Pediococcus pentosaceus pp90 for preventing and treating alcoholic nerve injury and application thereof

Abstract

The present application relates to a kind of pentosus PP90 for preventing and treating alcoholic nerve injury and its application, wherein pentosus PP90 is preserved in China Microbial Culture Collection Center, and the preservation number is CGMCC NO.32034.The fermentation product of the pentosus PP90 can degrade the alcoholicity of alcohol, can produce ethanol dehydrogenase and acetaldehyde dehydrogenase, can improve the viability of nerve cell, can regulate the expression of cytokine, can inhibit the activation of astrocyte, can regulate the expression of brain-derived neurotrophic factor, can improve the level of disturbed neurotransmitter, and can be used for preparing product for preventing and treating alcoholic nerve injury or improving alcoholic nerve injury.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a Pediococcus pentosaceus PP90 for preventing and treating alcoholic nerve damage and its application. Background Technology

[0002] The main component of alcoholic beverages is ethanol. Ethanol has a small molecular weight and is both hydrophilic and lipophilic, allowing it to cross the blood-brain barrier and exert direct toxic effects on nerve cells. Free radicals and other metabolites generated during alcohol metabolism also exacerbate nervous system damage, leading to imbalances in the secretion of neurotransmitters such as dopamine (DA), gamma-aminobutyric acid (GABA), and serotonin (5-HT), resulting in an imbalance between excitatory and inhibitory neurotransmitters and causing a series of adverse consequences, including memory loss, difficulty concentrating, anxiety, and insomnia. Ethanol exposure triggers the activation of immune signaling pathways in nerve cells, activating glial cells such as astrocytes and producing inflammatory factors such as tumor necrosis factor-α (TNF-α). Therefore, long-term alcohol abuse and excessive drinking severely impact physical and brain health. Most studies suggest that even low-dose alcohol intake can impair motor control, judgment, and reaction time in the brain. Long-term heavy drinking leads to alcohol dependence and alcoholic brain damage, ultimately causing both structural and functional changes in the central nervous system. It is reported that about 50% of chronic drinkers suffer from chronic neurological deficits, which lead to a range of neurological diseases, including alcohol dependence, peripheral neuropathy, alcoholic cerebellar degeneration, alcoholic dementia, and alcoholic optic neuropathy.

[0003] Therefore, screening for strains that can alleviate alcohol-induced neurological damage is of great significance for improving alcohol-related neurological diseases and preventing alcohol-induced neurological damage in drinkers. Summary of the Invention

[0004] The purpose of this invention is to overcome the deficiencies in the prior art and provide a Pediococcus pentosaceus PP90 for preventing and treating alcohol-induced nerve damage and its application, which can improve and prevent alcohol-induced nerve damage.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A Pediococcus pentosaceus PP90 strain for preventing alcohol-induced nerve damage has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32034, located at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, on September 23, 2024.

[0007] As a further technical solution, the Pediococcus pentosaceus PP90 can grow and reproduce in an alcohol-containing culture medium, wherein the volume concentration of alcohol in the alcohol-containing culture medium is 15%.

[0008] A bacterial agent for preventing alcohol-induced nerve damage, comprising Pediococcus pentosaceus PP90.

[0009] A fermentation product for preventing alcohol-induced nerve damage is prepared by fermentation of Pediococcus pentosus PP90.

[0010] As a further technical solution, the fermentation product can degrade alcohol content, produce alcohol dehydrogenase and acetaldehyde dehydrogenase, enhance the vitality of nerve cells, regulate the expression of cytokines, inhibit astrocyte activation, regulate the expression of brain-derived neurotrophic factor, and improve disordered neurotransmitter levels.

