Pediococcus pentosaceus PP90 for preventing and treating alcoholic nerve injury and application thereof

Abstract

The invention relates to Pediococcus pentosaceus PP90 for preventing and treating alcoholic nerve injury and application of the Pediococcus pentosaceus PP90, the Pediococcus pentosaceus PP90 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.32034. The Pediococcus pentosaceus PP90 can be used for preventing and treating alcoholic nerve injury. The fermentation product of the pediococcus pentosaceus PP90 can degrade the alcoholic strength of alcohol, can generate ethanol dehydrogenase and acetaldehyde dehydrogenase, can improve the activity of nerve cells, can regulate the expression of cytokines, can inhibit the activation of astrocytes, can regulate the expression of brain-derived neurotrophic factors, and can inhibit the growth of the brain-derived neurotrophic factors. The compound can improve the disordered neurotransmitter level, and can be used for preparing products for preventing or improving alcoholic nerve injury.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to Pediococcus pentosaceus PP90 for preventing and treating alcoholic nerve damage and application thereof. Background Art

[0002] The main component of alcoholic beverages is ethanol, which has a small molecular weight and is both hydrophilic and lipophilic. It can cross the blood-brain barrier and has a direct toxic effect on nerve cells. The free radicals and other metabolites generated in alcohol metabolism will also aggravate the damage to the nervous system, leading to disordered secretion of neurotransmitters such as dopamine (DA), γ-aminobutyric acid (GABA), and 5-hydroxytryptamine (5-HT), imbalance between excitatory and inhibitory neurotransmitters, and a series of adverse consequences such as memory loss, difficulty concentrating, anxiety, and insomnia. Ethanol exposure can lead to the activation of immune signaling pathways in nerve cells, activate some glial cells such as astrocytes, and produce inflammatory factors such as tumor necrosis factor (TNF-α). Therefore, long-term alcoholism and alcohol abuse seriously affect the health of the body and brain. Most studies believe that even low doses of alcohol intake can lead to impaired brain motor control, judgment, and reaction time. Long-term and heavy drinking can lead to alcohol dependence and alcoholic brain damage, and ultimately lead to dual changes in the structure and function of the central nervous system. It is reported that about 50% of chronic drinkers suffer from chronic neurological deficits, resulting in a series of neurological diseases, including alcohol dependence, peripheral neuropathy, alcoholic cerebellar degeneration, alcoholic dementia, alcoholic optic neuropathy, etc.

[0003] Therefore, screening strains that can alleviate alcoholic nerve damage is of great significance for improving alcohol-induced neurological diseases in drinkers and preventing nerve damage caused by drinking. Summary of the invention

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

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

[0006] A Pediococcus pentosaceus PP90 for preventing and treating alcoholic nerve damage is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.32034. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the deposit time is September 23, 2024.

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

[0008] A bacterial agent for preventing and treating alcoholic nerve damage comprises the Pediococcus pentosaceus PP90.

[0009] A fermentation product for preventing and treating alcoholic nerve damage is prepared by fermenting the Pediococcus pentosaceus PP90.

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

[0011] The use of the Pediococcus pentosaceus PP90, the bacterial agent, or the fermentation product in the preparation of products for preventing and / or improving alcoholic nerve damage.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention screens a large number of microbial strains and finds that Pediococcus pentosaceus PP90 can grow and reproduce using alcohol as a carbon source, and its fermentation supernatant can degrade alcohol content, and has the ability to produce ethanol dehydrogenase and acetaldehyde dehydrogenase, thereby improving the vitality of nerve cells, regulating the expression of pro-inflammatory cytokines (TNF-α), inhibiting the activation of astrocytes, regulating the expression of brain-derived neurotrophic factor, and improving the disordered neurotransmitter level, thereby achieving the effect of improving and preventing alcoholic nerve damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the graph of the alcohol degradation ability of Pediococcus pentosaceus PP90;

[0015] Figure 2 This is the ability diagram of Pediococcus pentosaceus PP90 to produce ethanol dehydrogenase;

