results showed that probiotics can reduce the levels of IKKβ, p65, p-p65, and p-p65 / p65 in mouse spinal cord lysates, but the change in p65 levels was not significant. This suggests that probiotics can inhibit the abnormal activation of the NF-kB pathway and downregulate the levels of inflammatory factors (IL-1β, IL-6, and TNF-α) to exert anti-inflammatory effects.

[0142] Experimental Example 2: Metabolomics Analysis of Mouse Feces Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

[0143] 1. Metabolomics analysis

[0144] Experimental plan: Mice were divided into a control group and an experimental group. The control group was fed a normal diet, while the experimental group received a probiotic product containing 4.5 billion inactivated bacterial cells and 30 mg of metabolites. After 90 days of feeding, fecal samples from both the control and experimental groups were placed in 400 μL of extraction solution (acetonitrile:methanol = 1:1). After vortexing for 30 seconds, the samples were extracted with low-temperature ultrasonication for 30 minutes (5°C, 40 kHz). The samples were allowed to stand at -20°C for 30 minutes and then centrifuged at 13,000 rpm for 15 minutes in a 4°C centrifuge. The supernatant was collected, dried with nitrogen, and reconstituted with 120 μL of reconstitution solution (acetonitrile:water = 1:1). The samples were extracted with low-temperature ultrasonication for 5 minutes (5°C, 40 kHz), centrifuged at 13,000 rpm for 5 minutes in a 4°C centrifuge. An appropriate amount of the supernatant was collected for analysis. 10 μL of the sample supernatant was separated on a BEH C18 column and analyzed by mass spectrometry. The separation flow rate was 0.40 mL / min and the column temperature was 40°C. After completion, the LC-MS raw data were transferred to the computer for processing and analysis. The MS mass spectrum information was matched with the public database to obtain the main metabolite information such as Figure 8 and as shown in Table 3.

[0145] Table 3 Main metabolomics analysis of probiotic-fed mice

[0146] Metabolomics name Content of control group (%) Content in experimental group (%) Quercetin 0.04 0.34 <![CDATA[Vitamin B6]]> 0.01 0.23 Vitamin D 0.02 0.31 potassium 0.02 0.29

[0147] Quercetin: As a potent scavenger of reactive oxygen species (ROS), quercetin possesses strong cytoprotective properties. It can significantly reverse the cytotoxicity of UVB radiation mediated by keratinocytes, reduce intracellular ROS formation, and inhibit ROS-mediated cell membrane and mitochondrial damage. Quercetin can also reduce oxidative damage caused by excess iron by chelating iron. Furthermore, it can inhibit the expression of inflammatory factors such as IL-6 in cells.

[0148] Vitamin B6: ALS patients often have elevated plasma homocysteine ​​levels. Vitamin B6 can convert homocysteine ​​into sulfur amino acids, potentially playing a protective role in neurodegenerative diseases. Vitamin B6 also plays an important role in the development and regulation of the central nervous system and metabolism.

[0149] Vitamin D: Due to increased production of reactive oxygen species, mitochondrial dysfunction leads to mild but chronic inflammation, resulting in qualitative and quantitative deterioration of skeletal muscle, which is considered one of the main causes of sarcopenia. Reports show that vitamin D supplementation can improve mitochondrial oxidative phosphorylation function in patients' skeletal muscle, increase mitochondrial oxygen consumption rate, and activate pyruvate dehydrogenase.

[0150] Potassium: Potassium has the function of maintaining neuromuscular excitability. The neuromuscular system can only maintain normal excitability when the blood potassium concentration is maintained at a certain level.

[0151] 2. Mechanism of action research

[0152] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes the selective loss of motor neurons in the spinal cord, brainstem, and motor cortex. Neuronal survival is limited and affected by a variety of conditions, such as mitochondrial dysfunction, neurotoxic stimulation, and oxidative stress. Oxidative stress is a state of imbalance between oxidation and reduction in the body. Its pathological process includes inflammatory cell infiltration, increased secretion of oxidative proteases, and the production of a large number of reactive oxygen free radicals, such as superoxide (O2 - ), hydroxyl (OH - ) and hydrogen peroxide (H2O2), etc. Therefore, scavenging reactive oxygen free radicals can protect neurons and is an effective way to treat neurodegenerative diseases.