[0011] The application of the aforementioned Pediococcus pentosaceus PP90, the aforementioned inoculum, or the aforementioned fermentation product in the preparation of products for preventing and / or improving alcohol-induced nerve damage.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] This invention screened a large number of microbial strains and found that Pediococcus pentosaceus PP90 can grow and reproduce using alcohol as a carbon source. Its fermentation supernatant can degrade alcohol content and has the ability to produce alcohol dehydrogenase and acetaldehyde dehydrogenase. It can improve the vitality of nerve cells, regulate the expression of pro-inflammatory cytokines (TNF-α), inhibit astrocyte activation, regulate the expression of brain-derived neurotrophic factor, and improve disordered neurotransmitter levels, thereby achieving the effects of improving alcohol-induced nerve damage and preventing alcohol-induced nerve damage. Attached Figure Description

[0014] Figure 1 The graph shows the alcohol degradation capacity of Pediococcus pentosaceus PP90.

[0015] Figure 2 A graph showing the ability of Pediococcus pentosacchari PP90 to produce alcohol dehydrogenase;

[0016] Figure 3 A graph showing the ability of Pediococcus pentosacchari PP90 to produce acetaldehyde dehydrogenase;

[0017] Figure 4 Figure showing the effect of Pediococcus pentosaceus PP90 on alcohol-induced astrocyte viability;

[0018] Figure 5 Figure showing the effect of Pediococcus pentosaceus PP90 on alcohol-induced inflammatory factors in astrocytes;

[0019] Figure 6 Figure showing the effect of Pediococcus pentosaceus PP90 on alcohol-induced astrogenic factor;

[0020] Figure 7 Figure showing the effect of Pediococcus pentosaceus PP90 on alcohol-induced astrogenic neurotransmitters;

[0021] exist Figure 7 Medium, (A) DA; (B) GABA; (C) 5-HT. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0024] In this invention,

[0025] Pediococcus pentosaceus PP90 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.32034 and deposit date of September 23, 2024.

[0026] Lactobacillus rhamnosus LGG: This is a publicly available strain.

[0027] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0028] Example 1: Application of Pediococcus pentosaceus PP90 in the degradation of alcohol

[0029] I. Experimental Methods

[0030] Frozen tubes of *Pediococcus pentosaceus* PP90 and *Lactobacillus rhamnosaceus* LGG strains were continuously activated for three generations. The activated strains (PP90 and LGG) were inoculated at a 4% inoculum into MRS liquid medium containing 15% ethanol, with a blank control of MRS liquid medium containing ethanol (without bacterial culture). The ethanol concentration and bacterial growth were measured using a refractometer at 0 h and 24 h of incubation (expressed as OD). 610nm (Absorbance value is used as the indicator). Each group was divided into three replicates. The alcohol degradation capacity was calculated based on the alcohol concentration. The results are shown in […]. Figure 1 and Table 1;

[0031] The method for calculating the alcohol degradation capacity is: (blank control - experimental group) / blank control × 100%;

[0032] II. Test Results

[0033] Table 1 Growth of individual strains

[0034] Group <![CDATA[0hOD 610nm ]]> <![CDATA[24hOD 610nm ]]> Blank control 0.129 0.130 PP90 0.212 0.583 LGG 0.318 0.341

[0035] From Table 1 and Figure 1 Data shows that both *Pediococcus pentosaceus* PP90 and *Lactobacillus rhamnosus* LGG are tolerant of alcohol and have the ability to degrade alcohol. Among them, *Pediococcus pentosaceus* PP90 has a better ability to degrade alcohol, with PP90 reducing alcohol content by 30% compared to the control group. This is because *Pediococcus pentosaceus* PP90 can grow and reproduce in an alcohol-containing environment, which is also why *Pediococcus pentosaceus* PP90 can degrade alcohol in MRS medium containing alcohol.

