CN110499263B - A kind of Lactobacillus plantarum X7022 and its application - Google Patents

Abstract

The invention discloses Lactobacillus plantarum X7022 and its application in preparing food, health care product, medicine and medicine combination for relieving and improving senile memory decline. The invention also discloses the application of Lactobacillus plantarum X7022 in the preparation of intestinal microecological preparations. The invention also discloses a microecological preparation with good intestinal colonization ability. The beneficial effects are: Lactobacillus plantarum X7022 has good intestinal colonization ability and good ability to relieve and improve senile memory, and has broad market application value.

Description

A kind of Lactobacillus plantarum X7022 and its application

technical field

The invention belongs to the technical field of functional food, and specifically relates to a Lactobacillus plantarum X7022 and its application.

Background technique

Lactic acid bacteria refers to a general term for a class of non-spore-positive, Gram-positive bacteria whose main product of fermented carbohydrates is lactic acid. Among them, the ones that are beneficial to human health are called probiotics. Probiotics are a huge group of bacteria. When probiotics are dominant, the human body will maintain a healthy state. Control endotoxin, inhibit the growth and reproduction of spoilage bacteria in the intestine and the production of spoilage products, produce nutrients, stimulate tissue development, and thus have an effect on the nutritional status, physiological function, tumor occurrence, and aging process of the body. The number of lactic acid bacteria will gradually decrease as people grow older. When people get old or sick, the number of lactic acid bacteria may drop by 100 to 1000 times, and the elderly will almost disappear at the end of life.

Senile memory disorder is mainly manifested as memory decline, cognitive impairment and low learning ability due to aging. There are often many factors in the pathogenesis of the disease. At present, there is still no ideal treatment method and drug for this disease, and the purpose of treatment is mostly concentrated. In order to control the accompanying psychopathological symptoms and improve cognitive function, the drugs used include antidepressant and psychiatric drugs; drugs that act on neurotransmitters and brain metabolism activation, etc., but such drugs often only target one of them. There are also certain difficulties in determining the cause of the disease. At present, population aging has become an extremely serious social problem in my country. Due to the rapid development of social economy and the continuous improvement of people's living standards, more and more people tend to adjust their diets to prevent senile memory decline in advance.

Due to the complex relationship between the gut microbiota and the host, gastrointestinal function can affect the brain. The gut microbiota produces or alters neurotransmitters through metabolites, activates immune signaling pathways, and acts on the vagus nerve and immune system. structure and function. Therefore, the intestinal flora forms a new pathway for two-way interactive communication with the enteric nervous system and the central nervous system. The intestinal flora interacts with the brain through the vagus nerve to connect the innervation of the intestine, both from the spinal cord and the vagal afferent nerve. In addition to the innervation of external nerves, there is innervation of intestinal neurons themselves. Multiple reflexes are formed between the extrinsic nerve and the intrinsic nerve to maintain the normal physiological activity of the intestine, regulate the response of the intestine to various stimuli, and transmit various information felt by the intestine to the brain. Studies have found that changes in the intestinal flora of animals, such as taking probiotics, aseptic feeding, intestinal pathogen infection, and antibiotic treatment, can all cause significant neurobiochemical changes in animals. The probiotic Lactobacillus rhamnosus can alter the normal expression levels of γ-aminobutyric acid receptors in the hippocampus, hypothalamus, amygdala and prefrontal lobe through the vagus nerve, thereby reducing anxiety behavior and memory impairment in stressed mice, germ-free animals Excessive stress response is associated with abnormal expression of brain-derived neurotrophic factor in related brain regions; Citrobacter intestinalis infection leads to increased expression of c-FOS protein in vagus ganglia and increased anxiety behavior in mice; antibiotic treatment can increase Exploratory behavior in mice and increased levels of BDNF in the hippocampus and amygdala. Therefore, changes in the gut microbiota are not only related to the development and function of the central nervous system, but may also be closely related to cognition and memory.

BDNF: Brain-derived neurotrophic factor. BDNF is a protein synthesized in the brain. It is mainly distributed in the cerebral cortex and hippocampus. It can prevent the death of neurons from injury, improve the pathological state of neurons, promote the regeneration of damaged neurons and differentiate the pathological state, and promote the damaged neurons. Regeneration and differentiation are important regulators of synaptic regeneration and synaptic plasticity mechanisms related to learning and memory in the central nervous system, and are involved in regulating long-term potentiatio (LTP).

DCX: Double adrenocortical hormone. As a microtubule-associated protein, it is widely expressed in the development of the mammalian nervous system and is an essential element of neuronal migration and differentiation. In neurogenesis in adult animals, the expression of DCX begins with the generation of nerve cells and is closely related to cognition.

iNOS: nitric oxide synthase. iNOS is an isoenzyme, which is located in immune cells, glial cells, neurons and vascular smooth muscle cells. It is not expressed under ground state conditions. The activation of iNOS can continuously generate a large amount of NO and exert a cytotoxic effect.

CREB-1: Cyclic adenylate response element binding protein. BDNF binds to its receptor TrkB, TrkB acquires catalytic activity, activates its own tyrosine kinase, leading to autophosphorylation, and the activated receptor can stimulate signal transduction and trigger intracellular signaling cascades, including its downstream signaling molecule CREB, Thereby increasing synaptic connections and improving learning and memory. Activation of CREB is a key step in the MAPK signal transduction pathway, which can initiate the expression of proteins involved in the maintenance of long-lasting LTP and long-term memory. At least 100 downstream target genes are known to be regulated by p-CREB, so it is believed that CREB may play a switch role in learning and memory. The abnormal expression and activity changes of CREB are involved in the pathophysiological process of Alzheimer's disease and are related to the neuroprotective mechanism. And some studies have shown that improving the learning and memory ability of mice is related to the induction of CREB expression.

