Saccharide mixture containing psicose with improved sweetness quality and crystallization
11202460 · 2021-12-21
Assignee
Inventors
- Ji Won PARK (Seongnam-si, KR)
- Go-Eun Kim (Gwangju, KR)
- Sung Won PARK (Daejeon, KR)
- Hye Jung KIM (Daejeon, KR)
- Chong Jin PARK (Daejeon, KR)
- Kang Pyo Lee (Seoul, KR)
Cpc classification
A23L29/30
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a mixed saccharide composition containing psicose, glucose and fructose with improved sweetness quality and crystallization, and a method for preventing crystallization of a mixed saccharide composition containing a psicose.
Claims
1. A mixed saccharide composition with non-crystallization, comprising psicose, glucose and fructose, wherein the glucose content is 24 parts by weight or lower, the fructose content is about 1 to 10 parts by weight, and the psicose content is about 60 to 98 parts by weight, based on 100 parts by weight of a solid content of all saccharides contained in the mixed saccharide composition, wherein a saccharide crystal does not precipitate in the mixed saccharide composition, when 0.5 wt. % of a solid content of glucose based on the solid content of all saccharides contained in the mixed saccharide composition is further added to the mixed saccharide composition and the mixed saccharide composition is stored at 4° C. for 4 days, and wherein the mixed saccharide composition is a mixed saccharide solution.
2. The mixed saccharide composition of claim 1, wherein the total content of fructose and psicose is higher than 70 parts by weight, based on 100 parts by weight of the total solid content of all saccharides contained in the mixed saccharide composition.
3. The mixed saccharide composition of claim 1, wherein the psicose is D-psicose, glucose is D-glucose, and fructose is D-fructose.
4. A method for preventing production of a saccharide crystal in a mixed saccharide composition comprising psicose, glucose and fructose, by adjusting a glucose content to be 24 parts by weight or lower, based on 100 parts by weight of a solid content of all saccharides contained in the mixed saccharide composition, wherein the fructose content is about 1 to 10 parts by weight, and the psicose content is about 60 to 98 parts by weight, based on 100 parts by weight of the solid content of all saccharides contained in the mixed saccharide composition, wherein a saccharide crystal does not precipitate in the mixed saccharide composition, when 0.5 wt. % of a solid content of glucose based on the solid content of all saccharides contained in the mixed saccharide composition is further added to the mixed saccharide composition and the mixed saccharide composition is stored at 4° C. for 4 days, and wherein the mixed saccharide composition is a mixed saccharide solution.
5. The method of claim 4, wherein the total content of fructose and psicose is higher than 70 parts by weight, based on 100 parts by weight of the total solid content of all saccharides contained in the mixed saccharide composition.
6. The method of claim 4, wherein the adjusting a glucose content to be 24 parts by weight or lower glucose content is achieved by mixing a psicose crystallization mother liquor having a psicose content of 80% by weight or more and a glucose crystallization mother liquor having a glucose content of 80% by weight or more.
7. The method of claim 4, wherein the mixed saccharide composition is obtained by reacting a composition for producing psicose containing at least one selected from the group consisting of a psicose epimerase, a microbial cell of strain producing the epimerase, a culture of the strain, a lysate of the strain, and an extract of the lysate or culture, with a fructose-containing raw material.
8. The method of claim 4, wherein the mixed saccharide composition is obtained by mixing a psicose crystallization mother liquor having a psicose content of 80% by weight or higher and a glucose crystallization mother liquor having a glucose content of 80% by weight or higher.
9. The method of claim 8, wherein the psicose crystallization mother liquor comprises 80 to 99.9% by weight of psicose in the solid content of all saccharides and further comprises at least one selected from the group consisting of fructose, glucose, oligosaccharide and rare saccharide other than psicose.