[0016] Figure 3 This is the ability diagram of Pediococcus pentosaceus PP90 to produce acetaldehyde dehydrogenase;

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

[0018] Figure 5 This is a graph showing the effect of Pediococcus pentosaceus PP90 on alcohol-induced astrocyte inflammatory factors;

[0019] Figure 6 This is a graph showing the effect of Pediococcus pentosaceus PP90 on alcohol-induced astroglial neurotrophic factor;

[0020] Figure 7 This is a graph showing the effect of Pediococcus pentosaceus PP90 on alcohol-induced astrocyte neurotransmitters;

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

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0024] In the present invention,

[0025] Pediococcus pentosaceus PP90, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit number CGMCCNO.32034, deposit date September 23, 2024;

[0026] Lactobacillus rhamnosus LGG: It is a published strain and is available to the public.

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

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

[0029] 1. Test methods

[0030] Take the frozen tubes of Pediococcus pentosaceus PP90 and Lactobacillus rhamnosus LGG single strains and activate them for three generations. The activated single strains (PP90 and LGG) were inoculated with 4% inoculation volume in MRS liquid medium containing 15% alcohol concentration, and the MRS liquid medium containing alcohol without bacterial solution was used as a blank control. The alcohol concentration and strain growth (OD 610nm Each group was repeated three times. The degradation capacity of alcohol was calculated according to the alcohol concentration. The results are shown in Figure 1 and Table 1;

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

[0032] 2. Test results

[0033] Table 1 Growth of each strain

[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 The data show that both Pediococcus pentosaceus PP90 and Lactobacillus rhamnosus LGG can tolerate alcohol and have the ability to degrade alcohol. Among them, Pediococcus pentosaceus PP90 has a better ability to degrade alcohol. Compared with the control group, the alcohol content of PP90 is degraded by 30%. This is because Pediococcus pentosaceus PP90 can grow and reproduce in an environment containing alcohol. This is also the reason why Pediococcus pentosaceus PP90 can degrade alcohol in the alcohol-containing MRS culture medium.

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

[0037] 1. Test methods

[0038] Take the frozen tubes of Pediococcus pentosaceus PP90 and Lactobacillus rhamnosus LGG and activate them for three generations. Inoculate the activated single strains (PP90 and LGG) into MRS medium, culture for 16 hours, centrifuge and remove the supernatant, wash the bacteria three times with sterilized saline, and adjust the viable count to 5×10 6 CFU / mL was added with 1 ml of lysis buffer and ultrasonically disrupted (ice bath, 200 W, ultrasonic for 3 s; interval 10 s, repeated 30 times), centrifuged at 12000 rpm and 4 °C for 10 min, and the supernatant was taken for the determination of acetaldehyde dehydrogenase and alcohol dehydrogenase. The results are shown in Figure 2-3 ;

[0039] 2. Test results

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

[0041] Example 3: Application of Pediococcus pentosaceus PP90 in preventing and treating alcoholic nerve damage

[0042] 1. Test methods

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

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

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

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

[0047] The cultured astrocytes were adjusted to a cell density of 1.5×10 5 The cells were inoculated into 96-well plates at 100 μg / mL and incubated in a 37°C 5% CO2 incubator for 24 h. After the cells adhered to the wall, the supernatant was discarded and 100 μL of cell culture medium containing 100 mM alcohol was added to induce the astrocyte model induced by alcohol.

[0048] (3) Cell viability assay

[0049] After the above cell models were incubated for 1 hour, 100 μL of cell culture medium containing 100 mM alcohol was added as the model group, 100 μL of fermentation supernatant of Pediococcus pentosaceus PP90 or Lactobacillus rhamnosus LGG containing 100 mM alcohol was added as the experimental group, and 100 μL of cell culture medium without alcohol was added as the control group. After incubation for 24 hours, 10 μL of CCK-8 solution was added to each well, and the incubation continued for 2 hours. The OD value was measured at a wavelength of 450 nm on an ELISA reader, and the cell viability was calculated; cell viability (%) = (experimental OD value / control OD value) × 100%, and the 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 density of 1.5×10 5 / mL, 1.5mL was inoculated in a 24-well plate and incubated in a 37℃, 5% CO2 incubator for 24h. After the cells attached to the wall, the supernatant was discarded, and the cells were pretreated with cell culture medium containing 100mM alcohol for 1h. Then, 200μL of cell culture medium containing 100mM alcohol was added as the model group, and 200μL of fermentation supernatant of Pediococcus pentosaceus PP90 or Lactobacillus rhamnosus LGG containing 100mM alcohol was added as the experimental group. The control group was not intervened by alcohol and incubated in a 37℃, 5% CO2 incubator for 24h.