SUMMARY OF THE INVENTION

The inventor of the present case isolated a strain from the stinky tofu brine, which was identified as Lactobacillus plantarum X7022. In the process of researching its function, it was found that the strain could alleviate and improve the aging-related memory decline. problem, and has good intestinal colonization ability. Therefore, the first object of the present invention is to provide a Lactobacillus plantarum X7022. The second object of the present invention is to provide a kind of application of Lactobacillus plantarum X7022 in the preparation of food, health care product, medicine, and drug combination for relieving and/or improving senile memory loss; the third object of the present invention is to provide a kind of A product for relieving and improving senile memory loss to alleviate and improve the problem of senile memory loss; the fourth purpose of the present invention is to provide the application of Lactobacillus plantarum X7022 in the preparation of intestinal microecological preparations; The fifth objective is to provide a microbial preparation with good intestinal colonization ability.

In order to achieve the above object, the present invention provides the following technical solutions:

The first object of the present invention is to provide a Lactobacillus plantarum X7022, the preservation number of the Lactobacillus plantarum X7022 is CCTCC NO: M2016505.

The second object of the present invention is to provide an application of the above-mentioned Lactobacillus plantarum X7022 in the preparation of foods, health products, medicines and pharmaceutical combinations for relieving and improving senile memory loss.

The 3rd object of the present invention is to provide a kind of product that alleviates and improves senile memory decline, and this product takes the bacterium liquid or bacterium powder of described Lactobacillus plantarum X7022 as active component.

According to the present invention, the products for relieving and improving senile memory loss include food, health products, medicines and pharmaceutical compositions.

Further, the Lactobacillus plantarum X7022 bacterial powder is prepared by freezing and freeze-drying, contains a viable count of 1×10 ⁷ to 1×10 ¹¹ CFU/g, and has good environmental stability.

Further, the Lactobacillus plantarum X7022 bacterial liquid is obtained by diluting the Lactobacillus plantarum X7022 freeze-dried bacterial powder with sterile physiological saline.

Further, Lactobacillus plantarum X7022 bacterial powder is stable when the ambient temperature is normal temperature, 4°C, and -20°C.

The fourth object of the present invention is to provide an application of the Lactobacillus plantarum X7022 in the preparation of intestinal microecological preparations.

The fifth object of the present invention is to provide a kind of microbial preparation, and this microbial preparation has good intestinal colonization ability, and the bacterial liquid or bacterial powder of the above-mentioned Lactobacillus plantarum X7022 is used as active component.

Further, the Lactobacillus plantarum X7022 bacterial powder is prepared by freezing and freeze-drying, contains a viable count of 1×10 ⁷ to 1×10 ¹¹ CFU/g, and has good environmental stability.

Further, the Lactobacillus plantarum X7022 bacterial liquid is obtained by diluting the Lactobacillus plantarum X7022 freeze-dried bacterial powder with sterile physiological saline.

Further, Lactobacillus plantarum X7022 bacterial powder is stable when the ambient temperature is normal temperature, 4°C, and -20°C.

The beneficial effects of the present invention are:

1. Lactobacillus plantarum X7022 has good intestinal colonization ability and can be used to prepare microecological preparations, especially intestinal microecological preparations.

2. Lactobacillus plantarum X7022 has a good effect of relieving and improving senile memory loss, and can be used to prepare food, health care products, medicines and drug combinations for improving senile memory loss. Therefore, Lactobacillus plantarum X7022 has broad market application value as the main fermentation strain in the food industry.

Description of drawings

Fig. 1 is a graph showing the results of detecting positive bacteria in fecal samples after gavage of Lactobacillus plantarum X7022 in Example 3.

FIG. 2 is a graph showing the results of the behavioral trajectories of the Mirros water maze experiment on the first day of training and the sixth day of testing in each group of mice in Example 4. FIG.

Fig. 3 is a graph showing the change of the incubation period of the water maze training stage in Example 4 by gavage of Lactobacillus plantarum X7022, the control group and the model group.

Figure 4 is a graph showing the changes in the average exercise level during the training stages of the gavage of Lactobacillus plantarum X7022, the control group and the model group in Example 4.

FIG. 5 is a graph showing the results of HE-stained paraffin sections of mouse brain tissue in each group in Example 4. FIG.

FIG. 6(A) is a graph showing the results of the expression of each protein in the hippocampus of mice in each group of Example 4. FIG.

FIG. 6(B) is a graph showing the results of quantitative optical density analysis of BDNF protein in the hippocampus of mice in each group of Example 4. FIG.

FIG. 6(C) is a graph showing the results of quantitative optical density analysis of CREB1 protein in the hippocampus of mice in each group of Example 4. FIG.

FIG. 6(D) is a graph showing the results of quantitative optical density analysis of DCX protein in the hippocampus of mice in each group of Example 4. FIG.

FIG. 6(E) is a graph showing the results of quantitative optical density analysis of iNOS protein in the hippocampus of mice in each group of Example 4. FIG.

FIG. 7 is a graph showing the results of the determination of superoxide dismutase enzyme activity in the supernatant of mouse brain homogenate in each group of Example 4. FIG.

FIG. 8 is a graph showing the results of determination of glutathione content in the supernatant of mouse brain homogenate in each group of Example 4. FIG.

FIG. 9 is a graph showing the results of the determination of glutathione peroxidase enzymatic activity in the supernatant of mouse brain homogenate in each group of Example 4. FIG.

Figure 10 is a graph showing the results of the determination of the content of malondialdehyde in the supernatant of the mouse brain homogenate of each group of Example 4.

FIG. 11(A) is a heat map of species abundance clustering at the phylum level of the species annotation and taxonomic analysis of the gut microbes of each group of mice in Example 4. FIG.

FIG. 11(B) is a heat map of species abundance clustering at the genus level of the species annotation and taxonomic analysis of the gut microbes of each group of mice in Example 4. FIG.

