JPS6362184B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is an oligosaccharide obtained by allowing fructosyltransferase to act on sucrose,
Looking at it compositionally, it is a composition containing an oligosaccharide in which 1 to 4 molecules of fructose are bound to sucrose, and the composition contains at least twice as much of the oligosaccharide as sucrose in terms of weight ratio. The present invention relates to a sweetener and its manufacturing method. Hitherto, sucrose has been widely used in confectionery and foods, taking advantage of its superior properties such as good sweetness, body, and crystallinity. However, sucrose serves as a substrate for dextran eucrase produced by oral microorganisms, and as a result, continuous collection of sucrose produces a large amount of insoluble dextran in the mouth, promoting the formation of dental plaque, which can induce caries. It is said to be the cause. The present inventors made use of the excellent properties of sucrose and, as a result of intensive studies on sucrose-related carbohydrates that are less likely to cause dental caries, discovered an oligosaccharide group obtained by the action of fructosyltransferase on sucrose. A substance in which one molecule of fructose is bound to sucrose (hereinafter referred to as GF ₂ ), a substance in which two molecules of fructose are bound to sucrose (hereinafter referred to as GF ₃ ), a substance in which three molecules of fructose are bound to sucrose (hereinafter referred to as GF 3). (hereinafter referred to as GF ₄ ), a substance in which four molecules of fructose are bound to sucrose (hereinafter referred to as GF ₅ ), etc. are produced by Streptococcus mutans.
It has been found that not only is it not affected by the action of dextran eucrase produced by oral microorganisms, but it also has the effect of suppressing the production of insoluble dextran from sucrose by dextran eucrase. GF ₂ , GF ₃ , GF ₄ used here,
Oligosaccharides such as GF ₅ can be isolated and purified from a transferred sugar composition obtained by the action of fructosyltransferase on sucrose by carbon chromatography, ion exchange chromatography, etc., but this is not practical. It is preferable to use compositions of these oligosaccharides, and furthermore, cariogenic sugar alcohols such as sorbitol, mannitol, and maltitol, and artificial sweeteners such as dehydrochalcone and stevioside are added to such compositions. It can also be used. Further, after adjusting the pH of the sugar composition aqueous solution obtained by acting fructosyltransferase on sucrose to 7 to 9, nickel catalyst of 3 to 10% based on the solid matter was added, and the reaction temperature was 50 to 130°C. , reaction hydrogen pressure 50-120Kg/
These monosaccharides can be converted into sorbitol and mannitol by selectively catalytically reducing only glucose and fructose in the composition by carrying out catalytic reduction under conditions of ² cm2. Compositions containing sugar alcohols obtained as a result of this treatment include, for example, sorbitol 37%, mannitol 2%, sucrose 10%, GF ₂ 22%, GF ₃ 22
%, GF ₄ 7%, but since no insoluble dextran is observed to be produced by oral microorganisms, and little organic acid is produced, it is considered a sweetener with a more cariogenic effect. (See Test Example 4). The transferred sugar composition obtained by acting fructosyltransferase on sucrose contains unreacted sucrose, oligosaccharides such as GF ₂ and GF ₃ produced by the transfer reaction, and glucose by-produced by the transfer reaction. etc. However, such transferred sugar compositions are also less susceptible to the action of dextran euclase of oral microorganisms, and as a result, the amount of insoluble dextran produced is small. This is because even if sucrose is present in the composition, if oligosaccharides such as GF ₂ and GF ₃ are present at least twice the weight ratio of sucrose, the production of insoluble dextran from sucrose is suppressed, and GF ₂ and GF ₃ This is because insoluble dextran is not produced from such oligosaccharides. In this way, the composition contains an oligosaccharide in which one to four molecules of fructose are bonded to sucrose, and the sucrose content (wt%) in the total solid content of the composition is
The ratio of the total content (wt%) of oligosaccharides in which 1 to 4 molecules of fructose are bound to sucrose is 2.0
The composition described above has characteristics such as a small amount of insoluble dextran produced, which itself is considered to be a main factor in the development of dental caries, and that oligosaccharides such as GF ₂ and GF ₃ themselves suppress the production of dextran from sucrose. have. Furthermore, the present composition is not only resistant to caries, but also has excellent properties as a sweetener, such as good sweetness, moderate body feel, and moisturizing properties. As mentioned above, compositions containing oligosaccharides in which one to four molecules of fructose are bonded to sucrose in a specific ratio as described above (hereinafter referred to as cariogenic sweeteners) have the potential to induce caries. Although it is a sweet composition that is unlikely to cause caries, the present inventors have also conducted intensive studies on the industrial production method of this non-cariogenic sweetener, and have completed the second invention. The sweetener can be obtained by allowing fructosyltransferase produced by a microorganism of the genus Aspergillus or Fusarium to act on the prepared sucrose solution at a pH of 4.0 to 7.0. Fructosyltransferase used in this second invention mainly acts on sucrose to cleave the β-1,2 bond between fructose and glucose, then transfers the fructose to sucrose to produce GF ₂ , and further It has the effect of transferring fructose to GF ₂ to generate GF ₃ .