[0053] (2) Sample collection and testing:

[0054] After 24 h of cell culture, the cell culture fluid of each group was collected into a sterile centrifuge tube and centrifuged at 4°C, 1000 r / min for 20 min. The supernatant was collected and tested according to the tumor necrosis factor-α (TNF-α), brain-derived neurotrophic factor (BDNF), dopamine (DA), γ-aminobutyric acid (GABA), and 5-hydroxytryptamine (5-HT) kits. The results are shown in Figure 5-7 .

[0055] 2. Test results

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

[0057] After treating astrocytes with 100 mM alcohol for 1 hour, different samples were given to treat the cells for another 24 hours. Figure 4 As shown in the figure, compared with the control group, the viability of astrocytes in the model group was significantly weakened, indicating that alcohol has a strong damaging effect on astrocytes. Compared with the model group, after adding the supernatant of Pediococcus pentosaceus PP90 and Lactobacillus rhamnosus LGG, the survival rate of astrocytes was significantly increased, among which Pediococcus pentosaceus PP90 had the best improvement effect. The results showed that Pediococcus pentosaceus PP90 can improve the viability of nerve cells and alleviate cell damage caused by alcohol.

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

[0059] When astrocytes are activated, they release a variety of pro-inflammatory cytokines and inflammatory mediators, promoting inflammatory responses and tissue damage, and producing toxic effects on neurons. Figure 5As shown in the results, the TNF-α secretion in the control group was 79.8pg / mL. In the astrocyte culture system of the model group, the content of inflammatory factor TNF-α in the cell culture fluid increased significantly due to the stimulation of alcohol, and the secretion was 141.3pg / mL. After adding the fermentation supernatant of different strains, the release amount decreased significantly. Among them, Pediococcus pentosaceus PP90 had the best improvement effect, with a TNF-α secretion of 102.4pg / mL. After intervention with Lactobacillus rhamnosus LGG, the TNF-α secretion was 116.9pg / mL. The above results show that Pediococcus pentosaceus PP90 can inhibit the activation of astrocytes by inhibiting the expression of proinflammatory cytokine TNF-α.

[0060] 3. Effect of Pediococcus pentosaceus PP90 fermentation supernatant on brain-derived neurotrophic factor in alcohol-induced 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, helps support the survival of existing neurons, and promotes the growth and differentiation of new neurons and synapses. In the brain, BDNF is active in the hippocampus, cortex, and basal forebrain, which are areas that are critical for learning, memory, and advanced thinking. In addition, various studies have shown that there may be a connection between BDNF and conditions related to synaptic transmission and long-term memory, which are related to some mental illnesses such as depression, schizophrenia, and Alzheimer's disease. Therefore, the present invention indicates its improvement effect on alcoholic nerve damage by studying the effect of Pediococcus pentosaceus PP90 on BDNF expression.

[0062] like Figure 6 As shown in the figure, the BDNF secretion in the control group was 111.2pg / mL. Compared with the control, the BDNF content in the astrocyte culture system of the alcohol-induced model group was significantly reduced, and the secretion was 74.6pg / mL. Compared with the model group, the release of BDNF increased significantly after intervention with the fermentation supernatant of different strains, among which Pediococcus pentosaceus PP90 had the best improvement effect, with a BDNF secretion of 92.6pg / mL, and Lactobacillus rhamnosus LGG had a secretion of 89.1pg / mL after intervention. The above results show that Pediococcus pentosaceus PP90 can improve alcoholic nerve damage by regulating the expression of brain-derived neurotrophic factor.

