JPH0513638B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing fermentation raw materials from molasses. Molasses is a byproduct produced in the sugar manufacturing industry, such as cane sugar or beet sugar.It is a sugar solution that mainly contains sucrose, but since it contains a large amount of impurities in addition to sugar, it is difficult to separate the sugar economically. Currently, it is used as a raw material for glutamic acid fermentation or alcohol fermentation. However, even when used as a fermentation raw material, large amounts of impurities, especially colored substances, are transferred directly to the fermentation liquid, so it is necessary to separate the fermentation products.
This poses a large cost burden in terms of purification and treatment of waste liquid. Furthermore, when used not only to remove impurities but also as a raw material for fermentation, especially when cane molasses is used, the fermentation yield is low and it cannot be said to be satisfactory in this respect as well. Therefore, the present inventors have conducted various studies with the aim of developing a method for economically and efficiently producing fermentation raw materials of quality that improves the quality, that is, the fermentation yield, from molasses. After sucrose is converted into glucose and fructose by the action of acid, invert sugar and impurities can be efficiently separated by chromatography using a cation exchange resin, and the sugar solution obtained in this way ( By using invert sugar solution), the yield of amino acid fermentation can be dramatically improved.
We also discovered that the fermentation products can be separated and purified extremely easily. (Patent application 116580/1982) As a result of further intensive research, after converting sucrose from molasses and replacing several types of cations contained in molasses with one type of cation using a cation-type cation exchange resin, (Hereinafter, this operation will be referred to as the softening process.) If chromatography is performed using the same cation-type cation exchange resin that was substituted with one type, it will be extremely easy to separate invert sugar and impurities. The present inventors have discovered that if the sucrose of molasses is converted after the softening process, the separation of invert sugar and impurities becomes extremely easy, and the present invention has been completed. In addition, by using this method, existing laws (patent application
57-116580 (Japanese Unexamined Patent Publication No. 59-6894)), it is possible to eliminate the drawback that the separation of invert sugar and impurities gradually decreases when repeated chromatography is performed. That is, the present invention converts sucrose into glucose and fructose by allowing invertase or acid to act on molasses, and also converts several types of cations contained in molasses into one by using a cation exchange resin.
A method for producing a fermented raw material characterized by performing chromatography using the same cation-type cation exchange resin as that used for the substitution to separate invert sugar. It is. The molasses used in the present invention is cane molasses, beet molasses, etc., and the sucrose contained in the molasses is converted into glucose and fructose using invertase or an acid. As invertase, commercially available enzymes as well as processed yeast cells having invertase activity can be used. To convert sucrose in molasses with invertase, molasses is heated to an appropriate concentration, e.g.
The solution may be diluted to 10 to 55 g/dl, the above invertase source added thereto, and kept at 20 to 60°C for 5 to 20 hours to carry out the enzymatic reaction. The conversion of sucrose with an acid is carried out according to a known method in which hydrochloric acid or sulfuric acid is added to molasses to adjust the pH of the molasses to 1.0 to 4.0, and the mixture is heated to a temperature of 80 to 110°C. After acid hydrolysis, add an alkali such as sodium hydroxide to neutralize. If a precipitate is generated in the acid decomposition-neutralization step, it is desirable to remove the precipitate, and the purification effect can be further enhanced. The cation exchange resins used in the softening process of the present invention include strongly acidic cation exchange resins such as Amberlite IR-120, Dowex-50, Diaion SK-1B, Amberlite IRC-50, Amberlite XE-80, and Diaion. A weakly acidic cation exchange resin such as WK-11 is used.
Use by changing to cationic type such as Na type or K type. In order to soften molasses using these ion exchange resins, the cation-type ion exchanger described above is packed into a column of an appropriate size, the molasses is supplied to this packed column, and the softened molasses that flows out is collected. Just collect it. The operating temperature is room temperature to 90°C, preferably 50 to 80°C, and the feed rate is 0.5 to 5SV (elution volume/resin volume x hours). When the ion exchange capacity of the ion exchanger is exceeded and the outflowing molasses is found to be contaminated with other cations by an ion equivalent ratio of 1 to 7%, the supply of molasses is stopped. The ion exchanger can be used any number of times if it is regenerated, and the regeneration method can be any conventional method, and any regenerating agent may be used. In addition, prior to the softening process, the solids contained in the molasses, so-called sludge, are removed using an ultra-high-speed centrifuge, such as Westphalia's SAOH model, and phosphoric acid or phosphates are added to remove turbidity. If you remove calcium salts,
The load on the ion exchanger during the softening process can be reduced. Cation exchange resins used for chromatography include strongly acidic cation exchange resins such as Amberlite IR-120, Dowex-50, Diaion SK-1B and SK-104S, Amberlite IRC-
50, Amberlight XE-80, Diamond Ion WK
A weakly acidic cation exchange resin such as -11 is used, and when used, it is changed to the same cation type as used in the softening step. In order to perform chromatography using these ion exchange resins, the above-mentioned cation-type ion exchanger is packed into a column of an appropriate size, and the invertase-treated molasses is supplied to this packed column.
The water is then eluted. The elution order is that colored substances and inorganic salts are eluted first, followed by glucose,
It is eluted in the order of fructose. After the chromatography is completed, sugar and impurities can be efficiently separated by collecting invert sugar, that is, a fraction containing glucose and fructose. This chromatographic separation can be performed stably no matter how many times it is repeated. On the other hand, if the softening treatment is not performed, invert sugar and impurities can be separated, but highly pure invert sugar cannot be obtained without loss of invert sugar. This decrease in separation becomes even stronger when chromatography is repeated, and the separation of invert sugar and impurities is significantly reduced. The sugars separated in this way are desirable as raw materials for fermentation and are used as raw materials for amino acid fermentation, including glutamic acid fermentation. Examples will be described below. Example 1 Add water to cane molasses to make sugar concentration about 50g/dl
To this, commercially available "Biocon" (dry yeast cell wall preparation manufactured by Biocon) was added as an invertase source at a ratio of 1.2mg to 1.0g of sugar.
The enzyme reaction was carried out by holding at 55°C for 10 hours. K-type cation exchange resin (Diaion SK-1B) 240
The jacket temperature of the column (30 φ x 300 mm) packed with 100ml of molasses was maintained at 50°C, and the invertase-treated molasses was adjusted to 50°C and supplied at SV2 (480ml/Hr), and the molasses that flowed out from the packed column was
ml was collected. Na, K, Mg,
Analysis of Ca and NH ₃ revealed that 98% of the ion equivalents were replaced by K. Jacketed column packed with 240ml of K-type cation exchange resin (Diaion SK104S) (Pyrex column manufactured by Kyowa Seimitsu Co., Ltd.)
30 φ
ml and then eluted with demineralized water kept at 60°C. The eluate was collected in 12 ml portions, and each fraction was analyzed by high performance liquid chromatography.
The results are shown in FIG. As a control, similar chromatography was performed on invertase-treated molasses without softening treatment. The elution pattern is shown in FIG. In each chromatography, the sugar concentration of the supplied molasses was adjusted to 40 g/dl with water. In Figures 1 and 2, the vertical axes indicate the concentration of glucose and fructose (g/dl), the chromaticity (absorbance at PH6.0400nm: Ai) as an index of impurities, and the concentration of impurities (g/dl). . The horizontal axis shows the fraction number. In addition, the chromaticity and impurity concentration (g/dl) of glucose, fructose, and impurities are -〇-, -, respectively.
Indicated by △-, -●-, -▲-. After completing the chromatography, collect the sugar fraction and collect 93%
of sugar was recovered. The impurity removal rate (removal rate of impurities other than sugar) of this sugar solution was 71%. In contrast, the impurity removal rate without softening treatment was
54%, which shows that the softening treatment can dramatically improve separability. After repeated chromatography,
Sugar fractions were collected for both the case of softening treatment and the case of not softening treatment, and the impurity removal rate of this sugar solution was determined. The results are shown in Table 1.

