JP2822240B2 - Novel saccharide and method for producing the same - Google Patents

Description

The present invention relates to a novel saccharide and a method for producing the same, and more specifically, to a highly functional oligosaccharide-containing isomerized liquid saccharide, The present invention relates to a highly functional high-purity oligosaccharide for separating fructose and glucose other than the oligosaccharide, if necessary, and a method for producing the same.

The oligosaccharide-containing isomerized liquid sugar and high-purity oligosaccharide can be used as a sweetener in foods and drinks, or as an active agent, as a raw material for cultivation of pharmaceuticals, bifidobacteria growth factor, low cariogenic sweetener, low calorie sweetener. It is a highly functional saccharide that is effective when used for such purposes.

<Conventional technology> Isomerized liquid sugar is obtained by isomerizing glucose, which is an enzymatic saccharified product of starch, into fructose by an immobilized enzyme. Usually, as an anhydride, 42% fructose, 52% glucose, and oligosaccharide Contains 6% and has about 95% sweetness of sugar. Further, fructose is separated from this isomerized liquid sugar by an ion exchange chromatography method to produce fructose liquid sugar (in general, 90% fructose, 7% glucose, and 3% oligosaccharide as an anhydride). And a ratio of about 7: 3 to give high fructose isomerized liquid sugar with the same sweetness as sugar and mild sweetness. This is an anhydride, generally 55% fructose, 40% glucose,
Although oligosaccharide is 5%, it is the mainstay of isomerized liquid sugar because its sweetness is equivalent to sugar.

On the other hand, research on saccharides from the aspect of functionality has been actively conducted. The function of this saccharide is generally called “nutrition function”
It is divided into three parts: "sensory function" and "biological control function". Nutritional function is fundamental in that sugars are one of the three major nutrients along with proteins and lipids, and fructose and glucose in isomerized liquid sugar are important sources of calories. Sensory functions indicate the properties of sweeteners such as taste, color, odor, and texture.The reason that isomerized liquid sugar is widely used in large quantities in beverages, frozen desserts, breads, canned foods, etc. First, it depends on the sensory functions, and on the properties of fructose and glucose in the isomerized liquid sugar. The bioregulatory function has been particularly attracting attention in recent years, and among sugars, oligosaccharides in which two to ten monosaccharides such as fructose and glucose are linked play an important role. That is, certain types of oligosaccharides have a function useful for maintaining human health, and in combination with the sweetness of consumers, a bioregulatory function has been particularly required as a sweetener. For example, sugars that are hard to become caries (low caries), sugars that are sweet but prevent obesity (insulin-independent), and sugars that have the effect of activating intestinal useful bacteria from the viewpoint of promoting health. It has characteristics.

However, some of the oligosaccharides contained in the isomerized liquid sugar are expected to have bioregulatory functions such as isomaltoligosaccharides, but also include maltooligosaccharides such as maltose, and the absolute content of oligosaccharides was originally low. Since there is little, there is no effect even if a bioregulatory function is sought, and isomerization liquid sugar can be obtained simply by increasing the conventional oligosaccharide component while demand for high-purity isomerization liquid sugar with a small amount of oligosaccharide is increasing. Was merely lowered. That is, the conventional isomerized liquid sugar had a nutritional function and a sensory function, but none had a biological regulation function.

<Problems to be Solved by the Invention> The present invention has been developed in view of the above circumstances, and an object of the present invention is to provide a novel saccharide having a bioregulatory function in addition to a nutritional function and a sensory function and a novel saccharide. Efficient sugars,
An object of the present invention is to provide a manufacturing method which can be economically produced.

[Configuration of the invention]

<Means for Solving the Problems> The novel saccharide of the present invention is obtained by allowing a complex enzyme preparation derived from Alpergillus niger to act on isomerized liquid sugar to obtain a homo-oligosaccharide comprising glucose-glucose and fructose-
Homo-oligosaccharides composed of fructose and hetero-oligosaccharides composed of glucose-fructose or isomerized saccharides containing at least 25% of oligosaccharides containing these as main components as anhydrides, and if necessary, from the isomerized liquid saccharides, fructose other than oligosaccharides or It is obtained by separating and removing glucose.