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

[0064] Alcohol intake can promote the release of dopamine DA in the brain, making the body addicted to alcohol. In contrast, alcohol intake can lead to a corresponding decrease in the content of γ-aminobutyric acid GABA. The intervention of the fermentation supernatant of the strain can regulate the disordered neurotransmitter levels, reduce the release of dopamine DA, promote the release of γ-aminobutyric acid GABA, and make the body's neurotransmitters tend to normal levels. The results showed that the DA secretion in the control group was 19.0pg / mL. Compared with the control, the DA release in the model group after alcohol stimulation was significantly increased, with a secretion of 25.0pg / mL. Compared with the model group, probiotics can significantly reduce DA secretion after intervention, among which Pediococcus pentosaceus PP90 had a better improvement effect, with a secretion of 17.9pg / mL after intervention, and Lactobacillus rhamnosus LGG had a secretion of 19.0pg / 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 in the control group was 4.17 μmol / L. Compared with the control group, the GABA release in the model group after alcohol stimulation was significantly reduced, and the secretion was 2.80 μmol / L. Compared with the model group, probiotic intervention can increase the secretion of GABA, among which the secretion after PP90 intervention was 3.44 μmol / L, and the secretion after LGG intervention was 3.20 μmol / L. The effect of PP90 is better than that of LGG. The secretion of 5-HT in the control group was 1.47 ng / mL. Compared with the control group, the release amount in the model group was reduced, and the secretion amount was 1.17 ng / mL. Compared with the model group, probiotic intervention can increase the secretion of 5-HT. The secretion amount after PP90 intervention was 1.34 ng / mL, and the secretion amount after LGG intervention was 1.23 ng / mL. Intervention with the fermentation supernatant of the strain can alleviate alcoholic nerve damage, which shows 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 the present invention has the following functions:

[0067] 1) It has the ability to degrade alcohol by up to 30%;

[0068] 2) It has the ability to produce ethanol dehydrogenase and acetaldehyde dehydrogenase, among which the ethanol dehydrogenase content of Pediococcus pentosaceus PP90 is 15nmol / min / 10 4 cell, the aldehyde dehydrogenase content level was 3.6nmol / 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 proinflammatory cytokine TNF-α, thereby inhibiting the activation of astrocytes; regulate the secretion of brain-derived neurotrophic factor BDNF, improve the level of neurotransmitters disordered by alcohol, and regulate the secretion of neurotransmitters such as dopamine (DA), γ-aminobutyric acid (GABA), and 5-hydroxytryptamine (5-HT), thereby achieving the effect of preventing and alleviating alcoholic nerve damage.

[0070] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in 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 in the General Microbiology Center of China Microorganism Culture Collection Administration with the accession number CGMCC NO.32034.

2. The Pediococcus pentosaceus PP90 for preventing and treating alcoholic nerve damage according to claim 1, characterized in that: The Pediococcus pentosaceus PP90 can grow and reproduce in an alcohol-containing culture medium, in which the volume concentration of alcohol is 15%.

3. A bacterial agent for preventing and treating alcoholic nerve damage, characterized in that: The method comprises the Pediococcus pentosaceus PP90 as claimed in claim 1.

4. A fermentation product for preventing and treating alcoholic nerve damage, characterized in that: The product is prepared by fermenting the Pediococcus pentosaceus PP90 as claimed in claim 1.

5. The fermentation product according to claim 4, characterized in that The fermentation product can degrade the alcohol content of alcohol, produce alcohol dehydrogenase and acetaldehyde dehydrogenase, improve the activity of nerve cells, regulate the expression of cytokines, inhibit the activation of astrocytes, regulate the expression of brain-derived neurotrophic factor, and improve the disordered neurotransmitter level.

6. Use of the Pediococcus pentosaceus PP90 according to claim 1, the bacterial agent according to claim 3 or the fermentation product according to claim 4 in the preparation of a product for preventing and / or improving alcoholic nerve damage.

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