KR20210047773A - New Leuconostoc citreum and Method for Producing Glucosyl Transfer Saccharide Using the Same - Google Patents

Abstract

The present application relates to a Leuconostoc citreum CJ-2B11 strain of accession number KCCM12588P, a composition comprising the Leuconostoc citreum CJ-2B11 strain of accession number KCCM12588P, and a method for producing glucose transfer saccharide, comprising allowing Leuconostoc genus microorganisms or a glucan sucrase derived from the Leuconostoc genus microorganisms to be in contact with a raw material containing sucrose. Accordingly, the present invention has a remarkable effect on the activity of glycotransferase.

Description

New Leuconostoc citreum and Method for Producing Glucosyl Transfer Saccharide Using the Same}

The present application relates to a novel leukonostock citium and a method for producing glucose-transferred saccharides using the same.

Glycosyltransferase (GTs; EC2.4) is a family that catalyzes the attachment of activated sugars to various receptor molecules important for biosynthesis, such as oligosaccharides, polysaccharides, and steviol glycosides, and plays an important role in the creation of glycosylated natural product complexes. Perform.

The above-described glycotransferase can be produced in a number of ways.As one of the production methods, a strain of the genus Leuconostoc is known to express glucan sucrase as a glycotransferase ( Leuconostoc mesenteroides NRRL B-1355, Appl Environ Microbiol. 1994 Aug; 60(8): 2723-2731.), there was a limitation in that the ability to produce sugar transfer enzymes using a strain of the genus Leukonostock was insufficient.

Therefore, in the present invention, a strain having increased production/expression ability of the sugar transfer enzyme (eg, glucan sucrase, etc.) as described above was developed.

Leuconostoc mesenteroides NRRL B-1355, Appl Environ Microbiol. 1994 Aug; 60(8): 2723-2731.

The present application is a novel Leuconostoc citreum strain capable of expressing a glycosyltransferase at a high level, and having remarkable activity of a glycosyltransferase compared to the wild-type Leuconostoc citium strain, a composition comprising the same, And it is intended to provide a method for producing glucose-transition saccharides using the same.

One aspect of the present application provides a Leuconostoc citreum CJ-2B11 strain of accession number KCCM12588P.

Another aspect of the present application provides a composition comprising a Leuconostoc citreum CJ-2B11 strain of accession number KCCM12588P.

Another aspect of the present application provides a flow Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock production of glucose transition saccharide which comprises contacting an (Leuconostoc) can glucan derived from microorganisms of the genus Klein dehydratase and sucrose.

Leuconostoc citreum CJ-2B11 (Luconostoc citreum CJ-2B11) strain (KCCM12588P) according to the present application can express glycotransferase at high levels, and the parent strain, wild-type Leuconostoc citreum As the strain is formed as a mutant through physical and/or chemical mutation, the activity of the sugar transfer enzyme is remarkable compared to the wild-type Leuconostoc citreum strain.

1 is a diagram showing an HPLC chromatogram of a glucose-transition saccharide prepared according to Example 1 of the present application.
2 is a diagram showing an HPLC chromatogram of a glucose-transition saccharide prepared according to Example 2 of the present application.
3 is a diagram showing an HPLC chromatogram of a glucose-transition saccharide prepared according to Example 3 of the present application.
4 is a view showing the results of evaluating the acid/heat resistance complex stability of the glucose-transition saccharide and isomaltooligosaccharide (IMO) of Example 2 according to Experimental Example 4 of the present application.
5 is a view showing the result of comparing the sensory profile of the glucose-transition saccharide of Example 2 and the starch sugar (F55) according to Experimental Example 5 of the present application.
6 is a view showing the result of comparing the sensory profile of the glucose-transition saccharide of Example 2 and isomaltooligosaccharide (IMO) according to Experimental Example 5 of the present application.
7 is a view showing a result of comparing the viscosity of the oligosaccharide composition of Example 2, isomaltooligosaccharide (IMO), and starch sugar (F55) according to Experimental Example 6 of the present application.

Hereinafter, the present application will be described in detail.

The present application provides a Leuconostoc citreum CJ-2B11 strain of accession number KCCM12588P.

The genus Leuconostoc is a twin or streptococcus, which is anaerobically and gram-positive.

The Leuconostoc citreum CJ-2B11 (Luconostoc citreum CJ-2B11) strain (KCCM12588P) is a novel strain having glucose transfer enzyme activity, maintaining a high dietary fiber content, and having excellent glucose transfer enzyme activity.

The sugar transfer enzyme may include glucan sucrase.

The glucan sucrase is an enzyme capable of transferring glucose of sucrose to other substances having free hydroxyl groups other than sucrose, and may be named by mixing with hexosyltransferase (EC number 2.4.1). The glucan sucrase may be dextransucrase (EC number 2.4.1.5), or alternative sucrase (Alternansucrases, EC number 2.4.1.140).

The Leuconostoc citreum CJ-2B11 (Luconostoc citreum CJ-2B11) strain (KCCM12588P) may be a strain formed by mutating a wild-type Leuconostoc citreum strain. Specifically, the wild-type leukonostock citium strains were selected as polysaccharide producing strains from the colonies produced after diluting and culturing juices derived from various kimchi, and strains of a large or viscous colony were selected as polysaccharide producing strains. For example, it is selected from strains having excellent expression of glucan sucrase or alternative sucrase). After selecting a wild-type leukonostock citium strain with relatively excellent glycotransferase expression, it is subjected to a physical mutation method by UV treatment and/or a chemical mutation method that treats an alkylating agent such as EMS (Ethyl Methane Sulfonate). Among the mutant strains derived by using, the strain having excellent activity of the sugar transfer enzyme can be finally selected.

In addition, the wild-type leukonostock citium strains may be isolated from various kimchi, but the separation range of the strains is not limited thereto.

