JPH078286A - Production of oligosaccharide with high panose content - Google Patents

Abstract

PURPOSE:To produce an oligosaccharide with a high panose content by the fermentation. CONSTITUTION:This method for producing an oligosaccharide by the fermentation comprises the steps of inoculating a yeast, belonging to the genus Moniliella and having the ability to produce the panose, preferably Moniliella tomentosa into a culture medium consisting essentially of maltose as a carbon source, aerobically culturing the yeast, producing and accumulating the panose in the culture medium and collecting the resultant panose.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inexpensively producing oligosaccharides having a high panose content by a fermentation method using maltose as a raw material.

[0002]

BACKGROUND OF THE INVENTION Branched oligosaccharides containing panose have properties such as bifidobacteria-enhancing action, anti-crystallinity, inferior aging, and moisturizing properties, and are attracting attention as important ingredients for the rich taste of sake and mirin. Widely used in medicine, pharmaceuticals, etc. In particular, panose not only does not become a raw material substrate of insoluble glucan produced by oral bacteria such as Streptococcus mutans in recent years, but it also inhibits the production of these glucans from sucrose, and further causes caries. It has been found that they are non-cariogenic, anti-cariogenic, non-fermentative oligosaccharides that do not serve as a substrate for acid production. Panose has been considered to be one of the safest oligosaccharides since it is a natural product contained in starch syrup, etc., but it has been difficult to mass-produce it by an inexpensive method until now, so it is an extremely expensive reagent. However, it is only used for elucidating the action mechanism of various amylase.

[0003]

Conventionally, it has been widely known that an oligosaccharide containing panose is prepared from a partial hydrolyzate of amylopectin and glycogen, but its panose content is low and it is not suitable for industrial production. It was unsuitable.
Recently, Bacillus stearothermophilus (Baci
llus stearothermophilus) or Bacillus subt
It has been reported that neopullulanase derived from (ilis) acts on starch and pullulan to obtain starch sugar containing panose and high-purity panose. (JP-A-1-171493,
J.Ferment.Bioeng.vol73, No.3,198-202.1992) However, these methods are still not suitable for industrial production because expensive pullulan must be used as a raw material and the productivity of the enzyme is low. I haven't arrived. Furthermore, when the action of neopullulanase preceding the starch, mainly isopanose and 6 ² -O-alpha-maltosyl maltose obtained (JP-5 over 95768 JP) rather than panose.

[0004] In addition, a transglucosidase (Methods in Carbohydrate Chemistry Vo
l.1, p319-324), a method for preparing isomaltose, panose, etc. from maltose is known, and further studies on these are described in JP-A-63-122.
It is disclosed in Japanese Patent No. 696. The method for producing panose using transglucosidase disclosed in the publication can use inexpensive maltose or starch as a raw material and is a relatively good method suitable for industrial production, but the produced panose does not decrease. , It is necessary to strictly control the degree of progress of the reaction, the cause of browning of the food in the reaction product, and the fact that a considerable amount of glucose, which is the best substrate for acid production of caries, is contained in the reaction product. There is room for.

[0005]

[Means for Solving the Problems] As a result of various studies to improve the above-mentioned drawbacks of the conventional method, the present inventors have found that Monilella tomentos (Monilella tomentos)
a) found that maltose was present as a carbon source and utilized it to efficiently produce panose,
Further research has been carried out to reach the present invention. That is, unlike the conventional method, the present invention relates to a method for producing an oligosaccharide having a high panose content by a fermentation method which does not require the preparation of an enzyme agent and strict control of reaction conditions.

More specifically, the present invention inoculates a medium containing maltose as a carbon source as a main component with a yeast capable of producing panose belonging to the genus Monillella and aerobically culturing it to produce panose in the medium. The present invention relates to a method for producing an oligosaccharide having a high panose content by fermentation, which is characterized by accumulating and collecting this.

The present invention will be described in detail below. In the present invention, Moniliella tomentosa IFO-9 is used as a representative example of yeast having panose-producing ability belonging to the genus Moniliella.
Strain 485 (Fermentation Research Institute No. 9485), Moniliella tomentosa IFO-9487 Strain (Fermentation Research Institute No. 948)
7) and Moniliella tomentosa IFO-9488 strain (Fermentation Research Institute No. 9488).

