CN103045571A - Fermentation process of quambalaria cyanescens strain - Google Patents

Abstract

The invention relates to a fermentation method of an excellent bacterial strain of C. indigo after mutagenesis selection, belongs to the technical field of bioengineering and enzyme preparation, and changes the cultivation temperature, shaking table speed, cultivation time and inoculation in the fermentation process parameters through a single factor experiment. The optimal fermentation conditions are finally determined based on factors such as the amount of fermentation and the amount of liquid. The verification experiment shows that the enzyme activity of chymosin in the fermentation broth can reach 610.52SU/mL, which is 96.71% higher than the initial enzyme activity of 310.36SU/mL.

Description

Fermentation method of echinospora blue strain

【Technical field】

The invention relates to a method for fermenting Echinococcus coeruleus through mutagenesis and selection, and belongs to the technical field of bioengineering and enzyme preparation preparation. the

【Background technique】

Rennet is the most critical enzyme in cheese production and has been widely used in the cheese industry since it was discovered. Cheese is widely loved by people all over the world because of its rich nutritional value and delicious taste. About 50 million calves need to be slaughtered every year in the world to meet the market demand for rennet. In recent years, with the improvement of people's living standards, the demand for cheese is increasing, and the supply of calf rennet is in short supply. Therefore, scientists are doing everything possible to find substitutes for calf rennet. the

Echinococcus blueus is a chymosin-producing strain isolated and purified from red yeast rice. But the chymosin activity produced by this strain is not high. We use C. coeruleus as the starting strain, and apply ultraviolet rays and diethyl sulfate composite factors to mutagenize the starting strain. The fine strain DU10 (microorganism preservation number: CGMCC 5423) selected by mutagenesis was used as the fermentation medium with glucose potato medium. The enzyme activity of chymosin in the fermentation liquid fluctuated around 110SU/mL, but the enzyme activity produced was still not enough. ideal. Therefore, the medium has to be optimized, and the optimal medium has been selected through optimization. The fermentation process parameters of DU10 still need to be further deepened to increase the activity of chymosin in the fermentation broth. The improvement of the fermentation process parameters needs to be related to the cultivation temperature, the rotation speed of the shaking table, the cultivation time and the liquid volume of the triangle bottle. the

【Content of invention】

The purpose of the present invention is to provide a method for optimizing the fermentation conditions of an excellent strain of C. coeruleus after mutagenesis and selection, so as to improve the activity of chymosin of C. coeruleus. The main process route: Utilize the mutagenized excellent strain of Echinococcus coeruleus DU10 (microbial guarantee number: CGMCC 5423), control its metabolism by changing the fermentation conditions, and produce a fermentation broth with high rennet activity. the

This technical solution is as follows

1) Aseptic operation Inject 10mL of sterile water into the preserved DU10 PDA slant, wash off the spores on the slant, then oscillate to disperse the spores, filter with a G3 glass funnel, count the hemocytometer and adjust the spore concentration to 10 ⁷ to 10 ⁸ /mL.

2) Inoculate 10 ⁷ ～10 ⁸ spore suspensions/mL prepared by inclined planes, respectively change the culture time, liquid volume, inoculum volume, culture temperature, rotation speed, and fermentation time, and conduct single-factor experiments to investigate the effect of each culture condition on Effects on the quality of fermented enzymes. The chymosin enzyme activity (MCA) and proteolytic activity (PA) of the fermented broth were measured. The higher the chymosin activity of the fermented broth and the larger the MCA/PA ratio, the better the rennet produced by fermentation.

3) Verification of optimal fermentation enzyme production conditions

Fermentation tank verification: According to the optimal fermentation conditions selected in the experiment, prepare 4L fermentation medium and put it into a 10L fermentation tank, and measure the enzyme activity of chymosin in the fermentation liquid after culturing under the optimal fermentation conditions. the

Above-mentioned steps 2) in the preferred formula of fermented medium is as follows, is percentage content:

Glucose 3.3-3.8%, peptone 0.38-0.42%, yeast extract 0.58-0.62%, CaCl ₂ 0.05%, MnSO ₄ 0.25%, Tween-800.1%, distilled water to make up 1000mL, pH natural, sterilized at 121°C for 20min.

