Animal coccus calves PD13 and application thereof in preparation of product for treating or relieving Parkinson's disease

Abstract

The invention discloses calf animal coccus PD13 and application thereof in preparation of a product for treating or relieving Parkinson's disease, and relates to the technical field of biology. The calf animal coccus PD13 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No.65890. The invention further discloses a preparation method of the calf animal coccus PD13. Experiments prove that the service life of NL5901 nematodes can be prolonged by the animal coccus calves PD13; the movement ability of the NL5901 nematode is improved; the aggregation of alpha-Syn on the body wall of the NL5901 nematode is reduced; and the fat content in the NL5901 nematode body is increased. Furthermore, the calf animal coccus PD13 has a good treatment or alleviation effect on the Parkinson's disease, and a new way is provided for developing related drugs for treating or alleviating the Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a strain of Zoococcus calvatus PD13 and an application thereof in preparing a product for treating or alleviating Parkinson's disease. Background Art

[0002] Mammaliicoccus vitulinus is a Gram-positive, facultative anaerobic coccus, an edible fungus. It was designated a new genus from Staphylococcus in 2020. It is commonly found on the skin, oral cavity, and intestinal tract of mammals (such as cattle and dogs) and is also found in the environment. It is primarily used in the food fermentation industry. By expanding the culture of Mammaliicoccus vitulinus, a starter culture is created, primarily used to improve the stability of fermented foods, shorten production cycles, and enrich the flavor of the product.

[0003] Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. Its primary pathological hallmarks are the abnormal aggregation of α-synuclein (α-Syn) and the degeneration and death of dopamine (DA) neurons. Abnormal α-Syn aggregation forms Lewy bodies (LBs), which, combined with the degeneration of DA neurons, lead to primary motor symptoms such as tremor, bradykinesia, and muscle rigidity. As the disease progresses, personality, behavioral, and cognitive symptoms such as anxiety, depression, dementia, and sleep disturbances may also develop. Parkinson's disease significantly impacts patients' ability to carry out daily activities, severely diminishes their quality of life, and places significant financial strain on society and families. Current clinical treatment options for PD are limited to alleviating symptoms in the early stages of the disease, failing to slow the progression of Parkinson's disease and associated with significant adverse effects. Therefore, current clinical treatment urgently needs a neuroprotective drug with significant efficacy, fewer side effects, and the ability to effectively prevent the natural course of the disease. Summary of the Invention

[0004] The present invention aims to provide a strain of Zoococcus calvae PD13 capable of treating or alleviating Parkinson's disease, the deposit number of which is GDMCC No.65890.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides use of a strain of Zoococcus calvae PD13 in preparing a product for treating or alleviating Parkinson's disease. The Zoococcus calvae PD13 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No. 65890.

[0007] Furthermore, the product includes medicine, food and / or health care product, and the viable count of the bovine animal coccus PD13 added to the medicine, food and / or health care product is 1×10 8 CFU / mL~1×10 10 CFU / mL.

[0008] The present invention also provides a product capable of treating or alleviating Parkinson's disease, wherein the active ingredients include the above-mentioned Zoococcus calvatus PD13 and acceptable adjuvants or auxiliary materials.

[0009] The present invention discloses the following technical effects:

[0010] The present invention provides the use of the aforementioned Zoococcus calvatus PD13 in the preparation of conventional foods and / or functional foods and / or medicaments for treating or alleviating Parkinson's disease: reducing α-Syn aggregation; improving behavioral cognition and motor abilities; and increasing body fat content in nematodes. This strain has been shown to have anti-Parkinson's disease (PD) effects and can be used in the preparation of probiotic preparations for the prevention and treatment of Parkinson's disease (PD). BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the implementation methods or the description of the prior art. Obviously, the drawings described below are only implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0012] Figure 1 This is a graph showing the effect of Zoococcus calvae PD13 on the lifespan of NL5901 nematodes;

[0013] Figure 2 This is a diagram showing the effect of Zoococcus calvatus PD13 on the motility of NL5901 nematodes; A, B, and C represent the nematodes being able to move freely without any contact, the nematodes being able to move freely after being stimulated by external stimulation (touching with platinum wire), and the nematodes being unable to move freely after being touched with platinum wire, respectively.

[0014] Figure 3Figure 2 shows the effect of Z. calvaria PD13 on α-Syn aggregation in NL5901 nematodes. A and B represent the α-Syn aggregation in NL5901 nematodes before and after Z. calvaria PD13 treatment at different stages, respectively. Image J was used to quantitatively analyze the fluorescence intensity of image A, and the relative fluorescence intensity of the Z. calvaria PD13-treated group relative to the control group was calculated.