Cultivation of this strain is preferably carried out by stirring culture under aerobic conditions using a liquid medium containing a carbon source, a nitrogen source, vitamins, inorganic salts and the like. A sugar containing maltose such as maltose and maltose syrup as a main component is used as a carbon source of the liquid medium, and the maltose purity in these sugars is 50 (W / W).
% Or more, particularly 80 (W / W)% or more is preferably used in consideration of the purity of panose accumulated in the fermentation liquid. Also, these carbohydrates are usually
It is added and used in the medium in a concentration range of 10 to 60 (W / V)%, preferably 30 to 50 (W / V)% in terms of solid content. The% used in the following description means weight (W / W)% in terms of purity and content in solids, and the content (concentration) of maltose and yeast extract in the medium is volume (W / V). ) Means%.

As the nitrogen source, nitrogen compounds available for the strain to be used, such as yeast extract, tryptone, malt extract, casamino acid, corn steep liquor and the like are used. Further, ammonium salts such as ammonium sulfate, ammonium acetate and ammonium nitrate, nitrates and the like can also be used. As the inorganic salts added to the medium, salts of phosphoric acid, magnesium, potassium, calcium, iron and the like are used. Furthermore, if necessary, various organic substances, inorganic substances, or commonly used defoaming agents necessary for yeast growth can be added.

The culture is carried out by directly inoculating the liquid culture medium of the above composition with the present bacterial cells or separately inoculating a seed culture solution obtained by preculture. This inoculum culture solution is prepared, for example, by inoculating one platinum loop into a liquid medium having a pH of 4 to 6 containing 30% maltose and 1% yeast extract, and 2 to 8 at a temperature of 26 to 36 ° C. at a temperature of 26 to 36 ° C. It is carried out by culturing for a day. The culture of the present invention is carried out within the range where the microorganism can grow, that is, at a culture temperature of 24 to 40 ° C, preferably 28 to 35 ° C. The pH of the medium is 3.5-7.
The range of 0, especially 4.0 to 6.0 is preferable. The culturing period varies depending on the culturing conditions, the type of medium used and the concentration of the sugar as the carbon source, but is usually about 3 to 6 days.

The culture in the present invention can be terminated by high performance liquid chromatography, thin layer chromatography or the like so that the amount of panose in the culture solution can be terminated when the production amount of panose in the culture solution reaches the maximum. It is desirable to carry out the measurement by a known method. The oligosaccharide containing panose accumulated in the culture solution is purified from the culture solution by a conventional method after the completion of the culture. That is, in such a case, well-known means usually used in the art, for example, operations such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction and the like are appropriately combined and used as necessary. As an example, bacterial cells are removed from the culture solution by filtration, centrifugation, etc., and then the solution is treated with activated carbon to remove coloring substances and the like, further deionized with an ion exchange resin, and then the solution is concentrated. It can be a syrup of oligosaccharides containing panose. In the present invention,
The obtained syrup can be subjected to gel filtration, ion exchange resin column chromatography, carbon column chromatography, etc. to obtain a highly pure oligosaccharide having a high panose content.

Next, the present invention will be described in detail with reference to examples. Example 1 (a) Preparation of seed culture Glucose 30 (W / V)%, yeast extract (manufactured by Difco, the same applies hereinafter) 1.0 (W / V)%, agar 1.5% (W
/ V) on a slant medium consisting of Moniliella tomentosa
Seed culture is obtained by applying IFO-9487 strain and culturing at 30 ° C. for 2 days. (B) Main culture Maltose 30 (W / V)% (Purity 99.0 (W / W)
%), Liquid medium 5 containing yeast extract 1.5 (W / V)%
Add 0 ml to a 500 ml Erlenmeyer flask,
Sterilize by autoclaving at ℃ for 15 minutes. The Moniliella tomentosa IFO-9487 strain prepared in (a) above was inoculated to one platinum loop and cultured at 30 ° C. and 220 rpm for 7 days. After completion of the culture, the sugar composition of the culture solution was examined by high performance liquid chromatography (HPLC). The results are shown below.

