JP2899619B2 - Novel food material containing isomaltosyl fructoside and / or isomeletitose - Google Patents

Description

The present invention relates to a novel food material containing isomaltosyl fructoside and / or isomeretose produced by a sucrosetransferase produced by a thermophilic bacterium, and particularly to a non-food material containing the same. The present invention relates to a novel food material having low, low, cariogenic and bifidobacterial growth promoting effects.

[Problems to be Solved by the Prior Art and the Invention] Conventionally, there is cyclodextrin glucanotransferase as a sucrosetransferase, and the present enzyme comprises glucose and / or glucose from α-1,4 glucan such as starch in the glucose part of sucrose. Or maltooligosaccharides such as maltose
It is used for the production of coupling sugar food materials because it forms sucrose such as erulose by binding at -1,4.

Β-fructofuranosidase and fructosyltransferase transfer the fructose moiety of another sucrose to sucrose, and produce various fructosyl sucrose by transferring to the F1, F6 and G6 positions of the sucrose molecule. can do. In particular, kestose, nystose, etc. in which fructose has been transferred to the F1 position are produced as fructooligosaccharides,
It is commercially available and is said to be widely used.

In addition, there are dextran sucrose and levansucrase, which are enzymes that form dextran by binding the glucose part of sucrose and form a polysaccharide such as levan by binding the fructose part.

Recently, it has been known that α-1,6 type oligosaccharides such as isomaltose are low-calorie food materials and can be used for anti-obesity, anti-, non- or low-cariogenic, bifidobacterial growth,
There has been an increasing demand for food materials having a function of preventing cholesterol accumulation, and sucrose having an isomaltose moiety has been expected to be used as a food material.

However, the glucose portion of sucrose is converted into many
No heat-resistant sucrose transferase capable of binding two to release fructose is known, and much less is expected of a technique for producing large amounts of isomaltosylfructoside and isomeretitol from sucrose.

Therefore, glucose is α in the glucose portion of sucrose.
It is well known that isomaltosyl fructoside (abbreviated as G16GF) with 1,6-linked isomeretose (abbreviated as GF61G) with α-1,6-linked glucose at the fructose portion of sucrose is known. However, it is generally impossible to work due to lack of established production methods.It is said that it has a slight caries-preventing effect on its properties, and its effect and other functions are almost unknown. None, and of course, these have not been made into food ingredients so far.

It is known that fructose does not serve as a substrate for insoluble glucan synthesis by glucosyltransferase possessed by cariogenic bacteria and further inhibits this insoluble glucan synthesis. In addition, since it has high sweetness and excellent taste, it can release the fructose moiety from sucrose and transfer the glucose moiety that affects caries development to the glucose and fructose moieties of sucrose. Is obtained, a very excellent sugar product can be obtained.

[Means for Solving the Problems] Accordingly, the present inventors search for bacteria that have a growth temperature of 65 ° C. or more and produce sucrose transferase together with bacterial cells from the soil of hot springs, and produce enzymes compatible with these. More than a dozen strains were found. Among them, three strains having a strong enzyme activity were selected, their microbiological properties were examined, and the enthusiastic studies were carried out to use them for producing sucrose-transferred sugar of the present invention.

Bacteria used in the present invention belong to the genus Bacillus because they form spores with bacilli and do not grow under anaerobic conditions and are aerobic.

The other properties are as shown in Table 1.
Bergey's Manual of Deter
minative Bacteriology, 8th edition), Cowan and Ste
Classification of el (Manual for the Identification of Medical Bacteria, 2nd editio
n, Cambridge Univ. Press, 1974) and The Genus Bacil
lus , Agriculture Handbook 427, USDept.of Agricultu
According to re, the growth temperature is high in the above properties,
Because the VP test is negative, they are closely related to Stearothermophilus. It is considered a different fungus. In addition, GC of DNA
The% values are 61, 52 and 58% respectively.

In addition, these bacteria have added the glucose portion of sucrose to α-1,6
It is clear that it is a novel bacterium because it produces an enzyme that transfers.

From these results, the present inventors recognized the three strains as novel strains and named them AIK90-30, 31, 50, respectively. These strains have been deposited with the National Institute of Microbial Industry and Technology, and their accession numbers are FERM BP-2927, 2928, and 2928, respectively.
2929.

The strain grows well when cultivated at an initial pH of 7.0 in a medium containing 2 to 10% of sucrose and 0.1 to 0.5% of peptone and / or yeast extract in tap water, and sucrose transferase when cultured at 65 ° C for 72 hours. Is produced in a state contained in the cells. The enzyme is produced even when the culture temperature is set to 65 ° C. or lower, for example, AIK9
In 0-31, the activity of 0.2 IU at 65 ° C and 0.1 IU / ml at 50 ° C is produced in the cells.

