Lactobacillus paracasei GP66 and application thereof

Abstract

The invention discloses a Lactobacillus paracasei GP66, which belongs to the technical field of microorganisms, the preservation number of the Lactobacillus paracasei GP66 is CGMCC (China General Microbiological Culture Collection Center) No.32952, the Lactobacillus paracasei GP66 is preserved in the China General Microbiological Culture Collection Center on December 20, 2024, and the strain is viable during preservation. The invention further discloses a fermentation product, a fermentation method, a probiotic preparation containing the casei paracasei GP66 and application of the probiotic preparation. According to the method disclosed by the invention, the fermentation condition for producing the GABA of the lactobacillus paracasei GP66 is optimized, and the content of the GABA in the fermentation liquor under the condition is 10.19 g / L.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and more specifically, to a Lacticaseibacillus paracasei GP66 and its application. Background Art

[0002] Lacticaseibacillus paracasei is a typical Gram-positive strain with a variety of probiotic functions. It is widely distributed in the human intestine, oral cavity, vagina, and dairy products, and is often used in industrial production of food fermenters, biopharmaceuticals, and functional probiotic supplements. Research shows that Lacticaseibacillus paracasei has a variety of physiological functions. For example, Lacticaseibacillus paracasei LP33 can grow and reproduce rapidly in the intestine, effectively inhibit the growth of harmful bacteria, and promote the proliferation of beneficial bacteria, thus better maintaining the stability of the intestinal microecology; Lacticaseibacillus paracasei IMC-4 can effectively reduce the risk of harmful substances entering the intestinal mucosa and causing inflammation, and can significantly enhance the tight junctions between intestinal epithelial cells and reduce intestinal permeability; Lacticaseibacillus paracasei LPB27 can significantly induce the expression of interleukin 10 (IL-10) and tumor necrosis factor-α (TNF-α) mRNA and the secretion of various cytokines in primary mouse splenocytes, and enhance the activity of immune cells. In addition, different Lacticaseibacillus paracasei strains have different tolerances to gastric acid and intestinal fluid, and their intestinal colonization abilities also vary. Generally speaking, Lacticaseibacillus paracasei can not only stabilize the intestinal flora, protect the intestinal barrier, but also play a role in immune repair and metabolic regulation.

[0003] γ-aminobutyric acid (GABA) is a four-carbon non-protein amino acid and an important part of most biological free amino acids, with a variety of physiological functions such as antidepressant, diuretic, and sedative effects. As an inhibitory neurotransmitter, GABA plays a key role in regulating the balance of neuronal excitation and inhibition in the central nervous system. Its three receptors (GABA A 、GABA B and GABA C)Allosteric sites on it can precisely regulate the inhibitory level of neurons in brain-related regions, and these sites are also molecular targets of hypnotic drugs. Research has found that after mice were gavaged with a compound preparation of Ziziphus jujuba seed extract, GABA, longan pulp extract, and casein hydrolysate at different doses for 4 weeks, the compound preparations at low, medium, and high doses could significantly prolong the sleeping time of mice induced by sodium pentobarbital. Currently, there are few studies exploring other potential physiological functions of GABA. In recent years, more and more studies have shown that GABA also has significant improvement effects on obesity and its complications (such as hyperlipidemia, hyperglycemia, and fatty liver, etc.). Thanks to the development of microbiology, compared with chemical synthesis, the method of enriching GABA by microbial fermentation is safer and has lower costs. Currently, the microorganisms commonly used for producing GABA are yeasts and lactic acid bacteria. In addition, factors such as the strain species, source, and culture conditions of the strain will affect the GABA yield of the strain. There are a variety of currently discovered high-yield GABA lactic acid bacteria. For example, Patent CN118497086B isolated and screened a Lactiplantibacillus plantarum NJ01 from a pickled vegetable sample produced in Liaoning region, China, and found that this strain not only has high GABA production but also has various physiological functions such as cholesterol degradation and blood sugar reduction; at the same time, Patent CN115521889B screened a Lactiplantibacillus plantarum WL02 producing GABA from the intestines of people in the longevity village of Guangxi, with a GABA yield of 501.86 mg / 100 mL, and provided a preparation method of a cereal beverage rich in GABA. Thus, GABA-producing probiotics can be used as ideal functional food raw materials and added to various foods, such as yogurt, beverages, baked foods, etc., to develop innovative foods that help relieve anxiety, improve sleep, regulate blood pressure, etc. Moreover, GABA-producing probiotics can affect the GABA level in the brain by regulating the intestinal flora and increasing the production and absorption of GABA in the intestine, and are expected to play an important role in the food field.

