JPH04237496A - Production of cyclic inulo oligosaccharide - Google Patents

Abstract

PURPOSE:To profitably provide having high solubility and useful as a clathrate- forming agent, etc., by treating inulin, etc., with a cycloinulo oligosaccharide fructano transferase originated from Bacillus.circulans. CONSTITUTION:beta-2,1-Fructose oligosaccharide or inulin, etc., is treated with a cycloinulo oligosaccharide fructano transferase originated from Basillus.circulans MCI-2554 (FERM 11940) to produce the objective cyclic inulo oligosaccharide having six-eight fructose molecules combined with each other through beta-2,1-fructoside bonds. The bacillus.circulans MCI-2554 has a bar-like shape having a diameter of 0.4-0.5mum and a length of 1.5-3.5mum, exhibits the expansion of the cell due to the formation of elliptical spores at the center or terminal part of the cell and is a gram positive spore-forming rod separated from natural soil.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a method for producing cyclic inulooligosaccharide. Specifically, the present invention relates to a method for producing a cyclic inulooligosaccharide by allowing an enzyme derived from a specific microorganism to act on β-2,1-fructose oligosaccharide or inulin.

0002

[Prior Art and Problems to be Solved by the Invention] Cyclocyclodextrin, in which glucose is linked to α-1,4-glucoside, has been known as a cyclic oligosaccharide composed of several sugars. Cyclocyclodextrin is attracting attention as a food clathrating agent.
It contributes to adding value to foods by preserving flavor components, masking unique odors, removing bitterness, and preventing oxidation. However, its use is currently limited due to its high cost and low solubility in water. Therefore, it has been desired to develop a food-use clathration agent with high solubility, and there has been a desire to develop a new clathration agent not only for foods but also for a wide range of fields.

[0003] In recent years, Uchiyama et al. have added Bacillus circulans MZ no. reported that cyclic inulooligosaccharides were obtained by the action of an enzyme derived from Carboh et al.
ydrate Research, 192, 83-9
0,1989 and JP-A-2-252701). However, the yield of cyclic oligosaccharides is about 2.5% or 2.
0%, and there are still few examples of methods for producing cyclic inulooligosaccharides using microbiological methods, and there has been a desire to develop a method that can efficiently produce cyclic oligosaccharides. .

0004

[Means for Solving the Problems] The present inventors have carried out extensive research on a method for producing cyclic inulooligosaccharides from inulin with a high yield by microbiological methods, and have found that Bacillus circulans MCI-2554 ( cycloinulo-oligo derived from Microtechnical Research Institute No. 11940)
Saccharide fructanotransferase (cy
cloinulo-oligosaccharide
fructanotransferase, hereinafter referred to as “
CFTase. ) was found to efficiently produce cyclic inulooligosaccharides, leading to the completion of the present invention. That is, the gist of the present invention is to convert CFTase derived from Bacillus circulans MCI-2554 into β-2,1-
The present invention relates to a method for producing a cyclic inulooligosaccharide, which is characterized by acting on fructose oligosaccharide or inulin.

[0005] The present invention will be explained below. The CFTase used in the present invention is Bacillus circulans MCI-
2544 (Feikoken Bacteria No. 11940; hereinafter also referred to as this strain or MCI2554). Bacterium MCI2554 was developed by the present inventors,
This bacterium was isolated from natural soil, and its mycological properties are as follows.

1. Morphological properties ○Characteristics of colonies on ordinary agar medium, 40℃, 3rd day 1
) External shape: circular 2) Size: 2 to 3 mm 3) Surface ridges: convex 4) Surface shape: smooth 5) Gloss: dull 6) Color: yellowish white 7) Transparency: translucent 8) Periphery: Whole edge ○Soil extract agar medium (R.E.Go
rdon et. al. The Genus B
acillus. P. 101, Agriculture
Hand book NO. 427, 1973) Morphological properties during culture at 40°C for 48 hours 1) Cell morphology: diameter 0.4-0.5 μm, length 1.
5-3.5 μm rod-shaped 2) Motility: Yes 3) Spore formation: Swelling of the bacterial cells forming oval spores at the center or end of the cell is observed.

