Application of aspergillus niger LCCC30112 in glucamylase production and tobacco fermentation
A technology of LCCC30112 and glucoamylase, which is applied in the field of microbial applications to achieve the effects of shortening the fermentation cycle, accelerating degradation, and improving quality
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Embodiment 1
[0032] Example 1 Production of glucoamylase by fermentation of Aspergillus niger LCCC30112
[0033] Study on Fermentation Technology of Aspergillus Niger LCCC30112
[0034] After single-factor multi-level and multi-factor multi-level formula tests, the liquid enzyme production process of Aspergillus niger LCCC30112 was determined:
[0035] The preserved strains were transferred to the activation medium, cultured at 30°C for 3 days, and set aside.
[0036] The activation medium adopts PDA medium: potato 200g / L, glucose 20g / L, agar 15g / L, pH natural, sterilized at 121°C for 30min for later use.
[0037] Seed solution preparation:
[0038] Aspergillus niger LCCC30112 bacterial strain is transferred to the activated medium, cultivated for 4 days until a large number of spores are produced, the spores are repeatedly washed with 1% physiological saline, the spore suspension is collected, and inoculated into the seed liquid medium, the inoculum size is 1% (concentration 10 8 cfu...
Embodiment 2
[0043] Example 2 Glucoamylase separation and purification:
[0044] (1) Ammonium sulfate precipitation
[0045] Centrifuge the fermentation broth at 4°C and 6000r / min for 15min to remove the mycelium and obtain the supernatant;
[0046] Ammonium sulfate with 80% saturation was slowly added to the supernatant for salting out and precipitated, centrifuged at 8000 r / min for 30 min, and the precipitate was dialyzed with deionized water to obtain a crude enzyme solution.
[0047](2) DEAE Sepharose Fast Flow Anion Exchange Chromatography
[0048] Load the crude enzyme solution onto a DEAE anion exchange column (16mm*100mm) for enzyme protein purification. Equilibrate with 10mmol Tris-HCl buffer (pH 8.4, containing 2mmol calcium chloride), elute with Tris-HCl buffer at a flow rate of 2mL / min, collect the components with enzyme activity peaks, and dialyze with deionized water , freeze-dried to obtain enzyme powder.
[0049] (3) Superdex-50 gel filtration chromatography
[0050]...
Embodiment 3
[0052] Example 3 Application of Aspergillus niger LCCC30112 in Tobacco Leaf Fermentation
[0053] Aspergillus niger LCCC30112 was inoculated on tobacco leaves, and the inoculation amount of Aspergillus niger LCCC30112 was 10 5 ~10 7 cfu / g, the fermentation temperature is 20-40°C, the humidity is 40-70%, and the fermentation time is 5-10 days.
[0054] Or the fermentation supernatant liquid of Aspergillus niger LCCC30112 that embodiment 2 obtains, or its crude enzyme, or the glucoamylase after the purification of its final preparation is added in the tobacco leaf, for fermentation, the glucoamylase that adds is in tobacco leaf The enzyme activity is 10-20U / mg, and the fermentation conditions are: temperature 20-40°C, humidity 40-70%, and time 5-10d.
[0055] In the present invention, 2018 Chongqing B4F original tobacco is used as a control group, and 10 6 Aspergillus niger (Aspergillus niger) LCCC30112 of cfu / g is as treatment group 1, adds the glucoamylase enzyme powder t...
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