Isolated bacillus subtilis strains and uses thereof
A technology for Bacillus subtilis and isolates, which is applied in the field of Bacillus subtilis isolates and can solve problems such as joint inflammation and slow chondrocyte regeneration rate.
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example 1
[0076] Example 1. Preparation of Bacillus subtilis fermentation extract
[0077] (1). Preparation of Bacillus subtilis P12-3C bacterial strain and P32-7B bacterial strain:
[0078] After culturing the Bacillus subtilis 199 strain on a nutrient agar plate at 30°C for 3 days, wash the bacterial cells with sterile water, and adjust the bacterial cell concentration to be greater than 1×10 8 cfu / mL. Take 0.5mL bacterial solution and place it on a sterile plate, place the plate in a UV light box, treat it with 1% EMS (ethyl methanesulfonate), and then irradiate it with 100mJ of UV light, then serially dilute it with sterile water , smear 0.1mL of bacterial solutions with different dilution concentrations on nutrient agar plates or screening media, and culture them at 30°C for 1 to 2 days under dark conditions, and then select to obtain the Bacillus subtilis P12-3C strain.
[0079] Continue to mutate the P12-3C strain in the same manner as above, and select to obtain the Bacillus s...
example 2
[0095] Example 2. Activity of Bacillus subtilis fermented extract in treating arthritis
[0096] This example uses interleukin (IL-1β) to induce the expression of osteoarthritis-related cytokines such as IL-8, ROS and MMP-1, MMP-3 and MMP-13 in human chondrosarcoma cells (SW1353), And evaluate the above-mentioned fermented samples to regulate the inducing activity of IL-1β on SW1353, so as to understand the potential of the above-mentioned fermented samples for anti-arthritis and development of articular cartilage health care products.
[0097] (1). Human chondrosarcoma cell (SW1353) culture:
[0098] SW1353 cells (obtained from Food Industry Development Research Institute, number BCRC60548) were cultured in Leibovitz's L-15 medium (HyClone ), at 37°C, 0% CO 2 cell culture incubator. When the cells grow to about 8-9 points full, carry out subculture. The cells attached to the culture dish were first washed with 10 mL of PBS (purchased from Sigma, pH 7.4), washed repeatedly...
example 3
[0103] Example 3. Bacillus subtilis fermentation extract promotes chondrocyte proliferation and increases extracellular matrix production
[0104] The main purpose of this experiment is to use chondrocytes isolated from pig knee joints to evaluate the induction of articular chondrocyte regeneration and extracellular matrix (ECM) production (such as collagen II and glycosaminoglycan ) and the ability to stimulate the expression of ECM-related genes to evaluate the activity of fermented samples in inducing articular cartilage regeneration.
[0105] (1). Chondrocyte culture of pig knee joint:
[0106] The primary chondrocytes from the knee joint of the hind leg bone of the pig were added to DMEM medium (purchased from GIBCO) containing 10% FBS, and the number of cells was adjusted to about 10 5 cells / mL (generation P0), take 10 mL of cell suspension and place it in a 10 cm diameter petri dish, 2 After culturing in the incubator to about eight to nine minutes full, wash the chon...
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