Enzyme _{Nomenclature (} Academic
It is different from Inulosucrase [2.4.1.9] and Levansucrase [2.4.1.10] described in Press. 1978). This enzyme is of the genus Aspergillus,
For example, Aspergillus niger
Enzymes of microbial origin such as Fusarium niger ATCC 20611, FERMP-5886 or Fusarium genus, for example Fusarium lini IAM5008 are used. Fructosyltransferase of microbial origin is grown in a suitable medium, such as 5.0% sucrose, 1.0% peptone, 0.7% meat extract.
%, NaCl 0.3% for 24 to 96 hours at the optimum temperature for each microorganism, i.e., 25 to 30°C, and after the cultivation is completed, the microorganisms are separated from the culture solution by means such as filtration or centrifugation. Use the obtained bacterial cells or
Furthermore, the culture filtrate after bacterial cell isolation is further purified and purified using conventional enzyme purification methods such as ultrafiltration, ammonium sulfate salting out, solvent precipitation, gel filtration, and ion exchange chromatography. enzymes can be used. The desired non-cariogenic sweetener can be obtained by allowing the bacterial cells or enzymes obtained in this way to act on sucrose; however, as a result of various studies on industrial transfer reaction conditions, it has been found that the process is carried out under the following conditions. is preferred. That is, the sucrose concentration during the transfer reaction is set to 5 to 70%, preferably 30 to 60%. In addition, the reaction pH and reaction temperature vary depending on the origin of the enzyme, but PH4.0 to 7.0 and temperature 25 to 65℃, preferably
The temperature should be 50-60℃. The amount of bacterial cells or enzyme used is 5 to 200 units, preferably 20 to 80 units per gram of sucrose. Here, the unit of enzyme is
5% sucrose solution 1.0ml, PH5.0 buffer 1.0
When 0.5 ml of enzyme solution is added to 1 ml and reacted at 40°C for 60 minutes, one unit is the amount of enzyme that produces 1 μmole of glucose in 2.5 ml of reaction solution for 60 minutes. After the rearrangement reaction is completed, the enzyme is deactivated by heating, decolorized with activated carbon, desalted with an ion exchange resin, and then concentrated to obtain the desired product. Analysis of the transition composition should be carried out by high performance liquid chromatography using, for example, a Microbondapak CH column (manufactured by Waters Limited) and a solvent system of acetonitrile:water (80:20 (v/v)). I can do it. The composition of the cariogenic sweetener obtained in this way is, for example, 30% glucose, 30% sucrose,
11%, GF ₂ 28%, GF ₃ 25%, GF ₄ 5%, and GF ₅ 1%, but the composition of each constituent sugar can take various values depending on the reaction conditions. Oligosaccharide GF ₂ includes O-β-D-fructofuranosyl-(2→1)-O-β-fructofuranosyl-(2→1)-α-D-glucopyranoside, O-β-D-fructo Furanosyl (2→
6) -O-β-glucopyranosyl-(1→2)-
There are β-D-fructofuranoside, O-β-D-fructofuranosyl-(2→6)-O-β-fructofuranosyl-(2→1)-α-D-glucopyranoside, etc., and as GF ₃ is O-β-D-fructofuranosyl-(2→[1-O-β-D-fructofuranosyl-2] ₂ →1)-α-D-glucopyranoside, O-β-D-fructofuranosyl- (2
→6)-O[β-D-fructofuranosyl-(2
→2)]-O-α-D-glucopyranosyl-(1
→2) -β-D-fructofuranoside, etc.