[0004] Currently, the strains with high GABA production are mostly Lactiplantibacillus plantarum and Lactobacillus brevis. Patent CN116286519B screened a Lactobacillus paracasei KS3 from pickled vegetables and elaborated its application in anti-aging and digestive food and drugs. However, the GABA content of Lactobacillus paracasei KS3 is only 95.13 pg / mL, which is relatively low compared with Lactiplantibacillus plantarum. Therefore, the present invention starts from multiple aspects such as the strain source and culture conditions to screen a Lactobacillus paracasei GP66 with excellent probiotic functions and high GABA production. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a Lactobacillus paracasei GP66 and its application. This strain has the special physiological function of high GABA production and can successfully reduce the blood lipid and blood sugar of mice on a high-fat diet, and has multiple potential functions.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A Lacticaseibacillus paracasei GP66, the preservation number of the Lacticaseibacillus paracasei GP66 is CGMCC No. 32952, and the strain was viable at the time of preservation.

[0008] A fermentate prepared by fermenting the Lacticaseibacillus paracasei GP66.

[0009] A fermentation method, in which the Lacticaseibacillus paracasei GP66 is inoculated on an MRS solid medium and then a single bacterial liquid is picked for activation, and after activation, it is inoculated into a liquid medium for fermentation.

[0010] Further preferably: 3% of the Lacticaseibacillus paracasei GP66 is inoculated into the liquid medium after activation.

[0011] Further preferably: the fermentation time is 16 h; the fermentation temperature is 37 °C.

[0012] Further preferably: the liquid medium comprises the following components in the following contents: casein peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, glucose 20.0 g / L, and Tween 1.0 g / L.

[0013] Further preferably: the MRS solid medium comprises the following components in the following contents: casein peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, glucose 20.0 g / L, Tween 1.0 g / L, and agar 15 g / L.

[0014] A probiotic preparation comprising the Lacticaseibacillus paracasei GP66.

[0015] An application of a probiotic preparation in the preparation of a sleep-aiding drug; or

[0016] in the preparation of a lipid-lowering drug; or

[0017] in the preparation of a hypoglycemic drug.

[0018] An application of a probiotic preparation in the preparation of a drug for promoting the expression of GLUT4, IRS1, and / or PPARα; or

[0019] It is applied to the preparation of a drug for inhibiting the expression of SREBP-1C.

[0020] In summary, the present invention has the following beneficial effects:

[0021] (1) A strain of Lactobacillus paracasei GP66 was isolated from pickled vegetable water in the present invention. This strain has the ability to produce high yields of GABA, and has good acid and bile salt tolerance and antibacterial ability.

[0022] (2) The Lactobacillus paracasei GP66 in the present invention can significantly prolong the sleep duration of sleep-deprived mice, reduce the levels of inflammatory factors in the hypothalamus of sleep-deprived mice, and increase the level of 5-HT in the hypothalamus of sleep-deprived mice;

[0023] (3) The Lactobacillus paracasei GP66 in the present invention has the function of improving the glycolipid metabolism disorder in obese mice, and can reduce the blood glucose level and blood lipid level of obese mice;

[0024] (4) The Lactobacillus paracasei GP66 in the present invention can restore the expression of genes related to glycolipid metabolism, such as GLUT4, IRS1, and PPARα, and can significantly inhibit the expression of the SREBP-1C gene.

