TH6944C3 - Lactic acid production process by immobilized cell system - Google Patents

Abstract

The process of producing acid exchange by immobilized cell system consists of preparation of cell suspension. Lactic acid bacteria Preparation of bacteriophage immobilization cells, lactonic acid, L-network cell fixation. Ginate and alginate membranes And the production of lactic acid with a complete fermentation process at the flask level Or a complete cycle, reverse fermentation or a complete cycle, reverse fermentation Or a continuous cycle of water Fermented in a column-containing, alginate-immobilized cell, using Lactococcus lactis IO-1 TISTR 1401 as a model for the case study. The fermentation was controlled under the most suitable conditions. From the design of the experiment using the Taguchi method.

Claims (6)

1. Lactic acid production process by immobilization cell system Consisting of 1.1 Bacterial cell suspension preparation The master strains were Lactococcus sp p. Bacteria. The steps were as follows: a. The stock culture was placed in a test tube with cultured food. Liquid MRS (De Man-Rogosa-Sharps: MRS) was incubated at 37 ° C for 24 hours. B. Inoculation was transplanted into a test tube with MRS liquid culture medium. Temperature 37 ° C for 24 hrs to be used as seedling in cell culture at flask level C. The seedling obtained from item B, size 5%, volume / volume added to flanes, amount 2F. Lasks with MRS liquid cultured feed d. Were then incubated at 37 ° C for 6 h and separated. Cells with a low temperature centrifuge at a speed of 9,000 rpm for 10 minutes e. Wet cells obtained from the s. Wash with distilled water 1 time, then add distilled water to sterilize the cell suspension for use. In cell fixation of alginate entrapment and alginate membrane types. (alginate-membrane encapsulation) 1.2 preparation of lactic acid bacteria-fixing cells Which can be selected from one of the following methods: 1.2.1 Lactic bacteria immobilization cell "Alginate entrapment" has the following steps: a. Mix the suspension obtained from Section 1.1 (e) with percent sodium alginate solution (NaC6H7O6). B. Remove the resulting cell suspension mixture. Put into a 3 percent solution of calcium chloride (CaCl2) with a silicone hose and a tear needle. By using a pump to control the flow rate of the substance The cells were suspended at a constant rate of 0.06 l / h. C. Immobilized gel cell pellets obtained from item B in 3 percent calcium chloride (CaCl2) solution were incubated for 2 h at room temperature 30 ° C, then filtered. Through the sieve d. Remove the seed cell seed from the article C. Wash it with distilled water, sterilize 1 time, leave it for about 10 minutes at room temperature, 30 ° C. E. Take the germinated gel cell from Article D. To weigh wet by every step. Acted under aseptic conditions The immobilized cells obtained in the alginate network were used to produce lactic acid.1.2.2 Alginate-membranc encapsulation of the bacteria-lactic acid bacteria immobilized cells have the following steps: a. The wet cells in item 1.1 (e) were mixed with a solution containing 20 percent polyethylene glycol (PEG 6000) (weight / volume) and 2 percent calcium chloride CaCl2). The cell suspension mixture obtained from Article A was mixed into a 0.5 percent sodium alginate (NaC6H7O6) solution containing 0.1 percent polysorbate 80 (tween 80) (weight / volume) with a silicone hose. And needle using a pump to control the flow rate of the mixture Suspend the cells to remain at 0.06 liters / hour. C. Distilled water is added to the suspension mixture in item B. to sterilize it to Dilute the concentration of sodium alginate solution. Take the immobilized cell capsule obtained from the c. Strain through a sieve and rinse the capsule. Immobilized cells with distilled water e. Immobilized cell capsules obtained from Article D are immersed in 1 percent calcium chloride solution, CaCl2) for 30 minutes to freeze the cell capsules. Filter the immobilized cell capsule Sieve and rinse with distilled water, disinfect again, leave for 10 minutes. F. We weigh the immobilized cell capsule obtained from Article E. Take the cell capsule Freeze the containing alginate pulp into a glass column. To be used in the production of lactic acid. 1.3 Production of lactic acid. It can be selected from one of the following methods: 1.3.1 Production of lactic acid by batch fermentation at the flask level. The steps are as follows: a. The lactic acid bacteria-fixation cell is attached in the form of "alginate-membrane encapsulation" obtained from item 1.2.2 b. And put it into the flas with cultured food. MRS was cultured by controlling the fermentation conditions at 37 ° C and shaking rate of 100 cycles / min. 1.3.2 Production of lactic acid by batch fermentation process with broth recycle. Freeze Lactic Acid Bacteria The alginate-membrane encapsulation obtained from 1.2.2 b. Was used to produce lactic acid in a cycle with weight by using a column-containing reactor with an alginate membrane. With stirred tank fermentation with MRS liquid culture medium by controlling the fermentation conditions at 37 ° C, stirring rate 400 cycles / min and adjusting the pH 6.85 c. Fermented by MRS culture feed pump from stirred fermenter. With a flow rate of 16 ml / min. Enter the reactor on the top and pump out the fermentation. At the bottom, a column reactor of alginate cell membranes. To circulate back the weight into the glue fermenter d. The production of lactic acid completely reversed the fermentation by holding cells. 