GF ₄ is O-β-D-fructofuranosyl-
(2→[1-O-β-D-fructofuranosyl-
2) ₃ →1) -α-D-glucopyranoside, etc. In addition, O-β-D-fructofuranosyl-(2→1)-O-β-fructofuranosyl-(2→1)-α-D-glucopyranoside (hereinafter referred to as 1-kestose) of GF ₂ ), O out of GF ₃
-β-D-fructofuranosyl-(2→[1-O
-β-D-fructofuranosyl-2] ₂ →1) -α
-D-glucopyranoside (hereinafter referred to as nystose) and O-β-D-fructofuranosyl-(2→[1-O-β-D-fructofuranosyl-2] ₃ →1)-α of GF ₄ -D-glucopyranoside is estimated to be low in calories as shown in Test Examples 5 and 6 below. Next, the sweetener of the present invention and its components
The effects of GF ₂ , GF ₃ , GF ₄ , and GF ₅ will be explained in detail using experimental examples. Using dextran eucrase obtained by culturing Streptococcus mutans ATCC25175 strain
Table 1 in Post-Test Example 1 compares the amount of insoluble dextran produced from GF ₂ , GF ₃ , GF ₄ and GF ₅ with that from sucrose. As is clear from the table, no insoluble dextran was produced from _GF2 , _GF3 , _GF4 , and _GF5 . Similarly, we investigated whether GF ₂ and GF ₃ inhibit the production of insoluble dextran from sucrose by dextran euculase, and the results are shown in Table 2 in Test Example 2 below. As is clear from the table, it was found that both GF ₂ and GF ₃ suppressed the production of insoluble dextran from sucrose. In addition, sweeteners with different compositions were prepared under various transition conditions, and the amount of insoluble dextran produced from these compositions was compared with that of sucrose, as shown in Table 4 in Test Example 3 below. As can be seen from this table, the amount of insoluble dextran produced is lower in all compositions than in sucrose.
In particular, the content (% by weight) of sucrose in the total solid weight of the sweetener composition is
The total content (wt%) of oligosaccharides such as GF ₂ , GF ₃ , GF ₄ , etc. is at least twice that of the sucrose content (wt%) in the total solid content. If the ratio is 2.0 or more,
The amount of insoluble dextran produced is
It is 50% or less, which is preferable for practical purposes. Test Example 1 Streptococcus mutans ATCC25175 strain was cultured under anaerobic conditions in a medium containing glucose and tryptocase, and after removing bacterial cells, it was concentrated and purified by ultrafiltration method to obtain dextran. Euculase was prepared. Next, add 1.0ml of 1% sugar solution, 0.67M phosphate buffer (PH
7.0) Mix 1.5ml and 0.25ml of the above enzyme solution and incubate at 37°C.
After reacting for an hour, the water-insoluble dextran produced was centrifuged at 3000 rpm for 15 minutes to collect the precipitate, which was washed twice with 5 ml of 70% ethanol.
Dissolve phenol in 2.5 ml of 1M KOH solution.