[0025] The present invention optimized the fermentation conditions for GABA production by Lactobacillus paracasei GP66. Under these conditions, the GABA content in the fermentation broth was 10.19 g / L. The Lactobacillus paracasei GP66 of the present invention can significantly prolong the sleep time of mice and improve the glycolipid metabolism disorder in high-fat diet mice, and has good application prospects.

[0026] Depositing description:

[0027] Scientific name: Lactobacillus paracasei;

[0028] Taxonomic name: Lactobacillus paracasei Lacticaseibacillus paracasei;

[0029] Strain number: GP66;

[0030] Deposit number: CGMCC No. 32952;

[0031] Deposit date: December 20, 2024;

[0032] Depositary institution: China General Microbiological Culture Collection Center;

[0033] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing 100101. Description of the drawings

[0034] Figure 1 Results of the self-aggregation ability assay for strains 3M7, 3T3 (GP66), and 3MC4 in Example 1

[0035] Figure 2 Microscopic morphology of Lactobacillus paracasei GP66

[0036] Figure 3 GABA standard curve in Example 5

[0037] Figure 4 Effect of different inoculation amounts on the GABA content in the fermentation broth of Lactobacillus paracasei GP66 in Example 6

[0038] Figure 5 Effect of different fermentation times on the GABA content in the fermentation broth of Lactobacillus paracasei GP66 in Example 6

[0039] Figure 6 Effect of different fermentation temperatures on the GABA content in the fermentation broth of Lactobacillus paracasei GP66 in Example 6

[0040] Figure 7 Effect of Lactobacillus paracasei GP66 on the hypothalamic IL-6 level in sleep-deprived mice in Example 7

[0041] Figure 8 Effect of Lactobacillus paracasei GP66 on the hypothalamic IL-1β level in sleep-deprived mice in Example 7

[0042] Figure 9 Effect of Lactobacillus paracasei GP66 on the hypothalamic TNF-α level in sleep-deprived mice in Example 7

[0043] Figure 10 Effect of Lactobacillus paracasei GP66 on the hypothalamic 5-HT level in sleep-deprived mice in Example 7

[0044] Figure 11 Effect of Lactobacillus paracasei GP66 on the body weight of high-fat diet mice in Example 8

[0045] Figure 12 Effect of Lactobacillus paracasei GP66 on the food intake of high-fat diet mice in Example 8

[0046] Figure 13 Effect of Lactobacillus paracasei GP66 on the fasting blood glucose index of high-fat diet mice in Example 8

[0047] Figure 14 Effect of Lactobacillus paracasei GP66 on the fasting insulin index of high-fat diet mice in Example 8

[0048] Figure 15 Effect of Lactobacillus paracasei GP66 on serum TC level in high-fat diet mice in Example 8;

[0049] Figure 16 Effect of Lactobacillus paracasei GP66 on serum TG level in high-fat diet mice in Example 8;

[0050] Figure 17 Effect of Lactobacillus paracasei GP66 on serum LDL-C level in high-fat diet mice in Example 8;

[0051] Figure 18 Effect of Lactobacillus paracasei GP66 on liver TC level in high-fat diet mice in Example 8;

[0052] Figure 19 Effect of Lactobacillus paracasei GP66 on liver TG level in high-fat diet mice in Example 8;

[0053] Figure 20 Effect of Lactobacillus paracasei GP66 on liver LDL-C level in high-fat diet mice in Example 8;

[0054] Figure 21 Effect of Lactobacillus paracasei GP66 on mRNA levels related to glycolipid metabolism in the liver of high-fat diet mice in Example 8. Detailed implementation mode

[0055] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions indicated in the embodiments are usually carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0056] The experimental materials involved in the following examples are as follows:

[0057] The MRS solid medium and the MRS liquid medium are both sterilized at 121 °C for 15 min. The MRS solid medium and the MRS liquid medium are respectively composed of the following components:

[0058] MRS solid medium: casein peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, glucose 20.0 g / L, Tween 1.0 g / L, agar 15 g / L.

