JPS62228293A - Production of inulooligosaccharide - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the titled compound in high yield without forming colored materials or by-products, by reacting endo- form inulinase which is a mold of the genus Penicillium with inulin. CONSTITUTION:Endo-form inulinase which is a mold of the genus Penicillium is reacted with inulin, preferably at 60 deg.C reaction temperature and 4-5.5pH to hydrolyze the inulin and afford the aimed compound. The above-mentioned endo-form inulinase is obtained by cultivating Penicillium trzebinskii at 25-35 deg.C cultivation temperature, 5-7 initial pH and 2.5-7pH during cultivation, separating microbial cells from the culture fluid and treating the resultant supernatant liquid.

Description

[Detailed Description of the Invention] The present invention relates to the preparation of inulooligosaccharides by enzymatic decomposition of inulin, and more specifically, the preparation of inulooligosaccharides by hydrolyzing inulin using endo-type inulinase of molds of the genus Penicillium. It is about the method.

Prior Art Inulin is a linear fructose polymer, consisting of approximately 35 β-fructofuranose residues terminated by a glucose molecule in a β-1,2 bonding mode.

Inulin exists in various plants, especially in the roots of dahlia of the Asteraceae family, tubers of Jerusalem artichoke, and chicory, and can be easily extracted with hot water and purified by treatment with lime, etc. in the same way as beet sugar.

Obtaining fructose by decomposing inulin with acid or enzymes has been studied for some time, and has even been carried out industrially. When decomposing with acid, most of the objectives are achieved by hydrolyzing at pH 1-2 and temperature 70-90℃ for several hours, but the yield is low due to the formation of colored substances and by-products such as cyfructose anhydride. There are drawbacks such as. On the other hand, enzymatic decomposition does not have these drawbacks and is considered to be industrially advantageous.

Numerous enzymes that degrade inulin have been discovered in inulin-containing plants and microorganisms, and many examples of research have been conducted (Reference 1, below; references below).

A typical microbial enzyme is Kluyver in yeast.

myces fragilis and Candida ps
eudotropicalis, but among filamentous fungi there are eight supergillus, Fusarium, and Peni.
It is from cillium. Among them, Penicill
exo-type inulinase of the genus Ium (Reference 2), and As
Endo-type inulinase and exo-type inulinase (Reference 4) from Pergil-lus niger (Reference 3) have been purified and their properties have been investigated.

However, no endo-type inulinase is known for the genus Penicillium.

Regarding the endo-type inulinase of the Aspergillus genus mold, there is a document 5 that will be described later on Asp and ficuum, and the exo-type inulinase of the fungus is also described there, and the optimal mixing ratio of both enzymes for complete decomposition of inulin is also described. The findings are stated. (Reference 5゜) Exo-type inulinase releases fructose one by one from the glucose residue side of inulin and the opposite fructose side, while endo-type inulinase cleaves the inulin linear chain from any bond, and finally mainly 3 Produces ~6 types of oligosaccharides. Furthermore, it is easy to imagine that when the two coexist, the substrate for exo-type inulinase increases, so the reaction rate for producing fructose becomes faster, and even if inulooligosaccharide is produced, it quickly becomes fructose.

Nakamura et al. reported that no endotype was found in inulinase of the genus Penicillium (Reference 2).
Also, as mentioned above, there have been no reports to date denying this.

Research and utilization of inulinase has been carried out mainly for the purpose of producing fructose, and no attempt has been made to actively obtain a partial decomposition product of inulin, that is, inulooligosaccharide, using the enzyme. Furthermore, research on the preparation of inulooligosaccharides using acids is also hardly known.

With the aim of preparing inulooligosaccharides using endo-type inulinase, we have been searching for microorganisms with strong oligosaccharide-producing ability in soil, and as a result, we have found several types of microorganisms that meet our purpose. Ta.

Two of these molds belong to the genus Penicillium due to their mycological properties, and among them, P.

purprogenum var rubriscle
rotium and P. trze-binskii.

Since these molds have been deposited with legal institutions and their names are also listed above, a description of their mycological properties will be omitted.

Examples of research on the production of inulooligosaccharides using enzymes include, for example, in the culture of Aspergillus niger, endo-type inulinase purified and separated from inulin linear chains was used to convert inulin decomposition products into inulotriose, inulochitraose, and inulin decomposition products. It is known that (Reference 3). Furthermore, regarding Asp and ficuum, the existence of endo-type inulinase and the mixing ratio with exo-type inulinase in fructose production by inulin decomposition have been discussed (Reference 5).

However, the genus Penicillium that produces the enzyme for producing canine oligosaccharides as shown in the present invention is not known, nor is a method for actively producing canine oligosaccharides using it.

