Paenibacillus pabuli, culture medium thereof and application of paenibacillus pabuli in preparation of enteromorpha prolifera polysaccharide degrading enzymes
A technology of prolifera polysaccharide and bacillus, applied in the field of microorganisms, can solve problems such as rare reports of enzymatic degradation of prolifera polysaccharide
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Embodiment 1
[0107] Isolation and purification of Enteromorpha polysaccharide-degrading bacteria (Paenibacillus pabuli) EP-1.
[0108] Collect sea mud from the sea area near the No. 1 bathing beach in Qingdao City, Shandong Province, add sterile seawater to dissolve it, transfer it to the primary screening medium, and shake the flask for enrichment and culture for 2-3 days. The culture solution is spread on the solid medium plate containing the enteromorpha polysaccharide, and cultivated for 3-5 days. Stain with Lugol's iodine solution, transfer the colonies with obvious degradation circles to a new Enteromorpha polysaccharide solid medium plate for culture, and repeat the streaking until a pure culture is obtained. Primary screening medium composition (mass volume ratio): Enteromorpha polysaccharide 4%, NaCl 1.5%, NaNO 3 0.5%, MgSO 4 ·7H 2 O0.05%, CaCl 2 0.01%, Iron Phosphate 0.005%, pH 7.0.
[0109] Inoculate the pure culture separated from the primary screening into the fermentatio...
Embodiment 2
[0121] The media shown in Table 1 were prepared respectively, and the preservation number was CGMCC No. 12912. Paenibacillus for feed was activated and inoculated into the media in Table 1. The inoculum size is 5%, the culture temperature is 28°C, the shaker speed is 200rpm, cultured for 24h, and the OD of the bacterial solution is measured 600 value.
[0122] The obtained bacterial solution was centrifuged at 6000 rpm for 20 minutes to obtain a fermentation supernatant; at 4°C, the obtained fermentation supernatant was sterilized through a 0.22 μm filter membrane, concentrated by ultrafiltration, desalted by dialysis, and vacuum freeze-dried to obtain Enteromorpha polysaccharide degrading enzyme. Determination of enzyme activity. The detection method of enzyme activity is DNS method, and the definition of enzyme activity: 1mL enzyme solution produces 1μg reducing sugar in 1min as an activity unit.
[0123] Table 1, medium formula
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Embodiment 3
[0129] Adopt the medium with the best effect in Table 1 (the formula is Enteromorpha polysaccharide 0.2%, peptone 0.6%, yeast extract powder 0.7%, NaCl 0.5%, MgSO 4 ·7H 2 O 0.1%, Na 2 HP0 4 6mmol / L) to prepare Enteromorpha polysaccharide degrading enzyme by fermentation. Pre-treated and activated before fermentation.
[0130] 1) Pre-treatment: Streak Paenibacillus for feed with the preservation number CGMCC NO.12912 on the solid medium of Enteromorpha polysaccharide, and culture it at 25-35°C for 5-7 days;
[0131] 2) Activation: Inoculate 1 to 3 loops of the solid culture medium strain into a test tube of seed culture solution, and culture at 25 to 35° C. at 150 to 210 rpm for 12 to 24 hours.
[0132] 3) Fermentation: insert the seed culture medium into the fermentation culture medium, culture on a shaker for 24-48 hours, and obtain the fermented product.
[0133] Step 3 carries out the fermentation test of different parameters, and each parameter is as table 2:
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