WO2018093196A1 - Method for synthesizing stevioside glycoside using modified strains belonging to genus leuconostoc and novel stevioside glycoside prepared thereby - Google Patents
Method for synthesizing stevioside glycoside using modified strains belonging to genus leuconostoc and novel stevioside glycoside prepared thereby Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
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- the present invention relates to a method for synthesizing stevioside glycosides using a modified Leuconostoke strain, and a novel stevioside glycoside prepared by the present invention, and more specifically, using a glucan sucrase produced from a modified Leuconostoke strain.
- a method for synthesizing stevioside glycosides and novel stevioside glycosides prepared thereby.
- Stevioside is a kind of natural sweetener, low calorie compared to sugar, and its sweetness is about 200-300 times higher than sugar, so its demand is rapidly increasing.
- Stevioside is a sweetness extracted from Stevia rebaudiana BERTONI, a perennial herbaceous plant native to South America Paraguay. Baudiosides C, D, E, Dulcoside A and the like are known.
- Stevia which is used worldwide as a natural sweetener, is growing rapidly in the US food and beverage industry and has been selected as one of the major food ingredients by the US Food and Drug Administration.
- Each component is classified according to the combination of various forms of sugar (glucose, mannose) to Steviol, a diterpene-based substance, and each substance is known to have a slightly different sweetness and taste.
- the Rebaudioside family has better microstructure than Stevioside.
- steviosides have long-lasting aftertaste and have disadvantages such as bitterness, unpleasantness, low water solubility, etc. in addition to sweetness, and thus, there is a problem in that the amount of usage and use is limited, so it is necessary to improve the quality of steviosides.
- Dextransucrase (EC 2.4.1.5) is a generic term for enzymes that synthesize glucans from sugar and is produced mainly from the microorganisms of the genus Leuconostoc and S treptococcus .
- the reactor operation for the sugar of dextran sucrose is as follows.
- Korean Patent Application No. 1998-0024355 describes the production of novel oligosaccharides using dextran sucrose obtained from a mutant strain of Leuconostoc mesenteroides using sugar as a substrate and maltose, genthiobiose, raffinose or lactose as receptors. A method is disclosed.
- the present inventors have diligently studied to overcome the problems of the prior art, when synthesizing stevioside glycosides using glucan sucrase produced from a modified leukonostok strain, stevioside glycosides can be produced with high efficiency. As well as being able to synthesize novel stevioside glycosides, the present invention was completed.
- An object of the present invention is to provide a method for producing stevioside glycosides using a modified Leuconose stock strain capable of synthesizing stevioside glycosides with high efficiency.
- Another object of the present invention is to provide a novel stevioside glycoside using the method for producing a stevioside glycoside.
- the present invention to solve the above problems,
- the present inventors continued mutating the 512FMCM bacteria, and completed the present invention by finding an enzyme-producing bacterium 1.5 times higher than that of the bacterium, that is, 1500-fold or more enzyme-activating bacteria in glucose medium compared to industrial bacteria.
- strains of 1355 and 1299 strains were mutated to the high yielding enzymes for synthesizing indigestible oligosaccharides.
- Leukonostock mesenteroides B-512F / KM, leukonostock mesenteroides B-1299C / KM and leukonostock citreum B-1355C / KM according to the present invention were published on July 14, 2015. It is deposited in BRC with accession number KCCM11728P, accession number KCCM11729P and accession number KCCM11730P.
- the glucan sucrase of step A) may be used as it is without fermentation.
- stevioside glycosides were synthesized by using the glucan sucrase produced by such a mutant strain.
- glucansucrase is an enzyme that synthesizes glucan from sucrose and is also referred to as glycosyltransferase or dextransucrase.
- glucan sucrase is produced mainly from the Leuconostoc strain or the Streptococcus strain.
- the two strains are Gram positive and closely related to the anaerobic cocci, but the biggest difference between them is that the Leukotostock strain requires sugar for enzyme production, while the Streptococcus strain does not require sugar.
- the leukotostock strain induces dextran sucrase induction
- the Streptococcus strain continuously produces dextran sucrase.
- Leuconostoc mesenteroides produces an enzyme that biosynthesizes dextran, in which glucose is primarily linked to ⁇ 1 ⁇ 6, and the reaction mechanism by which the enzyme biosynthesizes dextran using sucrose is as follows:
- the main products of the enzymatic reaction are glucan and fructose of high molecular weight of about 10 7 ⁇ 10 8 Da, and glucose and leucose (5-O- ⁇ -D-glucopyranosyl -Dfructopyranose) are produced as by-products.
- strains of the genus Leuconostok according to the present invention produce different types of dextran sucrase, and the dextran synthesized thereby is different in form or extent of binding depending on the enzyme.
- dextran binding is mainly known as ⁇ 1 ⁇ 3, and ⁇ 1 ⁇ 2 and ⁇ 1 ⁇ 4 binding have also been reported.
- Most strains of leukonostock can produce dextran sucrase only by adding sucrose to an enzyme-producing substrate, but constitutive mutant strains have been developed that produce unique dextran sucrase without sucrose.
- the modified leukonostok strain is characterized by being modified by chemical mutations.
- the modified leukonostock strain may be carried out by chemical mutagenesis by mutagenesis treatment, physical mutation by UV treatment or by mutagenesis by genetic engineering manipulation, preferably by chemical mutagenesis by mutagenesis treatment It can be modified by law.
- the mutagen may be selected from at least one of NTG (N-Methyl-N'-Nitro-N-Nitrosoguanidine), EMS (ethylmethane sulfonate) and triethylene melamine (triethylene melamine).
- the term “modified” or “engineered” strain is produced by introducing genetic material into a selected host or parent to alter or alter cell physiology and biochemistry.
- the parent acquires new properties, for example the ability to produce new intracellular metabolites or higher amounts of intracellular metabolites.
- the introduction of genetic material into the parent cell results in a new or modified ability to produce enzymes.
- the genetic material introduced into the parent cell comprises a gene or part of a gene encoding one or more enzymes involved in the biosynthetic pathway for the production of enzymes, and additional components, such as promoters, for the expression or expression control of these genes It may also comprise a sequence.
- the sucrose contained in the strain culture medium may be included in a concentration of 300 to 1100mM, more preferably 700 to 1100Mm concentration. .
- the glucan sucrase can be used with an activity of 1 to 5 Unit / ml, preferably used with an activity of 3 to 5 Unit / ml It features.
- the glucan sucrase concentration (X1), stevioside concentration (X2), the step of sucrose contained in the medium in step A) The following relation holds between the concentration (X3) and the production rate (Y) of stevioside glycosides.
- Y 46.466740 + 12.802404 (X1) -0.226268 (X2) +0.045081 (X3) +0.030300 (X1) (X2) +0.007352 (X1) (X3) -0.000049 (X2) (X3) -2.131815 (X1) 2 +0.000087 (X2) 2 -0.000043 (X3) 2
- the production rate of stevioside glycosides in the glucan sucrase concentration, stevioside concentration and sucrose concentration range of the present invention is excellent overall, in particular, the enzyme activity is 5 Unit /
- the highest yield was obtained when the mL, stevioside concentration of 30 mM and sucrose concentration of 1100 mM.
- the relationship was able to confirm the validity through the fact that the expected value and the experimental value obtained by substituting the variables are almost similar.
- the alcohol having 1 to 4 carbon atoms is characterized in that 70 to 100% ethanol.
- the present invention also provides a stevioside glycoside represented by the following formula (1) prepared by the above method.
- the present invention also provides a stevioside glycoside represented by the following formula (2) prepared by the above method.
- the stevioside glycoside of Formula 1 according to the present invention has a malditope mass spectrometry m / z of 980 to 990, and the stevioside glycoside of Formula 2 has a malditope mass spectrometry of m / z of 1140 to 1160.
- the present invention also provides a sweetener comprising a stevioside glycoside represented by Formula 1 or Formula 2.
- the stevioside glycoside production method according to the present invention can not only produce stevioside glycosides with high efficiency but also can be used as a sweetener by synthesizing novel stevioside glycosides.
- 1 is a diagram showing the TLC results of stevioside receptor reaction products of leuconostock enzymes.
- FIG. 4 shows TLC results according to Example 3.
- Figure 7 shows the results of confirming the concentration of the three variables for the optimization of steviol glycosides.
- FIG. 8 shows the results of Maldi-tof analysis of Sample 1 according to Experimental Example 6.
- FIG. 9 shows a Maldi-tof analysis result of Sample 2 according to Experimental Example 6.
- L. mesenteroides B-512FMCM, L. mesenteroides B-1299C and L. citreum B-1355C according to Example 1 were inoculated in 1.2 mL of glucose medium and incubated for 24 hours, followed by centrifugation and 200 ⁇ l of the supernatant. . The isolated strains were then washed three times with 200 ⁇ l sterile 20 mM sodium-citrate buffer pH 6.0.
- Each strain according to the above example was incubated for 24 hours in a liquid medium containing glucose and sucrose, and then the supernatant was obtained by centrifugation and mixed with 1: 1 (v / v) with 100 mM sugar. Then, the mixture was reacted at a temperature of 28 ° C., and the resulting sugar glucan was collected by centrifugation, dissolved in 1 M NaOH, and developed by dropping in TLC.
- One unit of enzyme refers to the ability of an enzyme to produce 1 mM fructose from sucrose under one minute of reaction conditions.
- Glucose medium (IU ml -1 ) Sucrose medium (IU ml -1 ) B-512F 0 0.01 B-512FMCM 6.5 8.2 B-512F / KM 12.3 13.1 B-1299 0 0.01 B-1299C 0.1 0.3 B-1299C / KM 0.8 1.5 B-1355 0 0.02 B-1355C 0.32 0.6 B-1355C / KM 1.1 1.7
- the enzyme productivity of the modified Leukonostok strains B-512F / KM, B-1299C / KM and B-1355C / KM of the present invention is more than twice as high as the parent It was confirmed.