Further, the production method of the present invention comprises reacting a complex enzyme preparation derived from Aspergillus niger with the isomerized liquid sugar to produce a homo-oligosaccharide comprising glucose-glucose and fructose-
A homooligosaccharide composed of fructose and a heterooligosaccharide composed of glucose-fructose or an isomerized liquid sugar containing at least 25% as an anhydride of an oligosaccharide containing these as a main component, and if necessary, the isomerized liquid sugar is converted to fructose other than oligosaccharides. Alternatively, it separates and removes glucose.

 Hereinafter, the present invention will be described in detail.

The present inventors have studied the properties of various oligosaccharides and found that oligosaccharides obtained by reacting isomerized liquid sugar with a complex enzyme preparation derived from Aspergillus niger include novel transferases and hydrolases. The present inventors have found that the oligosaccharide obtained by the method has excellent properties not found in the present invention, and have reached the present invention. That is, the present invention is to produce a highly functional oligosaccharide-containing isomerized liquid sugar by synthesizing an oligosaccharide having higher functionality than fructose or glucose which is a component of the isomerized liquid sugar. As a result, the nutritional function and sensory function of the original isomerized liquid sugar can be further provided with a biological control function, and the added value of the isomerized liquid sugar can be significantly increased.

This highly functional oligosaccharide-containing isomerized liquid saccharide enables production of a highly functional, highly pure oligosaccharide by separating fructose or glucose other than the oligosaccharide, if necessary. That is, the present invention focuses on the oligosaccharides in the isomerized liquid sugar, and rather increases the amount of the oligosaccharides by the components of the isomerized liquid sugar itself, thereby generating new functionality in the isomerized liquid sugar. The present invention is the first of the present invention, which has no past example of such a technical idea that is contrary to the tendency of isomerized liquid sugar to have a high purity.

The present invention has succeeded in solving the problems of the prior art by actively applying oligosaccharides in isomerized liquid sugar, and the present invention will be described in more detail below.

(Regarding oligosaccharides present in isomerized liquid sugar) Isomerized liquid sugar is obtained by isomerizing glucose, which is an enzyme saccharified product of starch, into fructose by an immobilized enzyme. , Glucose 52% and oligosaccharide 6%. Further, fructose was separated from this isomerized liquid sugar by ion exchange chromatography to produce fructose liquid sugar.
To produce high fructose isomerized liquid sugar.

Oligosaccharides remaining in the isomerized liquid sugar are by-produced because starch is not completely saccharified under industrial saccharification conditions. The reasons are as follows. Glucose produced by the reverse reaction of saccharifying enzymes becomes isomaltose or maltose, and if enzymes that generate oligosaccharides from maltose by the transfer reaction are mixed in the saccharifying enzyme agent, isomaltose or panose, etc.
Maltose and glucose at the reducing end of oligosaccharides are converted to foreign substances, remain as multilose even after the action of saccharifying enzyme, and there are binding molecules in starch molecules that are difficult to decompose by saccharifying enzyme. It has been desired that the number of oligosaccharides in this isomerized liquid sugar be smaller than before, but in particular, in recent years, fructose separation has been greatly affected. That is, when the fructose separation is performed, the oligosaccharide is mainly contained in the glucose section, and this glucose section increases in oligosaccharide content every time the isomerization step and the fructose separation step are performed again. That is, glucose can be converted to fructose, but oligosaccharides remain and accumulate and their amount doubles.To improve the efficiency of the process for producing isomerized liquid sugar, starch that does not produce oligosaccharides as a by-product is used. There has been a strong demand for complete saccharification of glycerol and production of isomerized liquid sugar containing no oligosaccharide. The present invention focuses on oligosaccharides of isomerized liquid sugar and actively applies the oligosaccharides themselves.Since oligosaccharides accumulated by fructose separation can be used at the same time, it is possible to solve this problem effectively. Also.

(Method for Producing Oligosaccharides) Oligosaccharides may be chemically synthesized in addition to naturally occurring ones. There are other types of oligosaccharides depending on the combination of the types of constituent monosaccharides and the combination of bonding modes. It is broadly classified into homooligosaccharides composed of sugar residues and heterooligosaccharides composed of heterologous sugar residues. These are described in Taku Mizuno and Shun Nishizawa, "Handbook of Illustrated Carbohydrate Chemistry" (Kyoritsu Shuppan, 197
1, pages 59-104).

