(1) Add purified water (deionized water) to L-Lysine hydrochloride, the weight ratio of L-Lysine hydrochloride to purified water is 1:1, mix well and dissolve completely, set aside for use ;
(2) Column adsorption
Add the lysine obtained by step (1) into the ion exchange column equipped with cation exchange resin at a flow rate of 13 L/min until the resin cation exchange resin completely absorbs lysine, and the discharge of the column does not contain lysine , then wash with purified water by the resin in the exchange column, and wash to the effluent of the column without Cl (the pH of the effluent at this moment is 7);
(3) Elution
Then use 2mol/L NH4OH (ammonia) to elute L Lysine, NH4OH flows through the ion exchange column at a flow rate of 13L/min, when the pH value of the effluent of the ion exchange column rises to 8.0, the effluent begins to There is a ninhydrin reaction, that is, L-lysine starts to flow out, and the effluent is collected; when the pH value of the effluent of the ion-exchange column rises to 9.0-12, the color of the ninhydrin reaction is dark, and the L-lysine content is high at this time. Elute until there is no ninhydrin reaction in the effluent to stop the collection;
(4) Concentration under reduced pressure
Concentrate the collected solution under reduced pressure at 0.2Mpa and 60-62°C to catch ammonia, concentrate to 1/3 of the volume of the original collected solution, add activated carbon with a weight of 3.5% of the concentrated solution at this time to decolorize, filter, cool and crystallize, and obtain L ‑Lysine.
The purity of the product detected and calculated by a potentiometric titrator is 98.5%.
Preparation of free L-Lysine Supplements solid: Weigh 1.00kg of L-lysine hydrochloride and add it to 5.0L of purified water, stir to dissolve and clarify, then add the solution to a 7.20kg activated strong acid cation exchange resin column, rinse slowly After washing the resin column for 2-3 hours, continue to wash with purified water until it is neutral and free of chloride ions (0.1mol/L silver nitrate monitoring and extensive pH test paper). Add 11.1kg of 5% v/v ammonia water to the resin column, and slowly rinse the resin column for about 2-3 hours. When the pH value is detected to be greater than 10 during the washing process, start to collect the alkaline eluent until the pH value decreases to about 10. Stop collect. Concentrate the collected solution under reduced pressure at 60°C (-0.10～-0.08Mpa), condense out water and ammonia gas, until no liquid drops drip out, continue to concentrate and turn into a white solid, add 8L of absolute ethanol to the system, Continue beating and crystallization, beat for 3 hours, precipitate all solids, centrifuge and filter the solids until no liquid drops out, spread the filter cake on an enamel tray, and vacuum dry (60±5°C, -0.08～-0.1Mpa) for 8 hours, The product was cooled to room temperature, stored in a sealed bag and sealed in a cool place to obtain 776g of free L-lysine solid, yield 96.95%, purity: 99.62%, HNMR (D O): 3.223-3.188 (CHN), 2.848-2.794 ( 2H/CH2N), 1.542-1.523, 1.276 (-CH2CH2CH2-). LCMS (M+1): 147.1127.
The fermentation method of L-lysine comprises three steps of lysine primary seed cultivation, lysine secondary seed cultivation and lysine fermentation cultivation. The specific operation of each step is as follows:
1. Primary seed culture of lysine
Prepare lysine primary seed medium. Lysine primary seed culture medium comprises sucrose 20g/L in the present embodiment, magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 1.5g/L, ammonium sulfate 9g/L, yeast extract powder 5g/L, threonine 0.5g/L, methionine 0.5g/L, monosodium glutamate 7g/L, sodium pyruvate 0.5g/L, calcium acetate 1g/L.