4) Gram staining: positive to indeterminate5) Acid-fastness: negative2. Physiological properties 1) Growth under anaerobic conditions
: Negative 2) Growth in air
:Positive 3) Catalase
:Positive 4) Oxidase
: Negative 5) O
-F test
: Oxidation 6) Hydrolysis of gelatin
:Positive (ordinary agar +0
．． 4% gelatin plate) 7) Casein hydrolysis: Negative 8
) Nitrate reduction
: Negative 9) Methyl red test
: Negative 10) VP test
: Negative 11) Production of indole
: Negative 12) Generation of hydrogen sulfide
:Negative 1
3) Hydrolysis of starch
:Positive 14) Use of citric acid
:Positive (on Christensen medium) 15) Utilization of inorganic nitrogen source
:Positive (ammonium salt)

Positive (nitrate) 16) Urease
: Negative 17) DNase production
: Negative 18) Growth in 2% NaCl : Growth possible
Growth in 3% NaCl
: Not possible 19) Formation of pigment
:Negative 20
) Growth temperature range
:20-47℃ Optimum growth temperature
40℃21) Growth pH
:pH5.6-822
) Decomposition of Tween80
:Positive 23) Tyrosine degradability
: Negative 24) Lysozyme resistance : Negative (10,000 units/ml) 25) Generation of acid from carbon source

[0008]

[Table 1]

3. Chemical taxonomic properties 1) GC content in DNA
55% 2) Cell wall diamino-amino acids
meso-diaminopimelic acid (whole cell hydrolyzate) 3) Whole cell sugar composition
glucose
Ribose 4) Major menaquinones
MK-7 5) Bacterial fatty acids
branched type

Anteiso-C15:0

Iso-C16:04. Taxonomic considerations ○ Identification at the genus level This bacterial strain (MCI No. 2554) is 1) a Gram-positive rod, 2) motile, 3) forms spores, 4
) produces acid from glucose; 5) diamino-amino acids in the cell wall have meso-diaminopimelic acid;
6) Main menaquinone exhibits characteristics such as having MK-7.

[0010] From these characteristics, this strain is classified as Bergey.
's Manual of Systemat
ic Bacteriology Volume 2 (1986)
Gram-positive endospore-forming rods and cocci, described in
-Positive Rods and Coc
It was found that the bacteria belong to the genus Bacillus of group ci).

[0011] Identification at the species level The Bacillus genus currently includes approximately 50 species. These species are distinguished by the morphology and site of spore formation, various physiological properties, and chemical taxonomic properties. In particular, the GC content in DNA ranges over a wide range from 32 to 69%, and this difference has been taken up as a characteristic of the species. GC of this strain (MCI2554)
The content is 55%, Bergey's Mannu
al of Systematic Bacteri
When we searched for species based on Volume 2 of B. circ
ulans, B. macerans B. coagu
lans, B. polymyxa, and B. star
thermophilus and the like. Therefore, we compared these five species with respect to various physiological properties. As shown in Table 1, this strain is B. staroth
ermophilus, B. It was clearly distinguished from P. coagulans in terms of growth temperature range. Also, B. ma
cerans, B. Polymyxa did not match in physiological properties such as production of gas from sugar and ability to reduce nitrate. Therefore, this strain 1) has a GC content of 55%, 2) does not show growth at 50-55℃, 3) is negative for VP test, 4) ferments sugar well without gas generation, 5) Because it has characteristics such as no ability to reduce nitrate, B. It was identified as P. circulans.

[0012] As shown below, this strain is B. steam
rothermophilus, B. coagulan
It was clearly distinguished from S. in terms of growth temperature range. In addition,
Hereinafter, A is MCI2554 bacteria, B is B. circle
ans, C is B. coagulans, D is B. mac
erans, E is B. polymyxa, F is B. st
(B to F are literature values: Bergey's Manual), + is positive (90% or more), - is negative (10% or less), d is positive (11 to 89%), and v is depending on the culture conditions. Unstable properties, N
D indicates no data.