The above-mentioned reagents and medium formulations are conventional reagents and medium in the art unless otherwise specified. the

The assay method of chymosin activity in the above-mentioned step 2) is _: use 0.01mol/L CaCl Prepare 100g/L skim milk, place it for 40min to make the formed milk system tend to be stable, then take 5mL skim milk in a test tube Incubate at 36°C for 5 minutes. Centrifuge the fermentation broth at 5000r/min for 10min to precipitate the mycelium, take 0.5mL of the supernatant and add it to the insulated skim milk, quickly mix it on the vortex mixer and start timing. Time for the supernatant to coagulate the milk. Define the amount of rennet required to coagulate 1 mL of 100 g/L skim milk in 40 minutes as a Soxhlet unit (SU).

The formula for rennet activity is: $\mathrm{SU}=\frac{2400\×5\×\mathrm{D.}}{T\×0.5}$

In the formula: T-coagulation time/s; D-dilution multiple. the

The proteolytic activity of the above step 2) was measured by Folin reagent method, 10 mL of 0.1 N NaOH was used to dissolve the casein, and the pH6.2 phosphorus buffer was adjusted to 100 mL. The fermentation broth was incubated at 36°C, and the enzyme activity was measured colorimetrically at 660nm. One unit of protease activity (U) is defined as the amount of enzyme that hydrolyzes casein to produce 1 μg of tyrosine per 1 min at 36°C and pH 6.2. the

The formula for proteolytic activity is:

In the formula: A-protease activity determination OD value corresponding to the tyrosine content μg/mL on the standard curve. the

Tyrosine standard curve: Mix 1 mL of tyrosine with different concentrations, 1 mL of Folin reagent and 5 mL of 0.4mol/L Na ₂ CO ₃ , develop color in a water bath at 36°C for 20 minutes, measure OD value at 660 nm, and plot the difference between tyrosine with different concentrations and Standard curve of OD value

Beneficial effects of the present invention:

1. Echinococcus blueis is a new type of chymosin-producing bacteria, and it is of great research significance to optimize the conditions for its enzyme production;

2. By optimizing the fermentation conditions of the mutagenized C. coeruleus, the activity of chymosin in the fermentation liquid was improved. the

【Information on Preservation of Biological Materials】

The strain DU10, classified as Sporothrix Cyanescens, is preserved in the General Microbiological Culture Collection Management Center of the China Microbiological Culture Collection Management Committee, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC 5423, and the preservation date is November 3, 2011. the

【Detailed ways】

The method of the present invention will be further described below in conjunction with the examples, but not limited thereto. The C. coeruleus used in the examples is a chymosin-producing strain isolated and purified from red yeast rice. The starting strains were mutagenized by ultraviolet rays and diethyl sulfate compound factors, and the strains with high rennet production were screened out from the treated strains, and then re-screened. Finally, the genetic stability and the quality of fermented enzymes were analyzed for the re-screened strains. Select excellent bacterial strain DU10 (its microbial preservation number is: CGMCC 5423), on the basis of its culture medium optimization, the present invention improves fermented liquid chymosin activity by changing fermentation conditions. the

Example 1

1) The influence of liquid volume on enzyme production

A 250mL Erlenmeyer flask was used for the shake-flask test, and the maximum liquid volume of the culture medium was 100mL, and the minimum was 10mL. The amount of liquid in the culture medium affects the transfer of oxygen, resulting in changes in the quality of the fermentation broth: the amount of liquid in the culture medium is 10mL to 100m/L, and the enzyme activity of rennet in the fermentation broth fluctuates. When the liquid volume is 55-60mL, the quality of the fermented enzyme is relatively high. the

2) Effect of inoculum size on enzyme production

Inoculate 10 ⁷ to 10 ⁸ spores/mL spore suspension in a 250 mL Erlenmeyer flask containing 50 mL of medium, and the inoculation amount is 0.5% to 2.5%. The inoculation amount will lead to changes in the quality of the fermentation broth: the inoculum amount is 0.5% to 1.5%, which has a greater impact on the quality of the fermentation broth, and the enzymatic activity of rennet decreases after the increase; the inoculum amount is 1.5% to 2.5%, and the quality of the fermentation broth The impact is small, and the enzyme activity of chymosin gradually decreases. When the inoculum amount is 1.0-1.3%, the quality of the fermentation broth is relatively high.