Sugar composition of culture solution (content% based on solid content) 5 sugars or more branched sugars 7.8 branched tetrasaccharides 17.4 maltotriose 2.6 panose 31.0 maltose 21.6 isomaltose 1. 9 Glucose 0.5 Other 17.2

Example 2 50 ml of a liquid medium containing 30% maltose (purity 99.0%) and 5% corn steep liquor was placed in a 500 ml Erlenmeyer flask and sterilized by autoclaving at 120 ° C. for 15 minutes. The Moniliella tomentosa IFO-9487 strain prepared in Example 1 (a) was inoculated to one platinum loop, and 30
Culturing was carried out at 220 ° C. for 4 days. After completion of the culture, the sugar composition of the culture solution was examined by high performance liquid chromatography (HPLC). The results are shown below.

Sugar composition of culture solution (content% based on solid content) 5 sugars or more branched sugar 1.6 branched tetrasaccharide 9.6 maltotriose 3.7 panose 32.6 maltose 36.5 isomaltose 1. 4 Glucose 4.4 Other 10.2

Example 3 (a) Preparation of seed culture Glucose 30%, yeast extract 1.0%, agar 1.5%
Moniliella tomentosa IFO-9 on a slant medium consisting of
The bacterial cells of 485 strains are applied, and static culture is performed at 30 ° C. for 2 days to obtain seed culture. (B) Main culture Maltose 30% (purity 99.0%), yeast extract 1.
50 ml of a liquid medium containing 5% is put into a 500 ml Erlenmeyer flask and sterilized by autoclaving at 120 ° C. for 15 minutes. Moniliella tomentosa prepared in (a) above
IFO-9485 strain was inoculated to one platinum loop, 30 ° C, 220 rp
Culture was carried out for 7 days. After completion of the culture, the sugar composition of the culture solution was examined by high performance liquid chromatography (HPLC). The results are shown below.

Sugar composition of culture solution (content% based on solid content) 5 sugars or more branched sugars 3.3 branched tetrasaccharides 2.4 maltotriose 4.7 panose 20.4 maltose 62.5 isomaltose 1. 0 Glucose 1.1 Other 4.6

Example 4 (a) Preparation of seed culture Moniliella tomentosa IFO-9488 was added to a slant medium consisting of glucose 30%, yeast extract (Difco, 1.0%) and agar 1.5%. Apply the cells of the strain,
Seed culture is obtained by stationary culture at 30 ° C. for 2 days. (B) Main culture 50 ml of a liquid medium containing 30% maltose (purity 99.0%) and 5% corn steep liquor is put in a 500 ml Erlenmeyer flask and sterilized by autoclave at 120 ° C. for 15 minutes. Moniliella tomentosa IFO-9488 strain prepared in (a) above was inoculated to one platinum loop at 30 ° C, 22
Culture was performed at 0 rpm for 4 days. After completion of the culture, the sugar composition of the culture solution was examined by high performance liquid chromatography (HPLC). The results are shown below.

Sugar composition of culture solution (content% based on solid content) 5 sugars or more branched sugar 0.8 branched tetrasaccharide 9.0 maltotriose 3.3 panose 31.3 isomaltotriose 0.7 maltose 34.9 Isomaltose 2.4 Glucose 5.3 Others 12.3

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, relatively inexpensive maltose is used as a carbon source of a medium, a high concentration medium is inoculated with yeast having a panose-producing ability belonging to the genus Moniliella, and a high panose content is obtained by fermentation. It is possible to efficiently and inexpensively produce oligosaccharides. In addition, Moniliella tomentosa strain IFO-9485, Moniliella tomentosa
IFO-9487 Strain or Moniliella tomentosa IFO-
When the 9488 strain is used, the glucose itself, which is produced from maltose and is not involved in the fermentation reaction, is used by the bacterial cells themselves, which may cause browning of foods and acid production of dental caries without special purification steps. It is possible to produce an oligosaccharide having a high glucose content and a high panose content. Therefore, the present invention is a very useful method for industrially producing oligosaccharides having a high panose content.

Claims (2)

[Claims] 1. A medium containing maltose as a carbon source as a main component is inoculated with a yeast capable of producing panose belonging to the genus Moniliella and aerobically cultured to produce and accumulate panose in the medium, which is then collected. A method for producing an oligosaccharide having a high panose content by fermentation, which comprises: 2. The method for producing an oligosaccharide having a high panose content according to claim 1, wherein the yeast belonging to the genus Moniliella is Moniliella tomentosa.