FIG. 12(A) is a histogram of species distribution at the phylum level of the species annotation and taxonomic analysis of gut microbes in each group of mice in Example 4. FIG.

FIG. 12(B) is a histogram of species distribution at the genus level of the species annotation and taxonomic analysis of the gut microbes of each group of mice in Example 4. FIG.

FIG. 13 is the Venn diagram of the OTU analysis of the intestinal microbes of the mice in each group of Example 4. FIG.

FIG. 14 is the Shannon index curve of the Alpha diversity analysis of the intestinal microbes of the mice in each group of Example 4. FIG.

FIG. 15 is a PCA analysis diagram of the Beta diversity analysis of intestinal microorganisms in each group of mice in Example 4. FIG.

Figure 16 is a circle diagram of the chr genome of Lactobacillus plantarum X7022.

Figure 17 is a circle map of the plasmid1 genome of Lactobacillus plantarum X7022.

Figure 18 is a circle map of the plasmid2 genome of Lactobacillus plantarum X7022.

Figure 19 is a circle map of the plasmid3 genome of Lactobacillus plantarum X7022.

Figure 20 is a circle map of the plasmid4 genome of Lactobacillus plantarum X7022.

Among them, the plus sign (+) in Fig. 6(A), Fig. 6(B), Fig. 6(C), Fig. 6(D), Fig. 6(E), Fig. 7, Fig. 8, Fig. 9 and Fig. 10 Relevant treatment is given, and a minus sign (-) represents no treatment. For example, the control group was administered with D-galactose (-) by intragastric administration of X7022 (-), which means that neither D-galactose nor intragastric administration of X7022 was administered.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually carried out under normal conditions.

1. Lactobacillus plantarum X7022 (Lactobacillus plantarum X7022) of the present invention is isolated from stinky tofu brine, and has been patented at the China Type Culture Collection on September 21, 2016. The deposit number is CCTCC NO: M2016505, and the deposit address is Wuhan, China.

2. Materials and reagents for the following examples

(1) The formula of MRS substratum is: get 48.3g MRS (lactic acid bacteria substratum), add in 1L deionized water, distribute after dissolving, 115 ℃ of high pressure sterilization 20min standby.

(2) The formula of the PBS buffer is: potassium dihydrogen phosphate (KH ₂ PO ₄ ): 0.27 g; disodium hydrogen phosphate (Na ₂ HPO ₄ ): 1.42 g; sodium chloride (NaCl): 8 g; potassium chloride (KCl): 0.2g; add about 800mL of deionized water and stir to dissolve, then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally set the volume to 1L.

(3) c-FOS: proto-oncogene

(4) HE staining: hematoxylin-eosin staining

(5) 4% PFA: 4% paraformaldehyde

(6) SDS-PAGE: SDS-polyacrylamide gel electrophoresis

(7) Tris: tris(hydroxymethyl)aminomethane

(8) TBS: Tris-buffered saline

(9) TBST: TBS+Tween buffer

(10) TrkB: brain-derived neurotrophic factor receptor

(11) PMSF: phenylmethylsulfonyl fluoride

(12) PVDF membrane: polyvinylidene fluoride membrane

(13) BDNF: Brain-Derived Neurotrophic Factor

(14) DCX: Double adrenocortical hormone

(15) iNOS: nitric oxide synthase

(16) CREB-1: Cyclic adenylate response element binding protein

(17) AD: Alzheimer’s Disease

(18) LTP: (Long-term potentiatio) long-term potentiation

(19) NMDS: Non-metric Multidimensional Scaling Analysis

(20) SOD: superoxide dismutase

(21) GSH: Glutathione

(22) NADPH: reduced coenzyme II

(23) NADP ⁺ : Coenzyme II

(24) MDA: Malondialdehyde

(25) Heatmap: heat map

3. Other materials and equipment in the following examples are commercially available.

4. Whole-genome sequencing identification of Lactobacillus plantarum X7022: The whole genome of Lactobacillus plantarum X7022 was sequenced and identified by using Pacbio RSII high-throughput sequencing technology. The sequencing identification results are shown in Figure 16-20. Figure 16 is a circle map of the chr genome of Lactobacillus plantarum X7022. Figure 17 is a circle map of the plasmid1 genome of Lactobacillus plantarum X7022. Figure 18 is a circle map of the plasmid2 genome of Lactobacillus plantarum X7022. Figure 19 is a circle map of the plasmid3 genome of Lactobacillus plantarum X7022. Figure 20 is a circle map of the plasmid4 genome of Lactobacillus plantarum X7022. The abbreviations of the COG categories and the descriptions of the COG categories in Figure 16-20 are shown in Table 1.

Table 1 COG category abbreviations and COG categories

Example 1 Activation of Lactobacillus plantarum X7022

Pick out part of the bacterial liquid from the glycerol tube of Lactobacillus plantarum X7022, streak it on the MRS solid plate, cultivate at 37°C for 36-48h, pick a single colony on the plate and inoculate it in 10mL MRS medium, incubate at 37°C for 18h, press 2% of the inoculum was added to 10 mL of MRS culture for activation for 18 h, the obtained bacterial solution was centrifuged at 3000 rmp for 10 min, and the obtained bacterial cells were mixed with 10 mL of PBS buffer to obtain the test bacterial solution.