 This tendency is also observed in the other two strains.

As the medium, an optimal medium may be used in combination with various inorganic and / or organic carbon sources and nitrogen sources, salts, vitamins, amino acids, etc., but economically, a medium containing sucrose is used, and 60 to 70 ° C. Incubate with When culturing, it is not necessary to use a sealed jar fermenter, and if the air is blown into a warmed vessel or the culture is allowed to stand at a culture solution depth of 1 cm or less, the bacteria will proliferate, and AIK90-30 bacteria will grow. Bacterial collection is easy because of easy precipitation. Therefore, it is also possible to continuously take out the grown bacteria while adding the medium,
The bacteria used in the present invention are also advantageous for continuous culture.

Furthermore, the strain used in the present invention can be cultured using a gas permeable membrane such as silicon (Japanese Patent Application No. 2-47199). In this case, the fructose part of sucrose is replaced with the fructose part of another sucrose in a medium. -1,2 transfer, or α-1,4 transfer of the glucose part of other sucrose or maltose to the glucose part of sucrose, and the conventional commercial fructooligosaccharides such as kestose and erulose, the main component of coupling sugar. It can also be generated mainly.

Since the cells cultured in this way contain heat-resistant intracellular enzymes, they are advantageous for producing these sugars. In addition, if equal amounts of sucrose and maltose are mixed, erucose becomes the main component, so that a mixture of fructooligosaccharide and coupling sugar and a product of the coupling sugar main component can be obtained as necessary.

In addition to the strain of the present invention, the present inventors have also found the same kind of bacteria that mainly produce invertase that hydrolyzes sucrose. Since the enzyme has a high temperature stability of 65 ° C. or higher, it is suitable for producing isomerized sugar from sucrose, and can produce isomerized sugar containing sucrose at an arbitrary ratio.

These enzymes can be separated and used by, for example, crushing these cells, and further immobilized to use as immobilized enzymes for production of sucrose transfer sugar and sucrose hydrolysate.

The cultured cells are washed with water, added to a sucrose solution, and shake-reacted, whereby the sucrose undergoes a transfer reaction gradually. The above-mentioned enzyme-producing bacterial cells are subjected to normal reaction conditions, for example, a 40% sucrose solution, pH
7.0, an enzyme concentration of 10 IU / g substrate, a reaction rate of 30% when acted at 65 ° C. for 24 hours, G16GF and GF61G as main components and small amounts of isomaltose as transfer sugars. The reaction conditions are not limited to the above conditions, but the concentration of the sucrose solution is preferably in the range of 5 to 60%.

Also, when cultured in a silicon tube in a medium containing about 10% sucrose, a transfer sugar different from the above at 65 ° C and 48 hours has a transfer rate of 30%.
Kestose (GF12F), Elrose (G14GF)
Is a main component, and a sugar mixture containing a small amount of nistose (GF12F12F) is obtained.

In this reaction, there are no particular restrictions on conditions such as the concentration of the sucrose solution, but conditions should be set according to the purpose.

After the reaction, a continuous reaction can be carried out by removing the sugar mixture and adding sucrose again, but when the enzyme is removed from the cells, the bond can be reinforced with glutaraldehyde. Other immobilized enzyme preparation methods can be used to reinforce the enzyme bond.For example, sodium alginate, polyacrylamide, nylon, cellulose nitrate, polyethyleneimine, and two or more of these may be mixed or used alone. However, it can be used.

Thus, in the present invention, sucrose-transferred sugar can be produced in large quantities,
Furthermore, they have found that G16GF and GF61G have useful functions among these carbohydrates, and thus completed the present invention.

In the present invention, G16GF and / or GF61G are:
Both components are pure products or those having an arbitrary mixing ratio.

 The present invention is described below.

The present invention relates to a novel food material containing G16GF and / or GF61G. Specifically, (1) A novel food material containing G16GF and / or GF61G.

(2) A food material containing one or more selected from other sugars or their reduced products, or other natural sweeteners, together with G16GF and / or GF61G.

(3) A food material containing bifidobacteria together with G16GF and / or GF61G.

(4) A food material containing G16GF and / or GF61G and alcohol.

The present inventors have found that α-1,6 glucosyl sucrose is selectively assimilated by intestinal bifidobacteria and promotes its growth, and that insoluble glucan is caused by bacteria that cause tooth decay such as Streptococcus mutans. No formation and / or no acid was found, and it was found that the formation of insoluble glucan by sucrose could be effectively suppressed. Also, from the characteristics of the functional food materials so far,
It can easily be expected that it has useful properties such as being hardly digested and absorbed by the human body and having low calories.

