KR900008744B1 - Process for the production of a fermentation starting material - Google Patents

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Description

Process for preparing fermentation starting material

The present invention relates to a method for preparing a fermentation starting material from sugar cane molasses, in particular a method for converting sugar cane molasses to be more suitable as a carbon source used when producing L-glutamic acid by fermentation technology.

Sugar cane molasses is a sucrose-containing byproduct produced in the sugar-making industry and is used as a preferred carbon source when producing L-glutamic acid by fermentation technology.

The present inventors have studied to improve the sugar cane molasses to be used as a more preferable starting material for producing L-glutamic acid, and the sugar cane molasses is passed through a resin column filled with a cationic exchange resin and the water having a pH of 5 to 8 After passing through a resin column to obtain an eluate fraction containing sugars eluted with it, the fraction is used as a carbon source and fermented to produce L-glutamic acid. L-glutamic acid is compared to the case where raw sugar cane molasses is used as a carbon source It has been found that it can be produced in high yields.

Based on the findings, the present inventors have further studied the study of changing sugar cane wheat, adding preliminary conversion of sucrose by adding invertase or inorganic acid to sugar cane wheat before the above-mentioned chromatography process using ion exchange resin. As a result, the sugar recovery during the chromatography process was found to be increased.

Thus, the following inventions existed.

That is, the present invention (1) invertase or inorganic acid added to sugar cane wheat, and invertase or inorganic acid added sugar cane molasses for the time necessary for almost all of the sugar contained in the mentioned sugar cane molasses to be converted At an appropriate temperature and pH (inversion step), (2) the sugar cane mill treated with invertase or inorganic acid in step (1) was passed through a column filled with a cation type cation exchange resin and Water of pH 5-8 was then passed through the resin column mentioned in an amount sufficient to elute 97% or more of invert sugar (chromatography process), and (3) invert sugar-containing eluate formed in process (2). A fraction is obtained as a carbon source for producing L-glutamic acid by fermentation, and thus a method for producing a fermentation starting material.

In the above method for preparing a fermentation starting material, the process (4) (ion exchange process) of passing sugarcane molasses through a column filled with potassium cation exchange resin is carried out between the processes (1) and (2). In the process (2), if the potassium type cation exchange resin is used as the cation type cation exchange resin, sugar recovery efficiency is higher and L-glutamic acid productivity is higher.

To date, it is known that if invertase is used to invert sugars contained in sugar cane and L-glutamic acid is produced by fermentation using the obtained invert sugar, L-glutamic acid productivity is higher than that of non-sugars. to be. [Reference: Japanese Patent Application Laid-Open No. 85290/1981] However, purification of sugars in sugarcane molasses by chromatography using a cationic cation exchange resin by preliminarily converting sugars in sugarcane molasses using an invertase or an inorganic acid. It is a new discovery that efficiency can be increased. Furthermore, the sugar in sugarcane molasses is purified by chromatography using a strong acid cation exchange resin. Sayama et al., K. Sayama et al., Chromatographic Separation fo Molasses Constituents Part 1, Recovery of sucrose form Molasses, "Journal of Sugar Refining Technique Research Association", No. 29 1-9 (1980), but it is not known to utilize sugars so purified and to enhance the productivity of L-glutamic acid. Furthermore, it has been known to obtain purified glucose and fructose by chromatography using molasses acid hydrolyzate sulfite-type or hydrogen sulfide-type ion exchange resins (Japanese Patent Publication No. 28680/1980). When producing L-glutamic acid by fermentation techniques using glucose and fructose purified by the method, L-glutamic acid was not produced as efficiently as using invert sugars purified by cation exchange resins.

As the invertase, commercially available yeast microorganism cells having enzyme and invertase activity and their processed products can be used.

Examples of yeast include Saccharomyces, Candida, Mycotorula, Debaryomyces, Pichia, Hansenula, and Tolulopsis. Yeast belonging to the genus Torulopsis is used, and yeast microbial cells obtained by culturing these yeasts in a medium containing molasses, glucose, pulp producing waste, soybean whey, food waste, juice and the like are also used. As a yeast microbial cell treated product, dried microbial cells obtained by only drying the microbial cells (e.g., yeast of brewers, brewing liquor yeast, etc.): residues after removing useful components such as yeast extract, RNA, glutathione, etc. from yeast microbial cells ; Autosomal digested microbial cell residue; Physically destroyed microbial cells: microbial cells treated with lysozyme, toluene, surfactants, and the like are used.

