Culture method of marine-derived protease producing strain

Abstract

The invention discloses a culture method of a marine-derived protease producing strain, and belongs to the technical field of marine microorganisms. The culture method comprises the following steps: S01, preparing a culture medium; s02, activating a strain; s03, culturing a seed solution; and S04, protease fermentation culture: adding an activator component for improving the concentration of sodium chloride in a culture solution and carrying out ultrasonic treatment. According to the culture method of the marine-derived protease producing strain, the protease activity of the obtained fermentation liquor can be remarkably improved, the use amount of crude enzyme degradation protein in feed can be reduced, and the cost can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of marine microorganisms, and particularly relates to a method for culturing a protease-producing strain of marine origin. Background Art

[0002] The use of proteases in the feed industry has multiple benefits. First, it can break down large molecular proteins into small molecular peptides and amino acids that are easily digestible and absorbable, thereby improving the digestibility and bioavailability of proteins. This not only helps to improve the growth performance of animals, such as increasing daily weight gain and reducing feed-to-weight ratio, but also reduces nitrogen emissions and reduces environmental pollution. In addition, proteases can promote the formation of bioactive peptides, which have nutritional and physiological regulatory functions, such as antibacterial and antioxidant. Adding proteases to low-protein feeds can also improve intestinal health, increase the number of intestinal goblet cells, and increase the ratio of villus height to crypt depth, thereby optimizing the intestinal microbial environment. These effects not only improve the utilization efficiency of feed raw materials, but also bring economic benefits, especially in aquaculture, by improving the utilization rate of protein raw materials and reducing dependence on protein resources.

[0003] Marine protease producing strains have some unique characteristics, which make them have potential advantages in industrial applications. The present invention optimizes and improves the culture method of existing marine protease producing strains to increase the production of protease. Summary of the invention

[0004] The present invention discloses a method for culturing a marine protease-producing strain, comprising the following steps: S01, preparation of culture medium; S02, bacterial activation; S03, seed solution culture; S04, protease fermentation culture, including the steps of adding an activator component to increase the sodium chloride concentration in the culture solution and performing ultrasonic treatment.

[0005] In some embodiments of the present invention, in S01, the culture medium includes activation medium, seed culture medium and fermentation culture medium, wherein the concentration of sodium chloride in the activation medium is 30-40 g / L, the concentration of sodium chloride in the seed culture medium is 1-10 g / L, and the concentration of sodium chloride in the fermentation culture medium is 30-40 g / L.

[0006] In some embodiments of the present invention, in S01, the formula of the culture medium is: Activation medium: peptone 8.0-12.0 g / L, beef extract 2.0-4.0 g / L, magnesium chloride 1.0-3.0 g / L, magnesium sulfate 4.0-5.0 g / L, potassium chloride 0.5-1.0 g / L, calcium chloride 1.0-1.5 g / L, sodium chloride 30-4.0 g / L, appropriate amount of agar, pH 6.5-7.5; Seed culture medium: peptone 8.0-12 g / L, yeast extract powder 4.0-6.0 g / L, sodium chloride 20-30 g / L, pH 6.5-7.5; Fermentation medium: glucose 25.0-35.0 g / L, sucrose 25.0-35.0 g / L, peptone 15.0-25.0 g / L, ammonium sulfate 1.5-2.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, magnesium sulfate 0.5-1.5 g / L, manganese sulfate 0.05-0.15 g / L, zinc sulfate 0.005-0.015 g / L, sodium chloride 30.0-40.0 g / L, pH 6.5-7.5.

[0007] In some preferred embodiments of the present invention, in S01, the formula of the culture medium is: Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2.

[0008] In some embodiments of the present invention, in S02, the bacterial strain is taken out from the ultra-low temperature refrigerator, inoculated into an activation culture medium, cultured, and passaged.

[0009] In some embodiments of the present invention, in S02, after inoculation into the activation medium, the cells are cultured at 28-32° C. for 4-8 h and passaged 1-3 times.

[0010] In some embodiments of the present invention, in S03, the cells are inoculated with seed culture solution and cultured in a shaking platform.

[0011] In some embodiments of the present invention, in S03, an inoculation loop is used to inoculate one loop of the activated strain into the seed culture medium.

[0012] In some embodiments of the present invention, in S03, the culture is carried out at 28-32° C. at 100-200 rpm on a shaking incubator for 10-14 h.

[0013] In some embodiments of the present invention, in S04, the seed liquid is inoculated into the fermentation medium and cultured on a shaking table.