On the other hand, various types of functional oligosaccharides are currently on the market or under development, and these are summarized as follows according to the production method (origin of synthesis). By the reverse condensation reaction of monosaccharides, by thermal condensation of monosaccharides, by chemical change of sugar residues of existing oligosaccharides, by transfer reaction between oligosaccharides or between oligosaccharides and monosaccharides, by polysaccharides These include those derived from partial decomposition and those extracted from nature.

What is particularly important in the production of these oligosaccharides is the relationship between the target oligosaccharide and the raw materials used. The oligosaccharides obtained by the above method are isomaltooligosaccharides using glucose as a raw material (JP-A-61-124389), polydextrose using glucose and sorbitol as raw materials (US Pat. No. 3,766,165), and lactose Lactulose as a raw material (for example, Japanese Patent Publication No. 55-20679). Fructooligosaccharides made from sucrose (JP-B-59-53834), palatinose (JP-B-58-36959), coupling sugar made from starch and sucrose (JP-B-53-17660), lactose From a galactooligosaccharide (for example,
58-20266). Numerous oligosaccharides have been obtained by partial decomposition using starch as a raw material, and examples include soybean oligosaccharides (stachyose and raffinose) using soybean (JP-A-62-155082).

The present invention belongs to the production of isomerized liquid sugar as a raw material, and a homo- or hetero-oligosaccharide is produced by acting a complex enzyme preparation derived from Aspergillus niger. The present invention is the first as a technical idea to enhance the performance. As the reaction conditions in the above case, the composition ratio of isomerized liquid sugar, the substrate concentration, the enzyme concentration, the action temperature, the action pH, the action time, and the like are important. Isomerized liquid sugar composition ratio (anhydrous) fructose 5% or more, glucose 95% or less, oligosaccharide
It may be 15% or less, and isomerized liquid of glucose fraction (raffinate) obtained from fructose separation process, including 42% fructose isomerized liquid sugar and 55% fructose isomerized liquid sugar generally used in the market Sugar or the like is used as a raw material. The reaction is performed at a solid concentration of 45% or more from the viewpoint of yield, work efficiency, and prevention of contamination by microorganisms.

(Complex enzyme preparation derived from Aspergillus niger) There are many types of Aspergillus genus, each having a unique characteristic. Molds that represent the genus Aspergillus include Aspergillus oryzae, Alpergillus soya, Aspergillus tamari, Aspergillus niger, Aspergillus awamori, Aspergillus rubber, Aspergillus repens, Aspergillus chevalierzo, Aspergillus sp. is there. Since these Aspergillus species produce large amounts of various enzymes in large quantities and secrete them out of the cells, many enzyme preparations have been produced. Examples include amylase preparations, protease preparations, pectinase preparations, naringinase preparations, hesperidinase preparations, cellulase preparations, hemicellulase preparations, lipase preparations, tannase preparations, and anthocyanase preparations.

The present inventors have studied the effects of Aspergillus molds and the effects of enzyme preparations produced therefrom as enzyme preparations acting on isomerized liquid sugar. The effect of any of the manufactured enzyme preparations was observed, and among them, an enzyme preparation derived from Aspergillus niger, wherein the main component of the enzyme preparation was a transglucosidase preparation,
It has been found that pectinase preparations, cellulase preparations, and protease preparations are particularly effective, leading to the present invention.

These enzyme preparations have two characteristics due to the characteristics of Aspergillus.
A complex enzyme consisting of more than one kind of enzyme. For example, as the enzyme contained in the pectinase preparation, mainly pectinase, β-1,3-glucanase, protease, amylase, lipase, polygalacturonase, macerating enzyme, β-galactosidase, β-fructofuranosidase and the like is there.

The enzymes contained in the cellulase preparation are mainly cellulase, hemicellulase, lipase, ribonuclease, protease, β-1,3-glucanase, polygalacturonase, macerating enzyme and the like.

Enzymes contained in protease preparations are mainly proteases such as amylase, lipase and ribonuclease. The enzyme contained in the transglucosidase preparation is mainly transglucosidase and contains other amylase.