10. The method of claim 9, wherein the psicose crystallization mother liquor comprises 20% by weight or lower of fructose and 1.0% by weight or lower of glucose, based on the solid content of all saccharides.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
(4)
(5)
DETAILED DESCRIPTION OF THE EMBODIMENTS
(6) The present invention will be described in more detail by the following examples. However, the following examples are only preferable examples, and the present invention does not limited thereto.
Preparative Example 1: Preparation of a Psicose Crystallization Mother Liquor
(7) 1-1: Preparation of a Strain Producing Psicose
(8) An encoding gene of a psicose epimerase derived from Clostridiuim scindens (Clostridiuim scindens ATCC 35704) (DPE gene; Gene bank: EDS06411.1) was synthesized as a modified form of polynucleotide by optimizing for E. coli and called CDPE. The polynucleotide optimized for E. coli (SEQ ID NO: 2) and sod promoter and T7 terminator obtained from pET21a vector were obtained as each template through PCR, and these were linked as one template by overlap PCR method and cloned into pGEM T-easy vector through T-vector cloning, thereby confirming sod promoter (SEQ ID NO: 1), optimized CDPE sequence of SEQ ID NO: 8 and a sequence of a polynucleotide comprising T7-terminator.
(9) The entire confirmed polynucleotide was inserted into the same restriction enzyme site of an expression vector pCES208 (J. Microbiol. Biotechnol., 18:639-647, 2008) using restriction enzymes NotI and XbaI(NEB), and a recombinant vector pCES208/psicose epimerase (pCES_sodCDPE) was prepared. The cleavage map of the prepared recombinant vector (pCES_sodCDPE) was shown in
(10) Corynebacterium glutaricum was transformed with the prepared recombinant vector (pCES_sodCDPE) plasmid using electroporation. Colonies were picked and inoculated in 4 ml of LB medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L) supplemented with Kanamycin at a final concentration of 15 ug/ml, and then cultured for approximately 16 hrs at the culture conditions of 30° C. and 250 rpm. Then, 1 ml of the culture was collected and inoculated in 100 ml LB medium comprising 15 ug.Math.ml of Kanamycin, and the culture was continued for over 16 hrs. After lysis of cells cultured using beadbeater, only supernatant is obtained and mixed with a sample buffer as 1:1, and then heated at 100° C. for 5 minutes. The prepared samples were subjected to electrophoresis on a 12% SDS-PAGE gel (composition: running gel—3.3 ml H2O, 4.0 ml 30% acrylamide, 2.5 ml 1.5M Tris buffer (pH 8.8), 100 μl 10% SDS, 100 μl, 10% APS, 4 μl TEMED/stacking gel—1.4 ml H2O, 0.33 ml 30% acrylamide, 0.25 ml 1.0M Tris buffer (pH 6.8), 20 μl 10% SDS, 20 μl 10% APS, 2 μl TEMED) at 180V for approximately 50 minutes, and the protein expression was confirmed. After the expression of CDPE was confirmed on the SDS-PAGE gel, His-tag purification was proceeded using Ni-NTA resin for accurate measurement of expression level, and the expression rate was calculated using a calculation formula (expression rate (%)=(Purified protein (mg)/Total soluble protein (mg))×100). The prepared transformed Corynebacterium glutaricum produced 16.62 mg of the total water-soluble proteins and 1.74 mg of purified enzyme proteins.
(11) 1-2: Preparation of Psicose Syrup
(12) In order to prepare psicose from fructose using the recombinant strain producing psicose epimerase obtained from Preparative Example 1-1, cells were collected by centrifugation in the strain culture.
(13) Then, the cell suspension was treated with 0.05% (v/v) of en emulsifier (M-1695) in a final volume and treated at 35° C. (±5° C.) for 60 minutes. The microbial cells in which the reaction was completed were collected after the supernatant comprising the emulsifier was removed again using a centrifuge.