[0014] In some embodiments of the present invention, in S04, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 2-10% (v / v).

[0015] In some embodiments of the present invention, in S04, the culture is carried out at 28-32° C. at 100-200 rpm on a shaking incubator for 10-14 h.

[0016] In some embodiments of the present invention, the step S05 of preparing a crude protease solution is further included: the fermentation solution is centrifuged and filtered to obtain a crude protease solution.

[0017] In some embodiments of the present invention, in S04, the activator includes sodium chloride, and the concentration of sodium chloride added to the culture medium is greater than 40 g / L; preferably 43-47 g / L; more preferably 45 g / L; Or, in S04, the activator includes at least two of sodium chloride, Tween 80 and ethylene glycol, preferably includes sodium chloride, Tween 80 and ethylene glycol, and further preferably, the concentration of sodium chloride added to the culture medium is greater than 40 g / L, the concentration of Tween 80 is 0.4-0.6 mL / L, and the concentration of ethylene glycol is 0.005-0.015 mL / L.

[0018] In some embodiments of the present invention, in S04, the ultrasonic treatment is 50W ultrasonic treatment for 2-3s, with an interval of 10s, repeated for a total of 4-6 times.

[0019] Beneficial effects: The method for cultivating marine protease producing strains of the present invention promotes the improvement of the protease activity of marine bacteria in culture by increasing the concentration of sodium chloride and ultrasonic treatment. Studies have found that the effect of ultrasonic treatment depends on sufficient sodium chloride concentration.

[0020] The study also unexpectedly found that adding a composite activator of sodium chloride, Tween 80 and ethylene glycol can further enhance the activity of the protease.

[0021] The patented method for culturing marine-derived protease-producing strains can significantly improve the protease activity of the resulting fermentation broth, and can significantly reduce the amount of crude enzyme solution used in feed to degrade protein and reduce costs. DETAILED DESCRIPTION

[0022] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0023] The marine-derived protease producing strain is CGMCC 1.504.

[0024] Unless otherwise specified, the examples and comparative examples are parallel tests with the same parameters such as components, component contents, preparation steps, preparation parameters, and dimensions.

[0025] Example 1 A method for cultivating a protease-producing strain of marine origin: (1) Culture medium Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2; (2) Activation of bacterial strains Take the strain out of the ultra-low temperature freezer, use an inoculation loop to inoculate 1 loop into the activation medium, culture at 30°C for 6 h, and then subculture twice in the same way.

[0026] (3) Seed liquid culture Inoculate one loop of the inoculation loop into the seed culture solution (50 mL / 250 mL) and culture at 30°C with a shaker at 150 rpm for 12 h. (4) Protease fermentation culture The seed solution was added to the fermentation medium (45 mL / 250 mL) at a 5% (v / v) inoculation rate and cultured at 30°C at 150 rpm for 6 h on a shaker; The activator was added in the amount of activator 1 (10 g sodium chloride) per 1000 mL culture medium, and the culture was cultured at 30°C at 150 rpm for 0.5 h. The cells were ultrasonically treated at 50 W for 3 s with an interval of 10 s, and the process was repeated for a total of 5 times. The culture was continued for 17.5 h. (5) Preparation of crude protease solution The fermentation broth was centrifuged (10000 rpm×10 min), and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a crude protease solution.

[0027] Example 2 A method for cultivating a protease-producing strain of marine origin: (1) Culture medium Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2; (2) Activation of bacterial strains Take the strain out of the ultra-low temperature freezer, use an inoculation loop to inoculate 1 loop into the activation medium, culture at 30°C for 6 h, and then subculture twice in the same way.

[0028] (3) Seed liquid culture Inoculate one loop of the inoculation loop into the seed culture solution (50 mL / 250 mL) and culture at 30°C with a shaker at 150 rpm for 12 h. (4) Protease fermentation culture The seed solution was added to the fermentation medium (45 mL / 250 mL) at a 5% (v / v) inoculation rate and cultured at 30°C at 150 rpm for 6 h on a shaker; The activator was added in the amount of activator 1 (8 g sodium chloride) per 1000 mL culture medium, and the culture was cultured at 30°C at 150 rpm for 0.5 h. The cells were ultrasonically treated at 50 W for 2 s, with an interval of 10 s, and repeated for a total of 6 times. The culture was continued for 17.5 h. (5) Preparation of crude protease solution The fermentation broth was centrifuged (10000 rpm×10 min), and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a crude protease solution.