[0153] (1) Antioxidant effect

[0154] ①Effects on cell viability and proliferation

[0155] The cells were cultured in a medium containing 10% fetal bovine serum and 1% antibiotics. The medium was changed every 2 days and the cells were passaged when they reached 80% to 90% confluence. P3-P6 cells were used for subsequent experiments. The cells were treated with 200 μmol / L H2O2 to establish an oxidative stress injury model. The cells were divided into control group, model group (200 μmol / L H2O2), experimental group 1 (200 μmol / L H2O2 + 2.5 μmol / L quercetin), experimental group 2 (200 μmol / L H2O2 + 2.5 μmol / L vitamin B6), experimental group 3 (200 μmol / L H2O2 + 2.5 μmol / L vitamin D), experimental group 4 (200 μmol / L H2O2 + 2.5 μmol / L potassium), and experimental group 5 (200 μmol / L H2O2 + 2.5 μmol / L quercetin + 2.5 μmol / L vitamin B6 + 2.5 μmol / L vitamin D + 2.5 μmol / L potassium). The cells were seeded into 96-well plates (3 × 10 cells per well). 3 After the cells adhered, quercetin, vitamin B6, vitamin D, potassium, or quercetin + vitamin B6 + vitamin D + potassium were added to the culture medium with or without H2O2 for 24 hours. After the intervention, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was detected using a microplate reader.

[0156] ② Detection of total SOD content in cells

[0157] Cells were seeded into 6-well plates (3 × 10 5After the cells adhered, the cells were treated according to the above groupings for 24 hours. After the treatment, cell samples from each group were collected and the working solution was prepared according to the instructions of the SOD activity assay kit. After incubation at 37°C for 30 minutes, the absorbance at 450 nm was measured using a microplate reader. The SOD activity of each cell sample was calculated using the standard curve.

[0158] ③ Detection of total intracellular MDA content

[0159] Cell intervention was performed as described above. After the intervention, cell samples were collected from each group. The working solution was prepared according to the MDA assay kit instructions, mixed with the sample, heated at 100°C for 15 minutes, cooled to room temperature in a water bath, and centrifuged at 1000g for 10 minutes at room temperature. 200 μL of the supernatant was added to a 96-well plate, and the absorbance was measured at 532 nm using a microplate reader. The MDA content in each cell sample was calculated using a standard curve.

[0160] Table 4 Effects on cell activity

[0161] Group Cell viability control group 1.0±0.00 Quercetin 0.97±0.02 <![CDATA[Vitamin B6]]> 0.96±0.01 Vitamin D 0.98±0.03 potassium 0.98±0.03 Quercetin + Vitamin B6 + Vitamin D + Potassium 1.03±0.05

[0162] Table 5 Effects on H2O2-induced cell activity

[0163] Group Cell viability control group 1.0±0.00 Model Group 0.74±0.04 Experimental Group 1 0.88±0.01 Experimental Group 2 0.75±0.04 Experimental Group 3 0.76±0.02 Experimental Group 4 0.78±0.01 Experimental Group 5 0.92±0.02

[0164] The results in Table 4 show that quercetin, vitamin B6, vitamin D, and potassium had no obvious toxic effects on cells;

[0165] The results in Table 5 show that 200 μmol / L H2O2 significantly inhibited cell proliferation. Quercetin, vitamin B6, vitamin D, potassium, and quercetin + vitamin B6 + vitamin D + potassium were able to alleviate the inhibitory effect of H2O2 on cell proliferation. This suggests that quercetin, vitamin B6, vitamin D, potassium, and quercetin + vitamin B6 + vitamin D + potassium have a protective effect on cells under oxidative stress.

[0166] Malondialdehyde (MDA) is a major oxidative stressor that can induce the formation of reactive oxygen species (ROS). Superoxide dismutase (SOD) is an antioxidant metalloenzyme present in organisms. It catalyzes the dismutation of superoxide anion radicals to produce oxygen and H2O2, playing a crucial role in the body's oxidative and antioxidant balance.

[0167] like Figure 9As shown, by detecting the intracellular MDA content, it was found that the MDA content increased significantly after H2O2 intervention, while quercetin, vitamin B6, vitamin D, potassium, and quercetin + vitamin B6 + vitamin D + potassium all reduced the intracellular MDA content, and quercetin + vitamin B6 + vitamin D + potassium had the best effect. In addition, it was found that H2O2 significantly reduced the intracellular SOD activity, while quercetin, vitamin B6, vitamin D, potassium, and quercetin + vitamin B6 + vitamin D + potassium increased the intracellular SOD activity after intervention, and quercetin + vitamin B6 + vitamin D + potassium had the best effect, indicating that quercetin + vitamin B6 + vitamin D + potassium has a good antioxidant effect.

