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Title:
FERMENTATION OF L-LYSINE USING ACID-TREATED PRODUCT OF SOYBEAN MEAL
Document Type and Number:
WIPO Patent Application WO/2006/004371
Kind Code:
A1
Abstract:
Disclosed is a method for fermentation of L-lysine using acid hydrolyzate of soybean meal as organic nitrogen source. The method comprises adjusting pH of the acid hydrolyzate of soybean meal to pH 2˜4 before preparing a culture medium. Such adjusted pH values allows the fermentation level and yield per sugar of L-lysine to be increased by at least 8% and at least 7%, respectively, as compared to other pH values.

Inventors:
LEE JAE-WON (KR)
LEE BYUNG-HAN (KR)
SHIN HYUN-CHUL (KR)
KIM DEUG-HOI (KR)
HEO YOUNG-SEUP (KR)
SUH HAI-CHANG (KR)
Application Number:
PCT/KR2005/002140
Publication Date:
January 12, 2006
Filing Date:
July 05, 2005
Export Citation:
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Assignee:
CJ CORP (KR)
LEE JAE-WON (KR)
LEE BYUNG-HAN (KR)
SHIN HYUN-CHUL (KR)
KIM DEUG-HOI (KR)
HEO YOUNG-SEUP (KR)
SUH HAI-CHANG (KR)
International Classes:
C12P13/08; (IPC1-7): C12P13/08
Foreign References:
US5302521A1994-04-12
US5179010A1993-01-12
KR810001746B11981-11-11
KR20000020629A2000-04-15
Other References:
EKWEALOR L A & EBELE O A: "Preliminary study of L-lysine production by Bacillus species using various agricultural by-products.", NAHRUNG, vol. 47, no. 4, 2003, pages 226 - 227
Attorney, Agent or Firm:
Lee, Duck-rog (YEiLPAT Bldg. 669-17,, Yorksam-don, Kangnam-ku Seoul 135-915, KR)
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Description:
[DESCRIPTION] [Invention Title) FERMENTATION OF L-LYSINE USING ACID-TREATED PRODUCT OF SOYBEAN MEAL [Technical Field] The present invention relates to a method for fermentation of L-lysine using Corynebacterium glutamicum as microorganism for fermentation and acid hydrolyzate of soybean meal as organic nitrogen source, characterized in that pH of the acid hydrolyzate of soybean meal is adjusted to a predetermined level when preparing a culture medium so as to improve the productivity by the fermentation of L-lysine. [Background Art] L-lysine is a kind of essential amino acid and is used as additive for livestock feed, food additive and pharmaceutical material. Particularly, L-lysine held the market of livestock feed additives in an amount of up to about 700,000 tons on 2004. Thus, it is understood that L-lysine is a macro-scaled biotechnological product. L-lysine as livestock feed additive has been increasingly on demand by 8 ~ 10% per year up to date. Furthermore, since China experiences rapid growth in economy and restriction on livestock excrement becomes strict more and more, the market of L-lysine is expected to experience rapid expansion. Therefore, it is necessary to improve the international competitive power of L- lysine products through technical research and development into fermentation processes and strains used therein. At the present time, L-lysine is produced industrially via a direct fermentation method using a carbon source, a nitrogen source and minerals as materials for a culture medium. Among the constitutional elements of the culture medium, nitrogen sources (for example, yeast extract, peptone, corn steep liquor and soybean meal hydrolyzate) have been intensively studied for their kinds and treatment in order to save the cost. Additionally, it is known that nitrogen sources have a plenty of room for improvement hereafter. Meanwhile, soybean meal is referred to as cheap byproducts remaining after recovering soybean oil from soybeans. Such soybean meal is used in a culture medium after it is hydrolyzed with a protease or acid such as hydrochloric acid or sulfuric acid. There are two types of processes for such acid hydrolysis. The Korean Laid-Open Patent No. 1996-0017840 discloses use of hydrolyzate of soybean meal obtained by treating soybean meal with a protease. Methods for acid hydrolysis of soybean meal are also known. One example of such methods includes the steps of: adding an acid to soybean meal, subjecting the mixture to a reaction at a high temperature of 110~ 120°C for a long time of 10~24 hours; cooling the reaction mixture; and providing the resultant product as material for a culture medium after filtering off solids. The above-mentioned method for hydrolysis of soybean meal using an enzyme is a relatively stable process. However, there is a significant limitation in practical use for industrial production of L-lysine. For example, most industrial fermentation plants use a continuous sterilizer for sterilizing a culture medium and need a plurality of pipelines for carrying out essential steps. However, hydrolyzate of soybean meal obtained by an enzyme contains many kinds of peptides or non- hydrolyzed protein solids, which may occur deposition of scale, thereby causing a severe problem. Meanwhile, although the above-mentioned acid hydrolysis method needs acid-resistant equipments due to the treatment of sulfuric acid or hydrochloric acid, it ensures the process stability by virtue of complete hydrolysis of soybean meal. Thus, a part of amino acid production companies including Ajinomoto Co. uses such acid hydrolyzate of soybean meal. [Disclosure] [Technical Problem] Accordingly, the present invention has been made to solve the above- mentioned problems occurring in the prior art. It is an object of the present invention to provide a method for preparing L-lysine by Corynebacterium glutamicum or E. coli producing L-lysine and acid hydrolyzate of soybean meal so as to obtain higher fermentation productivity. After we studied about advantages of acid hydrolyzate of soybean meal, we found that it is