- Sucrose (S) was purchased from sigma, stevioside was purchased from Daepyung Co., Ltd. (Gyeonggi-do, Korea), and the enzyme was derived from the Leukonostock Citrium B-1355C / KM strain according to Experimental Example 1.
- lanes 1 to 3 are all converted to stevioside glycosides ((Frc; fructose. STV; stevioside receptor, lane 1; receptor reaction products using B-1299C / KM derived enzyme) , Lane 2; receptor reaction product using B-1355C / KM derived enzyme, lane 3; receptor reaction product using B-512F / KM derived enzyme).
- HPLC High Pressure Liquid Chromatography
- Example 3 the sample prepared in Example 3 was purified using an HPLC-RI system equipped with NH 2 column (5 ⁇ m, 150 x 4.6 mm), wherein the purification conditions are shown in Table 2 below. The results are shown in FIG.
- Y 46.466740 + 12.802404 (X1) -0.226268 (X2) +0.045081 (X3) +0.030300 (X1) (X2) +0.007352 (X1) (X3) -0.000049 (X2) (X3) -2.131815 (X1) 2 +0.000087 (X2) 2 -0.000043 (X3) 2 It was confirmed.
- Example 1 and Sample 2 The molecular weight (Sample 1 and Sample 2) of steviol glycosides was confirmed using a Maldi-TOF MS (MALDI-TOF MS, Matrix-Assisted Laser Desorption Ionization -Time-of-Flight Mass Spectrometer) and the results are shown in FIGS. 8 and FIG. 9 is shown.
- MALDI-TOF MS Matrix-Assisted Laser Desorption Ionization -Time-of-Flight Mass Spectrometer
- steviol glycosides (3 mg / mL) of Samples 1 and 2 were dissolved in deionized water and then molecular weight was determined using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster, CA, USA). Mass spectra was obtained in a positive reflector mode, with an average of 300 laser shots and an acceleration voltage of 25 kV. The results are shown in FIGS. 8 and 9.
- Sample 1 (Compounds 1) is m / z 989.5 (M + Na) + is a sample combined with one glucose
- Sample 2 (Compounds 2) is m / z 1151.4 (M + Na) + 2 Dog glucose was found to bind to stevioside.
- Example 1 Each purified stevioside glycoside (10 mg) (Sample 1 and Sample 2) was dissolved in 600 ⁇ L DMSO-d6 and prepared in 5 mm NMR tubes. NMR spectra were analyzed at 25 ° C with AVANCEIII system (Bruker, Germany) at 850 MHz for 1H NMR and 125 MHz for 13C NMR.
- the binding structure was confirmed by homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC), and the results are shown in Tables 4 and 5 below.
- COSY homonuclear correlation spectroscopy
- TOCSY total correlation spectroscopy
- HSQC heteronuclear single-quantum coherence
- HMBC heteronuclear multiple-bond correlation
- Glucosyl-stevioside of Sample 1 is 13-O- ⁇ -sophorosyl-19-O- ⁇ -isomaltosyl-steviol as shown in the following formula 2
- Glucosyl-stevioside of Sample 2 is As shown in Chemical Formula 1, 13-O- [ ⁇ -D-glucosyl (1 ⁇ 2) - ⁇ -nigerosyl] -19-O- ⁇ -isomaltosyl-steviol was identified as a novel compound.
- the method for producing stevioside glycosides according to the present invention can not only produce stevioside glycosides with high efficiency, but also can be effectively applied as a natural sweetener by synthesizing novel stevioside glycosides. .
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Abstract
The present invention relates to a method for synthesizing a stevioside glycoside using modified strains belonging to the genus Leuconostoc and a novel stevioside glycoside prepared thereby. More specifically, the present invention relates to a method for synthesizing a stevioside glycoside by using glucansucrases, which are produced from modified strains belonging to the genus Leuconostoc, and a novel stevioside glycoside prepared thereby. The method for producing a stevioside glycoside according to the present invention regulates the type and concentration of glucansucrase, which are produced from modified strains belonging to the genus Leuconostoc by controlling the concentration of sucrose contained in a medium, and can produce a novel stevioside glycoside with high efficiency by controlling the concentration of stevioside reacting therewith. In addition, the resulting stevioside glycoside can be used as a sweetener.
Description
본 발명은 변형 류코노스톡속 균주를 이용한 스테비오사이드 배당체의 합성 방법 및 이에 의하여 제조된 신규한 스테비오사이드 배당체에 관한 것으로, 더욱 구체적으로 변형 류코노스톡속 균주로부터 생산된 글루칸수크라아제를 이용하여 스테비오사이드 배당체를 합성하는 방법 및 이에 의하여 제조된 신규한 스테비오사이드 배당체에 관한 것이다.The present invention relates to a method for synthesizing stevioside glycosides using a modified Leuconostoke strain, and a novel stevioside glycoside prepared by the present invention, and more specifically, using a glucan sucrase produced from a modified Leuconostoke strain. A method for synthesizing stevioside glycosides and novel stevioside glycosides prepared thereby.
최근 설탕의 과잉 섭취와 기존의 당류의 다량 섭취로 생기는 충치, 비만, 당뇨병, 성인병 등의 문제점을 보완하기 위해서 생물공학 기술을 통해 천연식품 소재의 새로운 종류의 대체 당질이 개발되고 있다. 천연 고감미료 소재의 주요 시장인 한국·미국(유럽)에서 모두 사용이 가능한 고감미료 소재는 총 8종 성분이 있으며, 이 중 천연 컨셉을 부여 가능한 제품은 단 2종(Stevioside 계열, Mogroside 계열)이 존재 한다. 각 소재의 원료(Stevia Leaf, Luo Han Guo Fruit)의 감미성분 함량,경작 환경 등을 분석한 결과, 기존의 설탕 혹은 합성감미료와 유사한 단위원가(가공식품에 적용 시 감미료가 차지하는 가격 수준)가 가능한 천연 고감미료 소재는 Stevioside 계열이 유일하다고 알려져 있다. Recently, a new kind of alternative sugars of natural food materials has been developed through biotechnology to compensate for problems such as caries, obesity, diabetes, and adult diseases caused by excessive intake of sugar and large amounts of existing sugars. There are 8 kinds of high sweetener materials that can be used in both Korea and the United States (Europe), which are the main markets for natural high sweetener materials, and among them, only two types (Stevioside series and Mogroside series) are available. exist. As a result of analyzing sweetness content and cultivation environment of raw materials of each material (Stevia Leaf, Luo Han Guo Fruit), unit cost (price level of sweetener when applied to processed food) can be similar to existing sugar or synthetic sweetener. It is known that Stevioside is the only natural high sweetener material.
스테비오사이드는 천연 감미료의 일종으로 설탕에 비하여 저칼로리이며 감미도는 설탕의 약 200-300배로 높아 그의 수요가 급속하게 높아지고 있다. 스테비오사이드는 남미 파라과이가 원산지인 국화과 다년생 초본인 스테비아 레바우디아나 베르토니 (Stevia rebaudiana BERTONI)로부터 추출한 감미성분으로 스테비아의 단맛 성분으로는 스테비오사이드(C38H60O18), 레바우디오사이드 A(C44H70O23), 레바우디오사이드 C, D, E, 둘코사이드 A 등이 알려져 있다. Stevioside is a kind of natural sweetener, low calorie compared to sugar, and its sweetness is about 200-300 times higher than sugar, so its demand is rapidly increasing. Stevioside is a sweetness extracted from Stevia rebaudiana BERTONI, a perennial herbaceous plant native to South America Paraguay. Baudiosides C, D, E, Dulcoside A and the like are known.
천연 감미료로서 전세계적으로 사용되고 있는 스테비아는 미국의 식품음료산업에서 빠르게 성장하고 있으며, 미국식품의약국(FDA)에 의해 주요 식품원료 중 하나로 선정되었다. 각각의 성분은 모두 diterpene계 물질인 Steviol에 다양한 형태의 당(포도당, 만노스)이 결합된 형태에 따라서 분류되며, 각각의 물질은 조금씩 다른 감미도 및 미질을 가지는 것으로 알려져 있다. 상대적으로 Stevioside에 비하여 Rebaudioside 계열의 구조가 보다 미질이 우수한 것으로 알려져 있다. 하지만, 스테비오사이드는 뒷맛이 오래 남으며 단맛 이외에 쓴맛, 불쾌감, 낮은 수용성 등이 있는 단점을 지니고 있으며, 그로 인해 사용량 및 용도의 한계가 발생하는 문제점이 있어 스테비오사이드의 미질을 개선할 필요가 있다. Stevia, which is used worldwide as a natural sweetener, is growing rapidly in the US food and beverage industry and has been selected as one of the major food ingredients by the US Food and Drug Administration. Each component is classified according to the combination of various forms of sugar (glucose, mannose) to Steviol, a diterpene-based substance, and each substance is known to have a slightly different sweetness and taste. It is known that the Rebaudioside family has better microstructure than Stevioside. However, steviosides have long-lasting aftertaste and have disadvantages such as bitterness, unpleasantness, low water solubility, etc. in addition to sweetness, and thus, there is a problem in that the amount of usage and use is limited, so it is necessary to improve the quality of steviosides.