At this time, the sugar transfer enzyme activity can be evaluated by evaluating the rate of decomposition of sugar and measuring the amount of fructose (fructose) produced.

The activity of the glucose transfer enzyme of the Leuconostoc citreum CJ-2B11 strain (KCCM12588P) obtained as above is 1 IU/mL or more, 1.5 IU/mL or more, 2 IU/ML or more, 2.5 IU /mL or more, 3 IU/mL or more, 3.5 IU/mL or more, 4 IU/mL or more, 4.5 IU/mL or more, 5 IU/mL or more, 5.5 IU/mL or more, 6 IU/mL or more, and 1 IU /mL or more, 1.5 IU/mL or more, 2 IU/ML or more, 2.5 IU/mL or more, 3 IU/mL or more, 3.5 IU/mL or more, 4 IU/mL or more, 4.5 IU/mL or more, 5 IU/mL Or more, 5.5 IU/mL or more, one lower limit selected from 6 IU/mL or more and/or 20 IU/mL or less, 19 IU/mL or less, 18 IU/mL or less, 17 IU/mL or less, 16 IU/mL or less Or less, 15 IU/mL or less, 14 IU/mL or less, 13 IU/mL or less, 12 IU/mL or less, 11 IU/mL or less, 10 IU/mL or less, 9 IU/mL or less, 8 IU/mL or less, It may be a range consisting of one upper limit selected from 7 IU/mL or less.

When the Leuconostoc citreum CJ-2B11 (Luconostoc citreum CJ-2B11) strain (KCCM12588P) has the above-described glycotransferase activity, it is about several tens of times more than the wild-type Leuconostoc citium strains, which are the parent strains. Enzymatic activity can be improved.

Another aspect of the present application provides a composition comprising at least one selected from a Leuconostoc citreum CJ-2B11 strain of accession number KCCM12588P and a culture thereof.

The composition is the Leuconostoc citreum CJ-2B11 (Luconostoc citreum CJ-2B11) strain (KCCM12588P) of 10 ⁷ cfu/mL or more, 10 ⁸ cfu/mL or more, 10 ⁹ cfu/mL or more, 10 ¹⁰ cfu/mL It may include more than one, specifically 10 ⁷ cfu / mL or more, 10 ⁸ cfu / mL or more, 10 ⁹ cfu / mL or more, ^{one lower limit selected from 10 10} cfu / mL or more and / or 10 ¹³ cfu / mL Hereinafter, 10 ¹² cfu/mL or less, 10 ¹¹ cfu/mL or less, and 10 ¹⁰ cfu/mL or less may be included in an amount in a range consisting of one upper limit selected from, for example, 10 ⁷ to 10 ¹³ cfu/ mL, 10 ⁷ to 10 ¹² cfu/mL, or 10 ⁸ to 10 ¹² cfu/mL.

In addition, the flow Pocono stock sheet Solarium inoculated CJ-2B11 (Leuconostoc citreum CJ- 2B11) culture of the strain (KCCM12588P) is the flow Pocono stock sheet Solarium CJ-2B11 (Leuconostoc citreum CJ- 2B11) strain (KCCM12588P) into the medium It can mean the result of cultivation.

The culture is a culture obtained by culturing the strain of the present application in a medium (that is, the Leuconostoc citreum CJ-2B11 of the present application) strain (KCCM12588P), a medium or a metabolite of the strain. May include), a filtrate in which the strain is removed by filtration or centrifugation of the culture (eg, a supernatant after centrifugation), and the like.

In addition, the culture may include powdered after drying (eg, freeze drying) of the culture.

The culture is one lower limit selected from 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ And/or 35° C., 34° C., 33° C., 32° C., 31° C., 30° C., 29° C., and 28° C. may be performed in a temperature range consisting of one upper limit selected from, for example, 15 to 35° C. , 20 to 35°C, 25 to 30°C, or 28 to 30°C.

In addition, the culture is one lower limit selected from 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours and/or 48 hours, 47 hours, 46 hours. Select from, 45 hours, 44 hours, 43 hours, 42 hours, 41 hours, 40 hours, 39 hours, 38 hours, 37 hours, 36 hours, 35 hours, 34 hours, 33 hours, 32 hours, 31 hours, 30 hours It may be performed for a time in the range consisting of one upper limit, for example, 6 to 48 hours, 10 to 40 hours, or may be performed for 12 to 36 hours.

The medium may be used without limitation if it is a known medium for lactic acid bacteria. Specifically, the medium may include a carbon source and a nitrogen source, and more specifically, the carbon source may include at least one selected from the group consisting of sucrose, glucose, and fructose, and the nitrogen source is yeast extract, peptone, It may include at least one selected from the group consisting of beef extract, malt extract, corn steep liquor, ammonium citrate, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.

In addition, the medium may further include at least one selected from the group consisting of Tween-80, sodium citrate, potassium phosphate, sodium acetate, manganese sulfate, magnesium sulfate, calcium chloride, and distilled water.

The composition may further include a cryoprotectant, and more specifically, may include at least one selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder, and starch.

The cryoprotectant from 0.01% by weight, 0.02% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight based on the total weight of the composition One lower limit selected and/or 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10% by weight %, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, in a range consisting of one upper limit selected from 1% by weight It may be included, for example, 0.01 to 20% by weight, 5 to 20% by weight, or 2 to 10% by weight, 0.01 to 10% by weight, or 0.5 to 2% by weight, may be 0.1 to 1% by weight. Specifically, the cryoprotectant is 5 to 20% by weight for glycerol, 2 to 10% by weight for trehalose, 2 to 10% by weight for maltodextrin, 0.5 to 2% by weight for skim milk powder, and 0.1 to 1 for starch It may be included in an amount of weight %.

The composition may further include an excipient, and specifically, may include at least one selected from the group consisting of glucose, dextrin, and skim milk.

