JP3609210B2 - Purification method of raffinose solution - Google Patents

Description

【００２２】
【実施例２】
ケストース類を含むラフィノース液を塩酸でｐＨ６．２に調製し、実施例１の粗酵素液を等量加え、４０℃で８時間攪拌し反応させた。結果は下記表２のとおりであった。
【００２３】
【表２】

【００２４】
【実施例３】
実施例１の粗酵素液を、セラミック担体を充填し４０℃に保温したカラムにＳＶ０．１で通液して、酵素を固定化した。ケストース類を含むラフィノース液を塩酸でｐＨ６．２に調整して、この固定化カラムにＳＶ０．１で通液した。結果は下記表３のとおりであった。
【００２５】
【表３】

【００２６】
【発明の効果】
上記から明らかなように、本発明の酵素によるラフィノース液の精製処理法によって、クロマトグラフィーでラフィノースと分離が不良であったケストース類を分解し、ラフィノース純度を向上させるとともに、同収率を向上させた。その結果、食品分野におけるラフィノース需要の増加に対し、供給量を増やすことができる。
【００２７】
また、従来より甜菜糖の製造において不純物として扱われてきたラフィノースに対して、本発明はその有効利用の途を拓いたものであって、本発明はこの点においても卓越している。
本発明について、ケストース類を含有するラフィノース液であれば、甜菜製糖由来のラフィノース液に限定されることなく、すべてのラフィノース液に対して適用できることはいうまでもないことである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for purifying raffinose liquid, and more particularly, the present invention relates to purification of raffinose liquid, particularly raffinose liquid containing a relatively large amount of kestoses (fructosyl sucrose) when producing raffinose. It relates to processing.
[0002]
[Prior art]
Raffinose is contained in sugar beet molasses and has been conventionally regarded as an impurity in beet sugar production. However, in recent years, it has been discovered that raffinose has excellent physiological effects such as intestinal regulation, and the usefulness of raffinose has been reviewed.
[0003]
Raffinose, a trisaccharide composed of galactose, glucose, and fructose, is produced by crystallizing sugar beet molasses (Kyoritsu Shuppan "Chemical Dictionary 9", p. 547). Specifically, conventionally, raffinose is produced by concentrating and crystallizing the raffinose fraction obtained by employing cation exchange resin chromatography to separate raffinose from the sugar-making juice. Further, in order to improve the recovery rate, the separation syrup at the time of crystallization is mixed with the chromatographic raw material and the crystallization raw material and circulated throughout the process.
[0004]
[Problems to be solved by the invention]
Separation and purification of raffinose, a trisaccharide from sugar beet sugar process juice, is significant from the aspect of effective utilization of impurities (by-products) in sugar beet sugar as described above. In the cation exchange resin chromatography used for the separation of raffinose, the separation of the trisaccharide kestoses (fructosyl sucrose) and raffinose is poor, and during crystallization to improve the recovery rate in the raffinose production process. When the syrup was mixed with the chromatographic raw material and the crystallization raw material and circulated in the process, kestoses accumulated in the process and the raffinose purity was lowered, making it difficult to recover more raffinose.
[0005]
In fact, when crystallization of Bx50, 50 ° C. syrup is cooled to 25 ° C., the raffinose crystallization rate reaches 82% when the syrup is raffinose 100%, whereas raffinose 60% and kestose 40 In the case of% syrup, the crystallization rate of raffinose falls to 64%.
Thus, efficient crystallization of raffinose in the coexistence of kestoses is extremely difficult, and the presence of kestoses is a major obstacle in the industrial production of raffinose.
[0006]
In the case of producing raffinose from sugar beet sugar process juice, the sugar beet sugar process juice contains kestose, so that raffinose cannot be produced efficiently, and a solution in this respect has been strongly demanded.
[0007]
[Problems to be solved by the invention]
The present invention has been made for the purpose of newly developing an efficient method for producing raffinose in response to such a demand in the industry.
As a result of studying from various directions, the present inventors have found for the first time that an inulin-degrading enzyme derived from Streptomyces rochei E87 has an action of degrading kestoses. As a result of further studies, it was discovered for the first time that the enzyme selectively decomposes only kestoses (fructosyl sucrose) and does not act on raffinose in the presence of raffinose.
[0008]
That is, the present inventors obtained a very useful new finding that the enzyme selectively decomposes only kestoses in the presence of raffinose, and raffinose is retained as it is, and based on this new finding, raffinose derived from sugar beet is obtained. When the enzyme was allowed to act on the liquid, the selective decomposition of kestoses was confirmed, and it was convinced that raffinose could be industrially produced, and the present invention was completed.
[0009]
That is, the basic technical idea of the present invention is to selectively decompose only kestose contained in a raffinose solution by purifying and separating raffinose by using an inulin-degrading enzyme.
Hereinafter, the present invention will be described in detail.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