[0059] MRS liquid medium: casein peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, glucose 20.0 g / L, Tween 1.0 g / L.

[0060] The structural formula of γ-aminobutyric acid (GABA) is as follows:

[0061]

[0062] Example 1: Isolation and identification of Lactobacillus paracasei GP66

[0063] Take 10 mL of the collected homemade pickled vegetable fermentation broth and place it in a sterile centrifuge tube. Use a disposable pipette tip to aspirate 2 mL of the pickled vegetable fermentation broth into 20 mL of liquid medium, and enrich and culture it at 37 °C for 30 min to obtain an enriched culture broth. The enriched culture broth is diluted 10-fold in gradients and diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 using 0.85% normal saline. After shaking and mixing evenly, aspirate 100 μL of the diluted solution and spread it on the surface of MRS solid medium, and anaerobically culture it at 37 °C for 48 h. Pick a single colony with a regular milky white edge and inoculate it on the surface of MRS solid medium for repeated streaking purification, and obtain pure bacteria after culturing at 37 °C for 24 h. Inoculate the strain into MRS liquid medium, after culturing for 24 h, centrifuge and take the supernatant, and use the colorimetric method to detect the GABA content in the supernatant. Ten GABA-producing strains were screened from 26 strains in the primary screening, as shown in Table 1.

[0064] Table 1 Screening results of GABA-producing strains

[0065]

[0066]

[0067] Example 2: Determination of the gastric acid and bile salt tolerance of Lactobacillus paracasei GP66

[0068] The gastric acid and bile salt tolerance of the 10 strains in Example 1 was determined in vitro. The activated bacterial solution was inoculated into the prepared artificial gastric juice and artificial intestinal juice containing 0.2% bile salt at a volume fraction of 10%, and after mixing evenly, cultured at 37 °C for 3 h, and the viable bacteria counts at 0 h and 3 h were measured respectively. The results are shown in Table 2.

[0069] Table 2 Determination results of the gastric acid and bile salt tolerance of GABA-producing strains

[0070]

[0071]

[0072] Example 3: Determination of the auto - aggregation ability of Lactobacillus paracasei GP66

[0073] Select strains 3M7, 3T3(GP66) and 3MC4 for the next experiment to determine their auto - aggregation ability. After adjusting the absorbance (OD600) of the bacterial suspension to 0.5, the bacterial suspension was cultured at 37 °C and the absorbance of the bacterial suspension was measured at 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h at OD 600 The absorbance value was used to calculate the auto - aggregation ability using the following formula:

[0074]

[0075] In the formula, A 0 and A 1 are the absorbance values before and after cultivation, respectively.

[0076] The auto - aggregation abilities of 3M7, 3T3(GP66) and 3MC4 are as Figure 1 shown.

[0077] Example 4: Determination of the antibacterial ability of Lactobacillus paracasei GP66

[0078] Select 3M7 and 3T3(GP66) for the antibacterial ability determination. Pour a thin layer of agar medium into a petri dish. After the medium solidifies, place 4 sterilized Oxford cups equidistantly. At the same time, pipette 200 μL of the pathogenic bacteria suspension into the non - solidified agar medium, and pour the agar medium containing the pathogenic bacteria into the petri dish with the Oxford cups. After the medium solidifies, remove the Oxford cups, inject 100 μL of the test bacterial suspension into the round holes, and culture at 37 °C for 24 h. After the culture, use a vernier caliper to measure the diameter of the antibacterial zone. The results are shown in Table 3.

[0079] Table 3 Results of the antibacterial ability determination of 3M7 and 3T3(GP66)

[0080]

[0081]

[0082] As can be seen from Examples 1 - 4, the GABA - producing ability, acid and bile salt tolerance ability, auto - aggregation ability and antibacterial ability of the 3T3(GP66) strain are relatively ideal. Therefore, Lactobacillus paracasei GP66 was selected as the experimental strain to improve the glycolipid metabolism of high - fat diet mice.

[0083] The microscopic morphological examination of Lactobacillus paracasei GP66 is as Figure 2 shown below.

