A strain of Lactobacillus johnsonii for alleviating anxiety and resisting depression and its postbiotics and applications

Abstract

The present invention discloses a strain of Lactobacillus johnsonii for relieving anxiety and anti-depression and its postbiotics and applications, belonging to the field of microbial fermentation technology. The present invention screens and obtains a strain of Lactobacillus johnsonii, obtains the bacterial body and its postbiotics by culture, and forms a complex with a dandelion extract. The complex exhibits a synergistic effect, which is specifically manifested in that the number of times mice enter the open arm in the elevated plus maze experiment increases, the residence time is prolonged, and higher curiosity and better anxiety relief ability are exhibited; in the tail suspension experiment, the immobility time of mice is reduced, showing a higher desire to escape and anti-depression ability. The present invention is not only of practical value for the comprehensive utilization of Lactobacillus johnsonii and its postbiotics, but also of important value for the body in relieving anxiety and anti-depression.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a strain of Lactobacillus johnsonii capable of relieving anxiety and resisting depression, and postbiotics and applications thereof. Background Art

[0002] Depressive disorder (also called depression) is a common mental disorder involving prolonged low mood, loss of pleasure or interest in activities. Anxiety is common in depression, and most patients with major depression have clinical anxiety. As they age, anxiety symptoms become a more obvious feature of major depression. There is currently no clear explanation for the cause of depression, and both social and biological stressors may contribute to the onset of depression.

[0003] Lactobacillus johnsonii ( Lactobacillus johnsonii ) is one of the important probiotics. Due to its high safety, my country has included it in the "List of Bacteria that can be used in food", allowing it to be used in food. The main biological activities of Lactobacillus johnsonii include enhancing the body's immunity, relieving intestinal inflammation, inhibiting intestinal pathogenic microorganisms and regulating the balance of intestinal flora.

[0004] Dandelion Taraxacum mongolicum Hand.-Mazz.) is a perennial herbaceous plant of the genus Taraxacum in the Asteraceae family. It is both cold-resistant and heat-resistant, has strong adaptability, and is widely distributed. The stems, leaves, and flowers of dandelion are all edible. At the same time, as a plant with both medicinal and edible properties, its dried whole herb can be used as medicine, which has the effects of clearing away heat and detoxifying, reducing swelling and dispersing knots, and promoting diuresis and relieving stranguria.

[0005] Although people in all walks of life have a certain understanding of the efficacy of Lactobacillus johnsonii and dandelion, there have been no reports of the combined use of the two substances for a long time, and the efficacy of adding Lactobacillus johnsonii and its postbiotics in relieving anxiety and anti-depression is also unknown. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a strain of Lactobacillus johnsonii for relieving anxiety and anti-depression and its postbiotics and applications.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a strain of Lactobacillus johnsonii for relieving anxiety and resisting depression. The Lactobacillus johnsonii is Lactobacillus johnsonii WZA-LJ18, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20241507.

[0009] The second aspect of the present invention provides the use of the Lactobacillus johnsonii in the preparation of a product for relieving anxiety and anti-depression, wherein the number of viable Lactobacillus johnsonii in the product is greater than 1×10 7cfu / mL.

[0010] The third aspect of the present invention provides a postbiotic of Lactobacillus johnsonii, wherein the postbiotic of Lactobacillus johnsonii is prepared by the following method:

[0011] The activated Lactobacillus johnsonii is inoculated into a fermentation medium for fermentation at a fermentation temperature of 30-45°C for 4-40 hours; the fermented product is centrifuged and the supernatant is freeze-dried to obtain the postbiotics of Lactobacillus johnsonii.

[0012] Furthermore, the fermentation medium is composed of: 5-15 parts of beef extract powder, 5-12 parts of peptone, 10-30 parts of glucose, 1-10 parts of yeast powder, 0.05-1 parts of magnesium sulfate, 1-5 parts of diammonium citrate, 1-5 parts of dipotassium hydrogen phosphate, 2-12 parts of sodium acetate, 0.01-1 parts of manganese sulfate, 0.5-5 parts of Tween 80, 800-1200 parts of distilled water, pH: 5-7.

[0013] The fourth aspect of the present invention provides the use of the postbiotics of Lactobacillus johnsonii in the preparation of products for relieving anxiety and anti-depression. The products for relieving anxiety and anti-depression are prepared by mixing the postbiotics of Lactobacillus johnsonii and dandelion extract in a mass ratio of (0.5-5):(0.5-5).