[0168] (2) Anti-inflammatory effect

[0169] ① Toxic effect on RAW264.7 cells

[0170] RAW264.7 cells in the logarithmic growth phase were seeded in 96-well culture plates at a cell density of approximately 5 × 10 5 / mL, 100μL cell suspension per well, the culture medium is DMEM (high glucose) culture medium containing 100mL / L fetal bovine serum, 200μL PBS is added to each well for four weeks as a moisturizing well, and cultured at 37℃ and 5% CO2 for 12 hours until it adheres to the wall and returns to a resting state. All culture medium was discarded, and the culture medium used subsequently was DMEM (high glucose) culture medium without fetal bovine serum. 100μL of DMEM (high glucose) culture medium containing different components was added to different groups, specifically: control group, DMEM (high glucose) culture medium was added; LPS group, LPS mother solution with a concentration of 100μg / mL was added to DMEM (high glucose) culture medium to make the final LPS concentration of 1μg / mL; experimental group 1, quercetin mother solution with a concentration of 100μg / mL was added to DMEM (high glucose) culture medium, and experimental group 2, quercetin mother solution with a concentration of 100μg / mL was added to DMEM (high glucose) culture medium. In experimental group 3, a 100 μg / mL vitamin B6 mother solution was added to DMEM (high glucose) culture medium. In experimental group 4, a 100 μg / mL potassium mother solution was added to DMEM (high glucose) culture medium. In experimental group 5, a 100 μg / mL quercetin + vitamin B6 + vitamin D + potassium mother solution was added to DMEM (high glucose) culture medium. The cells were filtered through 0.22 μm to a concentration of 10 μg / mL. Six replicate wells were added to each group and cultured for a further 24 hours. 20 μL of 5 mg / mL MTT was added to each group and cultured for a further 2 hours. The culture was terminated and all liquid was aspirated to avoid loss of adherent cells. 100 μL of DMSO was added to each group and shaken for 10 minutes to fully dissolve the intracellular crystals. The OD value of each well was measured at a wavelength of 520 nm using a microplate reader.

[0171] The results are as follows Figure 10 As shown, quercetin, vitamin B6, vitamin D, potassium, and quercetin + vitamin B6 + vitamin D + potassium had no toxic effects on RAW264.7 cells within 24 hours, and the OD values ​​were not significantly different from those of the control group.

[0172] ②Effects on NO secretion in RAW264.7 cells

[0173] RAW264.7 cells in the logarithmic growth phase were seeded in 6-well culture plates at a cell density of approximately 5 × 10 5 / mL, 2mL cell suspension per well, the culture medium is DMEM (high glucose) culture medium containing 100mL / L fetal bovine serum, cultured at 37°C, 5% CO2 volume fraction for 12h, until it adheres to the wall and returns to a resting state. The culture medium used subsequently is DMEM (high glucose) culture medium without fetal bovine serum. After the cells return to a resting state, all the culture medium is discarded, and 2mL of DMEM (high glucose) culture medium containing different components is added to different groups, specifically: control group and LPS group, DMEM (high glucose) culture medium is added; experimental group 1, quercetin stock solution with a concentration of 100μg / mL is added to DMEM (high glucose) culture medium, experimental group 2, vitamin B6 stock solution with a concentration of 100μg / mL is added to DMEM (high glucose) culture medium, experimental group In group 3, vitamin D stock solution at a concentration of 100 μg / mL was added to DMEM (high glucose). In experimental group 4, potassium stock solution at a concentration of 100 μg / mL was added to DMEM (high glucose). In experimental group 5, quercetin, vitamin B6, vitamin D, and potassium stock solution at a concentration of 100 μg / mL were added to DMEM (high glucose). The solution was filtered through 0.22 μm and concentrated to 10 μg / mL. Three replicates were prepared for each group and the cells were incubated for 4 hours. After pretreatment, all culture medium was aspirated and discarded. The control group was treated with DMEM (high glucose). The LPS group and each experimental group were treated with DMEM (high glucose) containing LPS (final concentration of 1 μg / mL) and incubated for 24 hours. Cell culture supernatants were collected at 6, 12, 18, and 24 hours, and NO levels were measured using a Griess assay kit.