The preparation of embodiment 2 Lactobacillus plantarum X7022 freeze-dried bacteria powder

In a sterile operating table, pick out a small amount of bacterial liquid from the glycerol tube of Lactobacillus plantarum X7022, streak it on the MRS solid plate, cultivate at 37°C for 36-48 hours, pick a single colony on the plate and inoculate it in 10 mL of liquid MRS medium , after anaerobic culture at 37°C for 18h, inoculated into a 10L fermenter containing MRS medium, anaerobic fermentation for 24h, and then centrifuged at 6000rmp for 10min to collect bacteria, and washed twice with sterile phosphate buffer (pH=6.4). Centrifuge and mix the resulting mash with skim milk powder containing 18%-22%, 4-9% trehalose, 2-4% sodium glycinate, 1-5% glycerol and 0.1-1% cysteine hydrochloride The sterilized lyophilized protective agent is mixed and emulsified, and the bacterial puree and the lyophilized protective agent are in different weight ratios. After the bacteria puree and the protective agent are fully suspended, the emulsified mixture is pre-frozen at -45°C for 4-10 hours, and then placed in a freeze dryer for 20-26 hours to obtain freeze-dried bacteria powder. When the obtained bacterial paste was mixed and emulsified with a sterilized freeze-drying protectant containing 20% skimmed milk powder, 6% trehalose, 2.5% sodium glutamate, 2% glycerol and 0.5% cysteine hydrochloride, the bacterial paste was emulsified. The weight ratio to the freeze-dried protective agent is 1:2. After the bacterial paste and the protective agent are fully suspended, the emulsified mixture is pre-frozen at -45 °C for more than 4 hours, and then placed in a freeze-drying machine for freeze-drying for 24 hours to obtain a freeze-dried solution. Dry bacterial powder, the viable bacteria count in the bacterial powder was determined to be 3×10 ¹⁰ CFU/g.

Example 3 Intestinal viability experiment of Lactobacillus plantarum X7022

1. Preparation of gavage bacterial solution

Weigh part of Lactobacillus plantarum X7022 bacteria powder in Example 2 and store at 4°C, set according to Table 2 respectively, dilute to required bacterial concentration with sterile physiological saline, obtain the required bacterial liquid for CFDA-SE fluorescent staining marking .

Table 2 Gavage settings

Gastric fluid name Bacterial concentration in gavage fluid (CFU/mL) Remark X7022 high concentration 1×10⁹ Experimental group A X7022 low concentration 1×10⁶ Experimental group B control group 0 Experimental group C

First, the cells were collected by centrifugation, and the cells were suspended in 15 mL centrifuge tubes with 1 mL of staining buffer. The bacterial concentrations were adjusted to 1×10 ⁹ CFU/mL and 1×10 ⁶ CFU/mL, and 500 μL of DMSO was used to dissolve the CFDA-SE fluorescent probe. Needle to obtain CFDA-SE staining storage solution, add 16 μL staining storage solution to 4 mL staining buffer to obtain staining working solution, add the above staining working solution to a 15 mL centrifuge tube, gently pipette and mix, incubate at 37 °C for 20 min, and centrifuge. Add 10 mL of PBS buffer to the tube, invert at room temperature to mix evenly, stop the labeling reaction, take the supernatant by centrifugation at room temperature, wash once with 5 mL of PBS buffer, add 5 mL of PBS buffer, centrifuge at room temperature to remove the supernatant, and add 1 mL of PBS buffer to resuspend the tube. Suspend to obtain the CFDA-SE fluorescent probe-labeled bacterial solution required for gavage.

2. Preparation of experimental animals

(1) The conditions of the animal room are as follows: keep the temperature at 20±2°C, the humidity at 50±5%, and change the bedding every three days. Control 12h light, 12h dark cycle standard, turn on the lights at 7 am every day. Mice were given free access to water and food during the experiment, and their body weights were measured every three days. Before the experiment, a one-week acclimation period was given, during which the basal diet was fed.

(2) Male ICR mice weighing 25-30 g were randomly divided into three groups, with 7 mice in each group, numbered as group A, group B, and group C, respectively.

3. Animal gavage method

The body weight of each mouse was weighed before the gavage, and the gavage was carried out according to the measurement of 1 mL/100g (bacteria liquid volume/mice body weight), and the gavage was regularly administered at 9:00 am every day for 7 days. The concentration of Lactobacillus plantarum X7022 in group A was 1×10 ⁹ CFU/mL, the bacterial concentration in group B was 1×10 ⁶ CFU/mL, and the concentration in group C was sterile saline. During the gavage period, except for fixed-point gavage, they were normally fed with regular feed and drinking water.

4. Animal waste collection

During the gavage period and within seven days after the gavage was stopped, fresh fecal samples were collected from the mice in each group. The sampling amount of each group was 10 g.

5. Flow cytometry to detect the proportion of Lactobacillus plantarum X7022 in stool samples

(1) Isolation of bacteria in fecal samples

Add 10 mL of sterile normal saline to the fresh fecal samples collected on the same day, shake until completely mixed, take three layers of gauze to filter the turbid liquid, repeat twice, take 5 mL of sterile normal saline to wash the upper layer residue of the filtered gauze, and centrifuge at 3000 rmp at room temperature for 15 min , discard the supernatant, add 10 mL of sterile normal saline to resuspend the obtained bacterial cells, centrifuge at 3000 rmp for 15 min, discard the supernatant liquid, and then separate the bacterial cells in the stool samples from the obtained sample to be tested.

(2) The proportion of positive bacteria labeled with fluorescent probes detected by flow cytometry

The flow cytometer collects part of the bacteria ⁽ 105) from the samples to be tested, and under the conditions of EX=494 nm and Em=521 nm, the proportion of positive bacteria labeled with fluorescent probes is obtained. The experimental results are shown in Figure 1.

Explanation of intestinal viability: In order to make the lactic acid bacteria ingested into the body to play a probiotic function, the bacteria should maintain a high survival rate in the intestinal tract.

It can be seen from Figure 1 that the proportion of positive bacteria in the bacteria isolated from the feces remained between 45% and 35% within 1-7 days during the gavage period of the high concentration group, and reached a peak on the second day, and the bacteria content gradually increased. will decrease, and it will remain between 25%-10% within four days after the gavage is stopped. It exists more stably in the intestinal tract and has better intestinal viability. In the low-dose group, feces were separated within 1-7 days of the high-concentration group during the gavage period. The proportion of positive bacteria in the obtained bacteria remained between 35% and 10%, and reached the peak on the second day.