[0013]
A B C D
E F────────────
──────────────────────── Cell diameter > 1.0 μm − −
− − −
-Globular spores
− − − −
− −Swelling of cells
＋＋v
＋ ＋ ＋Catalase ＋
+ + + d
+ Growth under anaerobic conditions −
d + +
+ -VP test
− − +
- + - pH
<6-
+ + + d
+ >7
− − −
− − −
A
B C D E
F────────────────────
────────────────Acid production D-glucose +
＋ ＋ ＋ ＋
+ L-arabinose
+ + d +
+ d D-xylose
＋＋d
+ + d D-mannitol + +
d + +
d Production of gas from glucose − −
− ＋ ＋
-Hydrolysis of casein-
d d − +
d Liquefaction of gelatin
+ d − +
＋ ＋Starch hydrolysis
＋ ＋ ＋
＋ ＋ ＋Use of citric acid ＋d
d d −
Decomposition of d-tyrosine −
− − −
− −Reduction of nitrates
- d d
+ + Generation of d-indole
− −
− − − −NaC
l and KCl requirements - -
- - - -Growth pH 6.8 +
＋ ＋ ＋ ＋
+5.7
＋d＋
＋ ＋ −
5.0 - -
＋ − −
-Influence of NaCl 2%
＋ND ＋ND
ND ND 5%
-d-
− − d
7% -
d − − −
-10%
− − −
− − −Growth temperature 5℃
− − − −
d-10℃
-d
- d + -
30℃+
＋ ＋ ＋
+ - 40℃
＋ ＋ ＋
＋ ＋ ＋
A
B C D
E F──────────────────
──────────────────── 50
℃ −
- + d -
+55℃
− − +
− − +65℃
− −
− − −
+Lysozyme resistance -
d − −
d-GC content (%)
55 31.6 44.3
44.9 41.0 43.5

-61.0 -56.0 -51.
0 -51.4 -62.2 Also, B. mac
erans, B. Polymyxa did not match in physiological properties such as gas production from sugar and nitrate reduction ability. Therefore, this strain 1) has a GC content of 55%, 2) does not show growth at 50-55°C, 3) has a negative VP test, 4) performs sugar fermentation well without gas generation, and 5) Because it has characteristics such as no ability to reduce nitrate, B. It was identified as P. circulans.

○ Subspecies level identification B. circulans are composed of composite species,
Nakamura and Swezey divided into 10 groups (Int.J.Sys.Bac.
Vol. 33, p. 703-708, 1983). Therefore, this strain and B. circulans and each group.

As shown below, this strain was found to fall within the range of groups 7 to 10 in terms of GC content. Furthermore, when various physiological properties were compared, the properties matched well with those of Group 9, except for growth under anaerobic conditions. In particular, it was suggested that it is closely related to group 9 because it has characteristics such as sensitivity to lysozyme and the ability to utilize citric acid. Hereinafter, A is this strain, 1 to 10 are B.
circulans (1 to 10 are literature values: Int.J.Sys.Bac.Vol.33, p
．． 703-708, 1983), +, - and d have the same meanings as above.

[0016]
A 1 2 3 4 5 6 7
8 9 10──────────────────
──────────────────Influence of NaCl 5% −
+ − − − d − − +
-d 7%
− − − − − − −
- - - - Growth at pH 5.6
＋ ＋ ＋ ＋ ＋ d d −
＋ ＋Lysozyme resistance
− − − ＋ − − − ＋ ＋
− + Growth under anaerobic conditions −
＋ ＋ − − ＋ ＋ − ＋
＋ ＋Starch hydrolysis ＋ ＋
− ＋ ＋ ＋ ＋ − ＋ ＋
+Hydrolysis of casein − −
+ − d d − − − − −
VP test - -
− − − − − − − −
− Reduction of nitrate −
− − − − − − − − + −
＋Use of citric acid ＋
− d − − − − − − +
-Acid production Glucose + +
＋ ＋ ＋ ＋ ＋ ＋ ＋
+ Arabinose +
＋ ＋ ＋ ＋ ＋ ＋ − ＋ ＋
+ xylose +
＋ ＋ ＋ ＋ ＋ ＋ ＋
＋ ＋ Mannitol
＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋
+ +GC content (%)
55 37 45 47 47 47 4
7 50 50 53 50
-39
-46 -49 -48 -50 -50 -52 -
52 -54 -53 Furthermore, B. nigra, which is known as a cyclic inulooligosaccharide-producing bacterium, circulans MZ
No. When compared with the properties of No. 31 (Japanese Unexamined Patent Publication No. 2-255086), they were clearly differentiated in terms of growth at 40°C, reduction of nitrate, utilization of citric acid, etc., as shown below. Hereinafter, A is MCI2554 bacteria, G is B. cir
culans MZ No. 31, +, -, ND
indicates the same meaning as above.