[0015] Figure 4 Figure 3 is the effect of Zoococcus calf PD13 on the fat content of NL5901 nematodes. A and B represent the Nile red-stained morphology of NL5901 nematodes in the control group and the Zoococcus calf PD13-treated group after feeding for 3 days, respectively. Image J was used to quantitatively analyze the fluorescence intensity of image A, and the relative fluorescence intensity of the Zoococcus calf PD13-treated group relative to the control group was calculated. DETAILED DESCRIPTION

[0016] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0017] If specific experimental steps or conditions are not specified in the examples, the procedures or conditions of conventional experimental steps described in the literature in the field can be followed. All raw materials and instruments used are commercially available, including but not limited to those used in the examples of this application.

[0018] Example 1 Cultivation and Synchronization of Nematodes

[0019] 1.1 Experimental animals: NL5901 nematodes, kept in the laboratory of the School of Bioengineering, Tianjin University of Science and Technology.

[0020] 1.2 Drugs and main reagents:

[0021] Uracil-deficient Escherichia coli (E. coli) OP50 is the food for Caenorhabditis elegans. E. coli OP50 is evenly spread on NGM (nematode growth medium) plates.

[0022] NGM plates: Prepare medium according to the recipe (0.3g NaCl, 0.25g peptone, 1.7g agar powder, 100mL water). Sterilize at 121°C for 20min. When the temperature drops to approximately 80°C, add 100μL cholesterol (5g / L), 100μL MgSO4 (1M), 100μL CaCl2 (1M), and 2.5mL KH2PO4 buffer (1M). Shake well and pour the medium onto sterilized 9cm culture plates while still hot (approximately 20ml / plate). Let cool before use.

[0023] M9 buffer: NaCl 5.0 g, Na2HPO4 6.0 g, KH2PO4 3.0 g, 1 mL MgSO4 (1 M) and water 1000 mL.

[0024] Lysis solution: 500 μL of 10% NaClO solution, 500 μL of 5M NaOH solution and 4 mL of membrane-permeabilized water were mixed.

[0025] 1.3 Synchronization of Nematodes: Transfer the revived transgenic nematodes to NGM medium coated with defective E. coli OP50 and culture in a constant temperature incubator at 20°C. Subculture the nematodes every 5 days. After 3-4 days of culture, gently rinse the nematodes from the NGM plate with filtered M9 solution and collect them in a 15 mL centrifuge tube. Centrifuge at 800 rpm for 2-3 minutes, discard the supernatant, add an appropriate amount of M9, and repeat the wash three times. Add 5 mL of nematode lysis buffer, mix thoroughly, and vortex for 2-3 minutes. Centrifuge and remove the supernatant. Repeat the wash three-five times with M9, then transfer the plate to a blank NGM plate and incubate overnight. Once all eggs have hatched, synchronized L1 NL5901 nematodes are obtained.

[0026] Example 2 Isolation, Identification and Preservation of Zoococcus calf PD13

[0027] The strain was isolated and purified from Daqu, and after DNA extraction, PCR amplification and agarose gel electrophoresis detection, the gene sequence obtained by sequencing was compared in the NCBI database. The strain was identified as Mammaliicoccus vitulinus PD13 and deposited in the Guangdong Provincial Microbial Culture Collection on February 24, 2025; the storage address is: Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences; the storage number is: GDMCC No. 65890.

[0028] Example 3 Preparation of Zoococcus calf PD13 bacterial solution

[0029] Zoococcus calf PD13 was inoculated into a 5 mL LB liquid test tube and cultured at 37° C. for 12 hours to prepare a Zoococcus calf PD13 bacterial liquid.

[0030] Example 4 Effect of Zoococcus calvae PD13 on the lifespan of NL5901 nematodes

[0031] Equal volumes of Zoococcus calvaria PD13 and E. coli OP50 were mixed and spread onto the surface of NGM culture medium supplemented with Fluorouracil, which inhibits nematode egg laying. Fifty NL5901 nematodes synchronized to the L4 stage were then transferred to NGM plates containing Zoococcus calvaria PD13. The culture temperature was set at 20°C, marking day 0 of the experiment. Nematode survival was recorded (escaped nematodes were excluded from the statistics), and statistics were stopped when all nematodes died. Nematode death and escape were determined by microscopic observation of nematodes that showed no movement or swallowing movements and showed no biological response after being touched with a platinum wire. Escape was defined as nematode burrowing into the NGM agar or as nematode death by crawling onto the side of the culture dish. The experiment was repeated three times. After every 1 day, the nematodes were transferred to a new NGM medium plate containing Zoococcus calf PD13 using a nematode-specific pick.

[0032] The toxin produced by α-Syn in NL5901 nematodes, the causative agent of Parkinson's disease, affects the lifespan of nematodes, such as Figure 1 The experimental results show that compared with nematodes cultured in a culture medium without the addition of calf zoococcus PD13, calf zoococcus PD13 can significantly extend the average lifespan of nematodes, with an extension rate of 15%.