Conclusion: Lactobacillus plantarum X7022 can exist stably in the intestine and has good intestinal survival ability; the proportion of positive bacteria in the high concentration group is higher than that in the low concentration group, indicating that the intestinal survival ability of the high concentration group is higher than that of the low concentration group, indicating that the plant The gut viability of Lactobacillus X7022 was dose-dependent.

Embodiment 4 Lactobacillus plantarum X7022 improves senile memory decline animal experiment

1. The strain of Lactobacillus plantarum X7022 activated in Example 1 was transferred in 10mL MRS medium by 2% inoculum, 37° C., 200rmp cultured for 18h, the bacterial concentration was 10 ^9-10 CFU/mL, the obtained bacterial liquid After centrifugation at 3000 rmp for 10 min, the supernatant was discarded, the cells were resuspended in 5 mL of sterile normal saline, and refrigerated at 4°C for intragastric administration on the same day for later use.

2. Preparation of experimental animals

30 20-week-old ICR male mice, 25±3g/mice, after 1 week of adaptation to the experimental environment, subcutaneous injection of D-galactose at a dose of 100 mg/kg, normal saline in the control group, once a day for 10 weeks, Behavioral tests were performed at 5 weeks after modeling to determine whether the senile memory decline model was successful. Twenty mice that were successfully modeled were selected and randomly divided into groups A and B. The experimental group of group A was given gastric gavage from the 5th week:

A group: Lactobacillus plantarum X7022 group, every day with the measurement of 1mL/100g (bacterial liquid volume/mouse body weight) to carry out gavage and continue to use D-galactose to carry out the subcutaneous injection of 100mg/kg;

Group B: model group (D-galactose model group), the mice with successful modeling were given 1mL/100g of sterile normal saline by gavage every day and continued to use D-galactose for subcutaneous injection of 100mg/kg;

Group C: control group, normal mice were given 1 mL/100 g of sterile normal saline by gavage every day.

During the experiment, the mice in each group were weighed and recorded every day, and the doses of gavage and injection were adjusted according to the body weight. The mice in each group were allowed to eat and drink freely.

3. Result analysis

(1) Behavioral test (Morris water maze)

The experimental device consists of a stainless steel circular pool, an escape platform and a data acquisition and analysis system. The diameter of the circular pool is about 1.5cm, the height is 60cm, and the diameter of the escape platform is 10cm. Fill the pool with water. The water temperature was maintained at 22-23 °C, and the water surface was covered with white floats to obstruct the escape of mice. The pool is equally divided into four quadrants according to the four directions of east, south, west and north, namely NE area, SE area, NW area and SW area. The escape platform is placed in the center of the second quadrant. The data acquisition and analysis system is used to record data such as the swimming trajectories of the mice during the experiment. In the training phase, the swimming time of the mice is set to 1 minute. The mice find the escape platform and stay on the escape platform for 5 seconds and then stop the timing automatically. Guide the mice to the escape platform and stay there for 10-15 s. In the test experiment, the swimming time of the mice was still set to one minute, but the stop time was no longer set, that is, the mice had to swim freely for one minute.

The first day of the experiment was the familiarization stage. The escape platform was 2 cm higher than the sleep platform, a flag was placed in the center of the escape platform, and the mice entered the water to swim freely facing the pool wall to familiarize themselves with the environment and adapt to the water temperature. If the mouse can successfully find the escape platform, make sure that the mouse stays on the platform for 10-15s. If the mouse does not find the escape platform, guide it to climb the escape platform and stay on the escape platform for 15-20s.

The second day of the experiment was the formal training phase, which was performed twice a day for a total of 5 days, and the training time period was fixed every day. Pull out the flag on the platform and enter the water in the N and W directions respectively. Similarly, if the mouse can find the escape platform within one minute and stop automatically after staying on the escape platform for 5s, this is the latency period for the mouse to find the escape platform. If the mouse does not find the escape platform within one minute, guide it to the escape platform and stay on the escape platform for 15-20s to record the surrounding environment. The sixth day of the experiment was the testing phase. The platform was removed, and the time was only set for one minute to test a series of parameters such as the number of times the mice entered the platform area and the residence time in the target quadrant. In the experiment, a video tracking and acquisition system was used for data acquisition and trajectory analysis. The result is shown in Figure 2-4.

Figure 2 shows the movement trajectories of the experimental mice in the control group, the D-galactose model group, and the model group by intragastric administration of X7022 on the first day of training and the movement trajectories on the last day of the test after the mice went through the platform to search for learning and memory.

The results in Figure 2 show that the behavioral trajectories of the mice in the control group on the first day of training were more cluttered. The behavioral trajectories presented in the testing phase on the last day after the platform searched for learning and memory were mostly concentrated in the second quadrant where the platform was located and crossed the escape platform many times. The content guided by the training stage has obvious learning and memory ability; the behavioral trajectory of the mice in the D-galactose injection model group on the first day of training is disorganized. Compared with the control group, the learning and memory ability of the guided behavior in the training phase was poor, so it can be judged that the model establishment was more successful; the mice in the model group were perfused The behavioral trajectory of the experimental group where Lactobacillus plantarum X7022 was located on the first day of training was more chaotic. After the mice searched for learning and memory on the platform, the behavioral trajectory in the testing phase was more targeted than that of the model group, and the trajectories were no longer in a chaotic state. The second quadrant where the escape platform is located and the second quadrant stays for a longer time, compared with the model group, the number of times that the model group crosses the escape platform increases significantly.

Conclusion: Figure 3 shows the change of the latency in the training phase of the water maze. The latency is the time before finding the escape platform. The shorter the latency, the stronger the learning ability of the mice and the clear memory of the platform orientation and path.