The excipients are 70% by weight, 71% by weight, 72% by weight, 73% by weight, 74% by weight, 75% by weight, 76% by weight, 77% by weight, 78% by weight, 79% by weight, 80% based on the total weight of the composition. One lower limit selected from wt%, 81 wt%, 82 wt%, 83 wt%, 84 wt%, 85 wt% and/or 95 wt%, 94 wt%, 93 wt%, 92 wt%, 91 wt% , It may be included in a content in a range consisting of one upper limit selected from 90% by weight, for example, 70 to 95% by weight, 75 to 95% by weight, 80 to 95% by weight, 85 to 95% by weight. have.

Another aspect of the present application provides a method of producing a glucose-transition saccharide.

Production method of the sugars glucose transition may include contact with a raw material including, Pocono stock flow (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) can glucan derived from microorganisms of the genus sucrose to Klein kinase.

The microorganisms in Leuconostoc can be used without limitation if they are microorganisms capable of producing glucan sucrase, but specifically Leuconostoc citreum , Leuconostoc mesenteroides , or It may be Leuconostoc kimchii, etc. In addition, the microorganisms of the genus Leukonostock may be microorganisms having an increased glucan sucrase activity compared to the microorganisms of the genus Leukonostock derived from nature, for example, may be a microorganism prepared through a physical and/or chemical method.

The microorganisms of the Leuconostoc genus may include a Leuconostoc citreum CJ-2B11 strain of accession number KCCM12588P. For example, the glucose-transferred saccharide may be obtained by contacting the raw material containing the sucrose with glucan sucrase expressed from the Leuconostoc citreum CJ-2B11 (Leuconostoc citreum CJ-2B11) strain (KCCM12588P). At this time, the above description may be applied in the same manner for the Leuconostoc citreum CJ-2B11 strain (KCCM12588P).

The sucrose is a disaccharide linked by a glycosidic bond between one molecule of fructose and one molecule of glucose, also called sucrose or sugar. It is produced naturally in plants and can be obtained by extracting/purifying sucrose from sugar cane or sugar beet.

The raw material containing sucrose may further contain maltose. Maltose is two molecules of glucose (glucose), also called maltose or maltose, and is a disaccharide linked by α-1,4 glycosidic bonds. In the isomer, isomaltose, two molecules of glucose are linked by α-1,6 glycosidic bonds. Such maltose is also produced when starch is decomposed using β-amylase, and is also found in germinating seeds.

The maltose may be any material containing maltose as a main component without limitation, and may be, for example, starch syrup, corn syrup, or hymaltose.

The starch syrup can be obtained by hydrolyzing starch with an acid or an enzyme, and contains maltose (syrup sugar) as a main component. Starch syrup is different from liquid fructose made by decomposing starch into glucose and isomerizing some of this glucose into fructose. Since the starch syrup is not completely decomposed into glucose and the decomposition stops in the middle, glucose (glucose), dextrin, and the like may be further included.

The starch syrup may contain maltose in an amount of 30% by weight or more and less than 50% by weight based on the total weight of the starch syrup. Specifically, the maltose is 30% by weight or more, 35% by weight or more, 40% by weight or more, 45% by weight or more, and/or less than 50% by weight, less than 45% by weight, less than 40% by weight based on the total weight of the starch syrup. It may be included in the content of.

The corn syrup is an edible syrup made from corn starch, and the content of maltose is not limited in the corn syrup.

The hymaltose is a type of starch syrup containing maltose as a main component, and the hymaltose may include maltose in an amount of 50% by weight or more based on the total weight of hymaltose. Specifically, the maltose is 60% by weight or more, 65% by weight or more, 70% by weight or more, 75% by weight or more, 80% by weight or more, and/or 99% by weight or less, 90% by weight based on the total weight of the hymaltose. It may be less than or equal to 80% by weight.

When the raw material containing sucrose further contains maltose, the mixed weight ratio of the sucrose and the dry solid content of maltose may be 1:1 to 15:1. For example, it may be 1:1 to 13:1, 1:1 to 6:1, 1:1 to 3:1, 2:1 to 13:1, or 2:1 to 6:1. As described above, when the mixed weight ratio of the dry solid content of sucrose and maltose satisfies the above range, the cloudiness of the glucose-transition sugar may be reduced and sweetness may be increased.

The flow Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) in it which contacts the glucan sucrase kinase derived from a microorganism and a raw material containing sucrose, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, With one lower limit selected from 30°C and/or one upper limit selected from 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41°C, 40°C It may be performed at a temperature in the configured range.

Also in contact with the raw material including sucrose the flow Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) can glucan of in microbial Klein dehydratase, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours , It may be performed for a time range consisting of one lower limit selected from 6 hours and/or one upper limit selected from 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, and 12 hours. .

When the above temperature range and/or time range are satisfied, the ability to produce glucose-transition saccharides becomes excellent.

The glucose-transition saccharide may include oligosaccharides. The oligosaccharide may include an oligosaccharide composed of glucose, and the oligosaccharide composed of glucose may include an oligosaccharide linked by an α glycosidic bond. Specifically, the oligosaccharide composed of glucose may include an oligosaccharide containing α-1,3 glycosidic bonds and/or α-1,6 glycosidic bonds. More specifically, it may include oligosaccharides in which α-1,3 glycosidic bonds and α-1,6 glycosidic bonds are alternately linked.

Oligosaccharides consisting of the glucose, is the raw material containing the sucrose-converting enzyme by contacting the stream Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) can glucan derived from microorganisms of the genus Klein dehydratase can be obtained.

The oligosaccharide composed of glucose is a form in which three or more glucose are connected, and may be expressed as a degree of polymerization (DP) of 3 to 10 depending on the number of connected monosaccharides. For example, if three monosaccharides are connected, the degree of polymerization (DP) is 3.