Homo- or hetero-oligosaccharides can be efficiently obtained by reacting these Aspergillus niger-derived complex enzyme preparations with isomerized liquid sugar.However, in the case of a batch reaction, the enzyme concentration is usually 0.1 to 0.1 per substrate solid content. 5%
Degree, action temperature is 40-65 ° C, action pH is 4.0-7.0,
The action time is about 20 to 100 hours.

(Preparation of immobilized enzyme) There are many methods for immobilizing the enzyme, but there is no particular limitation on the carrier of the column and the immobilization method for preparing the immobilized enzyme used in the present invention.

As the simple substance bonding method, there are a physical adsorption method using porous activated carbon or an ion exchange resin, an ion bonding method using DEAE-Sephadex, or a covalent bonding method using porous glass. Examples of the crosslinking method using a crosslinking agent such as glutaraldehyde and the entrapping method include a lattice type using carrageenan or the like and a microcapsule type using nylon or the like.

Either method may be used, but in order to perform oxygen immobilization on an industrial scale, the enzyme activity yield (activity expression rate) during immobilization must be excellent, and when used (during continuous enzyme reaction). It is important that the product has good stability, is economical, is easy to fix, and is safe in the food industry. A physical adsorption method using an adsorbent such as porous activated carbon or ion exchange resin is recommended. This physical adsorption method has the characteristics that the immobilization operation is easy, the safety is high, and the adsorption is performed reversibly.However, if a specific macroreticular type activated carbon or ion exchange resin is used, several tens of Since it has pores of Å to several thousand square meters and has a specific surface area of tens to tens of m ² / g, it is possible to use it for a long time without any elimination of enzymes.

As an immobilization operation, a pH buffer is added to ion-exchanged water to a predetermined pH, and then an enzyme agent and an adsorbent are added, and the mixture is immobilized while slowly stirring at a predetermined temperature. The ratio between the adsorbent and the enzyme agent used is preferably about 1: 0.1 to 10 in terms of volume ratio, and the ratio of the adsorbent and ion exchange water is preferably about 1: 5 to 20 in terms of volume ratio. Working temperature is 10-40 ° C, pH is 4-6, stirring is 30
~ 300r.pm is good.

This operation is completed in about 1 to 20 hours. The immobilization rate under the conditions described above is usually about 20 to 80.

The immobilized enzyme to which the immobilized enzyme is attached is washed with ion-exchanged water and used in a column. The basic reaction conditions for this immobilized enzyme column are that the composition ratio of the isomerized liquid sugar, the substrate concentration, the working temperature, the working pH, etc. are the same as in the batch reaction, but the flow rate is about 0.01 to 10 SV. Therefore, the reaction time is greatly reduced, and the control of the reaction is easily controlled.

(Production of Highly Functional, High-Purity Oligosaccharides by Chromatographic Separation) As a means for separating high-performance oligosaccharides in a large amount and at a low cost from highly functional oligosaccharide-containing isomerized liquid saccharides, chromatographic separation method is excellent As the filler, a strong acidic cation exchange resin is suitable. The strongly acidic cation exchange resin used in the present invention is a sulfonic acid type, for example, Diaion SK1B, Diaion SK106, Diaion SK110, Diaion SK112, Diaion SK116,
Diaion FR01 (trade name, manufactured by Mitsubishi Kasei) or Amberlite IR-116, Amberlite IR-118, Amberlite IR-120B, Amberlite XT1022E, Amberlite XT471E, Amberlite G102 (trade name,
Organo) or Dowex HCR-S, Dowex HCR-W2, Dowex HGR-W2, Dowex
50W, Dowex MSC-1, Dowex 88 (trade name, manufactured by Dow Chemical Company) or Levatit S-100, Lewatit S-109, Lewatit SP-112, Lewatit SP-1
2, (hereinafter trade name, manufactured by Bayer AG) and the like are suitable and used as an alkali metal type or an alkaline earth metal type before use. The separation device is composed of a stock solution tank, a developing solvent tank, a liquid sending pump, a separation tower, a detector, a separation and sorting mechanism, and a control mechanism, for efficiently separating fructose, glucose and oligosaccharides. As the operation, the concentration of the raw sugar was 30 to 80% by weight, the temperature was 30 to 80 ° C, and the flow rate was SV 0.01 to 1.
The condition of 0 is preferable. The separated high-functional high-purity oligosaccharides are separated by filtration, ion-exchange purification, activated carbon purification and concentration to obtain high-functional high-purity oligosaccharides. If necessary, powders can be obtained by spray drying.