The amount of dextran produced was determined by the sulfuric acid method. In addition, sucrose, GF ₂ , GF ₃ ,
1% solutions of each of GF ₄ and GF ₅ were used. _GF2 ,
GF ₃ , GF ₄ , and GF ₅ are obtained from a transferred sugar composition obtained by the action of fructosyltransferase on sucrose, which is fractionated and purified by carbon chromatography, and then isolated by thin layer chromatography. A fraction giving spots was used. Display the results.
Shown in 1.

[Table] As shown in Table-1, GF ₂ , GF ₃ , GF ₄ , GF ₅
No production of dextran was observed. Test Example 2 In the presence of sucrose using the dextran eucrase prepared in Test Example 1
It was investigated whether GF ₂ and GF ₃ inhibit the production of insoluble dextran from sucrose when GF ₂ and GF ₃ are added. The reaction conditions are sugar solution 1.0
ml (each containing carbohydrates listed in Table 2),
0.67M phosphate buffer (PH7.0) 1.5ml, enzyme solution 0.25ml
were mixed and reacted at 37° C. for 4 hours, and the amount of insoluble dextran produced in the reaction solution was determined in the same manner as in Test Example 1.

[Table] The numbers in parentheses in Table 2 indicate the amount of insoluble dextran produced from sucrose.
The index is shown when it is set to 100. Test Example 3 Sweeteners having the following composition were produced by reacting fructosyltransferase with sucrose under various conditions.

[Table] When the amount of insoluble dextran produced from the above transfer sugar composition was measured in the same manner as in Test Example 1, the results shown in Table 4 were obtained.

[Table] Test example 4 Streptococcus mutans Serotype C was mixed with 0.27% maltose, 0.01% L-cysteine hydrochloride, and L-cysteine hydrochloride 0.01%.
-Glutamate sodium salt 0.1%,
NH ₄ HPO ₄ 0.2%, MgSO ₄ ―7H ₂ O 0.02%,
NaCl0.001%, _MnSO4 0.01%, _FeSO4・_7H2O0.01
After culturing under anaerobic conditions in a medium containing 5%, the bacterial cells are collected by centrifugation and dispersed in 0.05M phosphate buffer at a concentration of 10mg/ml. Lactic acid production amount is 0.2M - Phosphate buffer 0.9ml, 22.5M -
Mix 0.4 ml of MgCl ₂ , 0.2 ml of 1.7% sugar solution, and 0.5 ml of bacterial cell dispersion, perform a shaking reaction at 37°C for 30 minutes, boil for 15 minutes to stop the reaction, and centrifuge the mixture. After removing the bacterial cells, the amount of lactic acid in the supernatant was determined by an enzymatic method.

[Table] Test Example 5 Y.Takesue from the small intestine mucosa of a rabbit weighing 3 kg
According to the method of (Journal of Biochemistry, Vol. 65, p. 545, 1969),
Small intestinal disaccharide degrading enzyme was prepared. This crude enzyme system contained 280 U/ml of cyclase activity, 540 U/ml of maltase activity, and 8 U/ml of trehalase activity. 1.0 ml of 5% substrate, 0.25 M phosphate buffer (PH
6.5) After adding 1.0 ml and 0.5 ml of the above crude enzyme solution and reacting at 37°C for 24 hours,
The decomposition rate was determined using high performance liquid chromatography using a Bondapack CH column. The solvent used was acetonitrile:water=75:25.
The results are shown in Table-6. In addition, the decomposition rate was calculated|required by the following formula. Decomposition rate (%) = 100 - Weight % of remaining substrate based on the total weight of solids in the reaction solution after 24 hours of reaction Table-6 Substrate degradation rate by rabbit small intestine disaccharide degrading enzyme Sucrose 100 Maltose 100 1-kestose 0 Nystose 0 1F-Fructofuranosyl-Nystose
0 As shown in Table 6, 1-kestose, nystose and 1F-fructofuranosyl-nystose were not completely degraded by rabbit small intestine disaccharide degrading enzyme. Test Example 6 Male Wistar rats weighing 170g (30 rats per group)
After fasting for 17 hours, various sugars were orally administered at a rate of 3 g/Kg.