(14) For the preparation of immobilized beads, the collected microbial cells were mixed with D.W. to a final microbial cell concentration of 5% (v/v), and 4% (v/v) of alginic acid dissolved in water and 5% (v/v) of the collected microbial cells were mixed at 1:1, and it was refrigerated at 4° C. to remove bubbles generated during mixing. The refrigerated mixture solution was injected through Neddel (inner diameter 0.20 to 0.30 mm) and formed into a droplet shape, and dropped by weight, and the dropped mixture solution was dropped into a previously prepared 100 mM calcium chloride (CaCl2) solution and cured, and spherical or elliptical beads (diameter 2.0 to 2.2 mm) were formed. The formed beads were soaked in a 100 mM calcium chloride solution and mixed evenly by a stirrer so as to be further cured.
(15) After all the mixture solution was injected, the beads were further cured while being refrigerated for 4 to 6 hrs, and then cured for approximately 6 hrs in a refrigerated state by replacing a new 100 mM calcium chloride solution. After beads completely cured were skimmed and moisture was completely removed, water was added 3 times as much as the volume of the beads and then it was stirred for 10 minutes, and the calcium chloride solution was removed by treating the beads with this process 3 times. The reaction substrate was adjusted to pH 6.8 to 7.2 with 3N NaOH, and liquid fructose or crystalline fructose can be the substrate depending on the kind of product. The beads replaced with the reaction substrate comprising 10 mM manganese were transferred to a reactor, and then reacted at the reaction temperature of 50° C. for approximately 30 to 60 minutes, and soaking of beads was completed with manganese and fructose. The beads completely soaked have a reduced diameter of approximately 1.6 to 1.8 mm and its strength was also increased. After removing the substrate of beads completely soaked, they were filled in an immobilized reaction column and then used for the production of a psicose syrup.
(16) <Immobilized Column Reaction Conditions>
(17) Reaction temperature: internal temperature of the column jacket 50° C.
(18) Substrate flow rate: 0.5 SV (space velocity L. h−1)
(19) Reaction substrate: crystalline fructose 40 brix, pH 6.8 to 7.2,
(20) Bead preparation: 2.5% (w/w) microbial cells, 2% (w/w) alginic acid mixture and 10 mM Mn2+ soaking
(21) To the immobilized reaction column, a raw material whose raw material solution comprised 75% of solid and the fructose content was 92 parts by weight when the total solid content was 100 parts by weight was provided, thereby preparing a psicose syrup, which is a saccharide mixture of two compositions. That is, the 25(w/w) % psicose syrup having glucose:fructose:psicose:oligosaccharide=6:67:25:2 in the ratio by weight of glucose:fructose:psicose:oligosaccharide was collected from the reaction solution, and used for the following Examples.
(22) 1-3: Preparation of a Psicose Crystallization Mother Liquor
(23) In order to remove impurities such as color and ion components, the psicose syrup obtained from Preparative Example 1-2 was passed through at a rate of 2 times volume of the ion exchange resin per hour in a column at the room temperature which was filled with resin in which anion exchange resin and cation and anion exchange resin were mixed and desalted, and then crystallization was proceeded by separating as a high-purity psicose solution using chromatograph which was filled with an ion exchange resin of the calcium (Ca2+) type and concentrating up to 80% by weight. The dewatered solution which was not crystallized in a dewatering process through a crystallizing process but dewatered was collected as a crystallization mother liquor, and the mother liquor was 65.1 Bx and comprised fructose 8.4% by weight and psicose 91.6% by weight as a solid composition.
Example 1: Crystallization Material of Fructose-Containing Syrup
(24) Crystals in commercially available liquid fructose 42 (fructose 39.5% by weight and glucose 53.2% by weight) and liquid fructose 55 (fructose 54.0% by weight and glucose 37.0% by weight) syrup were collected and centrifuged at 5,000 RPM for 30 minutes, and then the supernatant was discarded and the crystalline portion was taken. To completely remove the syrup component, it was vacuum filtered using a 5 μm membrane filter while washing with ethanol. The aqueous solution passed through the filter was discarded, and then crystals remaining in the filter were dried in a 60° C. oven for one day.