The oligosaccharide composed of glucose may mean an oligosaccharide having a polymerization degree of 3 (DP3) or higher, and the content of the oligosaccharide composed of glucose is 35 parts by weight, 40 parts by weight, 45 parts by weight, 50 parts by weight, based on 100 parts by weight of the saccharide, A range consisting of one lower limit selected from 53 parts by weight, 55 parts by weight, and 60 parts by weight and/or one upper limit selected from 80 parts by weight, 75 parts by weight, 70 parts by weight, 65 parts by weight, and 60 parts by weight It may be, for example, 35 to 80 parts by weight, 40 to 75 parts by weight, 50 to 70 parts by weight, 53 to 70 parts by weight, and 60 to 70 parts by weight.

When the content of the oligosaccharide composed of glucose (polymerization degree 3 (DP3) or higher oligosaccharide) satisfies the above range, the glucose-transition saccharide has the effect of improving the sweetness persistence and body feeling compared to the existing isomaltoligosaccharide. Moreover, the off-flavor is already lowered and the sweetness becomes excellent.

The glucose-transition saccharide may include indigestible oligosaccharides. The indigestible oligosaccharide may include oligosaccharides linked by α-1,3 glycosidic bonds. The indigestible oligosaccharide may further include an oligosaccharide linked by an α-1,6 glycosidic bond.

The indigestible oligosaccharide may include those that are not degraded by digestive enzymes among the oligosaccharides composed of glucose. In other words, it may mean that it is not decomposed by digestive enzymes in the human body, and these indigestible oligosaccharides cause a feeling of satiety in the stomach, and the rate of nutrient absorption in the small intestine decreases due to the rapid passage in the small intestine, which is helpful for diabetics. , It has the effect of preventing colon cancer.

The indigestible oligosaccharide is one selected from 6.2 parts by weight, 6.3 parts by weight, 6.4 parts by weight, 7 parts by weight, 8 parts by weight, 9 parts by weight, 10 parts by weight, or 12 parts by weight based on 100 parts by weight of the glucose-transition sugar. The lower limit of and/or 30 parts by weight, 25 parts by weight, 20 parts by weight, 19 parts by weight, 18 parts by weight, or 15 parts by weight may be included in a range consisting of an upper limit selected from, for example, 6.2 It may be included in an amount of to 30 parts by weight, 6.3 to 25 parts by weight, 6.4 to 20 parts by weight, 7 to 20 parts by weight, or 10 to 20 parts by weight.

When the content of the indigestible oligosaccharide satisfies the above range, there are effects such as preventing colon cancer, improving constipation, reducing blood cholesterol, helping diabetes, and reducing calories.

Glucose transition saccharides obtained a flow Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) in microorganisms, such as dangjeon glucan sucrase azepin-derived enzymes as described above is converted by the enzyme α-1,3-glycoside bond and It may include oligosaccharides in which α-1,6 glycosidic bonds are alternately linked. Since the sugar transfer enzyme as described above is used, the glucose-transfer saccharide may be one that does not contain isomaltooligosaccharide substantially as described above.

The isomaltooligosaccharide is an oligosaccharide produced by enzymatic conversion in which 1,2-glycosidic bonds between glucose and fructose are rearranged into 1,6-glycosidic bonds, whereas the glucose-transferred saccharides of the present application are sucrose. It may be obtained by converting into fructose (fructose) and oligosaccharides by glucan sucrase derived from microorganisms of the genus Konostok or microorganisms of the genus Leukonstock. The glucose-transition saccharides formed at this time may include oligosaccharides in which α-1,3 glycosidic bonds and α-1,6 glycosidic bonds are alternately connected as described above.

Meanwhile, when glucose is transferred using sucrose as a raw material, fructose may be produced as a by-product. Such fructose is a substance that gives off a sweet taste by itself, but may be used as a raw material for other sugars depending on the application.

The method for producing glucose-transferred saccharides further comprises producing a fructose epimer by contacting a fructose epimerase and a fructose derived from sucrose after the step of contacting the glucan sucrase with a raw material containing sucrose. can do.

The fructose epimerase is an enzyme belonging to an isomerase, and is an enzyme that catalyzes epimerization that reverses the steric configuration of only one asymmetric carbon among compounds having a plurality of asymmetric carbon atoms. For example, the fructose epimerase may include a fructose-3-epimerase or a fructose-4-epimerase, and specifically, the fructose-3-epimerase is It can be converted to allulose by reversing the steric configuration, and the fructose-4-epimerase can be converted to tagatose by reversing the steric configuration of the carbon at the 4th position of the fructose.

The glucose-transition saccharide has a water content of one lower limit selected from 18% by weight, 19% by weight, or 20% by weight based on the total weight of the glucose-transition saccharide and/or 25% by weight, 24% by weight, or 23% by weight. It may be a range consisting of one upper limit selected from, for example, 18 to 25% by weight, 19 to 24% by weight, or 20 to 23% by weight. When the water content satisfies the above range, it is possible to improve the softness and body feeling of glucose-transition saccharides, and to improve sweetness.

The viscosity of the glucose-transition saccharide is one lower limit selected from 400 cP, 420 cP, 440 cP, 460 cP, 480 cP, 500 cP and/or 5,000 cP, 3,000 cP, 2,000 cP, 1,000 cP at 25°C, It may be a range consisting of one upper limit selected from 800 cP, 700 cP, 650 cP, 600 cP, 550 cP, for example, the viscosity may be 400 to 5,000 cP at 25°C, and 420 to 3,000 cP, 440 to 1,000 cP, 460 to 800 cP, or 480 to 600 cP. When the viscosity of the glucose-transition saccharide satisfies the above range at 25° C., it has excellent viscosity and is suitable for dairy products and cereal bars requiring a mouthfeel.