[0084] The strain was sequenced using 16S rDNA universal primers, and the gene sequence of Lactobacillus paracasei GP66 is shown in SEQ ID No:1.

[0085] SEQ ID No:1

[0086] TTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAG

[0087] Lactobacillus paracasei GP66, isolated from the pickle fermentation broth produced in Hunan region of China, was cryopreserved at -80 °C in a glycerol tube. Generally, this strain was inoculated on the surface of an MRS solid medium plate and incubated in an anaerobic incubator at 37 °C in an inverted position for 24 h to obtain colonies. A single colony was picked and cultured in an MRS liquid medium for 24 h to obtain a fermentation broth.

[0088] Example 5: Determination of the ability of Lactobacillus paracasei GP66 to produce GABA

[0089] (1) Plotting of GABA standard curve: Prepare GABA standard solutions with concentrations of 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1.0 g / L. Use a GABA content detection kit (Laier Biotechnology Co., Ltd.) to detect the GABA content. Plot a standard curve with GABA concentration as the horizontal axis and absorbance as the vertical axis, as shown in the figure. Figure 3 shown.

[0090] (2) Fermentation and culture of Lactobacillus paracasei GP66: The frozen bacterial liquid was activated using MRS solid culture medium. After activation, a single colony was picked and cultured in MRS liquid culture medium for 24 h to obtain a second-generation bacterial liquid. The second-generation bacterial liquid of Lactobacillus paracasei GP66 was inoculated into liquid MRS culture medium with an inoculum size of 2% and cultured for 24 h. The fermented liquid was centrifuged at 6000 rpm for 10 min, and the supernatant was collected, and the GABA content in the supernatant was detected using a GABA detection kit. The GABA content in the supernatant after fermentation for 24 h was 8.73 g / L.

[0091] Example 6: Optimization of culture conditions of Lactobacillus paracasei GP66

[0092] (1) Effect of inoculation amount on GABA production of Lactobacillus paracasei GP66: 1%, 2%, 3%, 4%, 5%, and 6% bacterial solution were inoculated and cultured at 37°C for 24 hours. After the culture, the GABA content in the supernatant was measured to compare the effects of different inoculation amounts on GP66 GABA production. Figure 4 As shown, when the inoculation amount was 4%, the GABA content in the supernatant was the highest, which was 9.51 g / L. The inoculation amounts of 3%, 4%, and 5% were selected for the next orthogonal test.

[0093] (2) Effect of fermentation time on GABA production of Lactobacillus paracasei GP66: 2% GP66 bacterial solution was inoculated and cultured at 37°C for 8h, 12h, 16h, 20h, 24h, and 28h. After the fermentation, the GABA content in the supernatant was measured to compare the effects of different fermentation times on GABA production of GP66. Figure 5 As shown, the GABA content in the supernatant was the highest when the fermentation time was 20 h, which was 10.95 g / L. Fermentation times of 16 h, 20 h, and 24 h were selected for the next orthogonal test.

[0094] (3) Effect of fermentation temperature on GABA production of Lactobacillus paracasei GP66: 2% GP66 bacterial solution was inoculated and cultured at 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C for 24 h. After the fermentation, the GABA content in the supernatant was measured to compare the effects of different fermentation temperatures on GABA production of GP66. Figure 6As shown, when the fermentation temperature was 38°C, the GABA content in the supernatant was the highest, reaching 9.03 g / L. Fermentation temperatures of 37°C, 38°C, and 39°C were selected for the next orthogonal experiment.

[0095] (4) Orthogonal experiment: The inoculum size (3%, 4%, 5%), fermentation time (16 h, 20 h, 24 h), and fermentation temperature (37°C, 38°C, 39°C) were selected for three-factor and three-level orthogonal experiment analysis. The specific experimental results are shown in Table 4. The factor that had the greatest impact on the GABA content in the GP66 fermentation broth was factor B, i.e., the fermentation time, followed by C (fermentation temperature) and A (inoculum size). Therefore, based on the results of the orthogonal experiment, the optimal solution for the fermentation conditions was A1B1C1, i.e., the inoculum size was 3%, the fermentation time was 16 h, and the fermentation temperature was 37°C. To verify this result, Lactobacillus paracasei was cultured under these conditions, and the GABA content in the fermentation broth was measured to be 10.19 ± 0.08 g / L.

