JPS6345785B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to the production of sweeteners containing fructooligosaccharides, and will be explained in more detail as follows:
Using sugar beet molasses as a raw material, fructooligosaccharides are produced using fructosyltransferase, and unreacted sucrose, etc. are separated using a Ca-type reinforced acidic cation exchange resin column to produce fructooligosaccharides with high yield and at low cost. be. Fructooligosaccharide has a refreshing sweet taste, and unlike sucrose, it produces less insoluble dextrin, which is said to cause dental caries.Furthermore, fructooligosaccharide itself suppresses the production of insoluble dextrin from sucrose, so it has recently attracted attention as a cariogenic-resistant sweetener. It is a sugar that is Fructooligosaccharide is a sugar in which fructose is bound to sucrose.
It mainly consists of a mixture of sugars (hereinafter referred to as GF ₂ ), two molecules of fructose (hereinafter referred to as GF ₃ ), and sugars that have three molecules of fructose (hereinafter referred to as GF ₄ ). As described in JP-A-56-154969, the production method is as follows: it is obtained by reacting a fructosyltransferase enzyme with a sucrose solution, and sources of the enzyme fructosyltransferase include Aureobasidium sp. It is used as an enzyme source by cultivating strains of the genus Aspergillus and isolating or fixing them from the bacterial cells, or by isolating enzymes contained in plants such as asparagus and Jerusalem artichoke. be. It is usually produced by separating bacterial cells from a culture of microorganisms, immobilizing enzymes, and contacting them with a highly pure sucrose solution through a biochemical reaction, which inevitably results in an expensive product. It is possible to replace this with sucrose in molasses, which is inexpensive, but beet molasses containing a lot of sucrose contains oligosaccharides such as raffinose and galactinol, which may put pressure on the reaction or make separation difficult due to their similar physical properties. This is difficult and cannot be a very practical manufacturing method. This invention was made in view of the above circumstances, and as a result of research into a carious sweetener that is difficult to produce fructooligosaccharide at low cost and in good yield using cheap molasses as a raw material, it was discovered that fructooligosaccharide is a Ca-type strongly acidic cation exchanger. The first step is to purify sugar beet molasses and improve the purity of sucrose, focusing on the fact that it can be chromatographically separated using a resin column.The second step is to convert the sucrose solution obtained in the first step into fructooligosaccharide using fructosyltransferase. A third step in which the fructooligosaccharides obtained in the step and second step are separated using a Ca-type strongly acidic cation exchange resin column;
They solved the problem by mixing various sugars with the fructooligosaccharides separated during the process to create a cariogenic-resistant sweetener. The sugar beet molasses used in this invention is an inexpensive molasses containing impurities, preferably blackstrap molasses discharged during sugar production by the Steffen method or blackstrap molasses discharged during sugar production by the ion exchange resin method. Examples of their compositions are now shown in Table 1.

[Table] Unlike cane molasses, the main component of the molasses is sucrose, but since it contains many impurities, it is virtually impossible to recover it even if it is directly used in the production of fructooligosaccharides, and must be purified. The purpose of purification is to increase the sucrose content or remove impurities to improve purity, and various purification methods can be applied. Particularly preferred purification methods include raffinose and galactinol, which are contained in large amounts in sugar beet molasses. In this method, sucrose is hydrolyzed into sugars that can be easily separated from sucrose, the concentration of sucrose is increased, and the liquid is passed through a resin column made of Na-type strongly acidic cation resin to chromatically separate sucrose. As a method of hydrolyzing raffinose etc., for example, Absidea lignieri
It is preferable to culture the IFO8084 strain to prepare a bacterial enzyme and contact it with the above-mentioned sugar beet molasses. Raffinose etc. are hydrolyzed by the contact and become sucrose and galactose. Examples of ion-exchange molasses shown in Table 1 So it will look like Table 2.