[Table] As is clear from Table 1, when the softening treatment is performed, the impurity removal rate is stable and no decrease is observed even if chromatography is repeated. On the other hand, if softening treatment is not performed, as chromatography is repeated, the separation performance decreases and the removal rate of impurities decreases significantly, indicating that high separation performance can be stably maintained by softening treatment. . Example 2 "Biocon" was added to Cane Molasses diluted with water and adjusted to a sugar concentration of 55 g/dl, and an enzymatic reaction was carried out in the same manner as in Example 1. The precipitate was removed using a mold. Such invertase-treated molasses 30 was supplied to a column packed with Na-type cation exchange resin (Diaion SK-1B) 10, and water was then supplied to soften the sugar concentration 44.
Obtained 33 g/dl of molasses. When this softened molasses was analyzed for _NH3 , Na, Mg, and Ca by high performance liquid chromatography and atomic absorption,
97% of the ion equivalent of the cation was replaced by Na. The invertase-treated and softened molasses was diluted with water to a sugar concentration of 40 g/dl. Na type cation exchange resin (Diaion SK-104S)
Column packed with 240ml (with thermal jacket)
30φ×300mm), supply 30ml of this molasses to
It eluted with water. The temperature of the jacket was maintained at 70°C, and the supplied molasses and water were both maintained at 70°C. Moreover, the water supply rate was 12 ml/Hr. The eluate was analyzed by high-performance liquid chromatography, and fractions containing glucose and fructose were collected, and 93% of the sugars were recovered. The impurity removal rate of this sugar solution was 71%. On the other hand, chromatography was performed in the same manner on molasses that was not softened, and 94% of the sugar was recovered. The impurity removal rate of this sugar solution was 52%. The sugar solution separated by performing chromatography in this way was collected and concentrated to prepare a sugar solution with a sugar concentration (in terms of sucrose) of 50%, and 50 ml of this was mixed with 250 ml of a salt solution having the composition shown in Table 2. A 300 ml medium for glutamic acid fermentation was prepared.

[Table] Pour 300 ml of the L-glutamic acid production medium prepared in this way into a 1.0 volume fermenter, and
It was heat sterilized at ℃ for 10 minutes. Brevibacterium lactofermentum cultured in advance
ATCC13869 was inoculated and cultured at 31.5°C with aeration and stirring while keeping the pH at 7.8 with ammonia gas.
The sugar concentration in the culture medium in terms of sucrose is 3
%, the sugar solution used was added little by little to adjust the sugar concentration to 2 to 4%, and the cells were cultured for 36 hours. The amount of sugar solution added was unified to 80 ml in each experimental group. During the culture, when a predetermined amount of bacteria was reached, the surfactant "Tween 60" was added to the medium at a concentration of 0.6%. Table 3 shows the amount of L-glutamic acid accumulated in the culture solution and the sugar yield.

【table】 [Brief explanation of the drawing]

Figure 1 shows a chromatogram using Diamond Ion SK104S of cane molasses that was softened after invertase treatment, and Figure 2 shows a Diamond Ion chromatogram of cane molasses that was not softened after invertase treatment.
A chromatogram using SK104S is shown.

Claims (1)

[Claims] 1. After treating molasses with invertase or acid to convert sucrose into glucose and fructose, and using a cation-type cation exchange resin to replace several types of cations contained in molasses with one type of cation, A method for producing a fermented raw material, which comprises performing chromatography using an ionic cation exchange resin to separate invert sugar.