[0096] Table 4 Results of the orthogonal experiment for optimizing the fermentation conditions of Lactobacillus paracasei GP66

[0097]

[0098]

[0099] Example 7: Effect of Lactobacillus paracasei GP66 on the sleep of sleep-deprived mice

[0100] (1) Mouse feeding: Forty 8-week-old male C57BL / 6J mice (Wuhan Shulaibao Biological Co., Ltd.) were randomly divided into 4 groups (n = 10) after one week of adaptive feeding. The normal control group (Group C) and the negative control group were intragastrically administered sterile saline daily; the positive control group was intragastrically administered GABA at a dose of 100 mg / kg BW daily; the experimental group was intragastrically administered 1×10 9 CFU of Lactobacillus paracasei GP66 bacterial solution per mouse.

[0101] (2) Establishment of a sleep deprivation model: Except for the normal control group, the other four groups all used parachlorophenylalanine (PCPA) to block the synthesis of the monoamine neurotransmitter 5-hydroxytryptamine (5-HT) in mice to establish a sleep deprivation model, and PCPA solution was continuously intraperitoneally injected for two days. After the model was successfully established, GABA and Lactobacillus paracasei GP66 were used to intervene in the sleep-deprived mice, and the experimental period was 2 weeks.

[0102] (3) Pentobarbital sodium sleep experiment: After the intervention, pentobarbital sodium was intraperitoneally injected into each group of mice at a dose of 50 mg / kg BW. Timing started after the injection of pentobarbital sodium. If a mouse could not right itself within 60 s, the righting reflex was considered to have disappeared, and the time from the disappearance of the righting reflex to its reappearance was recorded as the sleeping time of the mouse. The sleeping time of the mice is shown in Table 5. Under the induction of PCPA, obvious sleep deprivation occurred in the mice, reducing the sleeping time of the mice, while the intervention of GABA and Lactobacillus paracasei GP66 significantly prolonged the sleeping time of the mice.

[0103] Table 5 Effects of Lactobacillus paracasei GP66 on the sleep duration of sleep-deprived mice

[0104]

[0105]

[0106] (4) The mice were sacrificed and their sera and hypothalami were collected. The contents of IL-6, IL-1β and TNF-α in the hypothalami of the mice were detected using an ELISA kit, and the level of 5-HT in the hypothalami of the mice was also detected. The results are as Figures 7 - 10 shown. The sleep deprivation induced by PCPA increased the levels of inflammatory factors in the hypothalami of the mice and decreased the level of 5-HT in the hypothalami of the mice, while the intervention of GABA and Lactobacillus paracasei GP66 restored the levels of inflammatory factors and 5-HT in the mice to normal.

[0107] Example 8: Improvement effect of Lactobacillus paracasei GP66 on glucose and lipid metabolism in high-fat diet mice

[0108] (1) Mouse feeding: Forty 8-week-old male C57BL / 6J mice (Wuhan Shulaibao Biological Co., Ltd.) were randomly divided into 4 groups (n = 10) after one week of adaptive feeding. The experimental groups were as follows: normal control group (Group C, fed with mouse growth and reproduction feed), high-fat model group (Group HFD, fed with a high-fat diet with 60% of energy from fat), positive control group (Group HFD+SIM, fed with a high-fat diet and intragastrically administered simvastatin solution at 4 mg / kg BW daily), experimental group (Group HFD+GP66, fed with a high-fat diet and intragastrically administered Lactobacillus paracasei GP66 bacterial solution daily). The experimental group needed to ensure that the daily intake of viable bacteria by each mouse was not less than 1×10 9 CFU / mouse. The experimental period was 8 weeks. The body weight and food intake of the mice were recorded weekly. After the end of the experimental period, the mice were sacrificed and relevant tissues were quickly collected for subsequent experimental analysis.