[0017]
B
A────────────────
──────────────────── Growth temperature range 15-35℃
20~47℃
(Weak growth at 10℃, no growth at 40℃) (Optimum temperature 40℃) Growth pH range
pH6-8
Growth under anaerobic conditions with pH 5.6-8
−
-catalase
+
+V
P test
−
-Methyl red test
+
- Reduction of nitrates
+
- Production of indole
−
-Generation of hydrogen sulfide-
−Hydrolysis of starch +

+Use of citric acid
−
+ Effect of salt
Unable to grow at 3% 3
Production of non-viable acids in % glucose
+
+ fructose
+
+ maltose
+
+ Galactose +

+ Mannitol
+
+ Sucrose
+
+ lactose
+
＋ Aesculin ＋

ND Sorbitol
−
- Adonitol
−
ND inositol −
−
ethanol
−
-GC content
N.D.
55% The Bacillus circulans MCI-2554 used in the present invention can be easily grown by culturing in a medium containing nutrients that can be used by ordinary microorganisms. As a carbon source,
Glucose, starch syrup, dextrin, sucrose, starch, inulin, molasses, animal and vegetable oils, etc. can be used. Further, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used as the nitrogen source. It is also effective to add, if necessary, inorganic salts that generate ions such as sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and others.

[0018] In the present invention, the CFTase of the present invention
is allowed to act on β-2,1-fucratose oligosaccharide in an aqueous solution. Furthermore, since the CFTase of the present invention acts on inulin to produce cyclic inulooligosaccharides, CFTase
Cyclic inulooligosaccharides can be obtained by reacting with extracts of plants with high inulin content, such as Jerusalem artichoke, burdock, and chicory, or in culture solutions containing inulin as the sole carbon source. In the present invention, the action of CFTase may be caused by the bacteria of the present invention itself,
Alternatively, CFTase may be extracted from the same bacteria and used as it is or after immobilization.

When obtaining a cyclic inulooligosaccharide from the culture supernatant obtained by culturing in a culture solution, for example, it contains about 1 to 10% inulin as a carbon source, and in addition, soybean flour, wheat germ, corn steep as a nitrogen source. Liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc., as well as sodium, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions as required. This bacterium is inoculated into a medium supplemented with inorganic salts, etc., and cultured with shaking. On this occasion,
The culture temperature is 20-50℃, and the culture time is 12-12℃.
0 hours is preferred. The obtained culture solution is sterilized by centrifugation, and the enzyme in the supernatant is inactivated by heat treatment. Concentration is then performed, for example, by adsorption onto an activated carbon column. After fructose, glucose, and other linear oligosaccharides are eluted with distilled water, elution is performed with a 50% methanol aqueous solution. Since cyclic inulooligosaccharide is obtained in this fraction, the desired cyclic inulooligosaccharide can be obtained by concentrating and drying the fraction.

[0020] When an enzyme is applied, for example, the culture solution cultured by the above method may be sterilized by centrifugation, and the obtained filtrate may be applied to the enzyme, or the filtrate may be further treated with ammonium sulfate (65%). The precipitate may be obtained by centrifugation, suspended in a small amount of water, and then subjected to dialysis, and the resulting crude enzyme solution may be allowed to act. For example, this culture filtrate or crude enzyme solution is allowed to react with inulin at a concentration of about 5% or more in a 0.01-0.1M phosphate buffer adjusted to pH 7.0 at 30-60°C for 30 minutes or more. The desired cyclic inulooligosaccharide can be obtained by et al. Of course, purified enzymes may be used, such as DEAE-Toyopearl 65
After purification by ion exchange chromatography using a 0M, SP-Toyoearl 650M column (trade name manufactured by Tosoh Corporation), ToyopearlHW55F (
By performing gel filtration with Tosoh Corporation (product name), SD
An enzyme preparation showing a single band can be obtained by S-polyacrylamide electrophoresis.

