JP2749217B2 - Method for producing trehalulose and palatinose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to Agrobacterium radiobacter MX-232 having the ability to produce trehalulose and palatinose (Microorganism Deposit No. 12397) and a microorganism belonging to the genus Agrobacterium, particularly Agrobacterium. -Radiobactor MX-232 (Microbial Deposit No. 1)
No. 2397) to produce a syrup containing trehalulose in high concentration from sucrose.

[0002]

BACKGROUND OF THE INVENTION Trehalulose, like palatinose, naturally exists as a component in honey. Palatinose has α-1,6 bonds between glucose and fructose, while trehalulose is a disaccharide having sweetness, although there is a difference that α-1,1 bonds between glucose and fructose. It is well known that palatinose is non-cariogenic dentally, but recently trehalulose has been confirmed to be non-cariogenic by in vitro and in vivo tests. Syrups containing trehalulose and palatinose have become widely used as low cariogenic sweeteners. Trehalulose and palatinose are generally produced from sucrose by a sugar transfer reaction by α-glucosyltransferase produced by microorganisms such as Protaminobacterium, Serratia and Erwinia. German Patent Specification 1,049,800 (1959) describes a method for converting sucrose to palatinose by the enzyme Protaminobacter rubrum. Tokiko 57-10
No. 720 discloses a method for producing palatinose by culturing a microorganism of the genus Protaminobacterium or Serratia under aerobic conditions in a sucrose solution, and isomaltulose with an immobilized enzyme described in Japanese Patent Publication No. 60-9797. Describes methods using Erwinia microorganisms, in which trehalulose is produced as a by-product.

[0003]

According to the conventionally known methods for producing palatinose using these microorganism-producing enzymes, the ratio of palatinose to trehalulose is 6: 1 to 1: 1.
The ratio is about 0: 1, and the production ratio of trehalulose is low.
Since palatinose easily crystallizes, the reaction solution can be easily purified and concentrated and crystallized to produce a product. However, since trehalulose does not crystallize, palatinose crystals remain in the final honey after recovery. The sugar composition of the final nectar from which palatinose crystals have been recovered is a mixture of palatinose or the like containing trehalulose as a main component, which is further purified and concentrated to obtain trehalulose syrup as a liquid product. However, in the enzymatic reaction described above, the target product, palatinose,
In addition to trehalulose, monosaccharides such as glucose, fructose, isomaltose, and isomeretose are produced as by-products. Among them, the production ratio of glucose and fructose as monosaccharides is about 5% of the sugar composition in the reaction solution. The production of these monosaccharides lowers the production efficiency of the target product, and in addition, the product is colored by repeated heating such as concentration during the manufacturing process, and the decolorization process is indispensable in the subsequent product recovery process. There was a problem in the manufacturing process.

[0004] Palatinose can be used as a sugar substitute in many foods, particularly in the production of low carious foods and drinks in the same manner as sugar, but palatinose has a slightly lower solubility in water than sugar. Therefore, foods and beverages that use high concentrations of sugar, if all are replaced with palatinose,
The amount of palatinose used was limited because crystals could precipitate in the final product. On the other hand, syrups containing trehalulose as a main component, which has high solubility, do not have such a problem, and the demand for the syrup is increasing due to the good quality of sweetness. Despite these circumstances, conventional production methods tend to lack trehalulose syrup due to the small amount of trehalulose produced, and due to these circumstances the production ratio of trehalulose is high and enzyme production with low monosaccharide production Bacteria were desired.

[0005]

Means for Solving the Problems The inventors of the present invention have been searching for a production bacterium which produces less monosaccharide and has a higher production ratio of trehalulose, but it has been found in a sugar factory in Kumpawapie group in Udon Province, Thailand. Agrobacterium radiobacteria isolated from the collected soil
acter), a new microorganism that produces little monosaccharide glucose and fructose from sucrose, produces trehalulose as a main component, and produces palatinose as a by-product. And produced the present invention. Conventionally, a method for producing trehalulose and palatinose by Agrobacterium has not been known. The trehalulose and palatinose-producing bacteria used in the present invention may be any microorganisms that belong to the genus Agrobacterium and have trehalulose and palatinose-producing ability. An example is Agrobacterium sp. Radiobacter MX-232 strain collected from soil in Udon, Thailand by the present inventors.