[0109] (2) Preparation of mouse tissue samples:

[0110] Preparation of mouse serum samples: Before sacrificing the mice, disinfect the fur around the eyes of the mice with an alcohol swab, gently press the skin around the eyes of the mice to make the eyeballs congested and protrude, use sharp curved forceps to pick up the protruding eyeballs of the mice and quickly remove them, collect the mouse blood in a sterilized centrifuge tube in the air, place it at room temperature for 30 - 60 min until the blood coagulates, centrifuge at 4°C and 2000 - 3000 rpm for 15 min, aliquot the supernatant equally and store it at -80°C, avoiding repeated freezing and thawing.

[0111] Preparation of mouse liver samples: Add 0.9 mL of sterile 0.85% normal saline (w / v) to 0.1 g of liver samples, use a handheld homogenizer to work for about 30 s to prepare liver homogenate, centrifuge it at 4°C and 3000 rpm for 20 min, and save the supernatant at -80°C for later use.

[0112] Preparation of mouse adipose tissue samples: After sacrificing the mice, isolate the epididymal fat, perirenal fat and mesenteric fat of the mice and weigh them.

[0113] (3) Detection of mouse indicators:

[0114] ① Routine indicators: The body weights, food intakes and fat weights of the mice in each group are as Figure 11 、 12 and shown in Table 6. Compared with group C, a high-fat diet led to a 20.54% increase in the body weight of the mice in the HFD group. However, under the intervention of Lactobacillus paracasei GP66, the body weight of the mice decreased significantly, and it only increased by 10.27% compared with group C. However, there was no significant difference in the food intakes of the four groups of mice, indicating that the decrease in the body weight of the mice caused by Lactobacillus paracasei GP66 was not due to differences in food intake. The weight of the mouse adipose tissue can reflect the obesity degree of the mice, and there will be certain differences in the fat weights of different tissues. As shown in Table 6, after 8 weeks of high-fat diet, the weight of the mouse adipose tissue increased significantly, with an average increase of 0.72 g of epididymal fat, 0.24 g of perirenal fat and 0.19 g of mesenteric fat compared with group C. Among the fats in each part, the most obvious change was in the epididymal fat. Under the intervention of Lactobacillus paracasei, the weight of the epididymal fat of the mice decreased significantly by 0.36 g, indicating that Lactobacillus paracasei GP66 can effectively alleviate the obesity degree of high-fat diet mice.

[0115] Table 6 Effects of Lactobacillus paracasei GP66 on the weight of adipose tissue in high-fat diet mice

[0116]

[0117] ② Serum biochemical indicators and liver biochemical indicators: Obesity caused by a high-fat diet not only leads to abnormal lipid metabolism in the body, but usually the occurrence of obesity is also related to impaired glucose metabolism in the body. As Figures 13 - 20As shown, under the action of a high-fat diet, the fasting blood glucose index (FBG) of mice increased significantly, and the fasting insulin index (FINS) also increased synchronously. However, the intervention of Lactobacillus paracasei GP66 significantly decreased both, demonstrating that Lactobacillus paracasei GP66 can improve blood glucose abnormalities and insulin resistance in mice caused by a high-fat diet and maintain glucose metabolism homeostasis in mice. Similarly, the lipid metabolism disorder in mice was also significantly improved under the intervention of Lactobacillus paracasei GP66, specifically manifested as a significant decrease in the levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) in serum and liver. These experimental results indicate that Lactobacillus paracasei GP66 can not only improve insulin resistance in mice caused by a high-fat diet, but also reduce the blood lipid level and liver lipid level of mice, relieve mouse obesity, and improve lipid metabolism disorder in obese mice.

[0118] ③ Quantitative fluorescence PCR test:

[0119] RNA extraction: Add 1 mL of TRIzol reagent to the liver tissue samples of mice. After thoroughly grinding the mouse liver on ice, use an RNA extraction kit (Thermo Fisher Scientific) to extract the RNA in the samples.