[0029] Example 3 A method for cultivating a protease-producing strain of marine origin: (1) Culture medium Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2; (2) Activation of bacterial strains Take the strain out of the ultra-low temperature freezer, use an inoculation loop to inoculate 1 loop into the activation medium, culture at 30°C for 6 h, and then subculture twice in the same way.

[0030] (3) Seed liquid culture Inoculate one loop of the inoculation loop into the seed culture solution (50 mL / 250 mL) and culture at 30°C with a shaker at 150 rpm for 12 h. (4) Protease fermentation culture The seed solution was added to the fermentation medium (45 mL / 250 mL) at a 5% (v / v) inoculation rate and cultured at 30°C at 150 rpm for 6 h on a shaker; The activator 1 (12 g sodium chloride) was added per 1000 mL of culture medium, and the culture was cultured at 30°C at 150 rpm for 0.5 h. The cells were ultrasonically treated at 50 W for 3 s with an interval of 10 s, and the process was repeated for a total of 4 times. The culture was continued for 17.5 h. (5) Preparation of crude protease solution The fermentation broth was centrifuged (10000 rpm×10 min), and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a crude protease solution.

[0031] Example 4 A method for cultivating a protease-producing strain of marine origin: (1) Culture medium Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2; (2) Activation of bacterial strains Take the strain out of the ultra-low temperature freezer, use an inoculation loop to inoculate 1 loop into the activation medium, culture at 30°C for 6 h, and then subculture twice in the same way.

[0032] (3) Seed liquid culture Inoculate one loop of the inoculation loop into the seed culture solution (50 mL / 250 mL) and culture at 30°C with a shaker at 150 rpm for 12 h. (4) Protease fermentation culture The seed solution was added to the fermentation medium (45 mL / 250 mL) at a 5% (v / v) inoculation rate and cultured at 30°C at 150 rpm for 6 h on a shaker; The activator was added in the amount of composite activator 2 (10 g sodium chloride, 0.4 mL Tween 80, 0.015 mL ethylene glycol) per 1000 mL culture medium, and the culture was cultured at 30°C at 150 rpm for 0.5 h. The cells were ultrasonically treated at 50 W for 3 s with an interval of 10 s, and the process was repeated for a total of 5 times. The culture was continued for 17.5 h. (5) Preparation of crude protease solution The fermentation broth was centrifuged (10000 rpm×10 min), and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a crude protease solution.

[0033] Example 5 A method for cultivating a protease-producing strain of marine origin: (1) Culture medium Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2; (2) Activation of bacterial strains Take the strain out of the ultra-low temperature freezer, use an inoculation loop to inoculate 1 loop into the activation medium, culture at 30°C for 6 h, and then subculture twice in the same way.

[0034] (3) Seed liquid culture Inoculate one loop of the inoculation loop into the seed culture solution (50 mL / 250 mL) and culture at 30°C with a shaker at 150 rpm for 12 h. (4) Protease fermentation culture The seed solution was added to the fermentation medium (45 mL / 250 mL) at a 5% (v / v) inoculation rate and cultured at 30°C at 150 rpm for 6 h on a shaker; The activator was added in the amount of composite activator 2 (10 g sodium chloride, 0.5 mL Tween 80, 0.01 mL ethylene glycol) per 1000 mL culture medium, and the culture was cultured at 30°C at 150 rpm for 0.5 h. The cells were ultrasonically treated at 50 W for 3 s with an interval of 10 s, and the process was repeated for a total of 5 times. The culture was continued for 17.5 h. (5) Preparation of crude protease solution The fermentation broth was centrifuged (10000 rpm×10 min), and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a crude protease solution.

[0035] Example 6 A method for cultivating a protease-producing strain of marine origin: (1) Culture medium Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2; (2) Activation of bacterial strains Take the strain out of the ultra-low temperature freezer, use an inoculation loop to inoculate 1 loop into the activation medium, culture at 30°C for 6 h, and then subculture twice in the same way.

[0036] (3) Seed liquid culture Inoculate one loop of the inoculation loop into the seed culture solution (50 mL / 250 mL) and culture at 30°C with a shaker at 150 rpm for 12 h. (4) Protease fermentation culture The seed solution was added to the fermentation medium (45 mL / 250 mL) at a 5% (v / v) inoculation rate and cultured at 30°C at 150 rpm for 6 h on a shaker; The activator was added in the amount of composite activator 2 (10 g sodium chloride, 0.6 mL Tween 80, 0.005 mL ethylene glycol) per 1000 mL culture medium, and the culture was cultured at 30°C at 150 rpm for 0.5 h. The cells were ultrasonically treated at 50 W for 3 s with an interval of 10 s, and the process was repeated for a total of 5 times. The culture was continued for 17.5 h. (5) Preparation of crude protease solution The fermentation broth was centrifuged (10000 rpm×10 min), and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a crude protease solution.