현재의 스테비오사이드의 감미질 개선 방법은 (1) 설탕, 포도당, 과당 등과 같은 천연 당질 감미료를 1종 또는 그 이상을 첨가하는 방법, (2) 아미노산 또는 아미노산의 염과 배합하는 방법, (3) 싸이클로덱스트린과 같이 포접능을 갖는 환형 당질에 물리적으로 결합시키는 방법 등이 있다. 그러나 이상의 방법은 첨가물을 상당히 많은 양을 첨가하여야 하며 결국 스테비오사이드가 저칼로리 감미료라는 특징을 잃어버리는 단점이 있다.Current methods for improving the sweetness of steviosides include (1) adding one or more natural sugar sweeteners such as sugar, glucose, fructose, and the like, (2) combining with amino acids or salts of amino acids, and (3) cyclo And physically binding to a cyclic saccharide having an inclusion ability such as dextrin. However, the above method requires the addition of a considerable amount of additives, which in turn loses the character of stevioside as a low calorie sweetener.
덱스트란수크레이즈(dextransucrase) (EC 2.4.1.5)는 설탕으로부터 글루칸을 합성하는 효소들을 포괄적으로 일컬으며 Leuconostoc과 Streptococcus 속의 미생물들로부터 주로 생산된다. 덱스트란수크레이즈의 설탕에 대한 반응기작은 다음과 같다.Dextransucrase (EC 2.4.1.5) is a generic term for enzymes that synthesize glucans from sugar and is produced mainly from the microorganisms of the genus Leuconostoc and S treptococcus . The reactor operation for the sugar of dextran sucrose is as follows.
수크로오스 → (글루코오스)n-m-w + n-m 프락토오스 + m 루크로오스(Leucrose) + w 글루코오스Sucrose → (glucose) n-m-w + n-m fructose + m leucrose + w glucose
한국특허출원 제1998-0024355호에는 설탕을 기질로 하고 말토오스, 겐티오바이오스, 라피노스 또는 락토오스를 수용체로 사용하여, Leuconostoc mesenteroides의 돌연변이 균주로부터 얻은 덱스트란수크레이즈를 사용하여 신규한 올리고당을 생산하는 하는 방법이 개시되어 있다.Korean Patent Application No. 1998-0024355 describes the production of novel oligosaccharides using dextran sucrose obtained from a mutant strain of Leuconostoc mesenteroides using sugar as a substrate and maltose, genthiobiose, raffinose or lactose as receptors. A method is disclosed.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구노력한 결과, 변형 류코노스톡속 균주로부터 생산된 글루칸수크라아제를 이용하여 스테비오사이드 배당체를 합성하는 경우, 스테비오사이드 배당체를 고효율로 생산할 수 있을 뿐만 아니라 신규한 스테비오사이드 배당체를 합성할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have diligently studied to overcome the problems of the prior art, when synthesizing stevioside glycosides using glucan sucrase produced from a modified leukonostok strain, stevioside glycosides can be produced with high efficiency. As well as being able to synthesize novel stevioside glycosides, the present invention was completed.
본 발명은 고효율로 스테비오사이드 배당체를 합성할 수 있는 변형 류코노스톡속 균주를 이용한 스테비오사이드 배당체의 생산방법을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a method for producing stevioside glycosides using a modified Leuconose stock strain capable of synthesizing stevioside glycosides with high efficiency.
본 발명은 또한, 상기 스테비오사이드 배당체의 생산방법을 이용한 신규한 스테비오사이드 배당체를 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a novel stevioside glycoside using the method for producing a stevioside glycoside.
본 발명은 상기와 같은 문제점을 해결하기 위하여, The present invention to solve the above problems,
A) 류코노스톡 메센테로이데스 B-512F/KM(KCCM11728P), 류코노스톡 메센테로이데스 B-1299C/KM(KCCM11729P) 및 류코노스톡 시트레움 B-1355C/KM(KCCM11730P)로 구성된 군으로부터 선택된 1종 이상의 변형 류코노스톡속 균주를 수크로오스(sucrose) 함유 배지에서 배양하여 글루칸수크라아제를 생성하는 단계; A) selected from the group consisting of leukonostock mesenteroides B-512F / KM (KCCM11728P), leukonostock mesenteroides B-1299C / KM (KCCM11729P) and leukonostock citrium B-1355C / KM (KCCM11730P) Culturing the at least one strain of leukonostok strain in a sucrose-containing medium to produce glucan sucrase;
B) 상기 배양에 의해 생성된 글루칸수크라아제(glucansucrase)와 수용체로서 스테비오사이드를 반응시키는 단계; 및B) reacting glucansucrase produced by the culture with stevioside as a receptor; And
C) 상기 수용체 반응에 의해 생성된 스테비오사이드 배당체를 회수하는 단계;를 포함하는 스테비오사이드(stevioside) 배당체의 고효율 생산방법을 제공한다. C) recovering the stevioside glycosides produced by the receptor reaction provides a high efficiency production method of stevioside glycosides comprising a.
본 발명자는 512FMCM균을 지속적으로 돌연변이 진행을 하였고, 이 균보다 1.5배 향상된 효소 생산성 균, 즉, 산업 균에 비해서 글루코오스 배지에서 1500배 이상의 효소 활성 생산 균을 찾아내어 본 발명을 완성하였다. 또한 난소화성 올리고당을 합성하기 위한 효소 고생산 균으로 1355, 1299 계통의 균을 돌연변이하여 모균보다 효소 생산성이 크게 우수한 균주를 개발하였다.The present inventors continued mutating the 512FMCM bacteria, and completed the present invention by finding an enzyme-producing bacterium 1.5 times higher than that of the bacterium, that is, 1500-fold or more enzyme-activating bacteria in glucose medium compared to industrial bacteria. In addition, strains of 1355 and 1299 strains were mutated to the high yielding enzymes for synthesizing indigestible oligosaccharides.
본 발명에 따른 류코노스톡 메센테로이데스 B-512F/KM, 류코노스톡 메센테로이데스 B-1299C/KM 및 류코노스톡 시트레움 B-1355C/KM은 2015년 7월 14일 자로 한국생명공학연구원 생물자원센터에 수탁번호 KCCM11728P, 수탁번호 KCCM11729P 및 수탁번호 KCCM11730P로 기탁되어 있다. Leukonostock mesenteroides B-512F / KM, leukonostock mesenteroides B-1299C / KM and leukonostock citreum B-1355C / KM according to the present invention were published on July 14, 2015. It is deposited in BRC with accession number KCCM11728P, accession number KCCM11729P and accession number KCCM11730P.
본 발명에서 상기 A) 단계의 글루칸수크라아제는 별도의 정제과정 없이 발효액을 그대로 사용할 수 있다.In the present invention, the glucan sucrase of step A) may be used as it is without fermentation.
본 발명에서는 이와 같은 변이 균주가 생성하는 글루칸수크라아제를 이용하여 스테비오사이드 배당체를 합성하였다. In the present invention, stevioside glycosides were synthesized by using the glucan sucrase produced by such a mutant strain.
본 명세서에서 사용된 용어 "글루칸수크라아제(glucansucrase)"는 수크로오스(sucrose)로부터 글루칸(glucan)을 합성하는 효소로서 글리코실트랜스퍼라제(glucosyltransferase) 또는 덱스트란수크라아제(dextransucrase)라고도 한다. The term "glucansucrase" as used herein is an enzyme that synthesizes glucan from sucrose and is also referred to as glycosyltransferase or dextransucrase.
일반적으로 글루칸수크라아제는 주로 류코토스톡속(Leuconostoc) 균주 또는 스트렙토코커스속(Streptococcus) 균주로부터 생산된다. 상기 두 균주는 그람 양성이며 통성 혐기성 구균으로서 서로 밀접한 관계가 있으나 이들 간에 가장 큰 차이점은 류코토스톡속 균주는 효소 생산을 위하여 설탕을 요구하는 반면 스트렙토코커스속 균주는 설탕을 필요로 하지 않는다. Generally, glucan sucrase is produced mainly from the Leuconostoc strain or the Streptococcus strain. The two strains are Gram positive and closely related to the anaerobic cocci, but the biggest difference between them is that the Leukotostock strain requires sugar for enzyme production, while the Streptococcus strain does not require sugar.
즉 류코토스톡속 균주는 덱스트란수크라아제를 유도적으로 생산하며 스트렙토코커스속 균주는 덱스트란수크라아제를 지속적으로 생산한다. 류코노스톡 메센테로이데스(Leuconostoc mesenteroides)는 글루코스가 주로 α1→6로 연결되어 있는 덱스트란을 생합성하는 효소를 생산하며, 상기 효소가 수크로오스를 이용하여 덱스트란을 생합성 하는 반응 기작은 다음과 같다:In other words, the leukotostock strain induces dextran sucrase induction, and the Streptococcus strain continuously produces dextran sucrase. Leuconostoc mesenteroides produces an enzyme that biosynthesizes dextran, in which glucose is primarily linked to α1 → 6, and the reaction mechanism by which the enzyme biosynthesizes dextran using sucrose is as follows:
수크로오스 글루칸(혹은 덱스트란) + 프럭토오스 + 류크로오스 Sucrose Glucan (or Dextran) + Fructose + Leucrose
상기 효소반응의 주된 산물은 약 107~108 Da 정도의 고분자량의 글루칸과 프럭토오스며 부산물로는 글루코오스와 류크로오스 (5-O-α-D- glucopyranosyl -Dfructopyranose) 가 생산된다. The main products of the enzymatic reaction are glucan and fructose of high molecular weight of about 10 7 ~ 10 8 Da, and glucose and leucose (5-O-α-D-glucopyranosyl -Dfructopyranose) are produced as by-products.
본 발명에 의한 각기 다른 류코노스톡속 균주는 다른 종류의 덱스트란 수크라아제를 생산하며 이에 의해 합성되는 덱스트란들은 그의 결합 형태나 정도가 효소에 따라 다르다.Different strains of the genus Leuconostok according to the present invention produce different types of dextran sucrase, and the dextran synthesized thereby is different in form or extent of binding depending on the enzyme.