[0174] NO is an important inflammatory mediator in the body. Inhibiting the synthesis of NO can improve inflammation. NO can be produced by macrophages under the induction of LPS and plays an important regulatory role in every stage of inflammation. Figure 11 As shown in the figure, after 12 hours of stimulation with inflammatory drugs, the NO secretion level of cells in the LPS group was significantly higher than that of the control group. After 6 hours of stimulation with inflammatory drugs, there was no significant difference in NO secretion between the experimental groups. However, after 12 hours of stimulation, the NO secretion level of each group was significantly lower than that of the LPS group. In particular, the NO secretion level of experimental group 5 after 24 hours of stimulation was very close to that of the control group.

[0175] ③Effects on TNF-α secretion in RAW264.7 cells

[0176] The cell treatment was the same as ②. After 6, 12, 18, and 24 hours of culture, the cell culture supernatant was collected and the TNF-α content of each group of samples was determined using a non-ready-to-use ELISA detection kit.

[0177] TNF-α can lead to the continuous aggravation of inflammatory response. Therefore, inhibiting the excessive release of TNF-α is one of the key measures to prevent and treat inflammation. Figure 12 As shown, TNF-α secretion in the LPS group was significantly higher than in the control group after 6 hours of stimulation with the proinflammatory drug, and a positive correlation was observed between stimulation time and TNF-α secretion within 24 hours. TNF-α secretion in each experimental group was significantly lower than in the LPS group at 6, 12, 18, and 24 hours of stimulation, but significantly higher than in the control group. Experimental group 5 showed the greatest effect in inhibiting TNF-α secretion.

[0178] ④Effects on the secretion of IL-1β, IL-6 and IL-10 by RAW264.7 cells

[0179] The cell treatment was the same as in step ②. After culturing for 24 hours, the cell culture supernatant was collected and the IL-1β, IL-6, and IL-10 levels in each group of samples were determined using a non-ready-to-use ELISA kit.

[0180] The results are as follows Figure 13 As shown, after 24 hours of pro-inflammatory drug stimulation, the secretion of IL-1β, IL-6, and IL-10 in the LPS group was significantly higher than that in the control group. After 24 hours of pro-inflammatory drug stimulation, the secretion of IL-1β, IL-6, and IL-10 in all experimental groups was significantly lower than that in the LPS group. Experimental group 5 showed the greatest effect in inhibiting the secretion of IL-1β, IL-6, and IL-10.

[0181] (3) Neuroprotection

[0182] Eleven-week-old mice were divided into WT mice and SOD1-G93A transgenic mice. WT mice served as the control group, and SOD1-G93A transgenic mice served as the model group and experimental group. The control and model groups were fed normally. Experimental group 1 had quercetin added to its food, experimental group 2 had vitamin B6 added to its food, experimental group 3 had vitamin D added to its food, experimental group 4 had potassium added to its food, and experimental group 5 had quercetin, vitamin B6, vitamin D, and potassium added to its food. The mice were fed once a day for five weeks. The test items were as follows:

[0183] ① The motor coordination of mice was assessed using a rotarod apparatus. The rotarod test was conducted for three consecutive days, each day consisting of a training phase and a testing phase. During the training phase, the mice were trained on the rotarod at a speed of 4 r / min for 5 minutes. Afterwards, the mice were returned to their cages and entered the testing phase 1 hour later. During the testing phase, the rotarod speed was uniformly accelerated from 0 r / min to 40 r / min over 5 minutes. The observer recorded the duration of the mouse's stay on the rotarod, with 300 seconds defined as the duration without falling. Testing was performed three times per day, with 30-minute intervals between each test. The results from day 3 were used for statistical analysis.

[0184] like Figure 14 As shown in (a), the SOD1-G93A transgenic model group mice stayed on the rotarod the shortest time, and the mice in each experimental group stayed on the rotarod longer than that in the model group, among which the mice in experimental group 5 stayed on the rotarod the longest.

[0185] ② Place the mouse on the cage lid. Turn the lid over and place it 50 cm away from the cage. Observe for 60 seconds and record the time the mouse remains on the cage lid. Any time the mouse remains on the cage lid exceeding 60 seconds will be counted as 60 seconds.