4) Effect of culture temperature on enzyme production

Most fungi can grow at room temperature, and the culture temperature is selected as 20°C to 40°C. Different culture temperatures will affect the quality of the fermentation broth: when the culture temperature is 20°C-30°C, the activity of chymosin in the fermentation broth gradually increases; when the culture temperature is 30°C-40°C, the enzyme activity of chymosin in the fermentation broth decreases sharply. When the culture temperature is 27-30°C, the quality of the fermented enzyme is better. the

5) Effect of rotational speed on enzyme production

The rotation speed is 120r/min-200r/min, different rotation speeds will affect the quality of fermentation enzymes: the rotation speed is 120r/min-180r/min, which has a greater impact on the quality of the fermentation broth, and the enzymatic activity of chymosin increases sharply; The rotation speed is 180r/min～200r/min, which has little effect on the quality of the fermentation broth, and the enzymatic activity of chymosin is basically the same. When the rotational speed is 170-180r/min, the quality of the fermented enzyme is better. the

6) Effect of culture time on enzyme production

The culture time is 48h-144h, and the culture time has an impact on the quality of the fermentation broth: the culture time is 48h-120h, the enzyme activity of chymosin in the fermentation broth gradually increases; the culture time is 120h-144h, the enzyme activity of the fermentation broth The value of MCA/PA is basically unchanged. When the culture time is 108-120h, the quality of the fermentation broth is better. the

Example 2

1) Aseptic operation Inject 10 mL of sterile water into the preserved DU10PDA slope, wash off the spores on the slope, then oscillate to disperse the spores, filter through a G3 glass funnel, count the hemocytometer and adjust the spore concentration to 10 ⁷ to 10 ⁸ / mL.

2) Inoculate the spore suspension containing 10 ⁷ -10 ⁸ spores/mL in a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium, at a 1% inoculum size, at 30° C., 170 r/min, and shake for 120 h.

3) Determination of chymosin enzymatic activity and proteolytic activity of the fermentation broth, the greater the ratio of MCA to MCA/PA in the fermentation broth, the better the rennet produced by fermentation. The measured chymosin activity of the fermentation broth was 586.21SU/mL, which was 88.88% higher than the initial activity of 310.36SU/mL. the

Example 3

1) Aseptic operation Inject 10 mL of sterile water into the preserved DU10PDA slant, wash off the spores on the slant, then oscillate to disperse the spores, filter with a G3 glass funnel, count the hemocytometer and adjust the spore concentration to 107-108/mL. the

2) Inoculate the spore suspension containing 107-108 spores/mL in a 250mL Erlenmeyer flask containing 50mL of culture medium, at a 1% inoculum size, at 30°C, 180r/min, and shake for 120h. the

3) Determination of chymosin enzymatic activity and proteolytic activity of the fermentation broth, the greater the ratio of MCA to MCA/PA in the fermentation broth, the better the rennet produced by fermentation. The measured chymosin activity of the fermentation broth was 595.30 SU/mL, which was 91.81% higher than the initial chymosin activity of 310.36SU/mL. the

It should be understood that the above-mentioned specific implementation is only an illustration, and those skilled in the art can improve or change according to the above description, and all these improvements and changes should belong to the scope of protection of the appended claims of the present invention . the

Claims (3)

1. the method that the mould strain excellent of blue thorn spore ferments behind the mutagenic and breeding, step is as follows:

1) aseptic technique is injected sterilized water to the DU10PDA inclined-plane of preservation, washes the spore on the inclined-plane, and then vibration disperses spore, and glass funnel filters, and blood cell plate counting and adjustment spore concentration are 10 ⁷～10 ⁸Individual/mL, wherein said DU10 is the mould strain excellent of blue thorn spore, and its microbial preservation number is CGMCC 5423;

2) in containing the triangular flask of substratum, inoculate by 10 of inclined-plane preparation ⁷～10 ⁸Individual/the mL spore suspension, liquid amount is 55～60mL, and inoculum size is 1.0～1.3%, and culture temperature is 27～30 ℃, and rotating speed is 170～180r/min, and incubation time is 108～120h;

Described substratum is made by following raw material: glucose 3.3～3.8%, peptone 0.38～0.42%, yeast extract paste 0.58～0.62%, CaCl ₂0.03-0.07%, MnSO ₄0.15-0.35%, tween-80 0.05-0.2%, distilled water is supplied 1000mL, pH nature, sterilization.

2. the method for the mould strain excellent fermentation of blue thorn spore behind the mutagenic and breeding according to claim 1 is characterized in that comprising the rennin enzyme work of fermented liquid and the determination step of proteolytic activity.

3. the application of the fermented liquid that obtains of method claimed in claim 1 in preparation cheese.