현재까지 덱스트란의 결합은 주로 α1→3가 알려져 있으며, α1→2 및 α1→4 결합도 보고되어 있다. 대부분의 류코노스톡속 균주는 수크로오스를 효소 생산 기질에 넣어 주어야만 덱스트란수크라아제를 생산할 수 있으나, 수크로오스를 기질에 넣지 않아도 독특한 덱스트란수크라아제를 생산하는 구성적 돌연변이 균주들이 개발되어 있다 (Kim, D, Robyt, JF(1995b) Enz. hficrob. TeGhnol. 17, 689). 즉, 수크로오스가 아닌 글루코오스와 프럭토오스 등을 탄소원으로 사용하여도 생장시 덱스트란수크라아제를 생산함으로써, 수크로오스를 포함하는 LM 배지에서 생산하는 경우 글루칸수크라아제가 생산된 덱스트란과 결합된 생태로 얻어지는 것에 비해, 덱스트란에 오염되지 않은 단백질 만의 효소를 얻는 것이 가능해 진다.To date, dextran binding is mainly known as α1 → 3, and α1 → 2 and α1 → 4 binding have also been reported. Most strains of leukonostock can produce dextran sucrase only by adding sucrose to an enzyme-producing substrate, but constitutive mutant strains have been developed that produce unique dextran sucrase without sucrose. Kim, D, Robyt, JF (1995b) Enz. Hficrob.Teghnol. 17, 689). That is, even when glucose and fructose, but not sucrose, is used as a carbon source to produce dextran sucrose during growth, when it is produced in LM medium containing sucrose, glucan sucrase is bound to the produced dextran. Compared with the ecological results, it is possible to obtain enzymes only of proteins that are not contaminated with dextran.
한편, 효소 고생산 균주의 개발은 산업적으로 매우 큰 의미를 갖는다. 효소 생산을 위한 배지의 비용이 전체 효소 생산 비용의 80%를 차지하고 있으므로, 같은 비용의 배지를 사용하여 더 많은 효소를 생산하는 것은 효소의 산업적 활용을 위해 매우 중요하다. 본 발명에 따른 스테비오사이드 배당체의 고효율 생산을 위한 변형 류코노스톡속 균주는 모균보다 효소 생산성이 현저히 증가함으로써, 효소의 산업적 활용 증대를 기대할 수 있게 한다.On the other hand, the development of high-enzyme strains of industrial significance is very significant. Since the cost of the medium for enzyme production accounts for 80% of the total enzyme production cost, it is very important for the industrial use of the enzyme to produce more enzymes using the same cost medium. Modified leukonostok strain for high efficiency production of stevioside glycosides according to the present invention can be expected to increase the industrial productivity of the enzyme by significantly increasing the enzyme productivity than the parent.
본 발명의 스테비오사이드(stevioside) 배당체의 고효율 생산방법에 있어서, 상기 변형 류코노스톡속 균주는 화학적 돌연변이에 의해 변형된 것을 특징으로 한다. In the high-efficiency production method of the stevioside glycoside of the present invention, the modified leukonostok strain is characterized by being modified by chemical mutations.
상기 변형 류코노스톡 균주는 돌연변이 유발물질 처리에 의한 화학적 돌연변이법, UV 처리에 의한 물리적 변이법 또는 유전공학 조작에 의한 돌연변이법에 의해 수행될 수 있으며, 바람직하게는 돌연변이 유발물질 처리에 의한 화학적 돌연변이법에 의하여 변형시킬 수 있다. 상기 돌연변이 유발물질은 NTG(N-Methyl-N'-Nitro-N-Nitrosoguanidine), EMS(ethylmethane sulfonate) 및 트리에틸렌 멜라민(triethylene melamine)으로부터 1종 이상 선택될 수 있다. The modified leukonostock strain may be carried out by chemical mutagenesis by mutagenesis treatment, physical mutation by UV treatment or by mutagenesis by genetic engineering manipulation, preferably by chemical mutagenesis by mutagenesis treatment It can be modified by law. The mutagen may be selected from at least one of NTG (N-Methyl-N'-Nitro-N-Nitrosoguanidine), EMS (ethylmethane sulfonate) and triethylene melamine (triethylene melamine).
본 명세서에서 사용된 용어 "변형된(modified)" 또는 “조작된(engineered)” 균주는 유전 물질을 선택된 숙주 또는 모균 내로 도입하여 세포 생리와 생화학을 변형하거나 변경함으로써 생산된다. 유전 물질의 도입을 통하여, 모균은 새로운 성질, 예를 들면, 새로운 세포내 대사산물 또는 더욱 많은 양의 세포내 대사산물을 생산하는 능력을 획득한다. 예를 들면, 유전 물질의 모균 내로의 도입은 효소를 생산하는 새롭거나 변형된 능력을 초래한다. 모균에 도입된 유전 물질은 효소의 생산을 위한 생합성 경로에 관여하는 하나 이상의 효소를 코딩하는 유전자 또는 유전자의 일부를 포함하고, 이들 유전자의 발현 또는 발현 조절을 위한 추가 구성요소, 예를 들면, 프로모터 서열을 포함할 수도 있다. As used herein, the term “modified” or “engineered” strain is produced by introducing genetic material into a selected host or parent to alter or alter cell physiology and biochemistry. Through the introduction of genetic material, the parent acquires new properties, for example the ability to produce new intracellular metabolites or higher amounts of intracellular metabolites. For example, the introduction of genetic material into the parent cell results in a new or modified ability to produce enzymes. The genetic material introduced into the parent cell comprises a gene or part of a gene encoding one or more enzymes involved in the biosynthetic pathway for the production of enzymes, and additional components, such as promoters, for the expression or expression control of these genes It may also comprise a sequence.
본 발명의 스테비오사이드(stevioside) 배당체의 고효율 생산방법에 있어서, 상기 균주 배양 배지에 포함되는 수크로오스는 300 내지 1100mM 농도로 포함할 수 있으며, 더욱 바람직하게는 700 내지 1100Mm 농도로 포함되는 것을 특징으로 한다.In the high efficiency production method of stevioside glycosides of the present invention, the sucrose contained in the strain culture medium may be included in a concentration of 300 to 1100mM, more preferably 700 to 1100Mm concentration. .
본 발명의 스테비오사이드(stevioside) 배당체의 고효율 생산방법에 있어서, 상기 글루칸수크라아제는 1 내지 5 Unit/ml의 활성으로 사용될 수 있으며, 바람직하게는 3 내지 5 Unit/ml의 활성으로 사용되는 것을 특징으로 한다. In the high efficiency production method of the stevioside glycoside of the present invention, the glucan sucrase can be used with an activity of 1 to 5 Unit / ml, preferably used with an activity of 3 to 5 Unit / ml It features.
본 발명의 스테비오사이드(stevioside) 배당체의 고효율 생산방법에 있어서, B) 상기 배양에 의해 생성된 글루칸수크라아제(glucansucrase)와 수용체로서 스테비오사이드를 반응시키는 단계에서 상기 스테비오사이드는 30 내지 150mM 농도로 포함될 수 있으며, 바람직하게는 30 내지 90Mm 농도로 포함하는 것을 특징으로 한다.In the high-efficiency production method of stevioside glycoside of the present invention, B) the stevioside in the step of reacting the glucan sucrase (glucansucrase) produced by the culture with stevioside as a receptor at a concentration of 30 to 150mM It may be included, preferably characterized in that it comprises a 30 to 90Mm concentration.
본 발명의 스테비오사이드(stevioside) 배당체의 고효율 생산방법에 있어서, 상기 B) 단계에서 반응하는 글루칸수크라아제 농도(X1), 스테비오사이드의 농도(X2), A) 단계에서 배지에 함유되는 수크로오스의 농도(X3), 및 스테비오사이드 배당체의 생산율(Y) 사이에는 하기의 관계식이 성립하는 것을 특징으로 한다.In the high efficiency production method of the stevioside glycoside of the present invention, the glucan sucrase concentration (X1), stevioside concentration (X2), the step of sucrose contained in the medium in step A) The following relation holds between the concentration (X3) and the production rate (Y) of stevioside glycosides.
[관계식 1][Relationship 1]
Y= 46.466740+12.802404(X1)-0.226268(X2)+0.045081(X3)+0.030300(X1)(X2) +0.007352(X1)(X3)-0.000049(X2)(X3)-2.131815(X1)2+0.000087(X2)2-0.000043(X3)2
Y = 46.466740 + 12.802404 (X1) -0.226268 (X2) +0.045081 (X3) +0.030300 (X1) (X2) +0.007352 (X1) (X3) -0.000049 (X2) (X3) -2.131815 (X1) 2 +0.000087 (X2) 2 -0.000043 (X3) 2
본 발명의 일 실험예에 따르면, 상기 본 발명의 글루칸수크라아제 농도, 스테비오사이드의 농도 및 수크로오스의 농도 범위 내에서 스테비오사이드 배당체의 생산율이 전반적으로 우수한 것을 확인하였으며, 특히 효소 활성이 5 Unit/mL, 스테비오사이드 농도가 30 mM 및 수크로오스 농도가 1100mM 일 때 가장 높은 생산율을 보였다. 또한, 상기 관계식은 변수들을 대입해서 얻은 예상값과 실험값이 거의 유사하다는 것을 통해 유효성을 확인할 수 있었다.According to an experimental example of the present invention, it was confirmed that the production rate of stevioside glycosides in the glucan sucrase concentration, stevioside concentration and sucrose concentration range of the present invention is excellent overall, in particular, the enzyme activity is 5 Unit / The highest yield was obtained when the mL, stevioside concentration of 30 mM and sucrose concentration of 1100 mM. In addition, the relationship was able to confirm the validity through the fact that the expected value and the experimental value obtained by substituting the variables are almost similar.