[0037] Example 7 A method for cultivating a protease-producing strain of marine origin: (1) Culture medium Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2; (2) Activation of bacterial strains Take the strain out of the ultra-low temperature freezer, use an inoculation loop to inoculate 1 loop into the activation medium, culture at 30°C for 6 h, and then subculture twice in the same way.

[0038] (3) Seed liquid culture Inoculate one loop of the inoculation loop into the seed culture solution (50 mL / 250 mL) and culture at 30°C with a shaker at 150 rpm for 12 h. (4) Protease fermentation culture The seed solution was added to the fermentation medium (45 mL / 250 mL) at a 5% (v / v) inoculation rate and cultured at 30°C at 150 rpm for 6 h on a shaker; The activator was added in the amount of composite activator 3 (10 g sodium chloride, 0.5 mL Tween 80) per 1000 mL culture medium, and the culture was cultured at 30°C at 150 rpm for 0.5 h. The cells were ultrasonically treated at 50 W for 3 s with an interval of 10 s, and the process was repeated for a total of 5 times. The culture was continued for 17.5 h. (5) Preparation of crude protease solution The fermentation broth was centrifuged (10000 rpm×10 min), and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a crude protease solution.

[0039] Example 8 A method for cultivating a protease-producing strain of marine origin: (1) Culture medium Activation medium: peptone 10.0 g / L, beef extract 3.0 g / L, magnesium chloride 2.0 g / L, magnesium sulfate 4.5 g / L, potassium chloride 0.8 g / L, calcium chloride 1.2 g / L, sodium chloride 35 g / L, appropriate amount of agar, pH 7.0-7.2; Seed culture medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 25 g / L, pH 7.0-7.2; Fermentation medium: glucose 30.0 g / L, sucrose 30.0 g / L, peptone 20.0 g / L, ammonium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 1.0 g / L, manganese sulfate 0.01 g / L, zinc sulfate 0.01 g / L, sodium chloride 35 g / L, pH 7.0-7.2; (2) Activation of bacterial strains Take the strain out of the ultra-low temperature freezer, use an inoculation loop to inoculate 1 loop into the activation medium, culture at 30°C for 6 h, and then subculture twice in the same way.

[0040] (3) Seed liquid culture Inoculate one loop of the inoculation loop into the seed culture solution (50 mL / 250 mL) and culture at 30°C with a shaker at 150 rpm for 12 h. (4) Protease fermentation culture The seed solution was added to the fermentation medium (45 mL / 250 mL) at a 5% (v / v) inoculation rate and cultured at 30°C at 150 rpm for 6 h on a shaker; The activator was added in the amount of composite activator 4 (10 g sodium chloride, 0.01 mL ethylene glycol) per 1000 mL culture medium, and the culture was cultured at 30°C at 150 rpm for 0.5 h. The cells were ultrasonically treated at 50 W for 3 s, with an interval of 10 s, and repeated for a total of 5 times. The culture was continued for 17.5 h. (5) Preparation of crude protease solution The fermentation broth was centrifuged (10000 rpm×10 min), and the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain a crude protease solution.