To convert sucrose in molasses using invertase, the molasses is diluted to an appropriate concentration (e.g. 10 to 50 g / dl) and the invertase source described above is added thereto and held at 60 ° C for 5 to 20 hours. To carry out the enzymatic reaction.

The conversion of sucrose into acid is carried out by the conventional method of adding hydrochloric acid or sulfuric acid to adjust the pH of molasses to 1.0 to 4.0 and heating to a temperature of 80 to 100 ° C. After acid hydrolysis, an alkali such as sodium hydroxide is added to neutralize. If a precipitate forms during acid decomposition and neutralization, it is desirable to remove the precipitate, thereby further enhancing the purification effect.

The cation exchange resin used in the present invention is preferably a strong acidic cation exchange resin such as Amberlite IR-120, Dafenx-50, Diion SK-IBS and the like.

The cation exchange resin is more preferably sodium or potassium type. After filling the above-mentioned ion exchange resin into a column of appropriate size, the coarse sugar cane molasses subjected to the conversion process is put into this column, and the invert sugar is eluted with water having a pH of 5 to 8. The elution sequence is as follows: The pigment and inorganic salts are eluted first, followed by glucose and fructose. After chromatography, fractions containing invert sugar (i.e., fructose fructose) can be harvested to recover sugars from extraneous matter.

The column used is preferably a column equipped with a heat retaining jacket and the temperature in the column is preferably maintained at 50 to 90 °. The concentration of invert sugar to be passed through the mentioned column is more preferable and economical if 15-50 g / dl is appropriate and 35-45 g / dl, calculated as sucrose. In addition, the solution passage rate is preferably 0.5 to 2 S. V.

The eluate by chromatography described above is divided into the following fractions.

First fraction: fraction from the initial eluate to the elution of solids (dirt of molasses)

Second fraction: The fraction from the end point until the concentration of foreign matter reaches 1/2 to 1/3 of the highest electrical conductivity measurement.

Third fraction: fraction from the end of the second fraction until the concentration of invert sugar reaches about 1-5 g / dl

4th fraction: The fraction from the end of the third fraction until the foreign substance concentration reaches 1/2 to 1/3 of the highest electrical conductivity measurement.

5th fraction: From the end of the 4th fraction, the fraction from invert sugar to about 1/2 to 1/3 of its maximum

6th fraction: The fraction from the end of the 5th fraction up to 1 g / dl of invert sugar

Among these fractions, the fifth fraction is a fraction containing invert sugar to be collected, and the third fraction is collected separately as a foreign matter-containing fraction. The second fraction is preferably fed to the resin column of the next chromatography batch before feeding the sugarcane molasses that has already been converted. In addition, it is preferable that the fourth and sixth fractions are fed to the resin column after supplying the sugar cane molasses which has already been converted to the resin column of the next batch. Moreover, the first fraction is preferably used again with water at pH 5-8 for elution.

By repeating this process, the equilibrium is reached after five or six times, which can safely separate foreign bodies and invert sugar. In other words, the fraction mainly containing foreign matter contained in molasses is recovered as the third fraction, and the invert sugar fraction containing only a small amount of foreign matter is recovered as the fifth fraction.

Prior to the above-mentioned chromatography, if the sugarcane molasses which has already been converted is passed through a column filled with potassium ion type cation resin, and if the cation type exchange resin during the chromatography process is potassium ion type, the purification efficiency of invert sugar is further increased. Is increased. The cation exchange resin is preferably a strong acidic cation exchange resin as in the chromatography process.

This process results in almost all cations in molasses becoming potassium ions. The idle temperature of the ion exchange process is from room temperature to 90 ° C., preferably from 50 to 80 ° C. and the feed rate is from 0.5 to 5 S. V. (Elution volume / resin volume per hour x).

The supply of molasses is stopped at the point that the excess ion exchange capacity of the ion exchange resin and the elution molasses are calculated as ion equivalents of other cations, resulting in about 17% incorporation.