[Table] As shown in Table 2, ruffinose decreases and sucrose increases relatively, but reducing sugar also increases, so this is passed through a Na-type strongly acidic cation exchange resin column to further increase the purity of sucrose. Good. The Na-type strong acid cation exchange resin used at this time is resin such as Amberlite IR-120, Dowetsukiss 50W x 4, and Diaion SK-1A (all trade names), especially with a degree of crosslinking of about 4 and 50 to 50%.
A resin with a particle size of 100 mesh is preferred. For liquid passage, the resin is packed in a long and narrow column, and the hydrolyzed molasses and extruded water are alternately passed through the column and extruded at a liquid passage rate (SV) of 0.5 to 2.5 to obtain a fraction containing a large amount of sucrose. Table 3 shows an analysis example of the sucrose fraction obtained by treating the molasses shown in Table 2 in this manner.

[Table] As is clear from Table 3, some raffinose coexists and transfers to the final product, but this amount does not significantly affect the product composition. Also, since the concentration of the solution is low and it is inconvenient to handle as it becomes a large amount as it is, it is preferable to concentrate it to around Bx50. The purified and concentrated molasses is then converted into fructo-oligosaccharides using fructosyltransferase in the second step. As the fructosyltransferase, known ones can be used. (as total sugar) and add to this
KH ₂ PO ₄ 0.1%, MgSO ₄ 7H ₂ O 0.05%, Urea 0.1
%, 0.1% NaNO _{3 ,} 0.3% polypeptone, and 0.1% coast liquor were added to prepare a culture medium, aeration culture was performed to grow the mycelia, the mycelium was centrifuged and washed, and the enzyme was fixed using a conventional method. It is recommended to use it as a bacterial enzyme with low invertase activity.
The bacterial enzyme thus produced usually has a fructosyltransferase activity of 20 to 40 units/mg dry matter. However, the active unit is 50g of sucrose.
Dissolve in 50ml of M/10 phosphate buffer (PH5.0),
After adding 5 g of bacterial enzyme and reacting at 50°C for 5 hours, glucose was quantified using high-performance liquid chromatography, and the amount of enzyme that produced 1 μMol of glucose per hour was defined as 1 unit, and 1 mg of dried bacterial cells was added. This is shown as a hit. The reaction is carried out using the purified and concentrated seurose solution.
Adjust the pH to around 5.0, heat to 40-60℃, and after heating, add about 500 raw bacterial enzymes with fructosyltransferase activity of 30 units/mg dry matter per 10.
g and maintain the temperature for 3 to 7 hours while stirring. Due to the retention, the reaction progresses and a large amount of fructooligosaccharide is produced, so when the reaction is almost completed, the bacterial enzyme is removed. Table 4 shows an example in which the solution was analyzed after reacting for 5 hours under the above conditions.

[Table] However, 〓 contains a small amount of raffinose in the fructooligosaccharide in proportion to the total sugar.
As is clear from Table 4, fructooligosaccharides account for approximately 60% of the total sugars, with GF ₂ being the most abundant, GF ₃ and GF ₄
About 40% of the unreacted sucrose and sugars mainly produced during the reaction, such as glucose and fructose, remain. One of the features of this invention is the use of a Ca-type strongly acidic cation exchange resin column in the third step to separate sucrose, etc. from the above reaction products. It is necessary to completely desalt and remove inhibiting ions using a strongly acidic cation exchange resin and a strongly basic anion exchange resin. Amberlite is the Ca-type strongly acidic cation exchange resin used in this invention.
IR-120, Dowetsukiss 50W x 6, Diamond Ion
A resin such as SK-1A (all trade names) with a degree of crosslinking of 4 to 6 and a particle size of 50 to 100 mesh is particularly preferred.
The resin is treated with a solution such as CaCl ₂ to form a Ca type, and packed into a long and narrow resin tower convenient for chromatographic separation.
Pass sugar solution and extruded water alternately at a temperature of ℃, SV0.1
Fractional separation is performed at a speed of ~2. Now Dawetzkiss
Convert 100 meshes of resin of 50W x 6 (trade name) to Ca type, fill 2.5 of it into a resin tower, and pass the treated sugar solution at SV0.3, and the first 800ml that flows out.
The sugar solution composition shown in Table 5 is obtained by discarding the liquid and then collecting 125 ml of the liquid that flows out.