[0036] Example 2: Enzyme production test of Pediococcus pentosaceus PP90

[0037] I. Experimental Methods

[0038] Cryopreserved single strains of *Pediococcus pentosaceus* PP90 and *Lactobacillus rhamnosaceus* LGG were continuously activated for three generations. The activated single strains (PP90 and LGG) were inoculated into MRS medium and cultured for 16 hours. After centrifugation and removal of the supernatant, the cells were washed three times with sterile physiological saline, and the viable count was adjusted to 5 × 10⁻⁶. 6 Add 1 ml of lysis buffer to CFU / mL, and sonicate (ice bath, 200 W, sonication for 3 seconds; 10-second interval, repeated 30 times). Centrifuge at 12000 rpm and 4°C for 10 minutes. Collect the supernatant for acetaldehyde dehydrogenase and alcohol dehydrogenase assays. Results are shown below. Figure 2-3 ;

[0039] II. Test Results

[0040] from Figure 2-3It can be seen that both Pediococcus pentosaceus PP90 and Lactobacillus rhamnosus LGG can produce alcohol dehydrogenase and aldehyde dehydrogenase. The alcohol dehydrogenase content of Pediococcus pentosaceus PP90 is 15 nmol / min / 104 cells, and the aldehyde dehydrogenase content is 3.6 nmol / min / 104 cells. Moreover, the production of both enzymes by Pediococcus pentosaceus PP90 is better than that of Lactobacillus rhamnosus LGG.

[0041] Example 3: Application of Pediococcus pentosaceus PP90 in the prevention and treatment of alcoholic nerve damage

[0042] I. Experimental Methods

[0043] 1. Effect of Pediococcus pentosaceus PP90 on cell viability

[0044] (1) Preparation of probiotic fermentation supernatant:

[0045] Activated Pediococcus pentosaceus PP90 and Lactobacillus rhamnosus LGG were inoculated into DMEM cell culture medium and cultured. The fermentation broth was collected after 16 h of culture and centrifuged at 8000 rpm and 4 °C for 10 min. The supernatant was collected and the pH was adjusted to about 7.0. It was then filtered through a 0.22 μm microporous membrane filter to obtain the fermentation supernatant.

[0046] (2) Establishment of an alcohol-induced cell model:

[0047] The cultured astrocytes were adjusted to a cell concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. After the cells adhered, the supernatant was discarded, and 100 μL of cell culture medium containing 100 mM ethanol was added to induce an ethanol-induced astrocyte model.

[0048] (3) Cell viability detection

[0049] After incubating the above cell models for 1 hour, 100 μL of cell culture medium containing 100 mM ethanol was added as the model group, 100 μL of fermentation supernatant of *Pediococcus pentosaceus* PP90 or *Lactobacillus rhamnosus* LGG containing 100 mM ethanol was added as the experimental group, and 100 μL of cell culture medium without ethanol was added as the control group. After incubation for 24 hours, 10 μL of CCK-8 solution was added to each well, and incubation continued for 2 hours. The OD value was measured at 450 nm using a microplate reader, and cell viability was calculated. Cell viability (%) = (experimental OD value / control OD value) × 100%. Results are shown in […]. Figure 4 .

[0050] 2. Effects of Pediococcus pentosaceus PP90 on alcohol-induced neurotransmitter release in astrocytes

[0051] (1) Fermentation supernatant intervention

[0052] The cultured astrocytes were adjusted to a cell concentration of 1.5 × 10⁻⁶. 5 Cells per mL were inoculated into 24-well plates at 37°C and 5% CO2 for 24 hours. After cell attachment, the supernatant was discarded, and the cells were pretreated with cell culture medium containing 100 mM ethanol for 1 hour. Then, 200 μL of cell culture medium containing 100 mM ethanol was added as the model group, and 200 μL of fermentation supernatant of *Pediococcus pentosus* PP90 or *Lactobacillus rhamnosus* LGG containing 100 mM ethanol was added as the experimental group. The control group was not treated with ethanol. The cells were incubated at 37°C and 5% CO2 for 24 hours.

[0053] (2) Sample collection and testing:

[0054] After 24 hours of cell culture, the cell culture medium from each group was collected into sterile centrifuge tubes and centrifuged at 1000 rpm for 20 minutes at 4°C. The supernatant was collected and analyzed according to the kits for tumor necrosis factor-α (TNF-α), brain-derived neurotrophic factor (BDNF), dopamine (DA), gamma-aminobutyric acid (GABA), and serotonin (5-HT). The results are shown in the table below. Figure 5-7 .