The glucose transitional saccharide has a viscosity at 30° C. of 240 cP, 260 cP, 280 cP, 300 cP, 320 cP, 340 cP, 350 cP and/or 1,000 cP, 800 cP, 600 cP, 500 cP , 400 cP may be a range consisting of one upper limit selected from, for example, the viscosity at 30 °C may be 240 to 1,000 cP, 280 to 800 cP, 300 to 600 cP, or 340 to 500 cP. . When the viscosity of the glucose-transition saccharide satisfies the above range at 35° C., it has excellent viscosity and is suitable for dairy products and cereal bars that require a mouthfeel.

The viscosity may be measured at a rotational speed of 70 Brix and/or 160 rpm of a glucose-transition saccharide.

The glucose-transition saccharide may further include fructose. The fructose is one lower limit selected from 10 parts by weight, 15 parts by weight, 20 parts by weight, 25 parts by weight, 27 parts by weight, and 30 parts by weight based on 100 parts by weight of the glucose-transition sugar and/or 50 parts by weight, 45 parts by weight. Parts, 40 parts by weight, 38 parts by weight, may be included in a range consisting of one upper limit selected from 35 parts by weight, for example, 10 to 50 parts by weight, 15 to 45 parts by weight, 20 to 45 parts by weight , Or may be included in an amount of 27 to 40 parts by weight.

When the fructose satisfies the above range, the sweetness of the glucose-transferred saccharide is improved, so that the body feel is excellent, and the sweet taste strength and the sweet taste persistence may be improved.

In addition, the glucose-transition saccharide may not substantially contain glucose. The meaning that the glucose-transition saccharides do not contain glucose substantially means that the glucose-transition saccharides do not contain glucose at all or are contained as impurities (less than about 0.1% by weight).

In the glucose-transition saccharide, an oligosaccharide having a degree of polymerization of 6 (DP6) or higher is 2 parts by weight, 2.5 parts by weight, 2.6 parts by weight, 3 parts by weight, 4 parts by weight, 5 parts by weight, 6 parts by weight, based on 100 parts by weight of the glucose-transition saccharide. One lower limit selected from 7 parts by weight, 8 parts by weight, 9 parts by weight, 10 parts by weight and/or selected from 40 parts by weight, 39 parts by weight, 38 parts by weight, 35 parts by weight, 30 parts by weight, 25 parts by weight It may be included in an amount of a range consisting of one upper limit, for example, 2 to 40 parts by weight, 2.5 to 39 parts by weight, and may be included in an amount of 2.6 to 35 parts by weight.

When the content of the oligosaccharide of the polymerization degree 6 (DP6) or more satisfies the above range, there is an effect of preventing the cloudiness of the glucose-transitioning saccharide, and already, the off-flavor is improved, so that the sweetness is improved, and the sweetness persistence can be improved.

Hereinafter, the present application will be described in more detail through examples. However, these examples are only intended to help the understanding of the present application, and the scope of the present application is not limited to these examples in any sense.

<Production Example 1>-Selection and identification of strains

1. Selection of strains

In order to separate strains with excellent sugar transfer enzyme production ability, the juice is diluted step by step from various kimchi, inoculated in a solid sucrose medium for separation, and cultured at 28°C. The strain was selected as a polysaccharide producing strain. Among them, about 545 species identified as strains of the genus Leuconostoc were derived through identification.

Glucose transfer enzyme activity and dietary fiber content were measured to select strains with excellent properties for 545 strains, and for comparison, standard strains ( Leuconostoc mesenteroides NRRL B-1355, Appl Environ Microbiol. 1994 Aug; 60(8) ): 2723-2731.) was used as a control. Specifically, the selection of strains with high sugar transfer enzyme production was performed in MRS medium (sucrose 20 g/L, Yeast extract 1.5 g/L, Ammonium citrate 2 g/L, dipotassium phosphate 2 g/L, Tween80 1 g/L, Magnesium sulfate heptahydrate 0.1 g/L). L, pH 6.5±0.2), incubated for 24 hours at 28°C and 180 rpm conditions, and reacted with 200 mM sucrose by taking the culture supernatant, and quantitatively analyze the rate of sugar decomposition, that is, the amount of fructose produced according to the reaction time by HPLC I did.

Comparison of enzyme activity and dietary fiber content of strains of the genus Leukonostock Strains
( Leuconostoc sp.) Enzyme activity* Dietary fiber content** fructose (g/L) HPLC (DP3+, area%) B-1355 (Standard strain) 2.16 16 JY164 6.78 24 Comparative strain 1 5.63 19 Comparative strain 2 5.98 18 Comparative strain 3 5.89 19 Comparative strain 4 5.17 18 Comparative strain 5 5.14 10 Comparative strain 6 0.12 12 Comparative strain 7 0.12 10 Comparative strain 8 4.45 18

*Enzyme activity: This is the rate of sugar decomposition, and the amount of fructose produced is measured.

** Dietary fiber content: HPLC DP3+ residual amount is measured after digestive enzyme treatment.

It is judged that JY164 selected in Table 1 has a relatively excellent expression ratio of alternative sucrase due to high sugar transfer enzyme activity and dietary fiber content. In addition, in order to develop a mutant strain in which the sugar transfer enzyme is highly expressed while maintaining the high dietary fiber content of JY164, a physical mutation method by UV treatment in JY164 and a chemical mutation method that treats alkylating agents such as EMS (Ethyl Methane Sulfonate). Was sequentially applied to induce mutations and repeated selection of mutant strains, and leuconostoc citreum 2B11 strain with remarkably improved glycotransferase expression was developed. The results of comparing the enzyme activity and dietary fiber content of JY164 and 2B11, a strain mutated therefrom, are shown in Table 2 below.