2 times of this alginate membrane in the column containing alginate immobilized cells f. 2nd production of the alginate by adding new culture medium. And control the operation of the complete fermentation system, recirculating the fermentation weight according to Article B.1.3.3 Production of lactic acid by the process of fermentation in a complete recirculation (repeated batch fermentation precess with broth recycle) a. The "alginate-membrane encapsulation" fixed cells obtained from Section 1.2.2 b. Complete lactic acid production, recirculating the fermented water in a column-containing cell reactor. Immobilize the alginate membranes for a total of 3 times by controlling the fermentation conditions at 37 oC, stirring rate 400 cycles / min, and adjusting the pH 6.85 C. After the 1st fermentation was completed, transfer The fermented water is completely removed from the system and then started to continue fermentation by adding new culture media. And control the work of Complete fermentation system, recirculating fermentation as per item B.1.3.4 Production of lactic acid by continuous fermentation process, recirculating fermentation (comtinuous fermentation process with broth recvcle) a. Lactic acid "Alginate-membrane encapsulation) obtained from clause 1.2.2 b. Continuous lactic acidification in the fermentation process. Operated by water Column-type reactor with alginate-fixed cell Connect to a stirred tank fermenter to form a fermentation tank. The fermentation conditions were controlled at 37 degrees Celsius, the stirring rate of 400 rpm and the pH value 6.85 C. The fermentation was started by the MRS culture feed pump from the spiral fermentation tank. The flow rate of 16 mL / min enters the reactor below the alginate cell column reactor d fermentation until the glucose concentration reaches a steady state (stationary state), thus changing the fermentation. Combined fermentation is continuous fermentation that controls the dilution rate (D) 0.5 hrs. E. Adding new culture media (MRS) to the fermentation tank used. As a storage tank of fermentation water With a flow rate of 8.33X60 ml / h, which is pump-controlled to stabilize the working volume of continuous fermentation. Under steady state 2. Production process of lactic acid by immobilization cell system according to claim 1, which contains Of the De Man-Rogosa-Sharpe (MRS) liquid culture media, including initial glucose concentration 45 g / l, yeast extract concentration 10 g / l, concentration of yeast extract. Beef extract 10 g / l, initial pH 6.85, calcium chloride (Cacl2) 10 g / l, and peptone 7.5 g / l. 3. Lactic acid production process by fixing cell system according to claim 1, where acid is produced Lactic with integral alginate immobilized cells By controlling the fermentation conditions at 37 degrees Celsius and the shaking rate of 100 cycles / minute. 4. Lactic acid production process by fixing cell system according to claim 1, where acid production Lactic with integrated alginate immobilized cells, circulating fermentation The fermentation conditions were controlled at 37 degrees Celsius, the stirring rate of 400 rpm and the pH value at 6.85. The culture medium from the cyclic fermentation tank to the columnar reactor of alginate cell membranes 16 ml / min. 5. Lactic acid production process by immobilized cell system according to claim 1, where acid production Lactic with integrated alginate immobilized cells, recirculating the fermentation water again. The fermentation conditions were controlled at 37 degrees Celsius, the stirring rate of 400 rpm and the pH value at 6.85. 6. Lactic acid production process by immobilized cell system according to claim 1, where acid production Lactic with continuous alginate immobilized cells, circulating fermentation The fermentation conditions were controlled at 37 degrees Celsius, the stirring rate of 400 rpm and the pH value at 6.85. The media from the agitated fermenter to the reactor, the column of the alginate cell, 16 ml / min, and the flow rate of the continuous fermentation culture was 8.33x60 ml / min. By controlling the dilution rate of 0.5 per hour under steady state.