Culture medium for producing protease through fermentation of mucor and application of culture medium
A technology for producing protease and culture medium, applied in the field of fermentation, can solve the problems of limited selection of food-grade commercial protease, high cost of commercial protease, low yield, etc., and achieves good application prospects, optimization of culture conditions, and reduction of production costs.
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Embodiment 1
[0053] The influence of embodiment 1 adding additive on producing protease
[0054] The effects of carbon source, nitrogen source, trace elements and other factors on protease activity were explored.
[0055] Determination results of Mucor protease species:
[0056] Mucor can secrete a variety of proteases after fermentation. In order to determine the type of high-yield protease, it was cultured at 28°C for 96 hours to fully ferment and produce enzymes in the basal medium (5g of yellow mushroom, 10g of bran, and 15g of deionized water). Finally, three protease enzyme activities of the crude enzyme liquid were measured, and the results are shown in figure 1 .
[0057] Depend on figure 1 It can be seen that the two kinds of mucormycetes can secrete alkaline protease, acid protease and neutral protease after fermentation and culture, and the enzyme activity of neutral protease is higher. vitality.
[0058] 1.1 Screening of external carbon sources
[0059] Add a certain amou...
Embodiment 2
[0079] The influence of embodiment 2 fermentation conditions on producing protease
[0080] 2.1 Screening of inoculum volume
[0081] Take five 250mL Erlenmeyer flasks, add the fermentation medium with the optimal ratio of 1.5 in Example 1, sterilize at 121°C for 30min, shake it while it is hot, and cool it to about 30°C at room temperature. The inoculum size of Mucor was 1×10 4 pcs / g, 1×10 5 pcs / g, 1×10 6 pcs / g, 1×10 7 pcs / g, 1×10 8 Each / g was cultured in a mold incubator at 28°C for 96 hours, and the protease activity was determined. The result is as Figure 5 shown. In the figure, 4: 1.0×10 4 pcs / g; 5: 1.0×10 5 pcs / g; 6: 1.0×10 6 pcs / g; 7: 1.0×10 7 pcs / g; 8: 1.0×10 8 pcs / g.
[0082] From Figure 5 It can be seen that the amount of inoculum has a great influence on the synthesis of protease. The general law of the change of protease activity with the inoculum size is that when the inoculum size is less than 1×10 6 Individual / g, the enzyme activity increased wit...
Embodiment 3
[0103] The research of embodiment 3 protease enzymatic properties
[0104] 3.1 Extraction of protease
[0105] 3.1.1 Preparation of crude enzyme solution
[0106] Add 5 times the volume of the fermentation product to sterile water, disperse evenly, extract at 4°C for 24 hours, filter with 8 layers of sterile gauze, centrifuge the filtrate (10000r / min, 20min), and obtain the supernatant as the crude enzyme solution.
[0107] 3.1.2 Fractional precipitation of ammonium sulfate
[0108] Take 7 samples of 20ml crude enzyme solution, and then slowly add solid ammonium sulfate successively, so that the mass fraction of ammonium sulfate is 20%, 40%, 60%, 80%, and 100% respectively. Centrifuge at ℃ (10000r / min, 30min) to remove the precipitate. The supernatant was taken, dialyzed with distilled water for 24 hours, and then refrigerated and centrifuged at 4°C (10000r / min, 30min). After the supernatant was adjusted to the same volume, the protein content and protease activity were mea...
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