Furthermore, the Penicillium genus inulinase is characterized by having inulooligosaccharide producing activity as the main ingredient in its culture solution, and very little fructose is produced in the inulin decomposition product using this enzyme, unlike the conventional Aspergillus genus inulinase. It is different from endo-type inulinase, which exists together with exo-type inulinase.

In carrying out the method of the present invention, Pen1c~i
The canine oligosaccharide producing enzyme of the genus 11ium is surface cultured or submerged in a solid or liquid culture medium containing nutrients of various compositions commonly used as a natural or artificial medium.

In countries producing canine oligosaccharide-forming enzymes, production of exo-type enzymes may also be active depending on the culture medium conditions, and in that case, the issulin degradation product becomes fructose, so efforts must be made to ensure that endo-type activity is predominant as much as possible. A consideration in this regard is to culture without using organic nitrogen as much as possible.

It is possible to separate only the endo-type from an enzyme mixture of the endo-type and exo-type using enzyme protein separation techniques such as ammonium sulfate fractionation and various gel chromatography, and to make it act on inulin to obtain inulooligosaccharide. It is extremely advantageous for the practice of the present invention that microorganisms exhibit primarily endo-type enzymatic activity.

Nutrient sources for culture include bran, soybean flour, peptone, meat extract, yeast extract, cornstarch liquor, etc., and main carbon sources include fructose and inulin, supplemented by inorganic nitrogen sources, phosphates, magnesium salts, and small amounts. A medium containing metal salts of Pen is used, but Pen
Organic nitrogen is required for the expression of strong inulinase activity in the genus I.i. 6).

It is generally known that the inulinase activity of Penicillium genus is induced by inulin, its partial hydrolyzate, fructose, etc., but in addition to these, maltose is also effective in inducing endo-type activity.

Culture temperature is 25°C to 35°C, initial pH is 5.0 to 7.0
However, the pH value during culture should be kept within the range of 2.5 to 7.0.If inulin and its hydrolyzate were used as the carbon source, a rapid drop in pH would be seen on the third day of culture; When it does not, endo-type inulinase activity appears mainly.

On the other hand, when maltose is used as a carbon source, the pH decreases more slowly.

The culture time varies depending on the culture conditions, but the culture should be stopped when the maximum titer is reached. The maximum titer is usually reached after 4 to 8 days of culture.

Since endo-type inulinase is an enzyme produced outside the bacterial cells, after the cultivation is completed, bacteria are removed by filtration or centrifugation, and the supernatant liquid or extract from the solid medium is recovered. If necessary, it is concentrated using an ultrafiltration membrane or the like, and the enzyme is recovered as a precipitate by salting out with ammonium sulfate or by adding an organic solvent such as acetone or ethanol.

In the decomposition operation of inulin, it is usually desirable to increase the concentration of inulin as much as possible and to keep the operation temperature at 55° C. at the lowest in order to avoid microbial contamination during decomposition.

In carrying out the present invention, the inulin concentration is around 20%, the reaction temperature is 55 to 65°C, preferably 60°C, and the pH is 3.
．． 5 to 7.0, preferably 4.0 to 5.5.

Report on inulinase of the genus Penicillium (Reference 6
), the optimal decomposition temperature for inulin is around 45°C for any type of inulinase.
All inulinases of the genus Penicillium in the present invention have an optimum temperature between 55°C and 58°C, which is different from conventional inulinases.

It can also be seen that it is different from conventional products in that it has good thermal stability and hardly loses its activity even in a thermal stability test at <60° C. for 10 minutes.

The concentration of inulin is related to the temperature during solution preparation and the molecular weight distribution of inulin, and can vary over a wide range, but if inulin is dissolved at a high temperature of 90°C or higher and kept at 60°C, a state of supersaturation will occur even at a concentration of about 25χ. can be maintained.

EXAMPLES The present invention will be described in detail with reference to Examples below.

Example I Penicillium purprogenum
var rubri-sclerotuim (HOK
-1, FERM P-8705) Preparation of inulooligosaccharide Inulin (separated and purified from chicory root) 0.5χ
, maltose 0.5χ, NH411zPO40,27χ
, K, HPO40,1χ, MgSO4,7HzOO,0
After adjusting 5χ, Tween 800.02χ with tap water to pH 7, add 100% to a 500ml Sakaro flask.
After sterilizing and inoculating the test strain with a gold loop for 1 day, it was incubated at 28℃ for 9 hours.
Incubate with shaking for 6 hours.

After filtering the culture solution to separate Kokutai, the inulin degrading activity was measured by the reducing power method, and the titer was 2.5 u/ml of the culture solution. However, the titer measurement method is as follows.

Add 0.5 ml of enzyme solution diluted with 1/ION acetic acid buffer (pH 5,0) to 0.5 ml of 1.5χ inulin solution,
3. The reducing sugar produced by enzymatic reaction at 50"C for 30 minutes.
Colorimetric determination was performed by measuring the absorbance at 500 nm using the 5-dinitrosalicylic acid method. The enzyme unit was expressed as the amount of enzyme that produced 1 μmole of reducing sugar per minute per ml of culture solution at 50°C.

