Application of Lactobacillus paracasei l.p R3-10 in the preparation of anxiety-relieving and sleep-improving drugs
A technology for improving sleep and Lactobacillus, applied in the field of microorganisms, can solve problems such as not necessarily suitable, few probiotics, and dependence on imports of probiotic strains, and achieve the effects of increasing resting time, reducing exercise distance, and relieving anxiety
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Embodiment 1L
[0024] Example 1 Isolation, culture and identification of the precursor strain L.p R3 (Lactobacillus paracasei) of L.p R3-10
[0025] (1) Sample source
[0026] Healthy infants aged 0-6 months were selected from school staff families as volunteers. Two weeks before sampling, normal diet was required and there was no recent history of intestinal infection and antibiotic use. The morning stool was taken on the day of sampling. After collection, the intelligent microbial separation system of Nanjing Famet Company was used to separate fecal bacteria. After the separation was completed, the crude fecal bacterial liquid was collected quickly, added with cryopreservation protection solution, and then placed in a -80°C ultra-low temperature refrigerator for future use.
[0027] (2) Isolation, culture and identification of L.p R3
[0028] Take 1mL of crude fecal bacteria solution and add it to 9mL of normal saline, mix well and then carry out gradient dilution. Aspirate dilution con...
Embodiment 2L
[0031] Example 2 Induction and identification of L.p R3-10
[0032] (1) Low nutrient gradient tolerance method induces L.p R3 to become L.p R3-10
[0033]Take out the frozen L.p R3 from the -80°C freezer, and put it in a 37°C warm water bath to thaw it quickly. The thawed bacterial liquid was poured into an anaerobic blood agar plate, and placed under anaerobic conditions at 37 °C for 48 h. Observe the growth of colonies in the plate and the formation of hemolytic rings. Observe the morphology of the strains under a Gram stain microscope. After confirming that there is no contamination, transfer them to MRS agar plates and place them in anaerobic conditions at 37 °C for 24 hours. Pick a single colony on the plate. Colonies were inoculated into 6 mL of MRS liquid medium, and cultured under anaerobic conditions at 37 °C for 16-18 h. The activated bacterial solution was inoculated into 100 mL of MRS broth with a 3% (v / v) inoculum, 120 rpm / min, and shaken at 37° C. for 16-18 h. ...
Embodiment 3
[0043] Example 3 Preparation of Lactobacillus paracasei L.p R3-10 fermentation supernatant (extracellular secretion), bacterial suspension (cell)
[0044] Lactobacillus paracasei L.p R3-10 was activated and cultured and inoculated into MRS liquid medium. After culturing at 37°C for 15h, the concentration of fermented bacteria was adjusted to 1×10 7 CFU / mL, 4°C, 6000r / min centrifugation for 10min to obtain the culture supernatant and bacterial cell precipitate, the supernatant liquid was filtered through a 0.22μm filter to obtain the fermentation supernatant (extracellular secretion); After washing, the cells were resuspended in PBS, and the cell concentration was adjusted to 1 × 10 7 CFU / mL to obtain bacterial suspension (cell). Fermentation supernatant (extracellular secretion) and bacterial suspension (cell) were heated at 121°C for 20 minutes to prepare heat-inactivated fermentation supernatant (extracellular secretion) and heat-inactivated bacterial suspension (cell) ). ...
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