(25) As the result of HPLC analysis of dried crystals, the main component of crystals was shown as glucose 80% by weight in Table 2 and
(26) TABLE-US-00002 TABLE 2 tetrasac- charide trisac- disac- classification or more charide charide glucose fructose ethanol Sugar 0.5% 0.4% 1.4% 80.6% 13.1% 4.0% composition
Example 2: Preparation of a Crystallization Syrup Composition
(27) A high-purity psicose syrup was prepared by mixing a crystallization mother liquor produced after crystallizing the syrup comprising 95% by weight or more psicose content and a crystallization mother liquor after crystallizing glucose through high-purity separation in Preparative Example 1 at a certain mixing ratio.
(28) Specifically, using the psicose crystallization mother liquor obtained in Preparative Example 1 (Samyang Genex, 65.1 Bx, solid composition: fructose 8.4% by weight, psicose 91.6% by weight) and the glucose crystallization mother liquor (hydrol) obtained in Preparative Example 2 (Samyang Genex, 65.3 Bx, solid composition: disaccharide or more 8.7% by weight, glucose 91.3% by weight), the syrup composition prepared by mixing various mixing ratios was prepared. The syrup composition was prepared to be the glucose content of 10% by weight, 20% by weight, 30% by weight, 40% by weight, or 50% by weight, respectively, and a concentrated syrup solution was prepared by concentrating to be 75Bx.
(29) The glucose crystallization mother liquor product was obtained by after forming glucose crystals (anhydrous crystals or hydrocrystals) by crystallizing glucose original solution for preparation of glucose, filtering products and collecting glucose crystals. The obtained hydrol was 65.3 Bx and comprised disaccharide or more 8.7% by weight and glucose 91.3% by weight as the solid composition.
(30) Anhydrous crystalline glucoses (Samyang Genex) were added to the concentrated syrup solution at a solid concentration of 0.5% by weight each, and the mixture was stored at 4° C. for 4 days, and the crystallization acceleration test was proceeded. The saccharide composition of the syrup solution obtained by mixing anhydrous glucose powder was shown in Table 3. Table 3 exhibited the solid composition of the psicose crystallization mother liquor and the glucose crystallization mother liquor composition of the present invention.
(31) The presence of absence of crystal precipitation (turbidity) was observed and evaluated according to the following standard, and the result was shown in Table 4 and
(32) TABLE-US-00003 TABLE 3 Mixture ratio (wt %) (Hydrol:Psicose disaccharides mother liquor) or more glucose fructose psicose 55%:45% 4.7% 50.0% 3.7% 41.1% 44%:56% 3.8% 40.0% 4.7% 51.6% 33%:67% 2.8% 30.0% 5.6% 61.3% 22%:78% 1.9% 20.0% 6.5% 71.2% 11%:89% 1.0% 10.0% 7.4% 81.4%
(33) The following Table 4 exhibited the crystallization acceleration test result of the psicose crystallization mother liquor and the glucose crystallization mother liquor.
(34) TABLE-US-00004 TABLE 4 Storage Glucose Glucose Glucose Glucose Glucose time(hour) 10% 20% 30% 40% 50% 0 h − − − − − 1 h − − − + + 2 h − − − + ++ 3 h − − − + ++ 4 h − − + ++ ++ 5 h − − + ++ ++ 6 h − − + ++ ++ 12 h − − + ++ ++ 24 h − − + ++ ++ 48 h − − + +++ +++ 72 h − − + +++ +++ 96 h − − + +++ +++ <Evaluation standard> −: No crystal ±: Crystalline nuclei were produced but practical +: Crystals were visible but had fluidity ++: Crystals were precipitated and there was turbidity +++: Crystals were precipitated and had no fluidity
(35) As can be seen in Table 4 and
Example 3: Preparation of Crystallization Syrup Composition
(36) In order to elucidate the concentration of glucose inducing crystallization of syrup composition in detail, a crystallization test was conducted by preparing a syrup composition by further subdividing the glucose concentration.