본 발명의 스테비오사이드(stevioside) 배당체의 고효율 생산방법에 있어서, C) 상기 수용체 반응에 의해 생성된 스테비오사이드 배당체를 회수하는 단계; 에서는 상기 수용체 반응액에 탄소수 1 내지 4의 알코올을 첨가하여 스테비오사이드 배당체를 분리하는 것을 특징으로 한다.In the high efficiency production method of the stevioside glycoside of the present invention, C) Recovering the stevioside glycoside produced by the receptor reaction; Is characterized in that the stevioside glycoside is separated by adding an alcohol having 1 to 4 carbon atoms to the receptor reaction solution.
본 발명의 스테비오사이드(stevioside) 배당체의 고효율 생산방법에 있어서, 상기 탄소수 1 내지 4의 알코올은 70 내지 100%의 에탄올인 것을 특징으로 한다.In the high-efficiency production method of the stevioside glycoside of the present invention, the alcohol having 1 to 4 carbon atoms is characterized in that 70 to 100% ethanol.
본 발명은 또한, 상기 제조방법으로 제조된 하기 화학식 1로 표시된 스테비오사이드 배당체를 제공한다.The present invention also provides a stevioside glycoside represented by the following formula (1) prepared by the above method.
[화학식 1][Formula 1]
본 발명은 또한, 상기 제조방법으로 제조된 하기 화학식 2로 표시된 스테비오사이드 배당체를 제공한다.The present invention also provides a stevioside glycoside represented by the following formula (2) prepared by the above method.
[화학식 2][Formula 2]
본 발명에 따른 화학식 1의 스테비오사이드 배당체는 말디토프 질량 분석 결과 m/z가 980 내지 990이며, 화학식 2의 스테비오사이드 배당체는 말디토프 질량 분석 결과 m/z가 1140 내지 1160인 것을 특징으로 한다.The stevioside glycoside of Formula 1 according to the present invention has a malditope mass spectrometry m / z of 980 to 990, and the stevioside glycoside of Formula 2 has a malditope mass spectrometry of m / z of 1140 to 1160.
본 발명은 또한, 상기 화학식 1 또는 화학식 2로 표시되는 스테비오사이드 배당체를 포함하는 감미료를 제공한다.The present invention also provides a sweetener comprising a stevioside glycoside represented by Formula 1 or Formula 2.
전술한 바와 같이, 본 발명에 따른 스테비오사이드 배당체 생산 방법은 스테비오사이드 배당체를 고효율로 생산할 수 있을 뿐만 아니라 신규한 스테비오사이드 배당체를 합성함으로써 감미료로 이용될 수 있다.As described above, the stevioside glycoside production method according to the present invention can not only produce stevioside glycosides with high efficiency but also can be used as a sweetener by synthesizing novel stevioside glycosides.
도 1은 류코노스톡 효소들의 스테비오사이드 수용체 반응 산물의 TLC 결과를 나타내는 도면이다.1 is a diagram showing the TLC results of stevioside receptor reaction products of leuconostock enzymes.
도 2는 실험예 3-1에 따른 TLC 결과를 나타낸다.2 shows TLC results according to Experimental Example 3-1.
도 3은 실험예 3-2에 따른 TLC 결과를 나타낸다.3 shows TLC results according to Experimental Example 3-2.
도 4는 실시예 3에 따른 TLC 결과를 나타낸다.4 shows TLC results according to Example 3. FIG.
도 5는 실험예 4에 따른 HPLC 결과를 나타낸다.5 shows the HPLC results according to Experimental Example 4.
도 6은 실험예 4에 따른 TLC 결과를 나타낸다.6 shows TLC results according to Experimental Example 4.
도 7은 스테비올 배당체의 최적화를 위한 3가지 변수의 농도를 확인한 결과를 나타낸다.Figure 7 shows the results of confirming the concentration of the three variables for the optimization of steviol glycosides.
도 8은 실험예 6에 따른 시료 1의 Maldi-tof 분석 결과를 나타낸다.8 shows the results of Maldi-tof analysis of Sample 1 according to Experimental Example 6. FIG.
도 9는 실험예 6에 따른 시료 2의 Maldi-tof 분석 결과를 나타낸다.9 shows a Maldi-tof analysis result of Sample 2 according to Experimental Example 6. FIG.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: 모균의 배양Example 1 Cultivation of Mother Cells
글루코오스 (18.8 g/L) 또는 수크로오스 (25 g/L), 박토펩톤 (4.2 g/L), 효모 추출물 (4.2 g/L), K2HPO4 (글루코오스의 경우 20 g/L, 수크로오스의 경우 16.7g/L), MgSO4 (0.17 g/L), NaCl (0.008 g/L), FeSO47H2O (0.008 g/L), MnSO42H2O (0.008 g/L), 및 CaCl22H2O (0.011 g/L)를 포함하는 배지를 준비하고, Leuconostoc mesenteroides B-512F, L. mesenteroides B-512FMCM, L. mesenteroidesB-1299,L. mesenteroidesB-1299C, L. citreum B-1355 및 L. citreum B-1355C 균주를 28℃에서 배양하였다.Glucose (18.8 g / L) or sucrose (25 g / L), bactopeptone (4.2 g / L), yeast extract (4.2 g / L), K 2 HPO 4 (20 g / L for glucose, for sucrose) 16.7 g / L), MgSO 4 (0.17 g / L), NaCl (0.008 g / L), FeSO 4 7H 2 O (0.008 g / L), MnSO 4 2H 2 O (0.008 g / L), and CaCl 2 Prepare a medium containing 2H 2 O (0.011 g / L), Leuconostoc mesenteroides B-512F, L. mesenteroides B-512FMCM, L. mesenteroides B-1299, L. mesenteroides B-1299C, L. citreum B-1355 and L. citreum B-1355C strains were incubated at 28 ° C.
실시예 2: 변형 류코노스톡속 균주의 제작Example 2 Preparation of Modified Leukonostok Strains
실시예 1에 따른 L. mesenteroides B-512FMCM, L. mesenteroides B-1299C 및 L. citreum B-1355C를 1.2mL의 글루코오스 배지에 접종하여 24시간 동안 배양한 후 200㎕를 원심분리하고 상등액을 제거하였다. 그 다음, 상기 분리된 균주들을 200㎕의 sterile 20mM sodium-citrate buffer pH 6.0로 3회 세척하였다. L. mesenteroides B-512FMCM, L. mesenteroides B-1299C and L. citreum B-1355C according to Example 1 were inoculated in 1.2 mL of glucose medium and incubated for 24 hours, followed by centrifugation and 200 μl of the supernatant. . The isolated strains were then washed three times with 200 μl sterile 20 mM sodium-citrate buffer pH 6.0.
세척된 균주에 화학적 돌연변이를 유도하기 위해 200㎕의 500㎍/ml-1N-methyl-N'-nitro-N-nitrosoguanidine (NTG) solution을 넣고 21℃의 온도에서 30분 동안 배양하였다. 상기 배양된 균주들을 20mM sodium citrate buffer pH 6.0로 3회 세척한 후 500㎕의 글루코오스 배지에 넣고 21℃의 온도에서 3시간 동안 배양하였다. In order to induce chemical mutations in the washed strain 200μl of 500μg / ml-1N-methyl-N'-nitro-N-nitrosoguanidine (NTG) solution was added and incubated for 30 minutes at a temperature of 21 ℃. The cultured strains were washed three times with 20 mM sodium citrate buffer pH 6.0 and placed in 500 μl glucose medium and incubated at 21 ° C. for 3 hours.
배양된 균주 배양액을 1:105으로 희석한 후, 100㎕를 글루코오스를 포함하는 아가(agar)배지 (2.5%)에 플레이팅 했다.After diluting the cultured strain culture to 1:10 5 , 100ul was plated on agar medium (2.5%) containing glucose.
실험예 1: 모균 및 변형 류코노스톡속 균주의 효소 생산성 비교Experimental Example 1 Comparison of Enzyme Productivity of Mother and Modified Leukonostok Strains
상기 실시예에 따른 각각의 균주들을 글루코오스 및 수크로오스를 포함하는 액체 배지에서 24시간 동안 배양한 후, 원심분리를 통해 상등액을 얻어 100mM 설탕과 1:1(v/v)로 혼합하였다. 그 다음, 28℃의 온도에서 반응시키고, 반응 후 생성된 당 글루칸을 원심 분리하여 모은 뒤 1 M NaOH를 이용하여 녹인 후 TLC에 점적하여 전개시켰다. Each strain according to the above example was incubated for 24 hours in a liquid medium containing glucose and sucrose, and then the supernatant was obtained by centrifugation and mixed with 1: 1 (v / v) with 100 mM sugar. Then, the mixture was reacted at a temperature of 28 ° C., and the resulting sugar glucan was collected by centrifugation, dissolved in 1 M NaOH, and developed by dropping in TLC.
전개가 끝나면, 실온에서 건조시킨 후에 에탄올:황산을 9:1로 섞은 발색 시약을 스프레이로 TLC판에 골고루 분사하여 120℃ 에서 15 내지 20분 동안 태워서 발색한 후 AlphaEase FC 프로그램을 이용하여 활성 여부를 계산하고, 그 결과를 표 1에 나타내었다. 효소 1유닛은 반응 조건 1분 하에서 1mM의 프럭토오스를 수크로오스부터 생산하는 효소의 능력을 의미한다.After the development, it was dried at room temperature, and then evenly sprayed onto the TLC plate with a ethanol: sulfuric acid 9: 1 color development reagent, followed by burning at 120 ° C. for 15 to 20 minutes to develop color activity using AlphaEase FC program. Calculations are shown in Table 1. One unit of enzyme refers to the ability of an enzyme to produce 1 mM fructose from sucrose under one minute of reaction conditions.