Filtered culture solution (10100O, inulinase activity 2.
Ammonium sulfate was added to 5 u/ml) and the 40-80% saturated precipitated fraction was centrifuged and dialyzed against distilled water. The obtained crude enzyme solution was 100 ml of 17.7 u/ml.

This crude enzyme solution was added to a 20% inulin solution at a rate of 3 units per 1 g of inulin and kept at pH 4.5 and temperature 60°C for 48 hours.
The reaction was carried out.

When the reaction product was analyzed by gel chromatography, it was found that inulooligosaccharide with a degree of polymerization (DP) of 3 to 9 and very small amounts of DP2 and DPI sugars were produced approximately 3 hours after the start of the reaction.
), then 24 hours (Figure 2), and 48 hours (Figure 3)
``il'ha DP1 force 1.5X, DP2 M3.3%,
DP3 is 31.4X, DP4 is 26.6X,
DP5-IJ<20. C4, DP6, '1 (13,3χ
, an inulooligosaccharide composition of 3.5χ or more was obtained.

These decomposition patterns over time are typical decomposition patterns of endo-type enzymes, and the final decomposition limit of this crude enzyme solution was approximately 50X, calculated assuming that the reducing sugar produced was fructose.

In this way, it has become possible to prepare inuloid inulinase from inulin using endo-type inulinase, which is completely different from the fructose-producing type of the Penicillium inulinase known so far.

The inulin decomposition product was purified by conventional methods using ion exchange resin and activated carbon, and then concentrated and dried to produce white inulooligosaccharide.

Example 2 P, Lrzebinskii (HOK-2, FERM
Preparation of inulooligosaccharide using endo-type inulinase of P-8706).

Inulin 1.0% obtained from chicory, (NH4)2HP
O40.15%, KH2PO40.1%, Tween
80 0.02%, MgSO4,7Hz00.05%,
A pH 7 culture medium prepared with tap water containing 1! Pour 300ml into an Erlenmeyer flask, sterilize it, and add P, trjebin.
Inoculate one platinum loop of P. skii with an amplitude of 7 C111 at 28°C.
Rotate and culture at 100 strokes.

After the 4th white of the culture, the pH starts to decrease and reaches pH 3 on the 6th day.
, 5, and the inulinase activity, which was initially fructose-producing, became oligosaccharide-producing.

On the 7th day, the endotype activity was 0.8u per ml of culture solution.
The culture was stopped when the number of microorganisms reached a maximum of nit, and the culture solution and the bacterial cells were separated by filtration.

The culture solution was salted out in the same manner as in Example 1, and the temperature was 4° to 80° with ammonium sulfate.
Obtain the
5,0) and then desalted using Biogel P-60G (manufactured by Bioland) to obtain a crude enzyme solution.

This crude enzyme solution was added to a 20% inulin solution at a rate of 3 units per 1 g of inulin, and po4. s, temperature 60℃
The reaction was carried out for 48 hours.

The results of gel chromatography analysis of the reaction products are shown in Figure 4. DPI is 1.3z, Kano 2 is 0.9z, D
P3 is 26.5χ, DP4 is 27.6χ, DP5 is 18
．． 5χ, DP6 is 14.2X, or higher 11.
A canine oligosaccharide composition of 0x was obtained.

When inulin was analyzed using the crude enzyme solution of this strain, the decomposition pattern over time showed a decomposition pattern due to endo-type enzymes, similar to Example 1.

(Reference 1) Vandamme, E, J, ;D
erycke, D.G., Adv.

Appl, Microbiol, 1983.29.13
9-L76 (Reference 2) Nakamura, T.
;Nakatsu, S, NipponNogeik
agaku Kaishi, 1977.51.681~
689 (Reference 3) Nakamura, T. et.
al, N1ppon Nogei-kagaku
Kaishi, 1978, 52.159-166 (Reference 4) Nakamura, T, et al.
N1ppon Nogei-kagaku Kaish
i, 1978, 52.581-587 (Reference 5)
L, Zittan, 5tarch, 19B1,33,3
73-377° (Reference 6) Nakamura,
T, et al, N1ppon Nogei-Kag
aku kaishi, 1969, 43, 599~
605

[Brief explanation of drawings]

The drawings from FIG. 1 to FIG. 4 are all gel chromatographs showing the enzymatic decomposition pattern of inulin in the present invention. Patent applicant: Mitsui Toatsu Chemical Co., Ltd. Figure 1 → 7 too! ;+321゜Dρ

Claims (1)

[Claims] A method for producing inulooligosaccharides, which comprises causing endo-type inulinase of a Penicillium fungus to act on inulin.