[0033] Example 5 Effect of Zoococcus calvae PD13 on the motility of NL5901 nematodes

[0034] Equal volumes of Zoococcus calf PD13 and E. coli OP50, a nematode food, were mixed and aseptically plated onto solid nematode culture medium supplemented with Fluorouracil, which inhibits egg laying in nematodes. Thirty synchronized L4 nematodes were transferred to NGM plates containing Zoococcus calf PD13 and incubated at 20°C. This was designated Day 0 of the experiment. Nematode movement was observed and recorded on Days 5 and 10. Nematode movement was categorized into three levels: Level A indicates free movement without any stimulation, Level B indicates free movement requiring external stimulation (e.g., platinum wire touch), and Level C indicates inability to move freely after touch. Experiments were repeated three times.

[0035] Harmful α-Syn aggregation in NL5901 nematodes can cause DA neuron toxicity, leading to movement disorders in nematodes and having certain side effects on the nematodes' motor ability. Figure 2 As shown, compared with the control group, the motility of nematodes cultured in NGM medium containing calf zoococcus PD13 at level A and B on the 10th day increased by 54%, demonstrating that calf zoococcus PD13 can significantly delay the weakening of the motility of NL5901 nematodes.

[0036] Example 6 Effect of Zoococcus calvae PD13 on α-Syn aggregation in NL5901 nematodes

[0037] Equal volumes of Zoococcus calf PD13 and E. coli OP50 (a nematode food) were mixed and aseptically applied to solid nematode culture medium supplemented with Fluorouracil, which inhibits nematode egg laying. Twenty L4 nematodes were placed in NGM medium containing Zoococcus calf PD13 and cultured at 20°C for 3, 5, and 7 days. Green fluorescent protein accumulation in the nematodes was observed using a laser confocal microscope and statistically analyzed.

[0038] α-Syn with YFP fluorescence signal is expressed in the body wall muscle cells of NL5901 nematodes. α-Syn is a disordered protein involved in the pathogenesis of PD. Figure 3 As shown in A and B, Zoococcus calvatus PD13 can reduce the aggregation of α-Syn.

[0039] Example 7 Effect of Zoococcus calf PD13 on the fat content of NL5901 nematodes

[0040] Equal volumes of Zoococcus calf PD13 and E. coli OP50, a nematode food, were mixed and aseptically plated onto solid nematode culture medium supplemented with Fluorouracil, which inhibits nematode egg laying. Thirty L4 nematodes were transferred to NGM plates containing Zoococcus calf PD13 and cultured at 20°C, marking day 0 of the experiment. Three days later, the nematodes were collected and cleaned of impurities by centrifugation using M9 buffer. Fixation was then performed overnight in fixative. The fixed nematodes were centrifuged at 800 rpm for 3 minutes, washed three times, and then brought to 1 mL with M9 buffer. 2 μL of Nile Red solution was added to the centrifuge tube. The tube was shaken at 20°C for 30 minutes. After centrifugation, the supernatant was removed and the tube was washed three times with M9 buffer to remove excess dye. The nematodes were then placed on a glass slide and photographed under an inverted fluorescence microscope with a 10× objective. Fluorescence quantification was performed using Image J software.

[0041] In NL5901 nematodes, α-Syn reduces the level of fat accumulation in the nematodes. Figure 4As shown in A and B, compared with the control group, the bovine animal coccus PD13 can increase the fat content of the nematodes by 48%, indicating that the bovine animal coccus PD13 significantly increases the fat content of the NL5901 nematodes.

[0042] In summary, Z. bovis PD13 can delay the lifespan of the nematode NL5901, a Parkinson's disease model; improve the decreased motility of the nematodes; reduce the aggregation of α-Syn in the nematodes; and increase fat content in the nematodes. This demonstrates that Z. bovis PD13 has a significant therapeutic and alleviating effect on Parkinson's disease and can be used to prepare drugs, foods, and health products for the treatment and / or alleviation of Parkinson's disease.

[0043] Obviously, the above examples are merely illustrative and are not intended to limit the embodiments. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A strain of Zoococcus calvatus PD13, characterized in that The Zoococcus calf PD13 is deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No.65890.

2. The use of the calf bacteria PD13 according to claim 1 in the preparation of a product for treating or alleviating Parkinson's disease, characterized in that: The products include medicines, foods and / or health products, and the number of viable bacteria of the bovine zoococcus PD13 added to the medicines, foods and / or health products is 1×10 8 CFU / mL~1×10 10 CFU / mL.

3. A product capable of treating or alleviating Parkinson's disease, characterized in that: The active ingredients include the calf zoococcus PD13 described in claim 1 and its acceptable adjuvants or excipients.

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