글루코오스 배지(IU ml-1)Glucose medium (IU ml -1 ) | 수크로오스 배지(IU ml-1)Sucrose medium (IU ml -1 ) | |
B-512FB- |
00 | 0.010.01 |
B-512FMCMB-512FMCM | 6.56.5 | 8.28.2 |
B-512F/KMB-512F / KM | 12.312.3 | 13.113.1 |
B-1299B-1299 | 00 | 0.010.01 |
B-1299CB-1299C | 0.10.1 | 0.30.3 |
B-1299C/KMB-1299C / KM | 0.80.8 | 1.51.5 |
B-1355B-1355 | 00 | 0.020.02 |
B-1355CB-1355C | 0.320.32 | 0.60.6 |
B-1355C/KMB-1355C / KM | 1.11.1 | 1.71.7 |
그 결과, 상기 표 1에서 확인할 수 있는 바와 같이, 본 발명의 변형 류코노스톡속 균주인 B-512F/KM, B-1299C/KM 및 B-1355C/KM의 효소 생산성이 모균보다 2배 이상 높은 것을 확인하였다.As a result, as can be seen in Table 1, the enzyme productivity of the modified Leukonostok strains B-512F / KM, B-1299C / KM and B-1355C / KM of the present invention is more than twice as high as the parent It was confirmed.
실험예 2: 류코노스톡 메센테로이데스 B-1299C/KM, B-512F/KM 혹은 류코노스톡 시트레움 B-1355C/KM 효소를 이용한 스테비오사이드 배당체 수용체 반응 및 생산 스테비오사이드 배당체 확인Experimental Example 2 Stevioside Glycoside Receptor Reaction and Production of Stevioside Glycosides Using Leuconosestock Mesenteroides B-1299C / KM, B-512F / KM or Leuconostok Citrium B-1355C / KM Enzyme
수크로오스(S)는 sigma로부터 구입하고, 스테비오사이드는 ㈜ 대평 (경기도, 한국)에서 구입하고, 효소는 상기 실험예 1에 따라 류코노스톡 시트레움 B-1355C/KM 균주에서 유래한 것을 이용하였다. Sucrose (S) was purchased from sigma, stevioside was purchased from Daepyung Co., Ltd. (Gyeonggi-do, Korea), and the enzyme was derived from the Leukonostock Citrium B-1355C / KM strain according to Experimental Example 1.
상기 실시예 2에서 제조한 류코노스톡 메센테로이데스 B-1299C/KM, 류코노스톡 시트리움 B-1355C/KM 균주로부터 생산된 효소 0.9 U/ml, 그리고 류코노스톡 메센테로이데스 B-512F/KM 균주로부터 생산한 효소 5 U/ml를 수크로오스(S) 600mM, 및 수크로오스(S) 600mM 및 스테비오사이드(Ste) 100mM 혼합물과 각각 28℃에서 6시간 동안 반응(R)시킨 후, 얇은막크로마토그래피(TLC)를 수행하였다. 얇은막크로마토그래피 전개 용매로는 nitromethane: n-propanol: water(2:5:1.5, v/v/v)를 사용하여 한 번 전개하였다. 그 결과는 도 1과 같다.0.9 U / ml of enzymes produced from the Leuconosestock mesenteroides B-1299C / KM, the leukonostock citrium B-1355C / KM strain prepared in Example 2, and leukonostock mesenteroides B-512F / 5 U / ml of the enzyme produced from the KM strain was reacted (R) with sucrose (S) 600 mM and sucrose (S) 600 mM and 100 mlM stevioside (Ste) at 28 ° C. for 6 hours, followed by thin layer chromatography. (TLC) was performed. Thin layer chromatography was developed once using nitromethane: n-propanol: water (2: 5: 1.5, v / v / v). The result is shown in FIG.
도 1을 참고하면, 상기 레인 1 내지 3 모두 스테비오사이드 배당체로 전환된 것을 확인할 수 있다((Frc; 프락토오스. STV; 스테비오사이드 수용체, 래인 1; B-1299C/KM 유래 효소를 이용한 수용체 반응산물, 래인 2; B-1355C/KM 유래 효소를 이용한 수용체 반응산물, 래인 3; B-512F/KM 유래 효소를 이용한 수용체 반응산물).Referring to Figure 1, it can be seen that lanes 1 to 3 are all converted to stevioside glycosides ((Frc; fructose. STV; stevioside receptor, lane 1; receptor reaction products using B-1299C / KM derived enzyme) , Lane 2; receptor reaction product using B-1355C / KM derived enzyme, lane 3; receptor reaction product using B-512F / KM derived enzyme).
실험예 3:. 류코노스톡 메센테로이데스 B-512F/KM의 효소 활성 온도에 따른 스테비오사이드 배당체 생산 비교Experimental Example 3: Comparison of Stevioside Glycoside Production According to Enzyme Activity Temperature of Leukonostock Mesenteroides B-512F / KM
3-1. 류코노스톡 메센테로이데스 B-512F/KM의 효소 반응 시간에 따른 스테비오사이드 배당체 생산 비교3-1. Comparison of Stevioside Glycoside Production According to Enzyme Reaction Time of Leukonostock Mesenteroides B-512F / KM
설탕 350 mM, 1% 스테비오사이드, 512F/KM 덱스트란수크라아제 (1-10 U/mL) 를 20mM Na-세테이트 buffer를 이용하여 pH 5.2로 준비하였다. Sugar 350 mM, 1% stevioside, 512 F / KM dextran sucrase (1-10 U / mL) was prepared at pH 5.2 using 20 mM Na-tate buffer.
스테비오사이드 배당체의 생산 반응은 28℃에서 1시간 내지 24시간 동안 진행하고, 그 결과를 도 2에 나타내었다. The production reaction of stevioside glycosides was carried out at 28 ° C. for 1 to 24 hours, and the results are shown in FIG. 2.
도 2를 참고하면, 1유닛의 효소를 이용한 경우 24시간 반응 후 초기 스테비오사이드(화살표로 표시함)는 모두 스테비오사이드 배당체로 전환됨이 확인되었다. 효소의 활성을 2유닛으로 증가시킨 경우 7시간 후에 거의 모든 스테비오사이드가 수용체 반응 후 스테비오사이드 배당체로 전환 됨이 확인되었다.Referring to FIG. 2, it was confirmed that the initial steviosides (indicated by arrows) are all converted to stevioside glycosides after the reaction for 24 hours using 1 unit of enzyme. When the activity of the enzyme was increased to 2 units, it was confirmed that after 7 hours, almost all steviosides were converted to stevioside glycosides after the receptor reaction.
3-2. 류코노스톡 메센테로이데스 B-512F/KM의 고정된 활성의 효소 반응에서 설탕 농도에 따른 스테비오사이드 배당체 생산 비교3-2. Comparison of Stevioside Glycoside Production According to Sugar Concentration in Enzymatic Reaction of Fixed Activity of Leuconostock Mesenteroides B-512F / KM
설탕 100mM - 1M, 2% 스테비오사이드, 덱스트란수크라아제 3U/mL을 20 mM Na-아세테이트 buffer를 이용하여 pH 5.2로 준비하였다. Sugar 100 mM-1M, 2% stevioside, dextran sucrose 3U / mL was prepared at pH 5.2 using 20 mM Na-acetate buffer.
스테비오사이드 배당체 생산 반응은 28℃에서 7시간 동안 진행하고, TLC를 이용하여 당과 스테비오사이드 배당체를 확인하고, 그 결과를 도 3에 나타내었다. 이때 TLC 전개 용매는 아세토니트릴(acetonitrile):물 = 85:15 (v/v), 그리고 이후 니트로메탄(nitromethane):엔-프로필 알코올(n-propyl alcohol):물 = 2:5:1.5 (v/v)를 이용하였다.Stevioside glycoside production reaction proceeds for 28 hours at 28 ℃, and confirmed the sugar and stevioside glycosides using TLC, the results are shown in FIG. Wherein the TLC developing solvent was acetonitrile: water = 85:15 (v / v), and then nitromethane: n-propyl alcohol: water = 2: 5: 1.5 (v / v) was used.
도 3을 참고하면, 7시간 반응 후 점적 한 결과 100 mM - 500 mM 설탕 모두에서 비슷한 분포의 스테비오사이드 배당체 합성이 잘 이루어졌음이 확인되었다.Referring to FIG. 3, after 7 hours of reaction, it was confirmed that the synthesis of stevioside glycosides of similar distribution was well performed in all 100 mM-500 mM sugars.
실시예 3: 효소 반응 후 반응액에서 단당과 glucosyl-stevioside 분리Example 3: Single Sugar and Glucosyl-stevioside Separation from Reaction Solution after Enzyme Reaction
준비한 효소 반응(설탕 500 mM, 류코노스톡 메센테로이데스 B-512F/KM의 효소 3 U/mL, Na-Ac buffer 20 mM, 반응시간 12 시간) 후 반응액에 90% ethyl alcohols을 넣어 올리고당 제거 한 상등액을 분리한다. 분리한 상등액을 Diaion HP- 20 resin 컬럼에 로딩하고, 물을 이용하여 단당을 제거하였다. 이후 남아 있는 glucosyl steviosides를 회수하기 위해서 80% ethyl alcohols을 이용하였다. 도 4와 같이 70% 내지 100%의 에탄올을 이용하였을 때 용출성분을 확인할 수 있었으며, 이에 따라 80% 에탄올을 이용하였다. After the prepared enzyme reaction (500 mM sugar, 3 U / mL of leukonostock mesenteroides B-512F / KM enzyme, Na-Ac buffer 20 mM, reaction time 12 hours), 90% ethyl alcohols were added to the reaction solution to remove oligosaccharides. Separate one supernatant. The separated supernatant was loaded on a Diaion HP-20 resin column, and monosaccharide was removed using water. Since 80% ethyl alcohols were used to recover the remaining glucosyl steviosides. When using 70% to 100% ethanol as shown in Figure 4 was able to confirm the elution component, accordingly used 80% ethanol.