Comparison of enzymatic activity of parental and modified leukonostock strains division Enzyme activity (IU/ml) Dietary fiber content (%) L.citreum JY164 0.10 24 L.citreum JY164_2B11 6.66 24

(1 unit of enzyme refers to the ability of the enzyme to produce 1 mM fructose from sucrose under 1 minute of reaction conditions.)

Wherein, according to Table 2, the flow Pocono stock sheet Solarium (Leuconostoc citreum) 2B11 strain according to the present application was confirmed to be more than 66 times the enzyme activity improved as compared with the parental strain the wild-type flow Pocono stock sheet Solarium (Leuconostoc citreum) JY164.

2. Identification of CJ-2B11 strain

The selected strains were identified with an API system (API System, La Balme-les-Grottes, France).

As a result of analyzing the 16s rRNA nucleotide sequence, the 16s rRNA of SEQ ID NO: 1 was derived, and it was confirmed that the 16s rRNA had 99% identity with Leuconostoc citreum, and the strain was Leuconostoc citreum ( Leuconostoc citreum ).

In this application, the strain was named as Leuconostoc citreum CJ-2B11 strain, and it was deposited with the Korean Culture Center of Microorganisms (KCCM) on September 2, 2019. (Accession number: KCCM12588P).

<Production Example 2>-Preparation of enzyme solution

Sucrose (70 g/L), yeast extract (10 g/L), K ₂ HPO ₄ (20 g/L), MgSO ₄ 7H ₂ O (0.10 g/L), MnSO ₄ 2H ₂ O (0.05 g /L), and CaCl ₂ · 2H ₂ O (0.01 g / L) in a medium containing Leuconostoc citreum CJ-2B11 (Leuconostoc citreum CJ-2B11) strain at 28 to 30 °C until carbohydrates are consumed. After incubation, the pH was adjusted to 6.0 (including 10% NaOH). Once the carbon source was depleted, the strain was removed by centrifugation at 12,200 xg and 4°C for 30 minutes. The culture supernatant was obtained by using an ultrafiltration 2,000 molecular weight cut-off (100 kD MWCO) membrane in a 10-fold volume of enzyme concentrate. (The Leuconostoc citreum CJ-2B11 strain caused by physical mutagenesis expresses glucan sucrase when grown in sucrose medium.)

<Examples 1 to 5>-Preparation of glucose-transition sugars

Sucrose (purity 98% by weight or more, CJ CheilJedang) and corn syrup (hymaltose) so that the weight ratio of the dry solid content of sucrose: the dry solid content of maltose is 1:1, 2:1, 3:1, 6:1, and 13:1, respectively. ; A mixed raw material substrate obtained by mixing a maltose content of 70% by weight or more, CJ CheilJedang) was dissolved in distilled water and placed in a polypropylene test tube with a 50 mL lid. Here, 1 IU/ml of the enzyme solution according to Preparation Example 2 was added to prepare a raw material solution having a final concentration of 50% by weight. This was put in a water bath set at 40° C., and reacted for 12 hours, and then a glucose transsaccharide sample was obtained from each tube.

<Experimental Example 1>

The degree of polymerization of the glucose-transferred saccharide samples of Examples 1 to 5 was analyzed by HPLC (High-Performance Liquid Chromatography) as described below, and the results are shown in Table 3 below, and HPLC chromatography for Examples 1 to 3 The gram (chromatogram) is shown in FIGS. 1 to 3.

Before the enzyme-reacted glucose-transferred saccharide sample was injected into the HPLC, it was diluted in distilled water to a concentration of 5 to 10% by weight, and filtered through a 0.20 micron filter. Oligosaccharide separation was performed twice at 85°C using a BioRad Aminex HPX-42A, 300X7.8 mm, column (Hercules, CA) and a refractive index detector. Water was used as the eluent at a flow rate of 0.4 ml/min.

(Unit: HPLC area%) ingredient Retention Time(min) Sucrose dry solid content: Maltose dry solid content weight ratio 1:1 2:1 3:1 6:1 13:1 Example 1 Example 2 Example 3 Example 4 Example 5 DP10+ 6.35 0 0 0 0 0 DP9 7.95 0 0 0 0 13.56 DP8 8.28 0 0 0 12.31 10.68 DP7 9.02 0 1.7 7.49 11.81 6.82 DP6 9.753 2.66 5.45 12.86 13.58 5.23 DP5 10.584 8.93 15.99 19.59 9.95 2.42 DP4 11.598 25.47 26.03 14.5 3.86 0 DP3 12.849 29.45 17.09 6.68 2.22 0 DP2 14.407 6.96 1.88 0 0 0 Leucrose 15.897 0 1.53 3.98 9.03 20.85 Glucose 16.547 0 0 0 0 0 Fructose 17.73 26.53 30.33 34.9 37.24 40.44 Total of DP3~DP10+ 66.51 66.26 61.12 53.73 38.71

According to Experimental Example 1, it was confirmed that the glucose-transitioned saccharides of Examples 1 to 3 had a relatively low mixing ratio of sucrose, so that the oligosaccharide content of polymerization degree 6 or higher (DP6+) was small, and the content of leucrose was small. there was.

<Experimental Example 2>

The results of measuring the turbidity of the glucose-transition saccharide samples of Examples 1 to 5 by the following method are shown in Table 4 below.

Turbidity measurement

1 mL of the oligosaccharide stock solution prepared in Examples 1 to 5 was added to 9 mL of deionized water, and adjusted to 10 mL, and used as a sample solution. 1 mL of the sample solution was taken and absorbance was measured at 720 nm using a spectrophotometer (UV mini-1240, Shimadzu, Tokyo, Japan). The results are shown in Table 4.

division Example 1 Example 2 Example 3 Example 4 Example 5 DP6 or higher content
(weight%) 2.66 7.15 20.35 37.71 36.29 Turbidity
(abs, 720 nm) 0.9 1.5 2.8 9.3 10.9

According to Experimental Example 2, it can be seen that the glucose-transition saccharides of Examples 1 to 5 have low turbidity (absorbance, 720 nm), and in particular, in the case of the glucose-transition saccharides of Examples 1 to 3, oligosaccharides having a polymerization degree of 6 (DP6) or higher It can be seen that the content is low and the turbidity (absorbance, 720 nm) is low, so that the cloudiness can be remarkably improved, and it can be confirmed that it is a very improved effect in terms of commercial production as it is possible to produce a substantially transparent syrup.