<Examples> Examples of the present invention will be described below, but the present invention is not limited to these examples, and various modifications can be made within the scope of the gist.

Example 1 A commercially available 42% isomerized liquid sugar (manufactured by Gun-ei Chemical Industry: 75% by weight of solid content) was added to a transglucosidase preparation derived from Aspergillus niger (Amano Pharmaceutical: transglucosidase "Amano", 300,000 u / g) Was added at a concentration of 3.0% per solid substrate and reacted at a temperature of 55 ° C. and a pH of 5.0 for 48 hours. After completion of the reaction, the mixture was heated at 85 ° C. for 5 minutes, filtered, ion exchange purified, treated with activated carbon, and concentrated to prepare a highly functional oligosaccharide-containing isomerized liquid sugar. HPLC analyzers for sugar components
[High Performance Liquid Chromatography: Waters Model 590,
Column Oligo-pw (Tosoh), Shimadzu LC4A type, Column Ami
de-80 (Tosoh)] and NMR (nuclear magnetic resonance apparatus). The homo-oligosaccharide composed of glucose-glucose obtained by the above-mentioned production method was a maltooligosaccharide such as maltose and maltotriose and an isomaltooligosaccharide such as isomaltose, nigerose, kojibiose, panose and isomalttriose. Homo-oligosaccharides composed of fructose-fructose are inuro-oligosaccharides and levan-oligosaccharides, but the amount of the production was trace. The hetero-oligosaccharide composed of glucose-fructose was mainly composed of α-glucosyl fructose such as maltulose, palatinose, turanose, α-glucosyl-1,1-fructose. Table 1 shows the above results.

Example 2 Reaction, purification, and concentration were performed under the same conditions as in Example 1 except that the 2% isomerized saccharide was changed to a commercially available 55% isomerized liquid sugar (manufactured by Gunei Chemical Industry, solid content: 75% by weight). The analysis was performed. Table 2 shows the above results.

Example 3 The glucose fraction (raffinate) isomerized liquid sugar obtained from the fructose separation step was adjusted to a solid content of 75% by weight, and a protease preparation derived from Aspergillus niger (Nagase Seikagaku Kogyo: Bioprase APS, 7000APUN / g) was added at 3.0% per solid substrate, and the reaction, purification, concentration and analysis were performed under the same conditions as in Example 1. Table 3 shows the above results.

Example 4 100 m of macroporous granular activated carbon (manufactured by Hokuetsu Carbon Industries, GS-A) and a pectinase preparation derived from Aspergillus niger (manufactured by Novo Industry: Pectinex Ultra SP-L, 2600 PG / g) in 1000 m of 0.1 M acetate buffer solution 100g
Was added thereto, and adsorption and immobilization were performed at pH 5.0, at a temperature of 30 ° C., and with stirring at 40 rpm for 5 hours. The immobilization rate was determined by the Kjeldahl method from the difference between the protein content of the original enzyme solution and the non-adsorbed protein content of the supernatant after the immobilization, and was 43%. The granular activated carbon to which the enzyme was adsorbed was packed in a glass column with an inner diameter of 20 mm and a length of 600 mm with a warm water jacket, and washed with 4000 m of ion-exchanged water to prepare an immobilized enzyme. The column-type reaction was carried out using the same 42% isomerized liquid sugar as in Example 1, with a substrate concentration of 75% by weight,
The system was operated continuously for 120 hours at a pH of 5.0, a temperature of 55 ° C., and a flow rate of SV 0.1. Purification, concentration, and analysis of the reaction solution were performed in the same manner as in Example 1. Table 4 shows the above results.