[0055] II. Test Results

[0056] 1. Effect of Pediococcus pentosaceus PP90 fermentation supernatant on alcohol-induced astrocyte viability

[0057] Astrocytes were treated with 100 mM ethanol for 1 hour, followed by further treatment with different samples for 24 hours. Results are as follows: Figure 4 As shown, compared with the control group, the astrocyte viability in the model group was significantly weakened, indicating that alcohol has a strong damaging effect on astrocytes. However, compared with the model group, treatment with Pediococcus pentosaceus PP90 and Lactobacillus rhamnosus LGG supernatant significantly increased the astrocyte survival rate, with Pediococcus pentosaceus PP90 showing the best improvement. These results indicate that Pediococcus pentosaceus PP90 can enhance nerve cell viability and alleviate cell damage caused by alcohol.

[0058] 2. Effects of Pediococcus pentosaceus PP90 fermentation supernatant on alcohol-induced inflammatory factors in astrocytes

[0059] When astrocytes are activated, they release various pro-inflammatory cytokines and inflammatory mediators, promoting inflammatory responses and tissue damage, and exerting toxic effects on neurons. For example... Figure 5As shown, the TNF-α secretion level in the control group was 79.8 pg / mL. In the astrocyte culture system of the model group, the content of the inflammatory factor TNF-α in the cell culture medium significantly increased due to alcohol stimulation, with a secretion level of 141.3 pg / mL. However, after treatment with fermentation supernatant from different bacterial strains, the release level significantly decreased. Among them, *Pediococcus pentosaceus* PP90 showed the best improvement effect, with a TNF-α secretion level of 102.4 pg / mL. After intervention with *Lactobacillus rhamnosus* LGG, the TNF-α secretion level was 116.9 pg / mL. These results indicate that *Pediococcus pentosaceus* PP90 can inhibit the expression of the pro-inflammatory cytokine TNF-α, thereby inhibiting astrocyte activation.

[0060] 3. Effects of Pediococcus pentosaceus PP90 fermentation supernatant on alcohol-induced brain-derived neurotrophic factor in astrocytes

[0061] BDNF is an important brain-derived neurotrophic factor in the nervous system. BDNF acts on certain neurons in the central and peripheral nervous systems, helping to support the survival of existing neurons and promoting the growth and differentiation of new neurons and synapses. In the brain, BDNF is active in the hippocampus, cortex, and basal forebrain, areas crucial for learning, memory, and higher cognitive functions. Furthermore, various studies have suggested a possible link between BDNF and conditions related to synaptic transmission and long-term memory, which are associated with some mental illnesses such as depression, schizophrenia, and Alzheimer's disease. Therefore, this invention investigates the effect of Pediococcus pentosaceus PP90 on BDNF expression to indicate its ameliorative effect on alcoholic neurological injury.

[0062] like Figure 6 As shown, the BDNF secretion level in the control group was 111.2 pg / mL. Compared with the control, the BDNF content in the astrocyte culture medium of the alcohol-induced model group was significantly reduced, with a secretion level of 74.6 pg / mL. However, compared with the model group, the release of BDNF significantly increased after intervention with fermentation supernatants from different bacterial strains. *Pediococcus pentosaceus* PP90 showed the best improvement, with a BDNF secretion level of 92.6 pg / mL, while *Lactobacillus rhamnosaceus* LGG showed a secretion level of 89.1 pg / mL. These results indicate that *Pediococcus pentosaceus* PP90 can improve alcohol-induced neurotrophic factor (ADNF) injury by regulating its expression.

