KR890003677B1 - Method for preparing hyaluronic acid by microorganism - Google Patents

Abstract

The method of culturing new microorganism for the production of hyaluronic acid is presented. Thus the fermenter containing 1.5% of tryptone, 0.5 peptone, 0.1 yeast extract, sodium chloride and phosphate buffer is autoclaved at 121 C for 40 minx. Glucose sterilized separately and preculture broth containing streptococcus equi are added to the fermenter. The culture is carried out at pH 7.0 for 20-30 hrs.

Description

Microbial culture method to obtain high concentration of hyaluronic acid

1 is a schematic diagram of repeated fed-batch cultivation used in the present invention.

* Explanation of symbols for main parts of the drawings

1 fermenter 2 medium storage tank

3: glucose storage tank 4,8,9 pump

5: pH control device 6: Wonsam separator

7: hyaluronic acid

The present invention relates to a new microbial culture method for obtaining a high concentration of hyaluronic acid (Hyaluronic acid).

Hyaluronic acid is widely distributed in cow's eye, chicken rice, animal hepatitis, placenta and cancer cells, and skin.

Hyaluronic acid forms a conjugate with protein and becomes a jelly state, which has a lubricating effect against physical friction and a protective effect against invasion of bacteria, etc., and is used as a skin protection agent because of its moisturizing properties.

In order to obtain hyaluronic acid, what was conventionally contained in human placenta, chicken rice, pig skin, etc. was obtained by the extraction method. The hyaluronic acid obtained at this time was mixed with a large amount of impurities, namely, chondriacin sulfate, glycosino glycan sulfate, and the like, and it was necessary to purify them with high purity in order to remove them.

On the other hand, in recent years, a fermentation technique has been developed, and a method of producing hyaluronic acid by culturing microorganisms such as pyogenes, Equi, and Jupitemicus in Streptococcus, Streptococcus, has been studied. (J. Biol. Chem. 244,236-246 (1969), Japanese Patent Application Laid-Open No. 58-56692 (1983), Ger offen.DE 3,517,659 (1985)) These methods are potted, and generally have low production yields and are difficult to use time and equipment efficiently. There was this. On the other hand, in the repetitive fed-batch method, cells can be recharged into the fermenter at regular intervals and only the filtrate is collected to obtain very high concentration of hyaluronic acid. Therefore, the present inventors have been able to obtain hyaluronic acid at a higher concentration of 10 times than in a conventional batch type by introducing a cultivation method by repeating fed-batch culture. Referring to the production method of hyaluronic acid used in the present invention in detail.

The strain used was Straptococcus Equi KCTC1873 strain, and the seed culture was 2.5% Biyl Infusion Broth. The seed was inoculated into the culture medium having the same composition as the comparative example, and cultured for 12-15 hours while maintaining pH, 7.0-7.5, temperature 30-37 ° C., and aeration of 0.1-1.0 VVM. At this time, the cells in the culture medium is in the late log phase (late log) state and produce a lot of hyaluronic acid, the cell growth factor (growth factor) due to the depletion of the cell growth is a state to stop.

At this time, the culture medium is collected, and only the cells are selectively refilled into the fermenter, and the fresh medium is continuously injected at regular intervals, and the cells continue to grow due to the influx of growth factors, thereby increasing the cell concentration in the fermenter. Hyaluronic acid is also concentrated in high concentrations in fermenters. In addition, if the glucose, which is the initial substrate of hyaluronic acid, is maintained at a constant level (3-6% (W / V)) in each fermentation step, more hyaluronic acid accumulates. Thus, high concentration of hyaluronic acid was obtained by the stepwise feeding of cell growth factors and the repetitive pollen culture and the repetitive fed-batch cultivation which supply glucose periodically.

The culture medium cultured every 12-15 hours by the above method is collected and centrifuged, and only the cell solids are refilled into the fermenter. In addition, the operation of refilling with fresh medium was repeated 4-5 times, and then 60% glucose solution was slowly injected into the culture medium using a pump to maintain the glucose concentration of the culture material at a constant level.

The above operation was repeated several times so that the concentration of hyaluronic acid in the culture was 10.0 g / l.

Comparative Example

9 l of medium containing tryptone 1.5%, peptone 0.5%, yeast extract 0.1%, saline and phosphate buffer solution was charged to a fermenter with a volume of 14 l, steam sterilized at 121 ° C. for 40 minutes, cooled to room temperature, and then sterilized separately. 1 liter of solution was added to a final concentration of 4%, and aeration volume was injected at 0.1 VVM by contacting 100 ml of the seed culture medium of Straptococcus quick (KCTC 1873) incubated in 2.5% Bi-Infusion Broth at 37 ° C for 12 hours. 25-30 hours were incubated while maintaining pH 7.0 with a pH adjuster.

By culturing in this manner and quantitatively analyzed by a known method, 3.0 g / L of cell dry weight and 1.5 g / L of hyaluronic acid were obtained.

Example 1

9 liters of medium was charged into a fermenter 1 having a capacity of 14 liters using a pump 4 from the medium storage tank 2 in which the medium was stored as in the comparative example, and then sterilized and cooled as in the comparative example, and then stored in a glucose storage tank (3). ), The glucose solution was sent to the fermenter 1 using the pump 8 to maintain the initial glucose concentration at 6%, inoculated with the same culture medium as in the comparative example, and ventilated with an aeration of 0.1 VVM, and a pH adjusting device (5). After culturing for 12-15 hours while maintaining the pH at 7.0), the culture solution was collected using a pump (9), and the supernatant was separated by a Wonsam separator (6). The supernatant was discarded and the solids of the cell were refilled into the fermenter (1) and fresh. Cultures were incubated with aseptically equal amounts of media. This operation was repeated several times in a batch batch culture to give a total fermentation time of 140-150 hours, yielding a cell dry weight of 15.0 g / l and hyaluronic acid of 6.0 g / l.

Example 2

In the method of Example 1 described above, when 78 hours have passed since the start of the cultivation with the repeated batch culture, a sterilized 60% glucose solution was injected at a rate of 200 ml / hr for 3 hours, and then every batch. After 6 hours after the start of each culture, the glucose solution was injected to obtain a cell dry weight of 20.0 g / L and hyaluronic acid of 8.5 g / L.

Example 3

In the method of Example 1 described above, when 24 hours after the start of the incubation with repeated batch culture, a sterilized 60% glucose solution was injected to maintain the glucose concentration in the culture solution at 3-6%. 24.0 g / L Cell dry weight and 10.0 g / l hyaluronic acid were obtained.

Claims (4)

Microorganism culturing method for obtaining high concentration of hyaluronic acid by culturing the straptococcus quick KCTC1873 and collecting the culture medium and selectively refilling and cultivating the cells when the cell state is post-logistic and the cell growth factor is almost depleted. The method of claim 1, wherein the culture method of stepwise supply only fresh medium after cell recharge. The culture method according to claim 2, wherein fresh medium and glucose are added together in a hepatic or continuous manner after cell refilling. The culture method according to claim 3, wherein the glucose concentration of the culture solution is maintained at 3-6%.

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