With the prepared lysine first-grade seed culture medium, adjust the pH value to 6.0 with 20% (volume ratio) potassium hydroxide solution, pass steam to heat up the culture medium to 121 ° C, sterilize for 20 minutes, and turn on the circulating water to cool down to 36°C and kept constant. Start stirring at 300rpm, introduce sterile air according to the ventilation ratio of 1:0.4, adjust the tank pressure to 0.05Mpa, adjust the pH to 6.7 with ammonia water and keep it constant, and adjust the dissolved oxygen to 100%.
The lysine-producing Escherichia coli shake flask seeds were received into the lysine primary seed medium according to 1‰ of the volume of the lysine primary seed medium for cultivation. Oxygen 30-50%, after cultivating for 10 hours, take a sample every 2 hours for microscopic examination and measure the total sugar. When the morphology of the microscopic bacteria is normal and the total sugar drops to 10g/L, the culture is stopped to obtain the mature first-grade seed liquid of lysine.
2. Lysine secondary seed culture
Prepare lysine secondary seed medium. Lysine secondary seed culture medium comprises glucose 70g/L in the present embodiment, magnesium sulfate 1g/L, potassium dihydrogen phosphate 1g/L, ammonium sulfate 10g/L, corn steep liquor hydrolyzate 1g/L, hair hydrolyzate 1g /L, beet molasses 10ml/L, threonine 0.4g/L, methionine 0.4g/L, calcium acetate 2g/L.
Adjust the pH value of the prepared lysine secondary seed medium to 6.2 with ammonia water, heat the medium to 121°C by steaming it, sterilize it for 20 minutes, and cool it down to 37°C with circulating water and keep it constant. Start stirring at 300rpm, introduce sterile air according to the ventilation ratio of 1:0.4, adjust the tank pressure to 0.05Mpa, adjust the pH value to 6.6 with ammonia water and keep it constant, and adjust the dissolved oxygen to 100%. The mature lysine primary seed liquid that step (1) obtains is received in the lysine secondary seed medium by 2% of the volume of the lysine secondary seed medium and cultivated. , tank pressure control dissolved oxygen 30-50%, after 10 hours of cultivation, take samples every 2 hours for microscopic examination and measure reducing sugar, stop culturing when the microscopic examination of bacteria is normal and the reducing sugar drops to 8g/L, to obtain mature lysine Secondary seed liquid.
3. Lysine fermentation culture
Preparation of lysine fermentation medium. In the present embodiment, the lysine fermentation medium includes glucose 20g/L, magnesium sulfate 0.5g/L, phosphoric acid 0.3/L, ammonium sulfate 9g/L, corn steep liquor hydrolyzate 0.5g/L, hair hydrolyzate 0.5g/L , beet molasses 10ml/L, betaine 0.5g/L, calcium acetate 1g/L.
Adjust the pH of the prepared lysine fermentation medium to 5.6 with 20% potassium hydroxide, raise the temperature of the medium to 121° C. with steam, sterilize it for 20 minutes, and cool it down to 36° C. with circulating water and keep it constant. Start stirring at 400rpm, introduce sterile air according to the ventilation ratio of 1:0.3, adjust the tank pressure to 0.07Mpa, adjust the pH value to 6.7 with ammonia water and keep it constant, and adjust the dissolved oxygen to 100%. The mature lysine secondary seed liquid that step (2) obtains is received in the lysine fermentation medium by 10% of the volume of the lysine fermentation medium for cultivation, and is controlled by adjusting the rotating speed, air volume and tank pressure during the cultivation process Dissolved oxygen 25-40%.
During the cultivation process, samples were taken every 2 hours to measure the reducing sugar concentration and ammonia nitrogen concentration of the fermented liquid. When the reducing sugar concentration in the fermented liquid dropped to 4g/L, a 60% (m/v) glucose solution was started to flow and maintain the reduction of the fermented liquid. The sugar concentration is about 4g/L. When the ammonia nitrogen concentration in the fermentation broth drops to 1g/L, start feeding 40% (m/v) ammonium sulfate solution and maintain the ammonia nitrogen concentration in the fermentation broth at about 1g/L.