[0014] Furthermore, the dandelion extract is prepared by the following method:

[0015] Take fresh young dandelion stems and leaves, wash them, dry them in a 60-80℃ oven, crush them to 100-600 mesh and sieve them to obtain dandelion stem and leaf powder. Extract the dandelion stem and leaf powder in 60-80% v / v ethanol at a mass volume ratio of 1g:(1-10)ml, the extraction temperature is 20-40℃, the extraction time is 5-20h each time, and the extraction is performed 1-3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 30-60℃, and then dried in a 60-80℃ oven to obtain dandelion extract.

[0016] Beneficial effects of the present invention:

[0017] (1) The present invention obtains Lactobacillus johnsonii by screening, which has the characteristics of acid resistance and bile salt resistance, and can relieve anxiety and resist depression.

[0018] (2) The present invention ferments Lactobacillus johnsonii, freeze-dries the supernatant to obtain postbiotics of Lactobacillus johnsonii, and forms a complex with dandelion extract, namely, the Lactobacillus johnsonii postbiotic complex.

[0019] (3) The present invention uses the Lactobacillus johnsonii postbiotic complex for experiments. The experimental results show that the complex group performs best: in the anti-anxiety experiment, the number of times the mice in the complex group entered the open arm was (8±3) s, and the residence time was (32.3±7.4) s. It can be seen that after the feeding treatment of the complex group, the health of the mice improved, the treatment of the complex group showed a synergistic effect, and the mice had higher curiosity and better anxiety relief ability; in the anti-depression experiment, the immobility time of the mice in the complex group was (82±7) s. It can be seen that after the feeding treatment of the complex group, the health of the mice improved, the treatment of the complex group showed a synergistic effect, and the mice had a higher desire to escape, showing that the mice had a higher anti-depression ability after the treatment. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0021] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0022] The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the field and can be purchased through commercial channels. The Kunming mice used were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., and animal welfare was guaranteed throughout the experiment.

[0023] The preservation information of Lactobacillus johnsonii used in the present invention is as follows:

[0024] Chinese name: Lactobacillus johnsonii WZA-LJ18;

[0025] Latin name: Lactobacillus johnsonii WZA-LJ18;

[0026] Deposit number: CCTCC NO:M 20241507;

[0027] Deposit date: July 8, 2024;

[0028] Depository: China Center for Type Culture Collection (CCTCC);

[0029] Deposit: Wuhan University, Wuhan, China.

[0030] Example 1: Screening of Lactobacillus johnsonii

[0031] 1. Isolation and initial screening of strains:

[0032] Collect 10 g of naturally fermented yogurt samples, take 1 g of each sample and put it into a 50 mL centrifuge tube containing 9 mL of sterile saline, shake for 15 min to mix it thoroughly, let it stand for 25 seconds, take 2 mL of suspension from each sample and place it in an 80 ℃ water bath for 10 min, take 1 mL for 10-fold gradient dilution, and then select the appropriate gradient suspension to spread on MRS agar medium (self-prepared) containing 0.3% CaCO3, and place it in a 37 ℃ incubator for constant temperature culture for 48 hours. Single colonies with calcium dissolution rings are streaked and purified, and after the single colony morphology is stable, they are numbered and stored.

[0033] 2. Strain screening: acid resistance and bile resistance

[0034] Probiotics must be able to survive in the intestines to exert specific biological activities, so they need to enter the intestines through the stomach containing acidic gastric juice. At the same time, the intestines contain high concentrations of bile acids, so bile acid tolerance is also an important selection criterion for probiotic microorganisms.

[0035] The strains were treated in simulated gastric fluid at pH = 3 for 0h, 2h, 3h, 4h, and 5h, and the survival rate was recorded; the strains were treated at bile salt concentrations of 0.1%, 0.3%, 0.5%, 0.75%, and 1%, and the survival rate was recorded. The strains with higher survival rates were selected for subsequent identification.

[0036] 3. Identification of strains:

[0037] The strain was sent to Henan Industrial Microbial Engineering Technology Research Center for 16S rDNA gene sequence identification. The nucleotide sequence is shown in SEQ ID NO: 1. The identification results showed that the strain belonged to Lactobacillus johnsonii ( Lactobacillus johnsonii ), and then the strain WZA-LJ18 was biologically deposited.

[0038] Example 2: Preparation of postbiotics of Lactobacillus johnsonii

[0039] The preparation method is as follows:

[0040] (1) The Lactobacillus johnsonii preserved in Example 1 was activated in an anaerobic environment to obtain an activated bacterial solution of Lactobacillus johnsonii. The activation temperature was 37°C and the activation time was 18 h. The composition of the activation medium was as follows: beef extract powder: 8 g, peptone: 10 g, glucose: 20 g, yeast powder: 4.5 g, magnesium sulfate: 0.2 g, diammonium citrate: 2 g, dipotassium hydrogen phosphate: 2 g, sodium acetate: 5.2 g, manganese sulfate: 0.04 g, Tween 80: 1 g, agar: 14 g, distilled water to make up to 1 L, pH: 5.7.