α-1,6 glucosyl sucrose (sugar in which glucose is α-1,6 linked to a sucrose molecule) is also relatively stable to high temperatures, acids, etc., and G16GF has no α-glucosidase action. , Because GF61G is relatively resistant to enzymatic action such as not receiving invertase action,
It can be used for a wide range of applications such as food and drink, feed, pharmaceuticals, and cosmetics. G16GF or GF61G can be used as is for these uses, but since their sweetness is lower than that of sucrose, if sweetness is required, other sugars or their reduced products, a kind of other sweeteners Or you may use together with 2 or more types. Examples of saccharides include sucrose, glucose, maltose, fructose, lactose, isomaltose, isomalttriose, panose, isomaltetrate, nigerose, kojibiose, cellobiose, gentiobiose, and the like.Examples of reduced saccharides Examples of other sweeteners such as sorbitol, isomaltitol, isomalttriitol, and panitol include honey, starch syrup, stevia, glycyrrhizin, saccharin, aspartame, and the like.
These materials include not only sweetness but also those which can be used as taste improving and physical property improving materials.

When sclerooligosaccharides are used in combination with other food materials, for example, sclerosaccharides (including sucrose, sucrose, sucrose, and sucrose) can be scro-honeyed, scrolemon, sucrose orange, sucrose grape, It can also be referred to as scro honey.

In the present invention, any one of these saccharides, saccharide reduced products, sweeteners and the like, or two or more thereof can be selected and used together with G16GF and / or GF61G.

G16GF and / or GF61G in the food material of the present invention
In order to exhibit the effects of promoting the growth of bifidobacteria and preventing caries, it must contain at least 5% by weight or more, preferably 10% by weight or more, and practically 25 to 50% Desirably the content. This is the same even when a component other than G16GF and / or GF61G in the food material is a saccharide such as sucrose that easily causes caries. Further, in a food material in which G16GF and / or GF61G is contained in a saccharide having an anti-cariogenic property and bifidobacterial activity, such as isomaltose, panose, and isomaltetrate, G16GF and / or GF61G are not necessarily 5%. It does not need to be more than
5% by weight or more of G16GF and / or GF61G, preferably
When it is 10% by weight, the effect increases synergistically.

In addition to the above, a filler such as dextrin, starch, lactose, a coloring agent, a spice, or the like can be added to the food material of the present invention, if necessary. To further increase the growth of bifidobacteria, dry cells of bacteria belonging to Bifidobacterium and vitamins such as pantethine, pantothenic acid, and riboflavin, which are said to be necessary for the growth of bifidobacteria, are added. You may.

Food materials of the present invention, depending on the intended use and purpose,
By appropriately combining known processing means such as mixing, dissolving, concentrating and drying, it can be obtained in the form of liquid, starch syrup, granule, powder and the like.

[Examples] Next, the selective promoting effects of G16GF and GF61G on the growth of bifidobacteria and the effect of suppressing the production of insoluble glucan will be described in detail based on the following experimental examples.

EXPERIMENTAL EXAMPLE 1 Utilization of Sugar by Bifidobacterium Fresh bacteria shown in Table 2 cultured on GAM agar medium were inoculated into an LB fermentation test medium supplemented with 0.5% of test saccharides, and incubated at 37 ° C under anaerobic conditions. After culturing for days, discoloration of the indicator accompanying assimilation fermentation was observed, and the results shown in Table 2 were obtained.

In this table, ++, +, ±, and-indicate the degree of discoloration, and indicate strong, weak, weak, and no change, respectively.

As is clear from this table, G16GF and GF61G were found to be selectively used by the Bifidobacterium group.

Experimental Example 2 Insoluble Glucan Synthesis Inhibitory Effect Glucosyltransferase (insoluble glucan synthase) prepared from Streptococcus sobrinus strain 6715 and a test sugar were added to an insoluble glucan synthesis measurement system, and the mixture was maintained at 37 ° C. for 6 hours. After the was produced, the produced insoluble glucan was suspended uniformly, and the turbidity at OD _{550 nm} was measured.

The amount of the enzyme was determined by keeping the control system using sucrose for 6 hours.
The OD ₅₅₀ was adjusted to 1.0. Further, the test sugar concentration was adjusted to the sugar concentration of palatinose showing a synthesis inhibition rate of 50%, and each was mixed and tested.

Synthesis inhibition rate, in the [control group OD ₅₅₀ - O in the test group
D ₅₅₀ ] × 100 / [OD _{550 in} control _plot ].
It is shown in the table.

Experimental Example 3 Production of Acid from Test Sugar 0.2 ml of viable cells of Streptococcus sobrinus 6715 strain or Streptococcus mutans cultured in BHI medium for 24 hours was treated with pH indicators such as colorophenol red, bromcresol purple and bromthymol blue. The solution was added to 3.8 ml of the added 1% test sugar solution (pH 7.0), incubated at 37 ° C. under anaerobic conditions, and the degree of discoloration of the pH indicator was observed. The results shown in Table 4 were obtained.