<Experimental Example 3>

1 mL of each of the glucose-transferred saccharides of Examples 1 to 5 and the conventional oligosaccharide IMO (Chungjungwon Oligosaccharide, Subject) samples were mixed with 10 μL of alpha amylase (Novozymes, NBSG4698) and reacted at 90° C. for 1 hour . The mixture was adjusted to pH 4.5 with 1 M acetate buffer, and then mixed with 30 μL of glucoamylase (Novozymes, AMS30092) and reacted at 60° C. for 1 hour. After digestion enzyme treatment, the reaction was stopped by boiling water at 100°C for 5 minutes. The content of residual oligosaccharides (DP3-DP9) by alpha amylase and glucoamylase enzymatic hydrolysis was measured by HPLC using a BioRad Aminex HPX-42A column, and distilled water was used as a mobile phase at 0.4 ml/min and 85°C. The content of indigestible oligosaccharide (dietary fiber) was expressed as the content (% by weight) of residual oligosaccharide after digestive enzyme treatment. The results are shown in Table 5 below.

division IMO Example 1 Example 2 Example 3 Example 4 Example 5 Before digestive enzyme treatment oligosaccharide 57.87 66.51 66.26 61.12 53.74 38.7 glucose 17.53 0.00 0.00 0.00 0.00 0.00 After digestive enzyme treatment oligosaccharide 6.09 6.45 9.15 14.14 17.51 19.21 glucose 83.96 69.15 64.19 56.48 49.68 45.28 digestibility(%) 89.48 90.30 86.19 76.87 67.42 50.36 Indigestible oligosaccharide content (% by weight) 6.09 6.45 9.15 14.14 17.51 19.21

According to Experimental Example 3, the glucose-transferring saccharides of Examples 1 to 5 contained a high content of indigestible oligosaccharides (dietary fiber) compared to the content (6.09% by weight) of indigestible oligosaccharides (dietary fiber) contained in the existing IMO. It was confirmed to be included.

<Experimental Example 4>

For the glucose-transferred saccharide samples of Examples 2 and 4, and the conventional oligosaccharide IMO (Chungjungwon Oligosaccharide, Co., Ltd.), the degree of polymerization was measured in the same manner as in Experimental Example 1, and is shown in Table 6 below. Example 2 and the conventional oligosaccharide IMO were evaluated for acid/heat-resistant composite stability and the results are shown in FIG. The acid/heat resistance composite stability was evaluated at pH 2 and 90°C to evaluate the residual rate (%) compared to the initial stage.

(Unit: HPLC area%) ingredient Example 2 Example 4 IMO DP9+ 0 0 0.75 DP8 0 12.31 1.69 DP7 1.7 11.81 1.16 DP6 5.45 13.58 1.71 DP5 15.99 9.95 5.53 DP4 26.03 3.86 15.34 DP3 17.09 2.22 30.05 DP2 1.88 0 23.35 Leucrose 1.53 9.03 0 Sucrose 0 0 0 Glucose 0 0 20.42 Fructose 30.33 37.24 0 Total of DP3~DP9+ 66.26 53.73 56.23

According to Experimental Example 4, compared to the conventional IMO, the glucose-transferred saccharide of the present application has improved sweetness, excellent body feeling, and improved sweet taste strength and sweet taste persistence.

According to FIG. 4, the glucose-transition saccharide of the present application maintains a residual rate of glucose-transition saccharide of a polymerization degree of 3 or more (DP3+) of 90% or more even under severe processing conditions of pH 2 and 90°C, so that the acid/heat resistance complex stability is It can be confirmed that the level is equivalent to the existing isomaltooligosaccharide (IMO). That is, it can be confirmed that it can be applied to various processed foods such as dairy products.

<Experimental Example 5>

Prepared in an aqueous solution having the same dry solid content of 15% by weight with respect to the glucose-transferred saccharide sample of Example 2 and the conventional oligosaccharide IMO (Cheongjeongwon Oligosaccharide, Daesang Co., Ltd.), and starch sugar F55 (CJ CheilJedang Co., Ltd.) Thus, the results of comparing and analyzing the sensory profiles for sweetness, body feeling, persistence of sweetness, taste/off-flavor, thickness, and bitter taste are shown in FIGS. 5 and 6.

Sensory evaluation

For 15 trained professional panelists, 6 items of intensity attributes (sweetness, body feeling, persistence of sweet taste, taste/off-flavor, thick, bitter taste), and 2 preference attributes (sweet taste, overall preference) were evaluated.

5 and 6, it was confirmed that the glucose-transferred saccharide of the present application has excellent sweetness persistence and body feeling compared to starch sugar (F55, liquid fructose), and has excellent sweetness compared to isomaltooligosaccharide (IMO). , It was confirmed that the weight average molecular weight is larger, so that the body feel is rich, the sweetness persistence is improved, and the content of indigestible oligosaccharides is high, so that the thickness is low, and the off-flavor is reduced.

<Experimental Example 6>

The oligosaccharide composition sample of Example 2 and the conventional oligosaccharide IMO (Cheongjeongwon Oligosaccharide, Daesang Co., Ltd.), and starch sugar F55 (CJ CheilJedang Co., Ltd.) were prepared in an aqueous solution of the same dry solid content of 70 Brix, and the temperature The results of comparative analysis of star viscosity are shown in FIG. 7.