[0041] Furthermore, in Examples 1-8, the shaking table is regulated by an intelligent control system, including: a setting module, a temperature control module, a remote control module, a sensing module and a timing module. When the shaking table is used for cultivation, the target parameters of the shaking table are obtained through the setting module, the target speed, the target temperature and the target duration are determined, and the temperature control module is used to regulate the shaking table according to the target temperature, and the temperature is maintained at a constant temperature after the shaking table temperature reaches the target temperature. Then, the sensing module is used to sense whether there is a culture vessel on the shaking table platform to obtain sensing information. When the remote control module is used to regulate the shaking table according to the sensing information, the target speed is adjusted. The shaking table platform is made to shake at a target speed. When there is no culture vessel on the shaking table platform, the shaking of the shaking table platform is suspended. In the process in which the temperature control module and the remote control module respectively control the target temperature and the target speed, the time statistics are performed through the timing module to obtain the control information of the temperature control module and the control information of the remote control module. The effective shaking time is analyzed according to the control information of the temperature control module and the control information of the remote control module to determine the duration in which the shaking table platform simultaneously meets the target temperature and the target speed during the shaking process, to obtain the effective shaking statistical time, and to give a timing reminder when the effective shaking statistical time reaches the target duration. The intelligent control system is used to realize intelligent control of the shaker, so that the shaker can cultivate the substances in the culture vessel according to the requirements of temperature and rotation speed, so that the substances in the culture vessel remain active, providing guarantee for seed liquid culture and protease fermentation culture, and the remote control module and the sensing module can be used to pause in time when other operations are performed on the culture vessel during the culture process to avoid waste of resources, and the shaking can be resumed in time when the culture vessel returns to the shaker platform, which saves resources and does not affect the normal use of the shaker. In addition, the shaking time of the shaker is counted through the timing module, so that when shaking according to the target time, a reminder can be issued when the target time is reached, providing convenience for relevant personnel, and the timing module is used to analyze the effective shaking statistical time to avoid misleading the shaker based on the shaker target parameters to affect the shaking effect.

[0042] Further, in Examples 1-8, when the supernatant is filtered with a 0.22 μm sterile filter membrane, a sterility test is performed on the sterile filter membrane in advance. When there are no bacteria on the sterile filter membrane, the supernatant is filtered with the sterile filter membrane. When there are bacteria on the sterile filter membrane, the bacteria on the sterile filter membrane are washed off and then the supernatant is filtered with the sterile filter membrane. In the process of filtering the supernatant with the sterile filter membrane, the sterile filter membrane is monitored for bacteria, the sterile filter membrane monitoring information is obtained in real time, and whether bacteria appear on the sterile filter membrane is analyzed based on the sterile filter membrane monitoring information. When bacteria appear on the sterile filter membrane, the filtration of the supernatant by the sterile filter membrane is suspended, the sterile filter membrane is washed with bacteria to remove the bacteria on the sterile filter membrane, and then the supernatant is filtered using the sterile filter membrane after the bacteria washing treatment, thereby obtaining a crude protease solution. By conducting a sterility test on the sterile filter membrane in advance, it is ensured that the sterile filter membrane is sterile when filtering the supernatant, thereby preventing bacteria on the sterile filter membrane from contaminating the filtration of the supernatant and affecting the quality of the crude protease solution. In addition, during the filtration of the supernatant using the sterile filter membrane, bacteria on the sterile filter membrane is monitored, so that the sterile filter membrane can always be filtered under sterile conditions during the filtration of the supernatant with a 0.22μm sterile filter membrane, thereby preventing bacteria from affecting the filtration effect, improving the production of the crude protease solution, and ensuring the quality of the crude protease solution.

[0043] Comparative Example 1 A method for culturing a marine-derived protease-producing strain, which differs from Example 1 in that: (4) activator 1 is not added during the protease fermentation culture.

[0044] Comparative Example 2 A method for culturing a marine protease-producing strain, which differs from Example 1 in that: (4) ultrasonic treatment is not performed during the protease fermentation culture.

[0045] Comparative Example 3 A method for culturing a marine-derived protease-producing strain, which differs from Example 1 in that: (4) during the protease fermentation culture, an activator is added in an amount of 1 activator (5 g of sodium chloride) per 1000 mL of culture solution.

[0046] Crude protease liquid performance test 1 Protease activity The crude protease solution obtained in the examples and comparative examples was used to determine the protease activity using the Folin phenol method in GB / T 23527-2009 Protease Preparations. The tyrosine standard curve formula is: y=0.0103x-0.0068, and the enzyme capacity is calculated according to the enzyme activity formula. The results are shown in Table 1.

[0047] Table 1 Performance test

[0048] The results showed that the protease activities of Examples 1-3 with the addition of sodium chloride activator and ultrasonic treatment were significantly higher than those of Comparative Examples 1 and 2, and there was no significant difference between Comparative Example 3 with the addition of 5 g of sodium chloride per 100 mL of culture medium and Comparative Examples 1 and 2. The protease activities of Examples 4-6 with the addition of sodium chloride, Tween 80 and ethylene glycol activators and ultrasonic treatment were significantly higher than those of Examples 1-3 and Examples 7 and 8.

[0049] 2 Degraded feather meal Equal amounts of feather powder were divided into centrifuge tubes, and the crude protease solutions of the embodiment and the comparative example were added respectively, treated at 40° C. for 30 min, and then heated in boiling water for 3 min. The prepared samples were separated by SDS-polyacrylamide gel vertical plate electrophoresis, stained with Coomassie brilliant blue staining solution, and decolorized with Coomassie brilliant blue decolorizing solution.