In this table, ++, ±, and-indicate the degree of discoloration, and are, respectively, strong, weak, and no change.

As is clear from this table, no acid formation was observed from G16GF, and slightly weak acid formation was observed from GF61G.

As is apparent from the above, both G16GF and GF61G produce insoluble glucan by bacteria causing caries,
Acid formation is significantly less. They are also useful as an anti-cariogenic food material because they suppress the production of insoluble glucan even when coexisting with other saccharides such as sucrose.

Example 1 Sucrose 41.3, G16GF9.5, GF61G obtained by allowing sucrose to react with the heat-resistant sucrose transferase produced by the method described above.
A sugar mixture having an ingredient composition of 8.7, 2.6 isomaltose, 23.4 fructose, and 14.5% glucose was commercialized as it was.

This product was an excellent sweetener because it contained fructose, and at the same time, was low in cariogenicity and had a significant bifidobacterial growth effect.

G16GF and GF61G can be separated from this product by column chromatography, yeast treatment, etc., depending on the application, and combined with other food materials.

Both G16GF and GF61G have a caries-preventing action and a bifidobacterial growth action, but the caries-preventing action is stronger with G16GF,
Bifidobacterium growth activity is stronger with GF61G.

Example 2 α-1.6 such as isomaltose and isomalt triose
G16GF (50 g) was mixed and dissolved in 100 g of a branched oligosaccharide syrup (water content: 25%) mainly composed of glucose disaccharide and trisaccharide having a bond to obtain a syrup-shaped sweetener. This product was suitable as a low cariogenic sweetener and had a bifidobacterial growth effect.

Example 3 Except having used GF61G, it carried out similarly to Example 2, and obtained the syrup-shaped sweetener. This product was a low cariogenic sweetener and at the same time was suitable as a sweetener for growing Bifidobacteria.

Example 4 90 parts by weight of the sweetener obtained in Example 1 and 10 parts by weight of dried bifidobacteria were mixed to prepare raw materials for various drinks containing Bifidobacterium, candy and the like.

Example 5 The sweetener obtained in Example 1 was allowed to act on yeast to convert sugars other than GF61G into alcohol, thereby obtaining a beverage mainly containing alcohol and GF61G.

[Effects of the Invention] G16GF and GF61G contained in the food material of the present invention hardly cause caries, and have an effect of suppressing caries even when combined with other saccharides such as sucrose which easily causes caries. It is useful as an anticariogenic and low cariogenic carbohydrate.

G16GF and GF61G are selectively assimilated by bifidobacteria and are hardly used by so-called harmful bacteria. This property is similarly exhibited when G16GF and GF61G are combined with other saccharides or their reduced products.

Therefore, the food material of the present invention containing G16GF and GF61G is useful as a food material having a bifidobacterium growth promotion effect.

In addition, G16GF and GF61G are expected to have an action of suppressing cholesterol accumulation, that is, an action of suppressing the accumulation of low-density cholesterol called so-called bad cholesterol in the serum and liver, and are less likely to be metabolized than sucrose and the like. Since it is a saccharide, it can also be used as a low-calorie food material.

The food material of the present invention can be used in a very wide range of fields, such as foods, pet foods, pharmaceuticals, cosmetics, toothpaste and other tretary products, and tobacco and other luxury goods, as well as food and drink.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12G 3/02 C12G 3/02 // C12P 19/00 C12P 19/00 (C12P 19/00 C12R 1:07) (72) Invention Person Shinichi Kunizaki 16-16-3 Futaba-cho, Ibaraki-shi, Osaka (72) Inventor Kyoichi Sugawa 1-1-1 Himuro-cho, Takatsuki-shi, Osaka (56) References Establishment of a method for assessing congenital properties Development of a method for preventing cancer using antineoplastic carbohydrate 1982 p. 24 (1983) (58) Field surveyed (Int. Cl. ⁶ , DB name) A23L 1/30

Claims (5)

(57) [Claims] 1. A novel food material containing isomaltosyl fructoside and / or isomeletitose. 2. The food material according to claim 1, which contains one or more selected from other sugars or reduced products thereof, or other sweeteners, together with isomaltosyl fructoside and / or isomeretitol. 3. The food material according to claim 1, which contains at least 5% by weight of isomaltosyl fructoside and / or isomeletitose. 4. The food material according to any one of claims 1 to 3, comprising a bifidobacterium together with isomaltosyl fructoside and / or isomeretitol. 5. A food material containing isomaltosyl fructoside and / or isomeletitose and an alcohol.