[0186] like Figure 14 As shown in (b), the SOD1-G93A transgenic model group mice spent the shortest time hanging on the cage cover. The time the mice in each experimental group spent hanging on the cage cover was longer than that in the model group, and the time the mice in experimental group 5 spent hanging on the cage cover was the longest. Application Example 1: Application Level for the Treatment of ALS

[0187] The recruitment targets are people with ALS in the community with an average age of 50 years old. As of April 2024, a total of 10 people have been recruited. All of the above people have signed a written informed consent form. If the participant is unable to sign, an agent (usually a family member) will sign it.

[0188] The content of ALS functional scoring is shown in Table 6. Each item is divided into 3 levels, 6-10 points for mild ALS, 11-20 points for moderate ALS, and 21-30 points for severe ALS. The results are shown in Table 7.

[0189] Table 6 ALS functional rating scale

[0190]

[0191]

[0192] Table 7 ALS functional scores

[0193] Serial number Rating / points category Dosage 1 8 Mild ALS Take 0.3 grams once a day before bed for 60 days 2 6 Mild ALS Take 0.3 grams once a day before bed for 60 days 3 8 Mild ALS Take 0.3 grams once a day before bed for 60 days 4 10 Mild ALS Take 0.3 grams once a day before bed for 60 days 5 12 Moderate ALS Take 0.3 grams once a day before bed for 90 days 6 8 Mild ALS Take 0.3 grams once a day before bed for 60 days 7 16 Moderate ALS Take 0.3 grams once a day before bed for 90 days 8 14 Moderate ALS Take 0.3 grams once a day before bed for 90 days 9 22 Severe ALS Take 0.3 grams once a day before bed for 180 days 10 24 Severe ALS Take 0.3 grams once a day before bed for 180 days

[0194] The above results show that:

[0195] Mild ALS: After taking the medicine for 60 days, 4 out of 5 people felt that their speech impairment and drooling caused by tongue muscle atrophy, slow writing caused by clumsy finger movements, and stiff walking caused by muscle weakness had improved. There was only 1 person who did not see any improvement after taking Lactobacillus fermentum.

[0196] Moderate ALS: After taking the product for 90 days, two of the three patients reported improvement in speech and eating disorders, drooling, clumsy finger movements that prevented them from writing for long periods of time, and muscle weakness that caused them to fall while walking. One patient did not see improvement after taking Lactobacillus fermentum.

[0197] Severe ALS: After taking the drug for 180 days, one of the two patients felt that the slurred speech, constant drooling, difficulty swallowing, and falls when walking caused by muscle atrophy in the trunk, neck, face, and throat had improved. One patient did not see any improvement after taking Lactobacillus fermentum.

[0198] The 16S rDNA sequence of the strain is as follows:

[0199] , see SEQ ID NO: 1.

[0200] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A strain of Lactobacillus fermentum AK223-12, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 8, 2024, with the deposit number GDMCC No: 64839.

2. A method for culturing Lactobacillus fermentum AK223-12 according to claim 1, characterized in that: The culture method includes: expansion culture, primary seed culture, seed tank culture and fermentation culture.

3. The culture method according to claim 2, wherein The culture medium for the expanded culture contains, per 1000 mL of water, 15-25 g of trehalose, 5-7 g of bovine colostrum powder, 5-7 g of beef extract powder, 0.5-0.8 g of sodium acetate, 0.3-0.7 mL of Tween-80, and 1.0-1.8 g of potassium dihydrogen phosphate. The pH value of the culture medium for the expanded culture is adjusted to 7.

0. The culture conditions for the expanded culture are: culture at 36-38° C. for 10-20 hours. The culture medium for the primary seed culture comprises, per 1000 mL of water, 8-12 g of trehalose, 5-7 g of peptone, 3-5 g of beef extract powder, 1.5-2.0 g of metasilicic acid, 0.5-1.2 g of sodium acetate, and 0.5-1.2 mL of Tween-80. The pH value of the culture medium for the primary seed culture is natural. The culture conditions for the primary seed culture are: culture at 36-38° C. for 20-30 hours. The inoculum size of the primary seed culture is 0.5%-0.6% by volume. The culture medium for the seed tank culture comprises, by weight percentage, 2-5% trehalose, 0.5-1.2% casein peptone, 0.1-0.3% dipotassium hydrogen phosphate, 1-5% corn peptide, 0.1-0.5% sodium acetate, 0.05-0.15% Tween-80, 0.05-0.15% polyether defoamer, and 90-92% tap water. The pH value of the culture medium for the seed tank culture is natural. The culture conditions for the seed tank culture are: a tank pressure of 0.04-0.06 MPa, culture at 36-38° C. for 10-20 hours, and an inoculum size of 1%-3% by volume. The fermentation culture medium comprises, by weight percentage, 2-3% trehalose, 0.5-1.2% casein peptone, 0.8-1.2% L-arabinose, 1-1.8% isomaltooligosaccharide, 2-5% yeast extract, 0.8-2.0% sodium acetate, 0.1-0.3% Tween-80, 0.5-1.0% arginine, 0.5-1.5% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 85-88% tap water. The pH value of the fermentation culture medium is adjusted to 6.