[Table] As is clear from Table 5, the sugars that first flow out are high molecular weight sugars such as fructooligosaccharides, and this also contains a small amount of raffinose, etc., which were contained in the molasses from the beginning. Next, the saccharides that flow out are sucrose, and finally, monosaccharides such as glucose flow out. Therefore, fractions 1 to 5 and fractions 1 to 9 are collected, concentrated to Bx50, decolorized with activated carbon, and further concentrated to Bx75 to obtain the sugar compositions shown in Table 6.

[Table] As can be seen from Table 6, most of the recovered fructooligosaccharides are GF ₃ , with a small amount of sucrose mixed in, with fructooligosaccharides having a purity of 95% in fractions 1 to 5 and 90.8 in fractions 1 to 9.
Contains % fructooligosaccharides. Therefore, by collecting the above fructooligosaccharide-rich fractions and concentrating them to a Bx of 60 to 80, a refreshing sweetener that can be stored for a long period of time can be obtained. Furthermore, the sweetness can be adjusted by mixing solutions of glucose, fructose, sorbitol, cupping sugar, etc. All of these can be used as non-cariogenic sweeteners and raw materials for the production of caries-preventing foods, and are particularly effective against caries. It is suitable as a sweetener for children who are easily spoiled. The present invention is made as described above, and has the advantage of producing fructooligosaccharide at a high yield using inexpensive sucrose in sugar beet molasses as a raw material, and can therefore be produced at low cost. This method also allows the use of sucrose in the biochemical production of maltoolgosylfructosides and other oligosaccharides that improve the cariogenic properties of starting materials. This will be explained below using examples. Example 1 Dowex 50W x 4 (trade name) resin with a particle size of 50 to 100 mesh was added to a jacketed stainless steel column with an inner diameter of 87 cm, a height of 250 cm, and a resin layer height of 170 cm.
The diluted sugar beet molasses solution 125 of the ion-exchange fat desalination method shown in Table 2 was filled and previously treated with α-galactosidase to hydrolyze 95% of raffinose.
The liquid was passed through the resin column at SV1.3 and extruded with hot water to recover the sugar solution in the sucrose section, and concentrated under reduced pressure to Bx50 to obtain 48 kg of concentrated liquid. After adjusting the pH of this concentrate to 5.0, 16 kg was placed in a 20-volume tank and heated to 55°C.Frozen cells of Aspergillus Oryze strain IAM2600 were treated with warm water at 55°C for 1 hour to form a fruct. 500 g of centrifugally dehydrated bacterial enzyme containing 30 units/mg dry matter of siltransferase was added, and the mixture was stirred and maintained at that temperature for 5 hours to react. After the reaction was completed, the bacterial cells and sugar solution were separated by centrifugation, and the bacterial cells were returned to the same tank and the enzymatic reaction was repeated three times. The entire amount of the enzyme treatment solution was then transferred to an H-type dowex.
The solution was desalted by passing it through a column of HCR-W ₂ (trade name) 2 and a column of OH type Revacit Ca9249 (trade name) 4. After desalination, a stainless steel column with a jacket of 15 cm in inner diameter and 220 cm in height was loaded with Ca-type Dowex 50W.
Filled with 35 ×6 (product name) (particle size 100 mesh),
Pour the desalted sugar solution at Bx30, SV0.3, temperature 60℃, charge amount 3.5, and then pour hot water at 60℃ in the same manner.
The sugar solution was supplied by SV, and a continuous operation was performed in which charging the sugar solution and supplying hot water were repeated, and a fraction containing a large amount of fructooligosaccharides was collected to obtain a sugar solution with a purity of 90%. The above sugar solution was concentrated to Bx50 under reduced pressure, decolorized by adding a small amount of activated carbon, and further concentrated to Bx75 after filtration.
The obtained fructooligosaccharide has a refreshing sweet taste and contains glucose, fructose, sorbitol,
It was suitable as a cariogenic sweetener that could not be mixed with other sweeteners such as sucrose.

Claims (1)

[Claims] 1 The first step of refining sugar beet molasses to improve the purity of sucrose, the second step of converting the sucrose solution obtained in the first step into fructooligosaccharide using fructosyltransferase, and the fructooligosaccharide solution obtained in the second step A method for producing a cariogenic sweetener that is less cariogenic than sugar beet molasses, characterized by a combination of a third step of chromatographically separating the sweetener using a Ca-type strongly acidic cation exchange resin column.