Agrobacterium Radiobacter MX-2
The bacteriological properties of the 32 strains are as follows. (a) Morphology After culturing on a broth agar slant medium at 28 ° C. for 2 days, observation with a phase-contrast microscope and flagella staining revealed that MX-232 had a periflagellate of 0.8 × 1.5 to 3.0 μm. Gram-negative bacilli, motile and do not form spores. (b) Growth on various media 1. Gravy agar plate culture: 26 ° C., 3-4 days culture for 2 days in diameter
Form a round, raised, full-edge colony of ~ 3 mm.
The surface is smooth, opaque and grayish white. 2. Meat juice liquid culture: The cells grow in the form of a film in the upper layer of the liquid by culturing for 3-4 days at 26 ° C., and a part of the cells in the film form sediment. 3. Culture in liquid medium containing sucrose: 26 ° C, 3-4
In the culture for one day, the culture solution becomes viscous due to production of a mucous substance. (c) Physiological properties 1.OF test: oxidative 2. Pigment productivity Fluorescent dye: none Pyocyanin: none Carotenoid: none 3. Growth temperature limit: 10-41 ° C 4. Cytochrome oxidase reaction: positive 5. Nitrate reduction Noh: Positive 6. Decarboxylation Arginine: Negative Lysine: Negative Ornithine: Negative 7. Denitrification: Negative 8. Degradability of gelatin (GEL): Negative 9. Degradability of starch: Negative 10. Degradability of Tween 80 : Negative 11. Use of carbohydrate (+: growing, ±: weak growing,-: no growing) D-glucose + D-fructose + D-galactose + L-arabinose + D-xylose + D-mannose + maltose + trehalose + Sucrose + raffinose + lactose + D-sorbitol + D-mannitol + inositol + glycerin + citric acid + acetic acid-succinic acid + 2-ke Togluconic acid ± L-alanine + β-alanine + 12. Degradation of esculin: positive 13. MR test: negative 14. VP test: negative 15. Indole production: negative 16. Urease activity: positive 17. Oxidase test: positive 18 Catalase test: Positive 19. Production of hydrogen sulfide: Negative 20. Attitude to oxygen: Aerobic 21. Production of 3-keto lactose: Positive 22. DNase test: Negative 23. GC content of DNA: 59.2% 24 . Mass onset due to phytopathogenicity test: negative

[0007] The mycological properties of the MX-232 strain were
Bergey's Manual of Systematic bacteriology Vo
As a result of comparison with lume1, the taxonomic position of this strain MX-232 was considered to belong to the obligate aerobic Gram-negative bacterium Agrobacterium radiobacter. Agrobacterium radiobacter MX-232 was sent to the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology.
No. 397 ". It has never been known that a species belonging to the genus Agrobacterium converts sucrose to trehalulose and palatinose, and the use of a strain belonging to this genus is an important feature of the present invention.

Hereinafter, a method for producing trehalulose and palatinose according to the present invention will be described. The enzyme that converts sucrose to trehalulose and palatinose, produced by this bacterium, is considered to be a type of glycosyltransferase. It is produced inductively by the presence of sucrose, fructose, and palatinose in the medium, and is present along with the cells. . Therefore, industrially, after culturing the cells in an enzyme-producing medium, an immobilized enzyme in which the cells are immobilized as it is is produced, filled in a bioreactor, and a sucrose solution is continuously passed therethrough to react. To produce trehalulose and palatinose,
The reaction solution is desalted, purified and concentrated to obtain the desired product.

The Agrobacterium bacterium used in the present invention generally has a readily changeable property and easily mutates. However, any mutant strain having the ability to produce palatinose and trehalulose can be used in the present invention. Can be used.

[0010] Culture is usually performed aerobically using a liquid medium. As the carbon source, for example, sucrose, molasses, molasses, glucose, maltose, glycerol, and organic acids can be used as appropriate as the carbon source in the medium composition for enzyme production, and sucrose, molasses, and molasses can be suitably used. . The quantitative ratio in the medium is 1 to 15% (w / v), particularly preferably 5 to 1%.
It is used in the range of 0% (w / v). As the nitrogen source, organic and inorganic nitrogen compounds such as yeast extract, meat extract, peptone, malt extract, corn steep liquor, urea, ammonium sulfate, ammonium nitrate, ammonium chloride and ammonium phosphate, which can be used by microorganisms, can be used. Yeast extract, corn steep liquor and the like are particularly preferred. As the inorganic salts, usually used are salts required for culturing bacteria, such as phosphoric acid, magnesium, calcium, potassium, and iron, alone or in appropriate combination. If necessary, other organic substances and inorganic substances are added to the medium.