Bacillus circulans-MCI-
The optimum pH of the enzyme specimen derived from Bacterium No. 2554 was 7.5, and the maximum activity was exhibited at 45°C. pH is 6.0-10
．． It is stable in the range of 0 and 50℃ after 30 minutes of heat treatment.
It was stable until then. The molecular weight determined by SDS-polyacrylamide electrophoresis was approximately 200,000. The molecular weight determined by gel filtration chromatography was approximately 600,000. By the above method, 6 to 8 fructose are usually converted into β-2,
A cyclic inulooligosaccharide with a 1-fluxid bond is obtained.

[0022]

[Examples] The method of the present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto unless it exceeds the gist thereof.

(Example 1) Inulin 4%, yeast extract 0.2%, sodium nitrate 0.5%, magnesium 0.05%, potassium chloride 0.05%, phosphoric acid
150 ml of a medium containing 0.05% potassium and 0.001% ferric chloride was adjusted to pH 7.0 and steam sterilized at 120° C. for 20 minutes. One platinum loop of Bacillus circulans MCI-2554 was inoculated into this sterilized medium.
The cells were cultured at 160 rpm at 30°C for 30 hours. The OD value of the culture solution at 660 nm was approximately 6. After completion of the culture, the bacterial cells were removed by centrifugation to obtain a culture filtrate. By heating the obtained culture filtrate at 100°C for 10 minutes,
The enzyme was inactivated and concentrated under reduced pressure to about 10 ml. Transfer this liquid to an activated carbon column (50 g of activated carbon and Celite No. 53).
5. 50g of the mixture was adsorbed in a 50% solution of methanol (filled with distilled water) and 1 liter of distilled water was passed through it, followed by elution with a 50% aqueous methanol solution. The eluted peaks were collected and concentrated to dryness under reduced pressure.
A powder of cyclic inulooligosaccharide was obtained. QAE-toyopearl made into ionic form with 0.5M ammonium sulfate
(trade name manufactured by Tosoh Corporation) in a column and equilibrated with 80% methanol.
% methanol and eluted with 80% methanol and fractionated, the amount of fructose was 6 to 8, molecularly bonded cyclic inulooligosaccharide (CI-6, CI-7,
CI-8) solutions were obtained. As a result of drying by vacuum concentration, 1.23 g of CI-6, 0.44 g of CI-7,
0.09g of CI-8 was obtained.

(Example 2) After concentrating 100 ml of the culture filtrate obtained in Example 1 to 10 ml by ultrafiltration to obtain an enzyme solution, it was added to 40 ml of 0.05 M phosphate buffer containing 10% inulin. In addition, the mixture was reacted at 50°C for 2 hours. After the reaction solution was heated to inactivate the enzyme, it was subjected to activated carbon column chromatography and eluted with 50% methanol to obtain a cyclic inulooligosaccharide in the eluate. After concentrating this under reduced pressure, the cyclic inulooligosaccharide was separated using QAE-toyopearl (trade name manufactured by Tosoh Corporation) in the same manner as in Example 1, and the cyclic inulooligosaccharide was concentrated to dryness under reduced pressure. As a result, 1.7 g of CI-6 and 1.7 g of CI-6 7
0.9 g of CI-8 and 0.11 g of CI-8 were obtained.

[0025]

[Effect of the invention] Bacillus circulans MC of the present invention
CF derived from I-2554 (Feikoken Bibori No. 11940)
Since Tase successfully produces cyclic inulooligosaccharide from β-2,1-fructose oligosaccharide or inulin, cyclic inulooligosaccharide can be produced at a high yield of about 80% or more. Cyclic inulooligosaccharides can be expected to be used as clathrate compounds not only in the food field but also in a wide range of fields.

Claims (3)

[Claims] [Claim 1] Bacillus circulans MCI-2
A method for producing a cyclic inulooligosaccharide, which comprises allowing cycloinulooligosaccharide fructanotransferase derived from No. 554 (Feikoken Bibori No. 11940) to act on β-2,1-fructose oligosaccharide. [Claim 2] Bacillus circulans MCI-2
A method for producing a cyclic inulooligosaccharide, which comprises allowing cycloinulooligosaccharide fructanotransferase derived from No. 554 (Feikoken Kyoiku No. 11940) to act on inulin. 3. The method for producing a cyclic inulooligosaccharide according to claim 1 or 2, wherein the cyclic inulooligosaccharide consists of 6 to 8 fructose molecules.