The results in Figure 3 show that the incubation period of the model group did not change significantly with the increase of the training period, while the control group decreased significantly, and the difference was extremely significant. The seek time is only 10-20s.

Conclusion: The Lactobacillus plantarum X7022 strain has obvious improvement effect on memory decline and learning ability decline.

Figure 4 is a graph of the average exercise level change during the training phase. The results in Figure 4 show that, compared with the model group, the model mice fed with lactic acid bacteria had a higher average exercise level and higher average exercise speed.

Conclusion: It is speculated that Lactobacillus plantarum X7022 strain can restore exercise ability and physical fitness well.

(2) Histopathology

The hippocampus is a key area of learning and memory, directly involved in the process of spatial positioning and learning. The head of the hippocampus is mainly folded from the CA1 area. Studies have shown that for long-term memory transferred to the cerebral cortex, if the hippocampus is inhibited in the memory retrieval stage The activity of the CA1 area of the body will directly lead to the generation of memory impairment, and the CA1 area is the most sensitive to damage such as hypoxia, and is also called the vulnerable area. Therefore, the head of the hippocampus is also the most prone to lesions.

Materials: Mice were anesthetized by intraperitoneal injection of 10% trichloroacetaldehyde at a dose of 0.1 mL/100 g, and then sacrificed by cervical dislocation. The skin of the abdomen of the mice was sterilized with alcohol cotton balls, and a needle was filled with PBS (1×). , another sucked 4% PFA fixative solution (4 ℃), set aside for use. Carefully insert a needle with PBS (1×) into the left ventricle and dissect the right ventricle to drain the fluid. Slowly but continuously infuse PBS (1×) into the heart (if perfusion is normal, blood rich organs such as liver, spleen and kidney will turn grayish white. When most blood has flowed out, remove the needle and aspirate 4% PFA fixative The needle was inserted into the same needle entry point of the left ventricle, and the mice were slowly perfused with 20 mL of fixative. After perfusion, the head of the mouse was taken, the skin was cut to expose the skull, the orbits on both sides were clamped with large forceps, and the midline of the skull was slightly cut out with ophthalmic scissors. , Gradually remove the skull and the meninges and blood vessels in the upper part of the whole brain from bottom to top, take out the whole brain from the hydrangea with forceps, put it into a labeled 10mL centrifuge tube containing 4% PFA fixative, and store at 4°C.

The mouse brain was subjected to the steps of fixation, washing and dehydration, clearing, dipping in wax, embedding, sectioning and patching, dewaxing, staining, dehydration, clearing, and mounting to prepare paraffin sections, and then HE (hematoxylin- eosin) staining. The results are shown in Figure 5.

As shown in Figure 5, after HE staining, it can be seen that there are 3 and 4 layers of pyramidal neurons in the hippocampal CA1 area of normal brain tissue, which are closely arranged and evenly distributed, with dark cytoplasm and clear nucleolus and large volume; D - After galactose injury, the arrangement of neurons is loose and disordered, the hippocampal cells are loose, the neurons either die or disappear, or their morphology changes, the cytoplasm is light in color and the nucleolus is small. After the model mice were administrated with Lactobacillus plantarum X7022, the neuron damage was recovered to a certain extent, and the hippocampal cells of the Lactobacillus plantarum X7022 group were clear, arranged in an orderly manner, and basically normal in shape.

CONCLUSION: Lactobacillus plantarum X7022 strain has obvious promoting effect on the recovery of neurons.

(3) Western blot

SDS-PAGE electrophoresis buffer (5×): Weigh 15.1 g of Tris, 94 g of glycine, and 5.0 g of sodium dodecyl sulfonate (SDS), and dilute to 1000 mL with ddH ₂ O and store for later use.

Transfer solution (10×): 151.1 g glycine, 30.3 g Tris, and make up to 1000 mL with ddH ₂ O;

TBS (10×): 24.2g Tris, 80g NaCl and make up to 1000mL with ddH ₂ O;

Transfer buffer: 100mL transfer solution (10×)+200mL methanol+700mL ddH ₂ O

TBST (1×): 100 mL (10×) TBS + 1 mL Tween-20 + 900 mL ddH ₂ O

Mice were anesthetized by intraperitoneal injection of 10% trichloroacetaldehyde at a dose of 0.1 mL/100 g, and then sacrificed by cervical dislocation. The skin of the mouse head was disinfected with alcohol cotton balls, and the mouse head was taken and cut out with ophthalmic scissors. From the midline of the skull, gradually remove the meninges and blood vessels in the upper part of the skull and the whole brain from bottom to top, remove the whole brain from the hydrangea with forceps, separate the left and right brains, and gradually peel off the excess brain tissue to obtain the mouse hippocampus, which was rapidly cooled in liquid nitrogen Store in -80℃ refrigerator and use within three months.

Mix PMSF and RIPA protein lysate at a ratio of 1:100, put it on ice for 30 minutes, weigh 50 mg of tissue, add it to a mortar and grind it into powder, transfer it to a 1.5 mL EP tube, and press tissue: lysate = Add lysate and PMSF at a ratio of 1:10, carefully pipette on ice, and repeat for about 200 times. Centrifuge at 12000rmp at 4°C for 15min, take the supernatant into a new EP tube, and record the volume of the supernatant taken. Measure the protein concentration by BCA method, add an appropriate amount of loading buffer, denature in a boiling water bath at 100 °C for 10 min, store in -80 °C refrigerator, and use within 1 month.

SDS-PAGE electrophoresis: After preparing the protein gel, fix the glass plate on the glass plate, fix it on the electrophoresis rack, and put it into the electrophoresis tank for leak detection. Calculate the sample loading amount according to the protein concentration. Adjust to 120v at the junction until the bromophenol blue runs near the bottom of the rubber.