The ion exchange resin can be regenerated and used again and the regeneration mentioned can be carried out in a conventional manner using a regenerant.

Furthermore, the load of the ion exchange resin during the ion exchange process is passed through the already processed sugar cane molasses through an ultrafast centrifuge (e.g., model SAOH from Westfalia) before the ion exchange process. It can be reduced by removing the contained solids (i.e., residues) or by adding phosphoric or phosphate to remove turbidity and at the same time removing calcium salts.

In order to produce L-glutamic acid by fermentation technique using the obtained sugar as a carbon source, a commonly used L-glutamic acid-producing microorganism can be used, and the culture medium and culture conditions used in the present invention are not specific.

Examples of commonly known L-glutamic acid-producible microorganisms are as follows:

Brevibacterium flavum ATCC 14067

Brevibacterium latofermentum ATCC 13869

Brevibacterium Saccharolyticum ATCC 14066

Brevibacterium thiogentalis ATCC 19240

Corynebacterium glutamicum ATCC 13032

Corynebacterium melassecola ATCC 17965

The carbon source contained in the medium is a sugar obtained by the method of the present invention, and may be used in the form of a mixture of the sugar with other carbon sources such as beet molasses, natural sugars, acids and enzyme hydrolysates of starch and the like.

By performing L-glutamic acid fermentation using a fermentation starting material prepared by the method of the present invention, L-glutamic acid can be produced with higher efficiency than when using sugarcane molasses. Moreover, such an effect of enhancing efficiency is not obtained by using beet molasses even if performed in the same process as the present method, so this is the specificity of cone molasses. The method of the present invention also makes it possible to obtain L-glutamic acid more easily from L-glutamic acid fermentation because the sugar recovery in sugarcane molasses is very high and more preferably refined sugars are obtained.

Example 1

1) phone fixed

(a) Telephone using invertase

To sugarcane molasses adjusted to a sugar concentration of 55 g / dl by dilution with water, a cell wall (dried product) of yeast microorganisms manufactured by commercially available "Biocon" (Biocon Co.) was added at a rate of 1.2 mg / g of sugar for 10 hours. The enzyme reaction is carried out by maintaining 55 °.

(b) telephone with mountains

Sulfuric acid was added to sugarcane molasses adjusted to a sugar concentration of 55 g / dl by adding water to adjust the pH of the molasses to 1.5, and the decomposition reaction was performed by maintaining the temperature at 100˚ for 20 minutes. The reaction mixture is then cooled, neutralized by addition of sodium hydroxide (pH 6.0) and filtered.

2) Chromatography Process

(a) cation exchange resin treatment

30 ml of molasses adjusted to a sugar concentration of 40 g / dl is placed in a column filled with 240 ml of potassium type cation exchange resin ("Diion SK-1Bs") and eluted with water. The temperature was maintained at 70 ° C. and the water feed rate was analyzed by high-performance liquid chromatography every 12 liters / hour and the fractions containing glucose, fructose and sucrose were recovered and 93% of sugar was recovered. The amount of sugar recovered as solids is measured and the sugar purity is calculated.

(b) anion exchange resin treatment

30 ml of molasses adjusted to a sugar concentration of 40 g / dl is placed in a column packed with 240 ml of sulfurous anion exchange resin ("Amberrite IRA-400") and eluted with water. The temperature is maintained at 40 ° and the water feed rate is measured every 12 liters / hour of sugar concentration and total solids concentration, and the sugar concentration of the sugar fraction with 93% sugar recovered is calculated.

3) L-glutamic acid fermentation

As the experimental composition of the following Table 1, as starting materials, the sugarcane molasses used was continuously treated under the conditions described above, and the sugar obtained was fermented. That is, the molasses obtained is concentrated to 50% sugar concentration, and 50 ml of the mixture is mixed with 250 ml of a salt solution having the composition of Table 1 to prepare 300 ml of glutamic acid fermentation medium.

TABLE 1 Composition of Salt Solution

(* Is added for sugar solution separated by chromatography)

300 ml of the medium prepared for the production of L-glutamic acid were put into each 1.0 L fermenter and sterilized by heating at 115 ° C. for 10 minutes. Brevibacterium lactofermentum ATCC 13869 that had been incubated beforehand was inoculated into each, and cultured by aeration stirring while maintaining pH 7.8 with ammonia gas at 31.5 ° C.