(37) Specifically, a crystallization acceleration test was conducted by preparing a concentrated syrup solution by mixing the psicose crystallization mother liquor and the glucose crystallization mother liquor in substantially the same manner as in Example 1. However, then, the composition of the high-purity psicose syrup was as same as Table 5. The result of observing the presence or absence of crystal precipitation (turbidity) was evaluated according to the same standard with Example 2 and shown in Table 6, Table 7 and
(38) TABLE-US-00005 TABLE 5 Mixture ratio (wt %) (Hydrol:Psicose disaccharides mother liquor) or more glucose fructose Psicose 23%:77% 2.1% 21.0% 6.5% 70.5% 24%:76% 2.2% 22.0% 6.4% 69.6% 25%:75% 2.3% 23.0% 6.3% 68.7% 27%:73% 2.3% 24.0% 6.1% 66.9% 28%:72% 2.4% 25.0% 6.0% 66.0% 29%:71% 2.4% 26.0% 6.0% 65.0% 30%:70% 2.5% 27.0% 5.9% 64.1% 31%:69% 2.6% 28.0% 5.8% 63.2% 32%:68% 2.7% 29.0% 5.7% 62.3% 33%:67% 2.8% 30.0% 5.6% 61.4%
(39) The following Tables 6 and 7 exhibited the crystallization acceleration test result of the psicose crystallization mother liquor and the glucose crystallization mother liquor composition.
(40) TABLE-US-00006 TABLE 6 Storage glucose glucose glucose glucose glucose time(hr) 21% 22% 23% 24% 25% 0 h − − − − − 1 h − − − − − 2 h − − − − − 3 h − − − − − 4 h − − − − − 5 h − − − − − 6 h − − − − − 12 h − − − − − 24 h − − − − − 48 h − − − − + 72 h − − − − + 96 h − − − − +
(41) TABLE-US-00007 TABLE 7 Storage glucose glucose glucose glucose glucose time(hr) 26% 27% 28% 29% 30% 0 h − − − − − 1 h − − − − − 2 h − − − − + 3 h − − − − + 4 h − − − − + 5 h − − − − + 6 h − − − − + 12 h − − − + + 24 h + + + + + 48 h + + + + + 72 h + + + + + 96 h + + + + ++
(42) As can be seen in Tables 6 and 7 and
(43) Based on the result of Example 3, since turbidity was not occurred even under a severe condition in case that the glucose content was 24% by weight or lower, the quality of products can be maintained when stored for a long time.
Example 4: Preparation of Crystallization Syrup Composition
(44) In order to demonstrate that the quality of products was maintained when stored for a long time in case that the glucose content was 24% by weight or lower shown in the result of Example 3, a syrup composition was prepared in which the glucose concentration was subdivided as same as Example 3 and was subjected to long-term storage crystallization test.
(45) Specifically, a crystallization acceleration test was conducted by preparing a concentrated syrup solution by mixing the psicose crystallization mother liquor and the glucose crystallization mother liquor in substantially the same manner as in Example 3. Then, the composition of the high-purity psicose syrup was as same as Table 5 of Example 3. In order to observe the presence or absence of crystal precipitation (turbidity), after stored in a low temperature chamber for 96 days, it was evaluated according to the same standard with Example 2, and the result was shown in Table 8, Table 9 and
(46) TABLE-US-00008 TABLE 8 Storage glucose glucose glucose glucose glucose time 21% 22% 23% 24% 25% 96 days − − − − +++
(47) TABLE-US-00009 TABLE 9 Storage glucose glucose glucose glucose glucose time 26% 27% 28% 29% 30% 96 days +++ +++ +++ +++ +++
(48) As can be seen in Table 8, Table 9 and
(49) In summary, the results of Examples 2, 3 and 4, since turbidity was not occurred even under a severe condition, when the glucose content was 24% by weight or lower, the quality of products can be maintained when stored for a long time.