Blood was collected from 6 mice at 180 minutes and 180 minutes, and the amount of glucose in the blood was determined using the glucose oxidase method. The results are shown in Table-7.

[Table] Runistose In addition, each value in the table is the blood glucose (mg/
dl) is set as 100. Blood sugar levels in the non-administration area were 61±4.3, 65±5.0, 67±2.3, 73±7.0, 64± at 30, 60, 90, 120, and 180 minutes, respectively.
It was 3.7. As is clear from Table 7, 1-kestose,
No increase in blood sugar was observed from nystose and 1F-fructofuranosyl-nystose.
This indicates that the fructooligosaccharide of the present invention is not absorbed in the body and therefore does not provide substantial calories. Example 1 10 ml of BS medium containing 5.0% sucrose, 1.0% peptone, 0.7% meat extract, and 0.3% NaCl was dispensed into two test tubes, and after sterilization at 120°C for 30 minutes, Aspergillus. Aspergillus niger ATCC20611, FERM
One loopful of P5886) was inoculated and cultured at 28°C for 24 hours. Pour the obtained culture solution into two Erlenmeyer flasks (sterilized at 120℃ for 30 minutes) each containing 200 ml of BS medium.
The cells were inoculated in 10 ml portions and cultured with shaking at 28°C for 24 hours to prepare a preculture. BS medium 20 was placed in a 30 jar fermenter, sterilized at 120°C for 30 minutes, cooled, and inoculated with 400ml of the preculture solution, followed by culturing at 300 rpm and 28°C for 72 hours. After the culture was completed, the culture solution was separated using a centrifuge to obtain 2800 g of viable bacterial cells and 20 g of culture filtrate. The enzyme activity of the bacterial cells was 95 units/mg. This culture filtrate 20 was concentrated and purified by ultrafiltration to obtain enzyme solution 2. The enzyme activity was 240 units/ml. Add 6.7 kg of water to 10 kg of seuclose to dissolve and adjust the pH.
After adjusting to 5.0, the enzyme is 48% per gram of sucrose.
Units were added and the transfer reaction was carried out at 50°C for 48 hours. After the rearrangement reaction was completed, the enzyme was inactivated by heating at 100°C for 15 minutes, and then activated carbon with a solid content of 0.5% was added to decolorize. Amber light after removing activated carbon
It was treated with IR.120B and Amberlite IRA.411 ion exchange resins and then concentrated to a 75% w/w concentration to yield 12 g of non-cariogenic sweetener. The sugar composition of the obtained sweetener was 26% glucose, 2% fructose, 18% sucrose, and GF ₂ 40
%, GF ₃ was 14%. In addition, 10 kg of seuucrose was dissolved by adding 6.7 g of water, the pH was adjusted to 5.0, and the bacterial cells obtained as described above (95 units/mg) were used as the enzyme, and 48 units of this enzyme were added per 1 g of sucrose. do,
The transfer reaction was carried out at 50°C for 48 hours. After the transfer reaction was completed, the enzyme was deactivated by heating at 100°C for 15 minutes, and then
Activated carbon with a solid content of 0.5% was added to decolorize. After removing the activated carbon, it was treated with Amberlite IR.120B and Amberlite IRA.411 ion exchange resins and then concentrated to 75% w/w concentration to obtain 12Kg of non-carious sweetener. The sugar components of the obtained sweetener were 26% glucose, 1% fructose, 13% sucrose, and GF ₂ 39
%, GF ₃ 19%, and GF ₄ 2%. Example 2 Fusarium lini (Fusarium lini IAM5008) was cultured by the method described in Example 1 to obtain 20 culture fluids, which were concentrated and purified by ultrafiltration to obtain enzyme solution 2. Enzyme activity is 200 units/ml
It was hot. Add 7 kg of water to 3 kg of seuclose and dissolve.