[0041] (2) The activated Lactobacillus johnsonii liquid obtained in step (1) is inoculated into a fermentation medium at an inoculum amount of 5wt% (g / g) of the fermentation medium to obtain a Lactobacillus johnsonii fermentation liquid. The fermentation temperature is 37° C., the fermentation time is 26 h, and the composition of the fermentation medium used is as follows: beef extract powder: 8.5 g, peptone: 9 g, glucose: 22 g, yeast powder: 4.5 g, magnesium sulfate: 0.2 g, diammonium citrate: 2 g, dipotassium hydrogen phosphate: 2 g, sodium acetate: 5 g, manganese sulfate: 0.04 g, Tween 80: 1 g, and distilled water is supplemented to 1 L, pH: 5.7.

[0042] (3) The Lactobacillus johnsonii fermentation broth obtained in step (2) was centrifuged at 25° C. and the supernatant was freeze-dried to obtain the postbiotics of Lactobacillus johnsonii.

[0043] The content of Lactobacillus johnsonii in the precipitate was detected by spectrophotometry. The number of viable bacteria was 5×10 8 The cfu / ml concentration was stored in a refrigerator at 4°C.

[0044] Example 3: Preparation of Lactobacillus johnsonii postbiotic complex

[0045] Dandelion extract preparation

[0046] Take fresh young dandelion stems and leaves, wash them, dry them in a 70℃ oven, crush them to 400 mesh and sieve them to obtain dandelion stem and leaf powder. Extract the dandelion stem and leaf powder in 70% v / v ethanol at a mass volume ratio of 1g:5ml, the extraction temperature is 30℃, the extraction time is 10h each time, and the extraction is performed twice in total; the extracts obtained each time are combined, concentrated under reduced pressure at 50℃, and then dried in a 75℃ oven to obtain dandelion extract.

[0047] Preparation of complexes

[0048] The postbiotics of Lactobacillus johnsonii prepared in Example 2 and the dandelion extract prepared in Example 3 were mixed in a mass ratio of 1:1 to obtain a Lactobacillus johnsonii postbiotic complex.

[0049] Experimental Example 1: Animal Model Experiment

[0050] Animal modeling and intervention methods

[0051] Six-week-old Kunming mice were randomly divided into groups, half male and half female, with 10 mice in each group, and three parallel groups were set up as follows: postbiotic group, the additive was the postbiotic of Lactobacillus johnsonii prepared in Example 2; dandelion group, the additive was the dandelion extract prepared in Example 3; probiotic group, the additive was the Lactobacillus johnsonii prepared and preserved in Example 2 (1 ml was regarded as 1 g); complex group, the additive was the postbiotic complex of Lactobacillus johnsonii prepared in Example 3; each additive group was gavage-fed according to the weight of the animal at a dose of 3 mg / kg, and the blank group and the control group were given an equal amount of normal saline. In addition to feeding with the same normal feed, each group was gavaged once a day until the end of the experiment.

[0052] Except for the blank group, mice in each group were subjected to chronic unpredictable mild stimulation for 4 weeks: (1) fasting for 24 hours; (2) water deprivation for 24 hours; (3) tilting cage (tilting the cage at 45℃ for 24 hours and returning the cage to normal state); (4) clamping the tail (clamping the plastic clamp 2 cm from the end of the tail for 3 minutes each time, 3 times a day); (5) cold water swimming (4℃, 5 minutes); (6) day and night reversal; (7) no bedding (the mouse was placed alone in a new cage without bedding for 24 hours and then returned to the original cage). In order to avoid the animals' adaptation to specific stimuli, thereby affecting the accuracy of the experimental results, these stimuli were randomly applied 1-2 times a day, and the stimulation was randomly applied 6 days a week. This method is designed to create a complex and varied stress environment to induce anxiety and depression behaviors and physiological responses in mice. After 4 weeks, the anxiety state test was performed by the elevated plus maze test, and the depression test was performed by the tail suspension test, as follows:

[0053] 1) Elevated plus maze test

[0054] The elevated plus maze uses the animals' exploration characteristics of novel environments and their fear of high-hanging open arms to form conflicting behaviors to examine the anxiety state of animals. The mice were introduced into the central area of ​​the four-walled maze, facing the open arms. After that, each mouse was placed in the same position. The two open arms (50×10cm) had no walls, and the two closed arms (50×10×30cm) had walls. The maze was one meter above the ground. The number of times the mouse entered the open arm and the time it stayed in the open arm within 5 minutes were recorded. Fewer times of entering the open arm and the time of staying in the open arm indicate more severe anxiety. After each experiment, the maze was cleaned with 75% ethanol to eliminate the effects of mouse urine, feces, and odor on subsequent experimental mice. The test results of the elevated plus maze experiment are shown in Table 1:

[0055] Table 1. Elevated plus maze test

[0056]

[0057] As shown in Table 1, compared with the blank group, the time and number of mice in the control group in the open arm were reduced, indicating that their hesitation and anxiety behaviors increased. Compared with the control group, the time and number of mice in the open arm in each experimental group were prolonged. Among them, the compound group performed best. In the compound group, the number of times the mice entered the open arm was (8±3) and the stay time was (32.3±7.4)s. It can be seen that after the compound group was fed, the health of the mice improved, and the compound group treatment showed a synergistic effect. The mice had higher curiosity and better ability to relieve anxiety.

[0058] 2) Tail suspension test

[0059] Use adhesive tape to stick the tip of the mouse's tail (2 cm from the tip of the tail) to a suspension rod (30 cm above the ground). When the mouse touches the suspension, it will instinctively struggle to escape from the predicament at first, but if it cannot escape, it will fall into despair and remain motionless, showing depressive-like behavior. The longer the immobility time, the more depressed it is. Monitor the movement activity of the mouse for 6 minutes, and record the immobility time of each mouse in the last 4 minutes of the 6-minute test (immobility is defined as no voluntary or escapist movement). The test results of the tail suspension experiment are shown in Table 2:

[0060] Table 2. Tail suspension test

[0061]

[0062] As shown in Table 2, compared with the blank group, the immobility time of the mice in the control group in the tail suspension test was significantly increased, showing a typical depressive state, reflecting a higher level of despair behavior. Compared with the control group, the immobility time of the mice in each experimental group in the tail suspension test was significantly reduced. Among them, the compound group performed best. In the compound group, the immobility time of the mice was (82±7)s. It can be seen that after the compound group was fed and treated, the health of the mice improved, and the compound group treatment showed a synergistic effect. The mice had a higher desire to escape, showing that the mice had a higher anti-depression ability after the treatment.

[0063] In summary, the compound group performed best: in the anti-anxiety experiment, the number of times the mice in the compound group entered the open arm was (8±3) and the residence time was (32.3±7.4)s. It can be seen that after the compound group was fed, the health of the mice improved, the compound group treatment showed a synergistic effect, and the mice had higher curiosity and better anxiety relief ability; in the anti-depression experiment, the immobility time of the mice in the compound group was (82±7)s, which shows that after the compound group was fed, the health of the mice improved, the compound group treatment showed a synergistic effect, and the mice had a higher desire to escape, showing that the mice had a higher anti-depression ability after this treatment.

[0064] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An application of a postbiotic of Lactobacillus johnsonii in the preparation of a product for relieving anxiety and anti-depression, characterized in that: The anxiety-relieving and anti-depression product is made from a mixture of postbiotics from Lactobacillus johnsonii and dandelion extract in a 1:1 mass ratio; The Lactobacillus johnsonii is Lactobacillus johnsonii ( Lactobacillus johnsonii ) WZA-LJ18, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO:M 20241507; The postbiotics of Lactobacillus johnsonii are prepared by the following method: The activated Lactobacillus johnsonii is inoculated into a fermentation medium for fermentation at a fermentation temperature of 30-45° C. for a fermentation time of 4-40 hours; the fermented product is centrifuged and the supernatant is freeze-dried to obtain postbiotics of Lactobacillus johnsonii; The dandelion extract is prepared by the following method: Take fresh young dandelion stems and leaves, wash them, dry them in a 60-80°C oven, crush them to 100-600 mesh and sieve them to obtain dandelion stem and leaf powder; extract the dandelion stem and leaf powder in 60-80% v / v ethanol at a mass volume ratio of 1g:(1-10)ml, the extraction temperature is 20-40°C, the extraction time is 5-20h each time, and the extraction is performed 1-3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 30-60°C, and then dried in a 60-80°C oven to obtain dandelion extract.

2. The use according to claim 1, characterized in that: The composition of the fermentation medium is as follows: 5-15 parts of beef extract powder, 5-12 parts of peptone, 10-30 parts of glucose, 1-10 parts of yeast powder, 0.05-1 parts of magnesium sulfate, 1-5 parts of diammonium citrate, 1-5 parts of dipotassium hydrogen phosphate, 2-12 parts of sodium acetate, 0.01-1 parts of manganese sulfate, 0.5-5 parts of Tween 80, 800-1200 parts of distilled water, pH: 5-7.

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