Viscosity evaluation

Each of the oligosaccharide, IMO, and F55 samples of Example 2 was contained in each of 42 g.

The viscosity was measured using a viscometer (Rapid visco analyser, PerkinElmer) at intervals of 10 minutes while gradually increasing the temperature from 25° C. to 70° C. at 160 rpm.

Referring to FIG. 7, the oligosaccharide of Example 2 showed a slight increase in viscosity compared to IMO and F55, but there was no significant difference.

That is, it was confirmed that the oligosaccharide of Example 2 was similar or further improved in terms of processability compared to the conventional oligosaccharides IMO and F55.

<Application Example> Conversion of fructose in glucose-transfer sugars

When glucose is transferred using sucrose as a raw material, fructose is produced as a by-product. Fructose is a substance that gives off a sweet taste by itself, but depending on the application, it can be used as a raw material for other sugars as follows.

Application Example 1: Conversion of fructose to allulose

The glucose-transferred saccharide sample of Example 2 was contacted with Kaistia granuli LIS1 strain (KCCM11916P) known in Korean Patent Laid-Open Publication No. 10-2018-0055737 at 55° C. for 2 hours to reduce fructose to allulose. By converting to ose, glucose-transition saccharides containing allulose were prepared.

Application Example 2: Conversion of fructose to tagatose

The glucose-transferred saccharide sample of Example 2 was contacted with a fructose-4-epimerase derived from Kosmotoga olearia known in Korean Patent Application Publication No. 10-2018-0111678 at 60° C. for 2 hours. By converting fructose to tagatose, a glucose-transition saccharide containing tagatose was prepared.

Korean Culture Center of Microorganisms (KCCM) KCCM12588P 20190902

<110> CJ CheilJedang Corporation <120> Oligosaccharide Composition and Preparation Method of Oligosaccharide Composition <130> 2019-DPCJ-0047 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1300 <212> DNA <213> Unknown <220> <223> Leuconostoc citreum CJ-2B11 <400> 1 gaacgggtga gtaacacgtg gataacctgc ctcaaggctg gggataacat ttggaaacag 60 atgctaatac cgaataaaac ttagtatcgc atgatataaa gttaaaaggc gctacggcgc 120 cacctagaga tggatccgcg gtgcattagt tagttggtgg ggtaaaggct taccaagaca 180 atgatgcata gccgagttga gagactgatc ggccacattg ggactgagac acggcccaaa 240 ctcctacggg aggctgcagt agggaatctt ccacaatggg cgcaagcctg atggagcaac 300 gccgcgtgtg tgatgaaggc tttcgggtcg taaagcactg ttgtatggga agaaatgcta 360 aaatagggaa tgattttagt ttgacggtac cataccagaa agggacggct aaatacgtgc 420 cagcagccgc ggtaatacgt atgtcccgag cgttatccgg atttattggg cgtaaagcga 480 gcgcagacgg ttgattaagt ctgatgtgaa agcccggagc tcaactccgg aatggcattg 540 gaaactggtt aacttgagtg ttgtagaggt aagtggaact ccatgtgtag cggtggaatg 600 cgtagatata tggaagaaca ccagtggcga aggcggctta ctggacaaca actgacgttg 660 aggctcgaaa gtgtgggtag caaacaggat tagataccct ggtagtccac accgtaaacg 720 atgaatacta ggtgttagga ggtttccgcc tcttagtgcc gaagctaacg cattaagtat 780 tccgcctggg gagtacgacc gcaaggttga aactcaaagg aattgacggg gacccgcaca 840 agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 900 cctttgaagc ttttagagat agaagtgttc tcttcggaga caaagtgaca ggtggtgcat 960 ggtcgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt 1020 attgttagtt gccagcattc agttgggcac tctagcgaga ctgccggtga caaaccggag 1080 gaaggcgggg acgacgtcag atcatcatgc cccttatgac ctgggctaca cacgtgctac 1140 aatggcgtat acaacgagtt gccaacctgc gaaggtgagc taatctctta aagtacgtct 1200 cagttcggac tgcagtctgc aactcgactg cacgaagtcg gaatcgctag taatcgcgga 1260 tcagcacgcc gcggtgaata cgttcccggg tcttgtacac 1300

Claims (10)

Leuconostoc citreum CJ-2B11 (Leuconostoc citreum CJ-2B11) strain of accession number KCCM12588P. The method according to claim 1,
The strain is to produce a sugar transfer enzyme 1 IU / mL or more, strain. The method according to claim 2,
The glycotransferase is a strain containing glucan sucrase. A composition comprising at least one selected from the Leuconostoc citreum CJ-2B11 (Leuconostoc citreum CJ-2B11) strain and a culture thereof of accession number KCCM12588P. Kono stock flow (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) method in the production of glucose transition sugars comprising contacting the microorganisms glucan derived Klein dehydratase and the raw material containing sucrose. The method of claim 5,
The microorganism of the leukonostock genus is a method for producing glucose-transferred saccharides comprising a leuconostoc citreum CJ-2B11 (Leuconostoc citreum CJ-2B11) strain of accession number KCCM12588P. The method of claim 5,
The glucose-transition saccharide is an oligosaccharide, a method of producing a glucose-transition saccharide. The method of claim 5,
In that the flow Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) in it which contacts the glucan sucrase kinase derived from a microorganism and a raw material including sucrose, is carried out at a temperature of 25 to 50 ℃, glucose transition sugars Method of production. The method of claim 5,
After the step of contacting the glucan sucrase with a raw material containing sucrose, a method for producing glucose-transferred saccharides, further comprising contacting a fructose epimerase and a fructose derived from sucrose to produce an epimer of fructose . The method of claim 9,
The fructose epimer comprises at least one selected from the group consisting of allulose, tagatose, and sorbose.

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