Nannochloropsis gaditana antibacterial peptide and application thereof
A technology of Nannochloropsis pseudochloropsis and antimicrobial peptide, which is applied in the field of biomedicine to achieve the effect of strong antibacterial activity, simple structure and wide antibacterial spectrum
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Embodiment 1
[0033] Embodiment 1, the heterotrophic cultivation of Nannochloropsis Gad
[0034] After activation and expansion of Nannochloropsis Gad, the strains of Nannochloropsis Gad were transferred to a 50L mechanical fermenter. Glucose was used as the carbon source and peptone was used as the nitrogen source for aeration and stirring fermentation. Under the condition of amount 2vvm, cultivate, when the residual sugar in the fermented liquid drops to 5g / L, carry out fed-batch fermentation with 900g / L glucose solution (sugar concentration is maintained on 4-6g / L). When the fermentation was terminated (the total fermentation time was 36 hours), the fermentation broth was centrifuged at 4°C and 4000rpm for 15min to collect algae cells, and freeze-dried at -86°C and 0.10mbar until constant weight.
Embodiment 2
[0035] Embodiment 2, the extraction of Pseudochlorococcus protein of Gad
[0036] Weigh the freeze-dried microalgae powder, use 0.01M, pH6.8 PBS buffer solution; prepare 200 mL of microalgae suspension according to the ratio of material to liquid 1:10 (w / w), and perform high-pressure homogeneous wall breaking under 1000 bar pressure, The wall-broken solution was collected, centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant was collected. Slowly add solid ammonium sulfate to 80% saturation concentration under stirring conditions, fully stir for 30 minutes, and stand at 4°C for 12 hours. After standing, centrifuge at 12,000 rpm for 30 minutes at 4°C to precipitate protein components.
[0037] Collect the precipitate, redissolve in 2 times the volume of 0.01M, pH6.8 PBS buffer, and then desalt the protein with Sephadex G25 desalting column (with Sephadex G25 5ml inside) (control the flow rate to 1.0 ml / min), freeze-dried algal protein (-86°C, 0.10mbar freeze-dried...
Embodiment 3
[0038] Example 3. Preparation and separation and purification of Nannochloropsis gaide hydrolyzed peptide
[0039] 1) Chronological hydrolysis of Nannochloropsis Gad
[0040] Weigh a certain amount of protein freeze-dried powder (obtained in Example 2), dissolve it in 0.01M PBS to prepare a protein solution with a concentration of 10mg / mL for enzyme reaction, adjust the temperature of the reaction system to 60°C, and use 0.01M NaOH to adjust the pH to 6.5 , adding 2% papain accounting for the mass of the protein freeze-dried powder, and the hydrolysis time is 0, 0.5, 1, 2, 4, and 8 hours respectively. After the hydrolysis in each period was completed, the hydrolyzate was collected and the enzyme was inactivated in a water bath at 85°C for 10 minutes, centrifuged at 4°C and 8000rpm for 10 minutes, and the supernatant was collected. By measuring the antibacterial activity, it was found that the activity of the hydrolyzate gradually increased with the increase of hydrolysis time...
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