possible to improve the fermentation productivity by adjusting the pH of preformed acid hydrolyzate of soybean meal to a predetermined level, when a culture medium is prepared, in such a manner that a microorganism utilizes hydrolysis products of soybean meal, other nutrients and minerals more efficiently. This is a novel technical solution that has never been known in the prior art. [Technical Solution] According to an aspect of the present invention, there is provided a method for improving fermentation productivity of L-lysine by Corynebacterium glutamicum or E. coli producing L-lysine and acid hydrolyzate of soybean meal. According to another aspect of the present invention, there is provided a method for producing L-lysine from a cultured product obtained by the above method for increasing fermentation productivity. Hereinafter, features and advantages of the present invention will be explained in more detail with reference the following Examples. [Advantageous Effects] As described above, the present invention relates to a method for fermentation of L-lysine using acid hydrolyzate of soybean meal as organic nitrogen source. According to the present invention, it is possible to increase fermentation level and yield per sugar of L-lysine by at least 8% and at least 7%, respectively, by adjusting the pH of hydrolyzate of soybean meal to pH 2~4 when preparing a culture medium, as compared to other pH values. [Description of Drawings] Figure 1 illustrates a process for preparing acid hydrolyzate of soybean meal; and Figure 2 is a graph showing the effect of pH of hydrolyzate of soybean meal on fermentation of L-lysine. [Best Mode] EXAMPLE 1: Preparation of acid hydrolyzate of soybean meal A solution of soybean meal with a concentration of 10~20% (w/v) was prepared by dissolving non-fat soybean meal having a total nitrogen content of 8% or more and a non-purified protein content of 50% or more into process water at 40 — 50 °C as solvent. The solution of soybean meal was treated with sulfuric acid adjusted to a final concentration of 20—30%, and then the mixture was further heat treated by heating it at 110- 120"C for 5 ~ 10 minutes. Next, the sulfuric acid-treated and heat-treated solution of soybean meal was cooled to 50~60°C and filtered with a filtering web having a size of 150~200 mesh. The resultant filtrate was used as material for a culture medium used in the following fermentation test (see, Fig. 1). Here, concentration of L-lysine was measured by HPLC (High Performance Liquid Chromatography), and cell counts in the culture medium were determined by centrifugal separation under 3,000 rpm for 15 minutes. If necessary, equivalent number was quantitively determined by the Bertrand method. [Mode for Invention] EXAMPLE 2: Comparison of fermentation productivity of L-lysine depending on pH of acid hydrolyzate of soybean meal The acid hydrolyzate of soybean meal obtained from Example 1 was subjected to seed culture (flask) in a shaking incubator with an L-lysine-producing microorganism, which is a strain deposited by the applicant in the accession number of KFCC 11043, and was subjected to seed culture in a 5L small fermentation tank. Then, a fermentation test was performed in a 30L fermentation tank. The following test was carried out in a fed-batch fermentation manner, wherein additional sugar and additives were added during culture. 1) Medium for seed culture: components and conditions (Flask) Sugar 50 g/L, peptone 10 g/L, yeast extract 10 g/L, KH2PO4 1.5 g/L, MgSO4JH2O 0.5 g/L, urea 5 g/L and biotin 50 //g/L (pH 7.0, before sterilization) were incubated at 220 rpm for 20 ~ 25 hours at 30 ~ 32 °C . 2) Medium for seed culture: components and conditions (5L Fermentation Tank) Sugar 70 g/L, yeast extract 5 g/L, (NH4)2SO4 10 g/L, KH2PO4 1.0 g/L, MgSO4.7H2O 0.5 g/L, biotin 300 //g/L, thiamine.HCl 1,000 //g/L, niacin amide 2,000 //g/L, pantothenic acid 500 //g/L, and anti-foaming agent 3 ml/L were incubated at 30~32°C under a pH of 7.0 ~ 7.3 (adjusted by gaseous ammonia) with an agitation speed of 450 — 600 rpm and an aeration rate of 0.5 ~ 1 wm (air volume/medium volume/minute) for 20 ~ 25 hours. 3) Medium for fermentation test: components and conditions (30L Fermentation Tank) Sugar 165 g/L, molasses (as reducing sugar) 165 g/L, acid hydrolyzate of soybean meal 30 g/L (prepared with different pH values by NaOH solution), (NEU)2SO4 50 g/L, KH2PO4 2.0 g/L, MgSO4.7H2O 1.5 g/L, FeSO4JH2O 50 mg/L, MnSO4.5H2O 50 mg/L, biotin 300 //g/L, thiamineΗCl 1,000 μg/L, niacin amide 2,000 //g/L, pantothenic acid 500 //g/L, and anti-foaming agent 3 m-β/L were incubated at 30~33 °C under a pH of 7.0 ~ 7.3 (adjusted by gaseous ammonia) with an agitation speed of 450 ~ 600 rpm and an aeration rate of 0.5 ~ 1 wm. The acid hydrolyzate of soybean meal was prepared with different pH values as shown in the following Table 1, and then used as material for a culture medium under the above-described conditions. As a result, when the pH of hydrolyzate of soybean meal was adjusted to pH 2—4, it was possible to increase fermentation level and yield per sugar of L- lysine by at least 8% and at least 7%, respectively, as compared to conventional methods carried out under a pH adjusted to 7.0 or other values (see, Figure 2). [Table 1] Effect of pH of acid hydrolyzate of soybean meal on fermentation

[Industrial Applicability] As described above, the present invention is very useful for the food industry, because adjusting pH of acid hydrolyzate of soybean meal to pH 2~4 makes it possible to increase the fermentation level and yield per sugar of L-lysine by at least 8% and at least 7%, respectively, as compared to other pH values.