When the sugar concentration measured as sucrose in the medium drops to 3% or less in culture, a small amount of the sugar solution used is added and incubated for 36 hours while adjusting the sugar concentration to 2% to 4%.

The amount of sugar solution added was 80 ml in all experiments. In addition, the surfactant "Tween 60" is added by 0.6% per medium at the point where the predetermined cell volume is reached during the culture.

4) Results

The experimental results performed are shown in Table 2. Table 2 shows the percent removal of impurities (non-sugar foreign matter), the amount of L-glutamic acid accumulated during the cultivation, and the yield of L-glutamic acid in the fermentation test (sugar-based). ).

[Table 2 Sugar Concentration and Fermentation Yield]

-: Do not perform

O: performed.

Example 2

Water is added to the sugar cane molasses so that the sugar concentration is about 50 g / dl, and commercially available "biocon" is added at a ratio of 1.2 mg per gram of sugar as an invertase source.

The mixture is then maintained at 55 ° C. for 10 hours to carry out the enzyme reaction. The jacket temperature of the column (30 ° 300 mm) packed with 240 ml K-type cation exchange resin (Daion SK-1B) was maintained at 50 ° C. and the invertase-treated molasses was adjusted to 50 ° C. to give 2S. Feed to V. and recover 500 ml of molasses eluted from the column. This molasses, which had already undergone ion exchange, was analyzed for Na, K, mg, Ca, and NH ₃ using atomic absorption spectroscopy and high performance liquid chromatography, indicating that 98% of the ionic equivalents were substituted with K.

The jacket column filled with 240 ml of K-type cation exchange resin (diion SK 104S) was kept at 60 ° C, and 30 ml of molasses maintained at 60 ° C through the invertase treatment and the above-described cation exchange process at 60 ° C. Supply. The eluate is partitioned into 12 ml portions and each batch is analyzed by high speed solution chromatography. As a control, the same chromatography was performed on molasses which had been subjected to invertase but not subjected to ion exchange. In any chromatography, the sugar concentration to be fed is adjusted to 40 g / dl.

After chromatography, the sugar fractions are collected and 93% of sugar is recovered. The impurities removal rate (% foreign matter removal rate other than sugar) of this sugar solution is 71%. In contrast, in the case of the control which does not perform the ion exchange treatment, the impurity removal rate is 54%. Therefore, it can be seen that the degree of separation increases when the ion exchange treatment is performed.

By repeating the chromatography, the sugar fraction is recovered and the percent impurity removal of each sugar solution is measured for both the ion exchange treatment and the ion exchange treatment. The results are in Table 3.

[Table 3 Changes in Impurity Removal Rate]

As apparent from Table 3, when the ion exchange treatment was performed, the removal rate of impurities was stable, and even when the chromatography was repeated, the removal rate did not decrease. On the contrary, when the ion exchange treatment was not performed, the repetition of the chromatography reduced the degree of separation and significantly reduced the removal rate of impurities. Therefore, it can be seen that the high degree of separation is stably maintained by the ion exchange treatment.

Claims (3)

(1) The temperature and pH appropriate for the conversion for the time necessary for adding the invertase or the inorganic acid to the sugar cane molasses and the sugar cane molasses with the addition of the invertase or the inorganic acid to almost all the sugars contained in the mentioned sugar cane molasses. (2) sugarcane molasses treated with invertase or inorganic acid in the process (1) was passed through a column filled with a cationic cation exchange resin, and then water having a pH of 5 to 8 was converted into 97 Pass the resin column mentioned in an amount sufficient to elute% or more, and (3) the invert sugar-containing eluate fraction formed in the process (2), as a carbon source for producing L-glutamic acid by fermentation. Characterized in that it is obtained, a process for producing a fermentation starting material. The process (4) according to claim 1, wherein between step (1) and step (2), the step (4) of passing the mentioned sugarcane molasses treated with invertase or inorganic acid through a column filled with potassium ion type cation exchange resin Wherein the cation type cation exchange resin in the step (2) is a potassium ion type cation exchange resin. The method of claim 1 wherein the inorganic acid is hydrochloric acid or sulfuric acid.