The cultivation is carried out at a temperature of 10 to 41 ° C. at which the cells grow, preferably in the range of about 25 to 35 ° C.
The medium pH is 5.0 to 7.0, preferably pH 6.0 to 7.0.
It is adjusted in the range of 0. In a culture using a normal culture tank, aeration of about 1/10 to 1 vvm and 100 to 600 rp
Stir for about m. The culturing time is about 48-80 hours. After the culture is completed, the culture solution is cooled, and various methods can be applied to use the immobilized enzyme for recovering the precipitated portion by centrifugation.For example, this is mixed with sodium alginate and dropped into a calcium chloride solution. To form a gel. This granulated enzyme is further treated with polyethyleneimine and glutaraldehyde to obtain an immobilized enzyme. This is packed in a column, and a sucrose solution having a concentration of 20 to 60% w / w is adjusted to a temperature of about 25 ° C. and a pH of 5.5, and the mixture is allowed to react by flowing. After filtration, the reaction solution is desalted with an ion exchange resin and then concentrated to obtain a product. The ratio of palatinose to trehalulose in the product of the present method is 1: 3 to 1:12. If necessary, a high-purity trehalulose product can be easily obtained by fractionating this mixed solution by ordinary chromatographic separation using an ion exchange resin or the like.

The trehalulose syrup thus obtained can be used for various foods and drinks because of its high solubility in water. Palatinose, reduced palatinose, maltitol, It can be used by mixing with various sugars such as liquid sugar or adding a sweetness enhancer such as aspartame, stevia, or acesulfame potassium as needed.

[0013]

In the production of palatinose and trehalulose, a new strain, Agrobacterium sp., Is used in place of the conventionally used P. rubrum and the like.
By using radiobacter) MX-232, it is possible to increase the production of trehalulose syrup. In the method of the present invention, the amount of monosaccharide fructose and glucose produced in the product is negligible. Therefore, the coloration of the reaction solution during the production process due to heating is small, so that the cleaning step can be simplified, and the fining with a low degree of coloring can be achieved. Trehalulose syrup is obtained. Furthermore, since the ratio of palatinose in the sugar composition of the reaction solution is lower than that of the conventional bacteria, crystallization of palatinose from the reaction solution can be avoided, so that the concentration of the raw material sucrose solution can be increased, and the contamination of microorganisms during the process can be avoided and high. Cost reduction by concentration raw material processing becomes possible.

Example 1 1) Preparation of enzyme 100 g of sucrose, 10 g of peptone, 1 g of meat extract, 1 g of yeast extract, 2 g of disodium phosphate and 3 g of sodium chloride were dissolved in water to make 1 liter, and the pH was adjusted to 6.0 with sodium hydroxide solution. The medium adjusted to 5 to 7.0 was used as a medium.
Sterilization conditions were an autoclave at a temperature of 120 ° C. for 20 minutes. Put 100 ml of medium in a 500 ml shake flask,
A. Inoculate the slant of radiobacter MX-232, 28 ℃,
What was cultured at 140 rpm for 24 hours was used as an inoculum. After 3 liters of the medium was placed in a 5.0 liter fermenter and sterilized and cooled to a temperature of 28 ° C., the inoculum was inoculated, and the cells were cultured for about 48 hours under aeration rate of 1/4 vvm and stirring at 460 rpm. During the culture, the temperature was kept at 28 ° C. The glycosyltransferase activity of the culture was 30 U / ml. 1U is pH 5.5 2
When reacted at 20 ° C. in a 0% sucrose solution, the amount of enzyme at which the initial rate of conversion of sucrose to product is 1 μmol / min is 1
Unit (U). 2) The enzyme-immobilized culture solution was cooled to 5 ° C., centrifuged at 9,000 G for 10 minutes, and the supernatant was discarded to recover the precipitated portion. The precipitate was mixed with a 4% sodium alginate solution at a ratio of 1: 1 (w / w), and the precipitate was mixed with a 0.5-mm nozzle through a dropping device.
The mixture was dropped into a 25 M calcium chloride solution to gel into granules, aged for 2 hours, and then washed with water to obtain granulated enzymes.
Next, the granulated enzyme was added to a 2% polyethyleneimine (PEI) solution adjusted to pH 5.5 by adding hydrochloric acid to 1: 1 (w / w).
And left to stand for 5 minutes. Immediately thereafter, filtration was performed to collect the granulated enzyme that had absorbed PEI. Subsequently, the mixture was cooled, poured into a 0.5% glutaraldehyde (GA) solution at 5 ° C., and gently stirred for 30 minutes. And
After thorough washing with water, 300 g of the immobilized enzyme was produced. The activity of the immobilized enzyme was 70 U / g. 3) Production of trehalulose and palatinose The immobilized enzyme of MX-232 was packed in a column having an inner diameter of 15 mm and a length of 300 mm, and a sucrose solution having a solid content of 50% (w / w) at a temperature of 25 ° C. and a flow rate of 8.5 ml / The liquid was passed in h. The sugar composition of the reaction solution discharged from the column was as follows. Reaction liquid sugar composition Fructose 0.1% Glucose 0.1 Sucrose 1.0 Palatinose 15.3 Trehalulose 83.5 Total 100% The above reaction liquid is filtered, passed through a cation exchange resin tower and an anion exchange resin tower, and desorbed. After salt purification and concentration, a syrup product containing trehalulose as a main component was obtained. This product is a syrup that contains less monosaccharides than conventional products, is clear, and has a high trehalulose content.