[0120] cDNA synthesis and reverse transcription reaction: Prepare the reaction solution according to Table 7 and react at room temperature for 30 minutes, then prepare the reverse transcription reaction solution according to Table 8 and perform the reverse transcription reaction in a PCR instrument.

[0121] Table 7 Reaction system

[0122] Reagent Dosage 5× gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0 μL Total RNA 1.0 μL RNase Free dH2O Make up to 10 μL

[0123] Table 8 Reaction system

[0124]

[0125] Real-time fluorescence quantitative PCR: The gene primer sequences of the present invention are shown in Table 9. The PCR amplification conditions are pre-denaturation at 95 °C for 30 s, followed by denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s for a total of 40 cycles. The results of fluorescence quantitative PCR are as Figure 21 shown. Lactobacillus paracasei GP66 significantly restored the dysregulation of genes GLUT4, IRSI, and PPARα in mice caused by a high-fat diet and significantly inhibited the overexpression of gene SREBP-1C, improving the glycolipid metabolism disorder in mice induced by a high-fat diet at the RNA level.

[0126] Table 9 Primers for fluorescence quantitative PCR analysis

[0127] Gene Forward primer (5′ - 3′) Reverse primer (5′ - 3′) GLUT4 TATCGGCATTCTGATCGCCC GTCAGGCGCTTCAGACTCTT IRS1 GTCAGGCGCTTCAGACTCTT GTCAGGCGCTTCAGACTCTT SREBP - 1C GCTACCGGTCTTCTATCAATGA CGCAAGACAGCAGATTTATTCA PPARα CCAGATGTGCCTGCTGCTTCC TGGTCGGTCTACAGAGTGAGTTCC β - actin GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT

[0128] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and modifications made without departing from the principle of the present invention should also be regarded as within the protection scope of the present invention.

Claims

1. A Lactobacillus paracasei GP66, characterized in that: The Lactobacillus paracasei GP66 is deposited with CGMCC No.32952.

2. A fermented product, characterized in that: It is prepared by fermenting the Lactobacillus paracasei GP66 according to claim 1.

3. A fermentation method, characterized in that: After the Lactobacillus paracasei GP66 described in claim 1 is inoculated on an MRS solid culture medium, a single bacterium is selected for liquid activation, and after activation, it is inoculated into a liquid culture medium for fermentation.

4. A fermentation method according to claim 3, characterized in that: After activation, 3% Lactobacillus paracasei GP66 was inoculated into the liquid culture medium.

5. A fermentation method according to claim 3, characterized in that: The fermentation time is 16 hours; the fermentation temperature is 37°C.

6. A fermentation method according to claim 3, characterized in that: The liquid culture medium comprises the following components: 10.0 g / L of casein digest, 10.0 g / L of beef extract powder, 4.0 g / L of yeast extract powder, 2.0 g / L of triammonium citrate, 5.0 g / L of sodium acetate, 0.2 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, 20.0 g / L of glucose and 1.0 g / L of Tween.

7. A fermentation method according to claim 3, characterized in that: The MRS solid culture medium comprises the following components: 10.0 g / L of casein digest, 10.0 g / L of beef extract powder, 4.0 g / L of yeast extract powder, 2.0 g / L of triammonium citrate, 5.0 g / L of sodium acetate, 0.2 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, 20.0 g / L of glucose, 1.0 g / L of Tween and 15 g / L of agar.

8. A probiotic preparation, characterized in that: The invention comprises the Lactobacillus paracasei GP66 as claimed in claim 1.

9. An application of a probiotic preparation, characterized in that: For use in the preparation of drugs for inducing sleep; or Used in the preparation of lipid-lowering drugs; or Used in the preparation of drugs for lowering blood sugar.

10. An application of a probiotic preparation, characterized in that: Used in the preparation of drugs for promoting the expression of GLUT4, IRS1 and / or PPARα; or The invention is used for preparing drugs for inhibiting the expression of SREBP-1C.

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