5. The fermentation culture conditions are: a tank pressure of 0.04-0.06 MPa, culturing at 36-38° C. for 25-40 hours, and an inoculum size of 4-6% by volume.

4. The culture method according to claim 3, wherein The culture medium for the expanded culture contains: 20 g of trehalose, 6 g of bovine colostrum powder, 6 g of beef extract powder, 0.7 g of sodium acetate, 0.5 mL of Tween-80, and 1.5 g of potassium dihydrogen phosphate per 1000 mL of water. The pH value of the culture medium for the expanded culture is adjusted to 7.

0. The culture conditions for the expanded culture are: culture at 37° C. for 16 hours. The culture medium for the primary seed culture comprises: 10 g of trehalose, 6 g of peptone, 4 g of beef extract powder, 1.8 g of metasilicic acid, 1 g of sodium acetate, and 1 mL of Tween-80 per 1000 mL of water; the pH value of the culture medium for the primary seed culture is natural; the culture conditions for the primary seed culture are: culture at 37° C. for 24 hours; and the inoculum size of the primary seed culture is 0.5% by volume. The culture medium for the seed tank culture comprises, by weight percentage, 4% trehalose, 1% casein peptone, 0.2% dipotassium hydrogen phosphate, 3% corn peptide, 0.3% sodium acetate, 0.1% Tween-80, 0.1% polyether defoamer, and 91.3% tap water. The pH value of the culture medium for the seed tank culture is natural. The culture conditions for the seed tank culture are: a tank pressure of 0.05 MPa, culture at 37° C. for 15 hours, and an inoculum size of 2% by volume. The fermentation culture medium comprises, by weight percentage, 2.5% trehalose, 1% casein peptone, 1% L-arabinose, 1.5% isomaltooligosaccharide, 3% yeast extract powder, 1.5% sodium acetate, 0.2% Tween-80, 0.8% arginine, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 87.4% tap water. The pH value of the fermentation culture medium is adjusted to 6.

5. The fermentation culture conditions are: a tank pressure of 0.05 MPa, culturing at 37° C. for 36 hours, and an inoculum size of 5% by volume.

5. A microbial agent, characterized in that: The invention comprises active ingredients, wherein the active ingredients include the inactivated bacteria of the fermented lactobacillus according to claim 1.

6. Use of the Lactobacillus fermentum AK223-12 according to claim 1 or the microbial agent according to claim 5 in preparing food.

7. Use of the Lactobacillus fermentum AK223-12 according to claim 1 or the microbial agent according to claim 5 in the preparation of a health food that helps regulate intestinal flora or aids digestion.

8. Use of the Lactobacillus fermentum AK223-12 according to claim 1 or the microbial agent according to claim 5 in the preparation of a drug for treating amyotrophic lateral sclerosis.

9. A method for preparing freeze-dried bacterial powder for treating ALS, characterized in that: include: The fermentation liquid obtained after the fermentation culture according to claim 2 is inactivated and centrifuged to obtain an inactivated wet bacterial sludge metabolite, and the inactivated wet bacterial sludge metabolite is freeze-dried, and then mixed with auxiliary materials to prepare a suspension, and the suspension is freeze-dried to obtain; The inactivation process refers to maintaining the fermentation liquid at 100-140°C for 5-8 seconds for UHT inactivation. The centrifugal speed is 8000r / min to 12000r / min, specifically centrifuged at a feed rate of 3 to 5kg / min; The drying conditions of the freeze drying are: vacuum degree 8-12 Pa, temperature 25-28° C., and drying time 8-12 h; The moisture content of the freeze-dried bacterial powder is ≤4% by mass.

10. The preparation method according to claim 9, characterized in that The excipients include whey peptides, pullulan, arginine and choline; The mass addition ratio of the inactivated wet bacterial mud metabolites to the auxiliary materials is 1:2-3.5.

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