Transfer membrane: after electrophoresis, take out the gel block, cut off the concentrated gel, select a suitable gel block according to the size of the target protein, put it into the transfer solution, cut out the PVDF membrane and filter paper, soak the PVDF membrane in methanol for 10 minutes, and 2 pieces on each side of the filter paper. A total of 4 sheets. Put the electric transfer clip into the electric transfer tank and place an ice box in the tank, fill up the pre-cooled film transfer liquid, adjust the current to 280mA, and transfer the film with constant current for 90min.

Elution: After transfer, remove PVDF and wash 3 times with TBST for 10 min each time;

Blocking: After elution, immerse the PVDF membrane protein side up in TBST containing 5% BSA, and block on a shaking table for 1 hour;

Elution: take out PVDF after blocking and wash 3 times with TBST for 10 min each time;

Incubate the primary antibody: Immerse the PVDF membrane protein side up in the primary antibody working solution prepared by diluting the primary antibody with blocking solution, overnight at 4°C;

Elution: After incubation, take out PVDF and wash 3 times with TBST for 10 min each time;

Incubate the secondary antibody: Immerse the PVDF membrane protein side up in the secondary antibody working solution prepared by diluting the secondary antibody with TBST, and incubate for 1 h on a shaking shaker;

ECL luminescence: Mix appropriate volumes of solution A and solution B to make a working solution, take out the PVDF membrane protein side up and add ECL luminescence solution for development.

The results are shown in Fig. 6(A), Fig. 6(B), Fig. 6(C), Fig. 6(D) and Fig. 6(E).

Experimental results: The results of Figure 6(A) and Figure 6(B) show that, compared with the control group, the content of BDNF protein in the hippocampus of the mice in the model group injected with D-galactose was significantly lower, while the content of BDNF protein in the hippocampus of the mice injected with D-galactose was significantly lower, while that of Lactobacillus plantarum X7022 Afterwards, its content was significantly increased and recovered well, and the difference was extremely significant compared with the model group (P﹤0.001). The increase of BDNF can effectively regulate human memory and emotions, and has an improvement effect on psychological diseases such as anxiety. The experimental results show that Lactobacillus plantarum X7022 can up-regulate the expression of BDNF and restore it to a normal level, improve the pathological state of neurons, Promote the regeneration and differentiation of injured neurons, and thus significantly improve memory decline and learning ability.

The results of Figure 6(A) and Figure 6(C) show that, compared with the control group, the expression of CREB1 protein in the hippocampus of the mice in the D-galactose model group decreased, and the content of CREB1 in the model mice after intragastric administration of Lactobacillus plantarum X7022 Significantly increased, and the difference was extremely significant (P﹤0.001). Lactobacillus plantarum X7022 contributes to the up-regulation of CREB expression. Studies have shown that improving the learning and memory ability of mice is related to the induction of increased CREB expression, indicating that Lactobacillus plantarum X7022 strain can effectively improve learning and memory ability through this approach.

The results of Figure 6(A) and Figure 6(D) show that, compared with the control group, the DCX content in the hippocampus of mice established by injection of D-galactose to establish a rapid aging model was significantly reduced. Its content was significantly increased, and the difference was extremely significant compared with the model group (P﹤0.001). It shows that Lactobacillus plantarum X7022 can promote the increase of DCX expression and significantly improve cognitive ability.

The results of Figure 6(A) and Figure 6(E) show that compared with the control group, the iNOS content in the hippocampus of mice was significantly increased after injection of D-galactose to establish a rapid aging model. , iNOS content was significantly reduced, and the difference was more significant, indicating that Lactobacillus plantarum X7022 can effectively reduce NO on delayed progressive neuronal damage.

Conclusion: Lactobacillus plantarum X7022 can reduce neuronal damage and promote the regeneration and differentiation of injured neurons by regulating the expression of memory-related key proteins BDNF, CREB1, DCX and iNOS, improving the pathological state of neurons, significantly improving memory decline, and improving learning and cognitive abilities.

(4) Changes of key enzymes in brain tissue supernatant

Determination of superoxide dismutase enzyme activity: Superoxide dismutase (SOD) plays a crucial role in the body's oxidation and antioxidant balance. SOD can scavenge superoxide anion free radicals to protect cells from damage. The amount of SOD corresponding to the SOD inhibition rate of 50% per mg of tissue protein in 1 mL of the reaction solution was one SOD activity unit (U).

The calculation formula is:

Determination of glutathione (GSH) content: Glutathione is a low molecular weight scavenger that scavenges superoxide anion, hydrogen peroxide and LOOH. Lack or depletion of GSH can induce or aggravate the toxic effects of many chemical substances or environmental factors. The amount of GSH is an important factor to measure the body's antioxidant capacity. Take the concentration of the standard product as the abscissa and ΔA ₄₁₂ /min as the ordinate, make a standard curve, and calculate the GSH content in the reaction system according to the sample ΔA ₄₁₂ /min and the standard curve:

Determination of glutathione peroxidase (GSH-PX): Glutathione peroxidase (GSH-Px) is an important enzyme that catalyzes the decomposition of hydrogen peroxide widely in the body. It specifically catalyzes the reduction reaction of reduced glutathione (GSH) to hydrogen peroxide, and plays a role in protecting the integrity of cell membrane structure and function. One unit of enzyme activity (1U) of GSH-Px can catalyze the conversion of 1 micromolar NADPH to NADP+ in 1 minute. The calculation formula is:

Determination of malondialdehyde (MDA) content: Malondialdehyde is a natural substance for lipid oxidation in organisms, and the level of lipid oxidation can be detected by detecting the level of MDA. Malondialdehyde can react with TBA at higher temperature and in an acidic environment to form a red MDA-TBA adduct with a maximum absorption peak at 535nm.