After adjusting the pH to 6.0, 16 units of enzyme was added per 1 g of sucrose, and the transfer reaction was carried out at 50°C for 24 hours. After the reaction was completed, the enzyme was deactivated by heating at 100℃ for 15 minutes, then decolorized by adding activated carbon with a solid content of 0.5%, and desalted with an ion exchange resin to 75% w/w.
3.7 kg of caries-resistant sweetener was obtained. The sugar composition of this sweetener is 38.2% glucose, 7.8% fructose, 17.2% sucrose, and 25.4 _{% GF2} .
%, GF ₃ was 11.4%. Example 3 Syuucrose 5%, peptone 1%, meat extract
Dispense 10 ml of BS medium containing 0.7% and 0.3% NaCl into two test tubes, sterilize them at 120℃ for 30 minutes,
One platinum loop of Aspergillus niger was inoculated into this and cultured at 28°C for 24 hours. Pour the obtained culture solution into two Erlenmeyer flasks (sterilized at 120℃ for 30 minutes) each containing 200 ml of BS medium.
The cells were inoculated in 10ml portions and cultured with shaking at 28°C for 24 hours to prepare a preculture. BS medium 2.0 was placed in a 30-jar fermenter, sterilized at 120°C for 30 minutes, cooled and inoculated with 400ml of the pre-culture solution, and cultured at 300 rpm and 28°C for 72 hours. After the culture was completed, the bacterial cells were removed by filtration to obtain a culture filtrate 20. This culture filtrate 20 was concentrated and purified by ultrafiltration to obtain enzyme solution 2. The enzyme activity was 240 units/ml. Add 3.3 kg of water to 5 kg of seuclose and dissolve.
After adjusting the pH to 6.0, 60 units of enzyme was added per 1 g of sucrose, and the transfer reaction was carried out at 50°C for 72 hours. After the rearrangement reaction was completed, the enzyme was inactivated by heating at 100°C for 15 minutes, and then activated carbon with a solid content of 0.5% was added to decolorize. After removing the activated carbon, it was treated with Amberlite IR-120B and Amberlite IRA-411 ion exchange resins and concentrated to 75% w/w concentration to obtain 6Kg of sweetener. The sugar composition of this sweetener was 37% glucose, 2% fructose, 10% sucrose, 22% _GF2 , ₂₃ % GF3, and 6% _GF4 . Add 700 ml of water to 1.3 kg of the above sweet composition, and add
Add 15ml of 10% Na ₂ HPO ₄ and adjust pH to 9.0 with 4% NaOH
Adjusted to. Add 50g of Raney Nickel to this,
The reaction was carried out at a reaction temperature of 80 to 90° C. and a hydrogen pressure of 60 to 120 Kg/cm ² with stirring for 50 minutes. After the reaction was completed, the nickel catalyst was removed, treated with Amberlite IR-120B and Amberlite IRA-411 ion exchange resins, and concentrated to 75% w/w to obtain 1 kg of product.
The sugar composition of this sweetener is 38% sorbitol, 2% mannitol, 9% sucrose, ₂₂ % GF2,
GF ₃ was 23% and GF ₄ was 6%.

Claims (1)

[Scope of Claims] 1. A composition containing an oligosaccharide in which 1 to 4 molecules of fructose are bound to sucrose,
A caries-resistant composition in which the ratio of the total content (wt%) of oligosaccharides in which 1 to 4 fructose molecules are bonded to sucrose to the sucrose content (wt%) in the total solid content of the composition is 2.0 or more. sexual sweetener. 2. Sucrose, which is characterized in that fructosyltransferase produced by a microorganism of the genus Aspergillus or Fusarium is applied to a sucrose solution adjusted to a concentration of 5 to 70% at a pH of 4.0 to 7.0. A composition containing an oligosaccharide in which 1 to 4 molecules of fructose are bonded to sucrose, and 1 to 4 molecules of fructose are bonded to sucrose relative to the sucrose content (wt%) in the total solid content of the composition. A method for producing a cariogenic-resistant sweetener in which the ratio of the total content (wt%) of oligosaccharides is 2.0 or more.