[0050] By visually observing the rubber plate, it can be seen that the bands of Examples 1-8 are clearer than those of Comparative Examples 1-3, and the enzymatic hydrolysis effect is good. Unlike the protease activity, there is no obvious difference between the bands of Examples 4-6 and Examples 1-3, indicating that there is no necessary logic between the protease activity of the crude enzyme solution and the degradation of feather powder. It may be that the crude enzyme solution contains multiple proteases, and only some of them are effective in degrading feather powder.

[0051] The preferred specific implementation modes and embodiments of the present invention are described in detail above, but the present invention is not limited to the above implementation modes and embodiments, and various changes can be made within the knowledge scope of those skilled in the art without departing from the concept of the present invention.

Claims

1. A method for culturing a protease-producing strain of marine origin, characterized in that: The following steps are involved: S01, preparation of culture medium; S02, bacterial activation; S03, seed solution culture; S04, protease fermentation culture, including the steps of adding an activator component to increase the sodium chloride concentration in the culture solution and performing ultrasonic treatment.

2. The method for culturing a marine-derived protease-producing strain according to claim 1, characterized in that: In S01, the culture medium includes an activation culture medium, a seed culture medium and a fermentation culture medium, wherein the concentration of sodium chloride in the activation culture medium is 30-40 g / L, the concentration of sodium chloride in the seed culture medium is 1-10 g / L, and the concentration of sodium chloride in the fermentation culture medium is 30-40 g / L.

3. The method for culturing a marine-derived protease-producing strain according to claim 2, characterized in that: In S01, the formula of the culture medium is: Activation medium: peptone 8.0-12.0 g / L, beef extract 2.0-4.0 g / L, magnesium chloride 1.0-3.0 g / L, magnesium sulfate 4.0-5.0 g / L, potassium chloride 0.5-1.0 g / L, calcium chloride 1.0-1.5 g / L, sodium chloride 30-4.0 g / L, appropriate amount of agar, pH 6.5-7.5; Seed culture medium: peptone 8.0-12 g / L, yeast extract powder 4.0-6.0 g / L, sodium chloride 20-30 g / L, pH 6.5-7.5; Fermentation medium: glucose 25.0-35.0 g / L, sucrose 25.0-35.0 g / L, peptone 15.0-25.0 g / L, ammonium sulfate 1.5-2.5 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, magnesium sulfate 0.5-1.5 g / L, manganese sulfate 0.05-0.15 g / L, zinc sulfate 0.005-0.015 g / L, sodium chloride 30.0-40.0 g / L, pH 6.5-7.

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4. The method for culturing a marine-derived protease-producing strain according to claim 1, characterized in that: In S02, the bacterial strain is taken out from the ultra-low temperature refrigerator, inoculated into the activation culture medium, cultured, and passaged.

5. The method for culturing a marine-derived protease-producing strain according to claim 1, characterized in that: In S03, the cells were inoculated with seed culture solution and cultured in a shaking incubator.

6. The method for culturing a marine-derived protease-producing strain according to claim 1, characterized in that: In S04, the seed solution is inoculated into the fermentation medium and cultured on a shaking table.

7. The method for culturing a marine-derived protease-producing strain according to claim 1, characterized in that: The method also includes the step S05 of preparing a crude protease solution: centrifuging and filtering the fermentation solution to obtain a crude protease solution.

8. The method for culturing a marine-derived protease-producing strain according to claim 1, characterized in that: In S04, the activator includes sodium chloride, and the concentration of sodium chloride added to the culture medium is greater than 40 g / L; Or, in S04, the activator includes at least two of sodium chloride, Tween 80 and ethylene glycol.

9. The method for culturing a marine-derived protease-producing strain according to claim 1, characterized in that: In S04, the activator includes sodium chloride, Tween 80 and ethylene glycol. The concentration of sodium chloride added to the culture medium by the activator is greater than 40 g / L, the concentration of Tween 80 is 0.4-0.6 mL / L, and the concentration of ethylene glycol is 0.005-0.015 mL / L.

10. The method for culturing a marine-derived protease-producing strain according to claim 1, characterized in that: In S04, the ultrasonic treatment is 50W ultrasonic treatment for 2-3s, with an interval of 10s, repeated for a total of 4-6 times.

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