이때 80% 에탄올로 용해되어 나오는 산물을 모두 회수하였다. 회수한 액은 rotary evaporator (Heidolph, Germany)를 이용하여 45°C에서 농축하고 freeze dryer (EYELA, Japan)를 이용하여 분말화하였다. 이 분말은 TLC plate (Silica Gel 60, Merck, Darmstadt, Germany)에 점적하여 nitromethan:n-propyl:water = 2:5:1.5 (v/v) 전개용매로 1회, 그리고 acetonitrile:water = 85:15 (v/v) 용매로 1회 전개 한 후, 0.3% (w/v) N-(1-naphthyl)-ethylene diamine과 5% (v/v) H2SO4 이 들어 있는 메탄올 용액에 담근 후 말리고 125 °C에서 5 min 간 구워 발색 하였다. At this time, all the products dissolved in 80% ethanol were recovered. The recovered solution was concentrated at 45 ° C using a rotary evaporator (Heidolph, Germany) and powdered using a freeze dryer (EYELA, Japan). This powder was added to a TLC plate (Silica Gel 60, Merck, Darmstadt, Germany), once with nitromethan: n-propyl: water = 2: 5: 1.5 (v / v) developing solvent, and acetonitrile: water = 85: Once developed with 15 (v / v) solvent, soaked in methanol solution containing 0.3% (w / v) N- (1-naphthyl) -ethylene diamine and 5% (v / v) H 2 SO 4 It was then dried and baked for 5 min at 125 ° C.
실험예 4: High Pressure Liquid Chromatography (HPLC)를 이용한 glucosyl stevioside 정제Experimental Example 4: Purification of Glucosyl Stevioside by High Pressure Liquid Chromatography (HPLC)
상기 실시예 3에서 준비한 glucosyl-stevioside를 정제하기 위하여 High Pressure Liquid Chromatography (HPLC)를 이용하였다.High Pressure Liquid Chromatography (HPLC) was used to purify the glucosyl-stevioside prepared in Example 3.
구체적으로, 상기 실시예 3에서 주비한 시료를 NH2 column(5μm, 150 x 4.6 mm)이 장착된 HPLC-RI system을 이용하여 정제하였으며, 이때 정제 조건은 하기 표 2와 같다. 그 결과를 도 5에 나타내었다.Specifically, the sample prepared in Example 3 was purified using an HPLC-RI system equipped with NH 2 column (5μm, 150 x 4.6 mm), wherein the purification conditions are shown in Table 2 below. The results are shown in FIG.
InstrumentInstrument | HPLC conditionHPLC condition |
columncolumn | Amino columnAmino column |
Mobile phaseMobile phase | A : 75% 아세토나이트릴A: 75% acetonitrile |
Flow rateFlow rate |
1.0 mL/min1.0 mL / |
Column temperatureColumn temperature | 40 °C40 ° C |
Injection vol.Injection vol. | 20 μL20 μL |
Analysis timeAnalysis time | 30 min30 min |
그 결과, HPLC에서 1 내지 3의 시료를 얻을 수 있었으며(도 5), 여기서 얻은 1 내지 3의 시료로 TLC 크로마토그램을 통해 glucosyl-stevioside를 확인하였다(도 6). As a result, a sample of 1 to 3 was obtained by HPLC (FIG. 5), and the glucosyl-stevioside was confirmed by TLC chromatogram with the samples of 1 to 3 obtained here (FIG. 6).
이후, 상기에서 얻은 1 내지 3의 glucosyl-stevioside를 정체하고 구조 결정을 확인하였다. Subsequently, the glucosyl-stevioside of 1 to 3 obtained above was stagnated and the structure was confirmed.
실험예 5: 스테비올 배당체 생산 최적화(반응표면분석: response surface methodology: RSM)Experimental Example 5: Optimization of Steviol Glycoside Production (response surface methodology: RSM)
반응표면분석(response surface methodology: RSM)을 이용하여 stevioside에서 glucosyl-stevioside가 생산되는 정도를 최적화 하고자 하였다. Response surface methodology (RSM) was used to optimize the production of glucosyl-stevioside from steviosides.
고려한 3가지 변수는 효소농도(x1), 설탕농도(x2) 및 스테비오사이드농도(x3) 였으며, 실험 조건은 하기 표 3의 조건에서 진행하였다.Three variables considered were enzyme concentration (x1), sugar concentration (x2) and stevioside concentration (x3), and the experimental conditions were carried out under the conditions shown in Table 3 below.
Run no. Run no. | Coded number Stevioside 전환 (%) Coded number Stevioside conversion (%) | ||||
X1 X1 | X2 X2 | X3 X3 | 실험값 Experimental value | 예상값 Expected value | |
Unit/mL Unit / mL | Stevioside(mM) Stevioside (mM) | Sucrose(mM) Sucrose (mM) | |||
1 One | 1 One | 150 150 | 300 300 | 40.42 40.42 | 39.37 39.37 |
2 2 | 3 3 | 90 90 | 700 700 | 80.39 80.39 | 77.25 77.25 |
3 3 | 1 One | 30 30 | 300 300 | 63.43 63.43 | 62.79 62.79 |
4 4 | 3 3 | 90 90 | 700 700 | 77.92 77.92 | 77.25 77.25 |
5 5 | 3 3 | 90 90 | 700 700 | 76.68 76.68 | 77.25 77.25 |
6 6 | 5 5 | 150 150 | 1100 1100 | 77.85 77.85 | 78.34 78.34 |
7 7 | 5 5 | 30 30 | 300 300 | 78.27 78.27 | 75.30 75.30 |
8 8 | 5 5 | 90 90 | 700 700 | 84.72 84.72 | 84.50 84.50 |
9 9 | 1 One | 30 30 | 1100 1100 | 57.86 57.86 | 55.93 55.93 |
10 10 | 5 5 | 150 150 | 300 300 | 64.63 64.63 | 66.42 66.42 |
11 11 | 1 One | 150 150 | 1100 1100 | 24.95 24.95 | 27.76 27.76 |
12 12 | 1 One | 90 90 | 700 700 | 52.16 52.16 | 52.96 52.96 |
13 13 | 3 3 | 150 150 | 700 700 | 72.36 72.36 | 68.30 68.30 |
14 14 | 3 3 | 90 90 | 700 700 | 79.90 79.90 | 77.25 77.25 |
15 15 | 3 3 | 90 90 | 700 700 | 75.77 75.77 | 77.25 77.25 |
16 16 | 3 3 | 90 90 | 700 700 | 74.00 74.00 | 77.25 77.25 |
17 17 | 3 3 | 90 90 | 1100 1100 | 74.00 74.00 | 71.72 71.72 |
18 18 | 5 5 | 30 30 | 1100 1100 | 91.05 91.05 | 91.96 91.96 |
19 19 | 3 3 | 90 90 | 300 300 | 66.33 66.33 | 69.19 69.19 |
20 20 | 3 3 | 30 30 | 700 700 | 82.20 82.20 | 86.83 86.83 |
3가지 변수, 효소농도(x1), 설탕농도(x2) 및 스테비오사이드농도(x3)와 스테비오사이드 전환율(Y) 와의 상관관계는 The three variables, enzyme concentration (x1), sugar concentration (x2), and stevioside concentration (x3) and stevioside conversion (Y)
Y= 46.466740 + 12.802404(X1)-0.226268(X2)+0.045081(X3)+0.030300(X1)(X2) +0.007352(X1)(X3)-0.000049(X2)(X3)-2.131815(X1)2+0.000087(X2)2-0.000043(X3)2 로 확인되었다. Y = 46.466740 + 12.802404 (X1) -0.226268 (X2) +0.045081 (X3) +0.030300 (X1) (X2) +0.007352 (X1) (X3) -0.000049 (X2) (X3) -2.131815 (X1) 2 +0.000087 (X2) 2 -0.000043 (X3) 2 It was confirmed.
이 식은 변수들의 대입에서 얻은 예상치와 실험치가 거의 동일함을 확인함으로써 유효성이 확인할 수 있었다.This equation was validated by confirming that the expected and experimental values from the substitution of the variables were nearly identical.
실험예 6: 말디토프(MALDI-TOF) 질량분석Experimental Example 6: MALDI-TOF Mass Spectrometry
말디토프 질량분석기 (MALDI-TOF MS, Matrix-Assisted Laser Desorption Ionization -Time-of-Flight Mass Spectrometer)를 이용하여 스테비올 배당체의 분자량(시료 1 및 시료 2)을 확인하고 그 결과를 도 8 및 도 9에 나타내었다. The molecular weight (Sample 1 and Sample 2) of steviol glycosides was confirmed using a Maldi-TOF MS (MALDI-TOF MS, Matrix-Assisted Laser Desorption Ionization -Time-of-Flight Mass Spectrometer) and the results are shown in FIGS. 8 and FIG. 9 is shown.