[0063] 4. Effects of Pediococcus pentosaceus PP90 fermentation supernatant on alcohol-induced neurotransmitter release from astrocytes

[0064] Alcohol intake can promote the release of dopamine (DA) in the brain, leading to alcohol addiction. Conversely, alcohol intake can reduce the content of gamma-aminobutyric acid (GABA). Intervention with the fermentation supernatant of the strain can regulate disordered neurotransmitter levels, reduce dopamine (DA) release, and promote GABA release, thus bringing the body's neurotransmitter levels closer to normal. The results showed that the DA secretion level in the control group was 19.0 pg / mL. Compared with the control group, the DA release level in the model group after alcohol stimulation was significantly increased, with a secretion level of 25.0 pg / mL. Compared with the model group, probiotic intervention can significantly reduce DA secretion. Among them, Pediococcus pentosaceus PP90 showed better improvement, with a secretion level of 17.9 pg / mL after intervention, and Lactobacillus rhamnosus LGG had a secretion level of 19.0 pg / mL after intervention.

[0065] Both 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) can regulate learning and memory functions. The results showed that the GABA secretion level in the control group was 4.17 μmol / L. Compared with the control group, the GABA release level in the model group after alcohol stimulation was significantly reduced, with a secretion level of 2.80 μmol / L. Compared with the model group, probiotic intervention can increase GABA secretion. Specifically, the secretion level after PP90 intervention was 3.44 μmol / L, and the secretion level after LGG intervention was 3.20 μmol / L. PP90 was more effective than LGG. In the control group, the 5-HT secretion level was 1.47 ng / mL. Compared with the control group, the release level in the model group was reduced, with a secretion level of 1.17 ng / mL. Compared with the model group, probiotic intervention can increase the secretion of 5-HT. Specifically, the secretion level after PP90 intervention was 1.34 ng / mL, and the secretion level after LGG intervention was 1.23 ng / mL. Intervention of the fermentation supernatant of the strain can alleviate alcohol-induced nerve damage. This indicates that Pediococcus pentosaceus PP90 can improve nerve damage caused by alcohol stimulation by regulating the secretion level of neurotransmitters.

[0066] In summary, the fermentation supernatant of *Pediococcus pentosaceus* PP90 of this invention has the following functions:

[0067] 1) It has the ability to degrade alcohol, with a degradation rate of up to 30%;

[0068] 2) It has the ability to produce alcohol dehydrogenase and acetaldehyde dehydrogenase, with Pediococcus pentosaceus PP90 producing alcohol dehydrogenase at a level of 15 nmol / min / 10 4 The acetaldehyde dehydrogenase content level in the cell was 3.6 nmol / min / 10. 4 cell;

[0069] 3) Pediococcus pentosaceus PP90 can enhance the vitality of nerve cells and alleviate cell damage caused by alcohol stimulation; inhibit the expression of pro-inflammatory cytokine TNF-α, thereby inhibiting astrocyte activation; regulate the secretion of brain-derived neurotrophic factor BDNF, improve the disordered neurotransmitter levels caused by alcohol, and regulate the secretion of neurotransmitters such as dopamine (DA), γ-aminobutyric acid (GABA), and serotonin (5-HT), thereby achieving the effect of preventing and alleviating alcohol-induced nerve damage.

[0070] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A Pediococcus pentosaceus PP90 for preventing and treating alcoholic nerve damage, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.32034.

2. The Pediococcus pentosaceus PP90 according to claim 1, characterized in that, The Pediococcus pentosaceus PP90 can grow and reproduce in an alcohol-containing culture medium with an alcohol volume concentration of 15%.

3. A bacterial agent for preventing alcohol-induced nerve damage, characterized in that, Includes Pediococcus pentosaceus PP90 as described in claim 1.

4. A fermentation product for preventing alcohol-induced nerve damage, characterized in that, It is prepared by fermentation of Pediococcus pentosaceus PP90 as described in claim 1.

5. The use of Pediococcus pentosaceus PP90 as described in claim 1, the bacterial agent as described in claim 3, or the fermentation product as described in claim 4 in the preparation of products that improve alcoholic nerve damage.

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