Example 2 In the production of trehalulose and palatinose in Example 1, a sucrose solution having a solid content of 50% (w / w) was passed at a temperature of 15 ° C. at a flow rate of 4.7 ml / h. The sugar composition of the reaction solution discharged from the column was as follows. Reaction liquid sugar composition fructose 0.1% glucose 0 sucrose 1.0 palatinose 8.5 trehalulose 90.4 total 100% trehalulose syrup obtained by the method of the present invention has about 50% sweetness of sucrose, It is also possible to add a natural or artificial high-intensity sweetener, for example, stevia sweetener, aspartame, alitame, acesulfame K, sucralose, etc. to enhance the sweetness, and in this case, to use the high-sweetness sweetener. It has the effect of improving the taste of the ingredients. [Manufacture of low carious foods] 1. Strawberry jam preparation example. Strawberry 300g Trehalulose syrup 300g Lemon juice a little 2. Example of mixing strawberry jam. Strawberry 300g Trehalulose syrup 300g Aspartame 5g Lemon juice a little 3. An example of mixing strawberry milk jelly. Strawberry 100g Milk 200ml Trehalulose syrup 80g Powdered gelatin 7g Water 30ml Lemon juice 5ml 4. Example of cream bavarois with strawberry. Powdered gelatin 4g Water 6ml Milk 100ml Trehalulose syrup 40g Egg yolk 30g Strawberry puree 70g Fresh cream 40ml 5. Mixing example of hot cake. Flour 200g Baking powder 6g Milk 180ml Egg 50g Trehalulose syrup 80g Water 45ml Butter 10g Syrup containing a high concentration of trehalulose is characterized by low caries-inducing, mild and refreshing sweetness and acidity. It is stable. It also enhances the flavor of other ingredients contained in the product. In the case of jams and jellies, the taste and aroma of the strawberry and other fruits used are suitably drawn, and the color and gloss of the final product are good.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hideaki Okui 3-3-5-105 Tendai, Chiba City, Chiba Prefecture (72) Inventor Kenzo Sawada 2-63-4 Itabashi, Itabashi-ku, Tokyo 801 (58) Investigated Field (Int.Cl. ⁶ , DB name) C12P 19/12 C12P 19/24 CA (STN)

Claims (4)

(57) [Claims] 1. A method for producing trehalulose and palatinose, comprising converting sucrose into trehalulose and palatinose using a microorganism belonging to the genus Agrobacterium or an enzyme produced by the microorganism. 2. The method according to claim 1, wherein the microorganism belonging to the genus Agrobacterium is Agrobacterium radiobacter MX-232 (Deposit No. 12397 of Microbiological Research Laboratories). 3. The method according to claim 1, wherein the microorganism of the genus Agrobacterium or the glycosyltransferase produced by the microorganism is immobilized. 4. Agrobacterium radiobacter MX-232 capable of producing trehalulose and palatinose from sucrose (Deposit No. 12397, deposited by Mie Kenken) and its mutant microorganism capable of producing trehalulose and palatinose.