구체적으로, 시료 1 및 시료 2의 스테비올 배당체(3 mg/mL)를 탈이온수로 녹인 다음 Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster, CA, USA)를 이용하여 분자량 결정하였다. Mass spectra는 positive reflector mode로 얻었는데, 평균 300 laser shots으로 하였고, acceleration voltage는 25 kV 로 하여 그 결과를 도 8 및 도 9에 나타내었다. Specifically, steviol glycosides (3 mg / mL) of Samples 1 and 2 were dissolved in deionized water and then molecular weight was determined using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster, CA, USA). Mass spectra was obtained in a positive reflector mode, with an average of 300 laser shots and an acceleration voltage of 25 kV. The results are shown in FIGS. 8 and 9.
분자량을 분석한 결과, 정제한 시료 1, 2에 대한 말디토프 질량분석기 (MALDI-TOP) 분석 결과로 얻은 분자량 결과로부터 스테비오사이드에 글루코오스가 1-2개 결합된 시료인 것을 확인하였다. As a result of analyzing the molecular weight, it was confirmed from the molecular weight result obtained by the Malditop mass spectrometer (MALDI-TOP) analysis of the purified samples 1 and 2 that the sample was bound to 1-2 glucose in stevioside.
또한, 시료 1(Compounds 1)의 분자량은 m/z 989.5 (M + Na)+로 한 개의 글루코오스가 결합된 시료이며, 시료 2(Compounds 2)는 m/z 1151.4 (M + Na)+으로 2개의 포도당이 stevioside에 결합된 것으로 확인되었다.In addition, the molecular weight of Sample 1 (Compounds 1) is m / z 989.5 (M + Na) + is a sample combined with one glucose, Sample 2 (Compounds 2) is m / z 1151.4 (M + Na) + 2 Dog glucose was found to bind to stevioside.
실험예 7: Nuclear Magnetic Resonance (NMR) 분석Experimental Example 7: Nuclear Magnetic Resonance (NMR) Analysis
각각 정제한 스테비오사이드 배당체 (10 mg) (시료 1 및 시료 2) 를 600μL DMSO-d6 에 녹이고 5 mm NMR tubes에 넣어 준비하였다. NMR spectra는 AVANCEIII system(Bruker, Germany) 1H NMR 경우 850 MHz로, 13C NMR경우 125 MHz로 25 °C에서 분석하였다. Each purified stevioside glycoside (10 mg) (Sample 1 and Sample 2) was dissolved in 600 μL DMSO-d6 and prepared in 5 mm NMR tubes. NMR spectra were analyzed at 25 ° C with AVANCEIII system (Bruker, Germany) at 850 MHz for 1H NMR and 125 MHz for 13C NMR.
결합구조는 homonuclear correlation spectroscopy(COSY), total correlation spectroscopy (TOCSY) heteronuclear single-quantum coherence (HSQC), 및 heteronuclear multiple-bond correlation (HMBC)로 확인하고 그 결과를 하기 표 4 및 표 5에 나타내었다.The binding structure was confirmed by homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC), and the results are shown in Tables 4 and 5 below.
또한, 시료 1 및 2의 구조를 확인한 결과, 시료 1의 Glucosyl-stevioside은 하기 화학식 2와 같이 13-O-β-sophorosyl-19-O-β-isomaltosyl-steviol이며, 시료 2의 Glucosyl-stevioside는 하기 화학식 1과 같이 13-O-[β-D-glucosyl(1 → 2)-β-nigerosyl]-19-O-β-isomaltosyl-steviol로서 신규 화합물임을 확인하였다. In addition, as a result of confirming the structure of Samples 1 and 2, Glucosyl-stevioside of Sample 1 is 13-O-β-sophorosyl-19-O-β-isomaltosyl-steviol as shown in the following formula 2, Glucosyl-stevioside of Sample 2 is As shown in Chemical Formula 1, 13-O- [β-D-glucosyl (1 → 2) -β-nigerosyl] -19-O-β-isomaltosyl-steviol was identified as a novel compound.
[화학식 1][Formula 1]
[화학식 2][Formula 2]
이상 설명한 바와 같이, 본 발명에 따르면 본 발명에 따른 스테비오사이드 배당체의 생산 방법은 스테비오사이드 배당체를 고효율로 생산할 수 있을 뿐만 아니라 신규한 스테비오사이드 배당체를 합성함으로써, 궁극적으로 천연감미료로서 효과적으로 적용할 수 있다.As described above, according to the present invention, the method for producing stevioside glycosides according to the present invention can not only produce stevioside glycosides with high efficiency, but also can be effectively applied as a natural sweetener by synthesizing novel stevioside glycosides. .
Claims (13)
- A) 류코노스톡 메센테로이데스 B-512F/KM(KCCM11728P), 류코노스톡 메센테로이데스 B-1299C/KM(KCCM11729P) 및 류코노스톡 시트레움 B-1355C/KM(KCCM11730P)로 구성된 군으로부터 선택된 1종 이상의 변형 류코노스톡속 균주를 수크로오스(sucrose) 함유 배지에서 배양하여 글루칸스쿠라아제를 생성하는 단계; A) selected from the group consisting of leukonostock mesenteroides B-512F / KM (KCCM11728P), leukonostock mesenteroides B-1299C / KM (KCCM11729P) and leukonostock citrium B-1355C / KM (KCCM11730P) Culturing one or more strains of the genus Leuconostok in a sucrose-containing medium to produce glucanscurase;B) 상기 배양에 의해 생성된 글루칸수크라아제(glucansucrase) 및 수용체로서 스테비오사이드를 반응시켜서 스테비오사이드 배당체를 생성하는 단계; 및B) reacting stevioside as a glucansucrase and a receptor produced by the culture to produce stevioside glycosides; AndC) 상기 수용체 반응액에서 생성된 스테비오사이드 배당체를 회수하는 단계;를 포함하는 C) recovering the stevioside glycosides produced in the receptor reaction solution;스테비오사이드(stevioside) 배당체의 고효율 생산방법.High efficiency production method of stevioside glycosides.
- 제1항에 있어서, The method of claim 1,상기 수크로오스는 300 내지 1100mM 농도로 포함되는 것인The sucrose is included in a concentration of 300 to 1100mM스테비오사이드(stevioside) 배당체의 고효율 생산방법.High efficiency production method of stevioside glycosides.
- 제1항에 있어서, The method of claim 1,상기 글루칸수크라아제는 1 내지 5 Unit/ml의 활성으로 사용되는 것인Wherein the glucan sucrase is to be used with an activity of 1 to 5 Unit / ml스테비오사이드(stevioside) 배당체의 고효율 생산방법.High efficiency production method of stevioside glycosides.
- 제1항에 있어서, The method of claim 1,상기 스테비오사이드는 30 내지 150mM 농도로 포함하는 것인The stevioside is to include at a concentration of 30 to 150mM스테비오사이드(stevioside) 배당체의 고효율 생산방법.High efficiency production method of stevioside glycosides.
- 제1항에 있어서, The method of claim 1,상기 B) 단계의 글루칸수크라아제 농도(X1), 스테비오사이드의 농도(X2), A) 단계의 수크로오스의 농도(X3)와 상기 C)단계의 분리되는 스테비오사이드 배당체의 생산량(Y)은 하기 관계식 1을 만족하는 것인 The glucan sucrase concentration (X1) of step B), the concentration of stevioside (X2), the concentration of sucrose (X3) of step A) and the production amount of the isolated stevioside glycoside of step C) (Y) are as follows. Satisfying relation 1스테비오사이드(stevioside) 배당체의 고효율 생산방법.High efficiency production method of stevioside glycosides.[관계식 1][Relationship 1]Y = 46.466740+12.802404(X1)-0.226268(X2) + 0.045081 (X3) + 0.030300 (X1)(X2)+0.007352(X1)(X3)-0.000049(X2)(X3)-2.131815(X1)2+0.000087(X2)2-0.000043(X3)2 Y = 46.466740 + 12.802404 (X1) -0.226268 (X2) + 0.045081 (X3) + 0.030300 (X1) (X2) +0.007352 (X1) (X3) -0.000049 (X2) (X3) -2.131815 (X1) 2 +0.000087 (X2) 2 -0.000043 (X3) 2
- 제1항에 있어서, The method of claim 1,상기 c)반응에 의해 생성된 스테비오사이드 배당체를 회수하는 단계에서는 상기 수용체 반응액에 탄소수 1 내지 4의 알코올을 첨가하여 스테비오사이드 배당체를 분리하는 것인In the step of recovering the stevioside glycoside produced by the reaction c) is to separate the stevioside glycoside by adding an alcohol having 1 to 4 carbon atoms to the receptor reaction solution스테비오사이드(stevioside) 배당체의 고효율 생산방법.High efficiency production method of stevioside glycosides.
- 제6항에 있어서, The method of claim 6,상기 탄소수 1 내지 4의 알코올은 50 내지 100%의 에탄올인 것인The alcohol having 1 to 4 carbon atoms is 50 to 100% ethanol스테비오사이드(stevioside) 배당체의 고효율 생산방법.High efficiency production method of stevioside glycosides.
- 제8항에 있어서, The method of claim 8,상기 스테비오사이드 배당체는 말디토프 질량 분석 결과 m/z 의 범위가 980 내지 990 인 것인 The stevioside glycosides are malditope mass spectrometry and the range of m / z is 980 to 990.스테비오사이드 배당체.Stevioside glycosides.
- 제9항에 있어서, The method of claim 9,상기 스테비오사이드 배당체는 말디토프 질량 분석 결과 m/z 1140 내지 1160 인 것인 The stevioside glycoside is that of malditope mass spectrometry m / z 1140 to 1160스테비오사이드 배당체.Stevioside glycosides.
- 제10항의 스테비오사이드 배당체를 포함하는 감미료.A sweetener comprising the stevioside glycoside of claim 10.
- 제11항의 스테비오사이드 배당체를 포함하는 감미료.A sweetener comprising the stevioside glycoside of claim 11.
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