TH17537Y - The process of preparing Pichia pastoris yeast starter cultures for large-scale liquid fermentation. - Google Patents

Abstract

------January 29, 2020------(OCR) Page 1 of 1 Abstract of Invention This invention concerns the development of a process for preparing Pichia postoris yeast starter cultures using a method that modifies cell growth activity. This process involves initial yeast cell culture, high-density cell culture, and preparation of a high-density starter culture bank in a freeze-dry solution. The resulting starter cultures are efficient and do not require a cell adaptation period for transplantation into various culture media. This reduces fermentation time and operating costs in biomass cell production or bioproduct production systems through fermentation. Furthermore, the yeast starter cultures obtained from this process are suitable for preparing efficient freeze-dry starter culture banks for storage and use in liquid fermentation, or for direct use in liquid fermentation. They are also suitable for use as starter cultures in high-density yeast fermentation at large and industrial scales to produce target bioproducts from P. postoris for use in the food, feed, and enzyme industries. ------------ Page 1 of 1 Abstract of Invention This invention concerns the development of a process for preparing Pichia postoris yeast starter cultures using a method that modifies cell growth activity. This process involves initial yeast cell culture and high-density cell culture. The preparation of high-cell density starter cultures in frozen solution form results in efficient starter cultures without the need for cell adaptation periods when transplanted into various culture media. This reduces fermentation time and operating costs in biomass or bioproduct production systems through fermentation. Furthermore, the yeast starter cultures obtained from this process are suitable for preparing efficient frozen starter cultures for storage and use in liquid fermentation, or for direct use in liquid fermentation. They are also ideal for use as starter cultures in high-density yeast fermentation at large and industrial scales to produce targeted bioproducts from P. pastoris for use in the food, feed, and enzyme industries.

Claims (3)

------January 29, 2020------(OCR) Page 1 of 2 pages. Claim 1. The process for preparing P. pastoris yeast starter cultures for large-scale liquid fermentation consists of the following steps: a) Primary yeast cell culture: Yeast cells are cultured in YPD liquid medium at 28-30°C with a shaking rate of 150-250 rpm for 14-18 hours. The resulting yeast cells are then cultured in a modified Hi-Innoc BSM medium at 28-30°C with a shaking rate of 150-250 rpm for 72-96 hours, or until the cells enter the reduction phase. b) High-density cell culture: The yeast cells obtained from step a) are transferred to a bioreactor containing the modified Hi-Innoc BSM medium. Modify Hi-Innoc BSM at a temperature of 28-30°C, stirring rate of 300-400 rpm, aeration rate of 1.0-1.5 L/L/min, oxygen solubility in the fermentation broth at 15-25% of saturated oxygen solubility, and pH of 5.5-6.5 for 16-18 hours. Then add a 50-70% by weight/volume glucose solution and add modified PTM1 solution at a rate of 13 ml/hour. Culturing until a cell concentration of 35-40 g dry cells per liter is obtained, and the culture is harvested aseptically. C) Preparation of a high-cell density yeast starter culture in freeze-dry solution form. 2. The preparation process for P. pastoris yeast starter cultures according to claim 1, where the modified i-Innoc BSM culture medium consists of the following components: glucose 8-20 g, phosphoric acid solution (85% by weight/volume) 26.7 ml, calcium sulfate 0.93 g, potassium sulfate 18.2 g, magnesium sulfate heptasulfate 14.9 g, potassium hydroxide 4.13 g, histidine amino acid 2.5-5.0 mg, glutamic acid amino acid 2.5-5.0 mg, leucine amino acid 2.5-5.0 mg, isoleucine amino acid 2.5-5.0 mg, and modified PTM1 solution 6.67 ml. 3. The preparation process for P. pastoris yeast starter cultures according to claim 2, where the modified PTM1 solution consists of... Copper sulfate pentahydrate 6.0 g, potassium iodide 0.08 g, manganese sulfate monohydrate 3.0 g, sodium molybdate dihydrate 0.2 g, boric acid 0.02 g, zinc chloride 20.0 g, iron trichloride 13.7 g, cobalt tetrachloride exahydrate 0.9 g, sulfuric acid solution 2 ml, amino acidsristidine 0.375 g, amino acidsglutamic acid 0.375 g, amino acidsleucine 0.375 g, amino acidsileucine 0.375 g, and biotin 0.3 g, and adjusted the volume with water to 1 liter. Page 2 of 2, page 4. The process of preparing P. pastoris yeast starter culture according to claim 1, where the said P. postoris yeast starter culture... It is stored in the form of a frozen solution, mixed with a glycerol solution at a concentration of 15-30 percent by weight/volume, in a ratio of yeast cell fermentation broth to glycerol solution of 1:1. ------------ Page 1 of 2 pages. Claims. 1. The process of preparing yeast starter cultures (P. pastoris) for large-scale liquid fermentation consists of the following steps: a) Primary yeast cell culture: Yeast cells are cultured in YPD liquid medium at 28-30°C, shaking at 150-250 rpm for 14-18 hours. The resulting yeast cells are then cultured in a modified Hi-Innock BSM medium at 28-30°C, shaking at 150-250 rpm for 72-96 hours, or until the cells enter the reduction phase. b) High-density cell culture: The yeast cells obtained from step a) are transferred to a bioreactor containing the modified Hi-Innock BSN medium at 28-30°C, stirring at 300-400 rpm, and aeration at 1.0-1.5 liters per liter per minute. The oxygen dissolution in the fermentation broth is maintained at 15-25% of the saturated oxygen dissolution value, with a pH of 5.5-6.5, for 16-18 hours. Then, a 50-70% by weight/volume glucose solution is added, along with a modified PTM 1 solution at a rate of 13 ml/hour. The culture is continued until a cell concentration of 35-40 g dry cells per liter is obtained, and the culture is harvested aseptically. C) Preparation of a high-density starter culture in the form of a freeze-dried solution: The yeast cell-containing fermentation broth obtained from step b) is mixed with a glycerol solution at a concentration of 15-30% by weight/volume, in a ratio of yeast cell-containing fermentation broth to glycerol solution of 1:1. 2. The process for preparing yeast starter cultures of P. pastoris according to claim 1, where the modified Hi-Innoc BSM medium is composed of the following: glucose 8-20 g, phosphoric acid solution (85% w/v concentration) 26.7 ml, calcium sulfate 0.93 g, potassium sulfate 18.2 g, magnesium sulfate heptasulfate 14.9 g, potassium hydroxide 4.13 g, histidine amino acid 2.5-5.0 mg, glutamic acid amino acid 2.5-5.0 mg, leucine amino acid 2.5-5.0 mg, osoleucine amino acid 2.5-5.0 mg, and modified PTM1 solution 6.67 ml. (Page 2 of 2) 3. Preparation of yeast starter cultures according to claim 2, in which a modified PTM 1 solution consisting of 6.0 g copper sulfate pentahydrate, 0.08 g potassium iodide, 3.0 g manganese sulfate monohydrate, 0.2 g sodium mosibdate dihydrate, 0.02 g boric acid, 20.0 g zinc chloride, 13.7 g iron trichloride, 0.9 g cobalt chloride hexahydrate, 2 ml silfuric acid solution, 0.375 g amino acidsistidine, 0.375 g amino acidsglutamic acid, 0.375 g amino acidsilicin, 0.375 g amino acidsileucine, and 0.3 g biotin, was adjusted in volume to 1 liter with water.