Calculation formula of MDA content:

The results are shown in Figures 7-10.

The results showed that, compared with the model group, intragastric administration of Lactobacillus plantarum X7022 could significantly increase the activities of SOD and GSH-Px, increase the content of GSH and reduce the content of MDA; the enzyme activity of SOD was significantly increased compared with the model group (P﹤0.05). Compared with the model group, the content of GSH was significantly increased (P﹤0.01), and the content of MDA was significantly lower than that of the model group (P﹤0.05).

Conclusion: Lactobacillus plantarum X7022 can improve memory decline and enhance the body's antioxidant capacity.

(5) Diversity of gut microbes

Three days after the behavioral test, the mice were anesthetized by intraperitoneal injection of 10% trichloroacetaldehyde at a dose of 0.1 mL/100 g, and then sacrificed by cervical dislocation. Samples were stored at -80°C in sterile EP tubes for analysis of microbial diversity.

Microbial diversity is based on the Illumina HiSeq sequencing platform, using the paired-end sequencing (Paired-End) method to construct a small fragment library for sequencing. By splicing and filtering the Reads, clustering OTUs (Operational Taxonomic Units), and performing species annotation and abundance analysis to reveal the species composition of the samples.

The results are shown in FIG. 11(A), FIG. 11(B), FIG. 12(A), FIG. 12(B), FIG. 13 , FIG. 14 and FIG. 15 .

Species heatmap analysis based on the species composition and relative abundance of each sample, Figure 11(A) and Figure 11(B) are Heatmap clustering analysis at the phylum and genus taxonomic levels, respectively. In the heat map clustering results, the color represents the abundance of species, and the vertical clustering represents the similarity of the abundance of different species in each sample. The closer the distance between the two species, the horizontal clustering represents the similarity of the abundance of each species in different samples. , the closer the distance between the two samples, the shorter the branch length. It shows that the abundance of each species in these two samples is more similar.

Figure 12(A) and Figure 12(B) are histograms of species distribution at each level, from top to bottom are phylum and genus levels, one color represents one species, and the length of the color block represents the relative abundance ratio of species.

Figure 13 shows the number of common and unique OUTs (classification operation units) between samples using the Venn diagram. Different samples are represented by different colors, and the number of overlapping parts between different color graphics is the number of OTUs shared between two samples.

Figure 14 shows the dilution curve in the Alpha diversity analysis, which is used to verify whether the amount of sequencing data is sufficient to reflect the species diversity in the sample and indirectly reflect the species richness in the sample. As the number of sequencing lines increases, if the curve shows a sharp rise, it means that a large number of species have been found in the community; when the curve becomes flat, it means that the sample sequence is sufficient and data analysis can be performed.

Figure 15 shows the non-metric multidimensional calibration method (NMDS) in Beta diversity analysis, which is a sorting method suitable for ecological research. Analysis and classification, the dots in the figure represent each sample, different colors represent different groups, and the distance between the points represents the degree of difference. When the Stress is less than 0.2, it indicates that the NMDS analysis has a certain reliability. The distance on the coordinate graph The closer the sample, the higher the similarity.

The results of Figure 11-Figure 12 show that the senile memory impairment caused by injection of D-galactose can change the composition of the intestinal flora of mice, and the disturbance of the intestinal flora may be closely related to the decline of memory and learning ability , the results in Figure 13-Figure 15 show that the composition of the intestinal microflora of the mice fed with Lactobacillus plantarum X7022 has a high degree of coincidence with that of the normal mice, while the composition of the intestinal microflora of the model group is similar to that of the normal mice. There are obvious differences.

Conclusion: Lactobacillus plantarum X7022 can significantly restore the diversity of intestinal flora to the normal level, significantly improve the health of the gastrointestinal tract, and then promote the improvement of memory and learning ability through the brain-gut axis.

The above are only examples of the embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principles of the present invention, several improvements and modifications can also be made. These improvements and Variations should also be considered within the scope of protection of the present invention.

Claims (10)

1. Lactobacillus plantarum X7022, wherein the preservation number of the Lactobacillus plantarum X7022 is CCTCC NO: M2016505.

2. Use of the lactobacillus plantarum X7022 according to claim 1 for the preparation of food, health care product, medicament, pharmaceutical composition for alleviating and improving senile memory decline.

3. A product for relieving and improving senile memory deterioration, which is characterized in that the product for relieving and improving senile memory deterioration takes the bacterial liquid or bacterial powder of lactobacillus plantarum X7022 of claim 1 as an active component.

4. The product for alleviating and improving senile memory decline according to claim 3, wherein the product for alleviating and improving senile memory decline comprises food, health products, medicines and pharmaceutical compositions.

5. The product for alleviating and improving senile memory deterioration as claimed in claim 3, wherein the Lactobacillus plantarum X7022 powder is prepared by freezing and lyophilizing, and comprises 1X 10⁷～1×10¹¹CFU/g viable count.

6. The product for alleviating and improving senile memory deterioration as claimed in claim 5, wherein the Lactobacillus plantarum X7022 bacterial liquid is obtained by diluting Lactobacillus plantarum X7022 lyophilized bacterial powder with sterile physiological saline.

7. Use of lactobacillus plantarum X7022 according to claim 1 for the preparation of an enteric microecological preparation.

8. A microecological preparation which comprises the bacterial liquid or bacterial powder of Lactobacillus plantarum X7022 according to claim 1 as an active ingredient.

9. The microecological preparation according to claim 8, wherein the Lactobacillus plantarum X7022 powder is prepared by freezing and freeze-drying, and comprises 1X 10⁷～1×10¹¹CFU/g viable count.

10. The microecological preparation according to claim 8, wherein the lactobacillus plantarum X7022 bacterial solution is obtained by diluting lactobacillus plantarum X7022 lyophilized bacterial powder with sterile physiological saline.

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