Serratia marcescens and application thereof in production of naringinase
A technology of Serratia marcescens and naringinase, which is applied in the field of microorganisms, can solve the problems of inability to meet industrial production and processing conditions, low stability of naringinase, etc., and achieves good application prospects, good fruit juice debittering effect and activity. and good stability
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Embodiment 1
[0040] Example 1: Serratia marcescens Serratia marcescens Isolation and identification of C10
[0041] The samples were collected from the soil of Daidaihua planting base (N25°41′29″; E110°35′44″) of Sanleng Company, Guilin City, Guangxi. Take 10 g of moldy soil and pomelo peel in 50 mL of sterile water, shake at 180 r / min at 30°C for half an hour. Then take 500 μL supernatant to 100 mL enrichment medium. Shake at 180 r / min at 30°C for 3 days. Perform serial dilutions to 10 -5 , take 10 μL and spread it on the plate, incubate at 30°C for 3 days, spray the surface of the plate with diethylene glycol lye, and the colonies will form a transparent circle, then streak and culture for 3 days. Finally, a single colony was fermented and cultured in liquid medium for 3 days at 30°C and 180 r / min, and then stored in a glycerol tube to obtain a new strain. The medium formula is as follows: liquid medium (g / 100mL): naringin 0.2, ammonium sulfate 0.1, magnesium sulfate 0.05, dipotass...
Embodiment 2
[0047] Example 2: Serratia marcescens Serratia marcescens Confirmation of C10 producing naringinase and determination of enzyme activity
[0048] 1. Preparation of Crude Enzyme Solution
[0049] The preserved glycerol bacteria were inoculated on solid medium, cultured at 30°C for 3 days, then inoculated into liquid medium, and cultured at 30°C and 180r / min for 3 days. Take the fermentation broth and centrifuge at 10,000 r / min, 4°C for 10 min, discard the supernatant, add 10 mL of 50 mM PBS buffer solution with pH 8.0, centrifuge at 10,000 r / min, 4°C for 5 min, repeat the operation twice, and discard the supernatant solution, and finally add PBS buffer according to 1:10 (g:mL) for sonication. Ultrasonic crushing conditions: 200 W, crushing 2 s, stop 2 s, time 10 min.
[0050] Solid medium (g / 100 mL): naringin 0.2, ammonium sulfate 0.1, magnesium sulfate 0.05, dipotassium hydrogen phosphate 0.1, calcium chloride 0.02, yeast 0.03, beef extract 0.03, agar powder 1;
[0051] Li...
Embodiment 3
[0056] Example 3: Serratia marcescens Serratia marcescens Analysis of Hydrolysis of Naringin Dihydrochalcone Catalyzed by Naringinase Produced by C10
[0057] Take the fermentation broth prepared in Example 2 and centrifuge at 10000 r / min at 4°C for 10 min, discard the supernatant, add 10 mL of 50 mM PBS buffer solution with pH 8.0, centrifuge at 10000 r / min at 4°C for 5 min, repeat the operation twice Discard the supernatant, and finally add PBS buffer at 1:10 (g:mL) for sonication. Ultrasonic crushing conditions: 200 W, crushing 2 s, stop 2 s, time 10 min. Take 2mL of 0.25mg / mL naringin dihydrochalcone + 1mL of 50 mM pH6 citrate buffer solution + 1mL of crude enzyme solution, react in a water bath at 50°C for 30min, then inactivate in a water bath at 100°C for 10min.
[0058] HPLC conditions for high performance liquid detection: Chromatographic column: Wondassil C18 analytical column (250 mm × 4.6 mm, 5 μm) mobile phase A: 0.1% formic acid water, mobile phase B: acetonitr...
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