In order to carry out the present invention, an inulin-degrading enzyme is used, but the origin of the enzyme is not particularly limited, and enzymes derived from all sources such as animals, plants, and / or microorganisms can be used.
Examples of the microorganism-derived enzyme include Streptomyces sp., For example, Streptomyces rochei or Streptomyces sp. (Kawamura et al. 1989) or from the genus Aureobacteria such as Aureobacterium sp. An inulin-degrading enzyme derived from MCI-2494 (Ohba et al. 1991) is exemplified.
[0011]
Among these enzymes, the inulin-degrading enzyme derived from Streptomyces rochei E87 is an exo-type hydrolase of inulin that attacks inulin in the form of exo to produce inulotetraose as the main product, and then inotriose It is an enzyme used for high-purity production of inulotriose (Tomita et al .: Letters in Applied Microbiology, 1995, 21, 330-333).
The above-described inulin-degrading enzyme used for high-purity production of inulotriose has been confirmed for the first time in the present invention to be able to be advantageously used as an enzyme that specifically degrades kestose and purifies raffinose. rochei E87 has been deposited as FERM P-15735 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.
[0012]
As an inulin-degrading enzyme, a purified enzyme can be used, but a crude enzyme, an enzyme-containing product containing the enzyme, and a processed product thereof can also be used.
Enzyme-containing materials include all those containing the enzyme. For example, the enzyme-producing microbial cells, the culture solution of the bacterium, the culture of the bacterium (the microbial cells, the culture solution and / or medium components, etc.) Mixtures) are also included in the enzyme-containing material.
Moreover, as a processed material of an enzyme, all the things which processed the refined enzyme, the crude enzyme, and / or the enzyme containing material suitably are pointed out, for example, a dried material-a paste-ized material-a concentrate-a dilution etc. are mentioned.
[0013]
When this enzyme is brought into contact with a kestose-containing raffinose solution, only kestoses are selectively decomposed into disaccharides and monosaccharides that are easily separated from raffinose by chromatographic treatment.
In this case, since this enzyme does not act on raffinose, raffinose is retained as it is without being decomposed even after treatment with this enzyme.
[0014]
Therefore, after treatment with this enzyme, it is sufficient to purify raffinose according to a conventional method, and to purify raffinose according to chromatographic treatment such as cation exchange resin chromatography and high performance liquid chromatography, crystallization treatment and other conventional methods. Can be separated.
These treatments may be performed batchwise or continuously using a reaction column. The enzyme treatment is the same, and the enzyme treatment can be carried out batchwise or continuously using an enzyme immobilized or not immobilized according to a conventional method.
Examples of the present invention will be described below.
[0015]
[Example 1]
Streptomyces rochei E87 (FERM P-15735) was cultured as follows using each medium of (1) to (3) described below. The medium was autoclaved at 121 ° C. for 10 minutes.
[0016]
(1) Slant medium composition glucose 10.0 g / l
Yeast Extract 1.0g / l
Meat extract 1.0g / l
NZ amine 2.0g / l
Cobalt chloride heptahydrate 0.02 g / l
Agar 15.0g / l
pH 7.3
[0017]
(2) Preculture medium (GPYS medium) Composition Glycerol 10.0 g / l
Polypeptone 10.0g / l
Yeast Extract 1.0g / l
Dipotassium hydrogen phosphate 1.0 g / l
Carboxymethylcellulose sodium salt 2.5g / l
Corn starch 30.0g / l
Agar 1.0g / l
pH 7.0
[0018]
(3) Enzyme production medium composition inulin 30.0 g / l
Sodium nitrate 2.0g / l
Magnesium sulfate heptahydrate 0.5g / l
Potassium chloride 0.5g / l
Potassium dihydrogen phosphate 0.5g / l
Iron nitrate heptahydrate 0.01g / l
Yeast extract 10.0 g / l
Meat extract 1.0g / l
Calcium carbonate 20.0g / l
pH 7.0
[0019]
After inoculation on slant medium, culture at 27 ° C for 3 days. One platinum loop from the slant was inoculated into 10 ml GPYS medium in a test tube and cultured with shaking at 27 ° C. for 1 day. 0.3 ml of the culture solution was inoculated into a 30 ml enzyme production medium in a 500 ml Sakaguchi flask, and cultured with shaking at 32 ° C. for 3 days. The obtained culture solution was centrifuged and the supernatant was used as a crude enzyme solution.
[0020]
After reacting a reaction solution having the following composition at 40 ° C. for 8 hours, the sugar concentrations were compared. The sugar concentration was analyzed by high performance liquid chromatography (Shodex Ionpack KS801, 80 ° C., water 1 ml / min, hereinafter HPLC).
2.5% sugar solution 0.1ml
0.1M phosphate buffer (pH 6.2) 0.5ml
Crude enzyme solution 0.4ml
The results were as shown in Table 1 below.
[0021]
[Table 1]

