[0049] Example 3, using the system of Example 1, a fermentation method using lactic acid bacteria for lactic acid fermentation production includes the following steps:
[0050] A. Pre-installation steps: Before starting the system, prepare 50% calcium hydroxide emulsion in the neutralizer storage tanks of the fermentation tank device 100 and the seed tank device 200 at all levels for standby, and sterilize each neutralizer feeding tube; Fill the culture tank of the first-level seed tank device 200 with fresh culture medium and sterilize it; proceed to step B;
[0051] B. Seed expansion step: use thermostable anaerobic lactic acid bacteria (Lactobacillus casei B9232) to carry out 1.6L seed culture in a shaker bottle, about 15 hours to reach the logarithmic phase, complete the seed culture, and transplant the seeds to the first The inside of the culture tank of the seed tank device;
[0052] Turn on the circulating pump corresponding to the first-stage seed tank device to make the neutralizer circulate in the pipeline, and pass through the automatic control system composed of pH electrode, programmable logic controller and control valve. According to the set pH value of 6.50, the automatic control valve automatically Turn it on or off and pump the neutralizer into the culture tank to stabilize the pH of the fermentation broth between 6.40 and 6.60;
[0053] After the seeds in the culture tank of the first-level seed tank device are cultivated to the logarithmic phase, start the corresponding seed transfer pump, and pump 16L of the first-level seed liquid into the cleaned, disinfected and sterilized, and filled with fresh medium The second-level seed tank device is sterilized and cultivated inside the culture tank; the pH value of the seeds in the second-level seed tank is also controlled to stabilize between 6.40 and 6.60; the culture tank of the second-level seed tank device 200 The second-stage seed in the tank is cultivated to the logarithmic phase, and its corresponding transplanting pump is started, and the 160L second-stage seed solution is pumped into the third stage that has been cleaned, disinfected, sterilized, filled with fresh medium and sterilized The seed tank device is cultivated in the culture tank; the pH value of the seeds in the third-level seed tank is also controlled to stabilize between 6.40 and 6.60; the third-level seed culture in the third-level seed tank is to be cultivated In the logarithmic phase, start its corresponding transplanting pump, and pump 1600L of the third-level seed solution into the culture tank of the fourth-level seed tank device that has been cleaned, disinfected, sterilized, filled with fresh medium and sterilized And cultivate; also control the pH value of the seeds in the fourth-level seed tank to stabilize between 6.40 and 6.60; until the fourth-level seeds in the culture tank of the fourth-level seed tank device are cultivated to the logarithmic phase; proceed to step C ;
[0054] C. Seed temporary storage steps: start the seed transfer pump corresponding to the fourth-level seed tank device, and pump 15000L of the fourth-level seed liquid into the fermentor device that has been cleaned, disinfected, sterilized, filled with fresh medium and sterilized The culture tank is inoculated in the tank for fermentation production, and the remaining 500L of the fourth-level seed liquid in the fourth-level seed tank is pumped through the return pump into the temporary storage tank that has been cleaned, sterilized, filled with fresh medium and sterilized (ie Cultivation in the third-level seed tank); proceed to step D and proceed to step E;
[0055] D. Fermentation step: Turn on the circulating pump corresponding to the fermentation tank device to make the neutralizing agent circulate in the pipeline, through the automatic control system composed of pH electrode, programmable logic controller and control valve, according to the set pH value of 6.50, automatic control The valve is automatically opened or closed, and the neutralizer is pumped into the culture tank to achieve a stable pH value of the fermentation liquid between 6.40 and 6.60; adjust the temperature and stirring speed and other culture conditions for lactic acid fermentation, and wait for the culture tank of the fermentation tank device After the fermentation is completed, the fermentation broth is discharged and the fermentation product is extracted; the culture tank is cleaned, sterilized, filled with fresh medium and sterilized; the current round of lactic acid fermentation production test results, the lactic acid content in the fermentation broth is 175g /L, record the fermentation period of 112 hours, where the fermentation period is the sum of all seed cultivation time and fermentation production time;
[0056] E. The fourth-level seed re-cultivation step: While performing step D, the culture tank of the fourth-level seed tank device is emptied, cleaned, disinfected and sterilized, filled with fresh medium and sterilized, and then temporarily The seed liquid in the storage tank is pumped back to the cultivation tank of the fourth-level seed tank device through its corresponding transplanting pump to inoculate, and the fourth-level seed is cultivated for the second time, and cultivated to reach the transplanting conditions;
[0057] F. Cycle steps: After steps D and E are completed, go to step C; the second cycle of step C is described below:
[0058] C. Seed temporary storage steps: (the second time) start the seed transfer pump corresponding to the fourth-level seed tank device, and pump 15000L of the fourth-level seed solution into the cleaned, disinfected and sterilized, and filled with fresh medium for the second time Inoculate the culture tank of the sterilized fermentation tank device for the second round of fermentation production. The remaining 500L of the fourth-level seed liquid in the fourth-level seed tank is pumped into the cleaned, disinfected, sterilized, and Cultivate in a temporary storage tank (that is, a third-level seed tank) filled with fresh medium and sterilized; proceed to step D and proceed to step E;
[0059] D. Fermentation step: (the second time) adjust the culture conditions for lactic acid fermentation. After the fermentation in the culture tank of the fermentation tank device is completed, the fermentation liquid is discharged and the fermentation product is extracted; the culture tank is cleaned, sterilized, and filled Add fresh medium and sterilize; the second round of lactic acid fermentation production test results, the lactic acid content in the fermentation broth of the tank is 178g/L, which is close to the content of the first round, and the fermentation cycle is 52 hours, which is 60 hours less than the first round. And it saves the cultivation cost of shake flask seeds and the first three seeds;
[0060] E. The fourth-level seed re-cultivation step: While performing step D, the culture tank of the fourth-level seed tank device is emptied, cleaned, disinfected and sterilized, filled with fresh medium and sterilized, and then temporarily The seed liquid in the storage tank is pumped back to the culture tank of the fourth-level seed tank device through its corresponding transplanting pump to inoculate the fourth-level seed for the third time, and cultivate it to reach the transplanting conditions;
[0061] F. Cycling steps: After steps D and E are completed, go to step C; continue to circulate until the production is stopped; and after each round of lactic acid fermentation production, it can save about 60 hours compared to the first round, shorten the fermentation cycle, and It saves the total fermentation cost and greatly improves the competitiveness of the company in the lactic acid industry.