Example 5 The highly functional oligosaccharide-containing isomerized liquid saccharide obtained by the operation of Example 4 was diluted to a predetermined concentration, passed through a strongly acidic cation exchange resin, and separated into A and B categories. The A category is a homooligosaccharide composed of glucose-glucose, a homooligosaccharide composed of fructose-fructose and a heterooligosaccharide composed of glucose-fructose, or a fraction containing 50% by weight or more per solid of an oligosaccharide composed mainly of these. Category B contains fructose and glucose as main components, and is a category to be removed from products. Category A was a target product, and purification, concentration, and analysis were performed in the same manner as in Example 1. Table 5 shows the results.

[Effects of the Invention] According to the present invention described in detail above, high-functional oligosaccharide-containing isomerized liquid sugar is produced from isomerized liquid sugar, and if necessary, fructose other than oligosaccharides and glucose are separated to provide high-performance Industrial production method for efficiently and economically producing large quantities of highly pure oligosaccharides of high quality.

The highly functional oligosaccharide-containing isomerized liquid saccharide and the high-purity oligosaccharide obtained by the production method of the present invention can be used as a sweetener in foods and drinks, as an active agent, as a raw material for cultivation of pharmaceuticals, etc., as a bifidobacterial growth factor. It is a novel saccharide that is effective in various fields such as low-cariogenic sweeteners and low-calorie sweeteners.

[Brief description of the drawings]

The drawing is an example of a liquid chromatogram of a reaction solution obtained by allowing a complex enzyme preparation derived from Aspergillus niger to act on the isomerized liquid sugar of the present invention. In the figure, a is a hetero-oligosaccharide composed of fructose-glucose obtained from isomerized liquid sugar, and b is a homo-oligosaccharide composed of glucose-glucose.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Toshio Hirohashi 622 Oyagi-cho, Takasaki City, Gunma Prefecture Inside Gunei Kagaku Kogyo Co., Ltd. (72) Inventor Takanao Kimura 622, Oyagi Town, Takasaki City, Gunma Prefecture Gunei Kagaku Kogyo Incorporated (58) Fields surveyed (Int.Cl. ⁶ , DB name) C12P 19/18 C12P 19/14 A23L 1/236

Claims (8)

(57) [Claims] 1. A complex enzyme preparation derived from Aspergillus niger is reacted with isomerized liquid sugar to obtain a homo-oligosaccharide composed of glucose-glucose, a homo-oligosaccharide composed of fructose-fructose and a hetero-oligosaccharide composed of glucose-fructose, or a mixture thereof. A novel saccharide obtained by obtaining an isomerized liquid saccharide containing 25% or more of an oligosaccharide as a component as an anhydride, and separating and removing fructose or glucose other than the oligosaccharide from the isomerized liquid saccharide as necessary. 2. A complex enzyme preparation derived from Aspergillus niger is allowed to act on isomerized liquid sugar to obtain a homooligosaccharide composed of glucose and fructose, a homooligosaccharide composed of fructose and fructose, and a heterooligosaccharide composed of glucose and fructose or a main component thereof. Isomerized liquid sugar containing 25% or more of an oligosaccharide as an anhydride, and fructose or glucose other than oligosaccharides is separated and removed from the isomerized liquid sugar as necessary. . 3. The method according to claim 1, wherein the main component of the complex enzyme preparation is any one of a transglucosidase preparation, a pectinase preparation, a cellulase preparation and a protease preparation, which is used as a soluble enzyme or an insolubilized immobilized enzyme. New sugars. 4. An isomerized liquid sugar having a sugar composition ratio of 5% or more of fructose, 95% or less of glucose, and 15% or less of oligosaccharide as an anhydride, and is used at a solid concentration of 45% or more. The novel saccharide according to claim 1. 5. The novel saccharide according to claim 1, wherein a strongly acidic cation exchange resin is used as the separating agent. 6. The method according to claim 2, wherein the main component of the complex enzyme preparation is any of a transglucosidase preparation, a pectinase preparation, a cellulase preparation, and a protease preparation, which are used as a soluble enzyme or an insolubilized immobilized enzyme. A novel saccharide production method. 7. An isomerized liquid sugar having a sugar composition ratio of 5% or more of fructose, 95% or less of glucose, and 15% or less of oligosaccharide as an anhydride, and is used at a solid concentration of 45% or more. A method for producing the novel saccharide according to claim 2. 8. The method for producing a novel saccharide according to claim 2, wherein a strongly acidic cation exchange resin is used as the separating agent.