[0022]
[Example 2]
A raffinose solution containing kestoses was adjusted to pH 6.2 with hydrochloric acid, an equal amount of the crude enzyme solution of Example 1 was added, and the mixture was stirred at 40 ° C. for 8 hours to be reacted. The results are shown in Table 2 below.
[0023]
[Table 2]

[0024]
[Example 3]
The crude enzyme solution of Example 1 was passed through a column packed with a ceramic carrier and kept at 40 ° C. with SV0.1 to immobilize the enzyme. The raffinose solution containing kestoses was adjusted to pH 6.2 with hydrochloric acid and passed through this immobilization column with SV0.1. The results are shown in Table 3 below.
[0025]
[Table 3]

[0026]
【The invention's effect】
As is apparent from the above, the method for purifying raffinose solution with the enzyme of the present invention decomposes kestose that was poorly separated from raffinose by chromatography, thereby improving raffinose purity and improving the yield. It was. As a result, the supply amount can be increased in response to an increase in raffinose demand in the food field.
[0027]
Further, the present invention has pioneered its effective use against raffinose, which has been conventionally treated as an impurity in the production of beet sugar, and the present invention is also excellent in this respect.
It is needless to say that the present invention is not limited to raffinose liquid derived from sugar beet sugar and can be applied to all raffinose liquids as long as it is a raffinose liquid containing kestoses.

Claims (8)

The use of exo-type inulin degrading enzymes, raffinose - Kesuto contained in vinegar - selectively degrades the scan compound is raffinose - scan solution purification method - scan is raffinose, characterized in that the retained. The method according to claim 1, wherein the exo-type inulin-degrading enzyme is derived from the genus Streptomyces. The method according to claim 2, wherein the exo-type inulin-degrading enzyme is derived from Streptomyces rochei. The method according to claim 3, wherein the exo-type inulin-degrading enzyme is derived from Streptomyces rochei E87. The method according to any one of claims 1 to 4, wherein the exo-type inulin-degrading enzyme is a purified enzyme, a crude enzyme, an enzyme-containing product, and / or a processed product. The method according to claim 5, wherein the enzyme-containing material includes the present enzyme-producing microbial cell, the microbial culture solution, and / or the microbial culture. The process according to claim 6, wherein the treated product is a dried product, a paste, a concentrate, and / or a diluted product. The method according to any one of claims 1 to 7, wherein the raffinose liquid is a sugar beet sugar process juice.