WO2021080356A1 - Oligosaccharide composition and method for producing oligosaccharide composition - Google Patents

Abstract

The present application relates to: an oligosaccharide composition comprising a saccharide containing an oligosaccharide composed of glucose, wherein the content of an indigestible oligosaccharide is 6.2 to 30 parts by weight on the basis of 100 parts by weight of the saccharide; and a method for producing an oligosaccharide composition comprising contacting a raw material containing sucrose and maltose with a microorganism of the genus Leuconostoc or a glucan sucrase derived from a microorganism of the genus Leuconostoc.

Description

Oligosaccharide composition and preparation method of oligosaccharide composition

The present application relates to an oligosaccharide composition and a method of preparing an oligosaccharide composition.

Sugar is currently the most widely used sweetener extracted and refined from sugar cane or sugar beet. Despite the widest availability, high sugar content in food is the most common cause of obesity and related health problems in humans. It is recognized as one of the important causes.

To compensate for this, a natural sweetener called oligosaccharide was developed. Oligosaccharides are polysaccharides in which several monosaccharides are bound, and are natural sweeteners that exist in nature. Isomaltooligosaccharide (IMO) is a representative oligosaccharide currently on the market.

Of these, isomaltooligosaccharide is made from corn starch, so the raw sugar (sugar) content is 0%. Therefore, it is possible to reduce the intake of sugar, and because it is strong against heat, it has the advantage of less destruction of beneficial ingredients even when heated. However, there is a problem that isomaltooligosaccharide is insufficient in dietary fiber and is not suitable for products requiring mouthfeel.

Sweeteners that can completely replace sugar are the subject of constant research, and studies to solve the problems present in sweeteners such as isomaltooligosaccharides currently available on the market as described above are continuously being conducted.

[Prior technical literature]

Korean Patent Application Publication No. 2015-0031959

Korean Patent Application Publication No. 2015-0027467

This application is to solve the problem of the conventional isomaltooligosaccharide used as a sweetener as described above, and ultimately to provide a sweetener capable of replacing sugar, while the content of the indigestible oligosaccharide is high, the cloudiness is improved, and It is intended to provide an oligosaccharide composition with improved sweetness (sweetness) by lowering the already (異味) and off-flavor (異臭).

In addition, it is intended to provide an oligosaccharide composition having acid resistance and heat resistance.

The present application provides an oligosaccharide composition comprising a saccharide containing an oligosaccharide composed of glucose, wherein the content of the indigestible oligosaccharide is 6.2 to 30 parts by weight based on 100 parts by weight of the saccharide.

In addition, the present application provides a process for the preparation of an oligosaccharide composition, comprising contacting the number glucan of the raw material containing sucrose and maltose flow Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) in Microbial Klein Kinase do.

The oligosaccharide composition of the present application has a high content of indigestible oligosaccharide compared to conventional isomaltoligosaccharide, so that it has effects such as preventing colon cancer and improving constipation, and has a calorie reduction effect.

In addition, the oligosaccharide composition of the present application maintains the same level of acid-resistance and heat-resistance stability, while reducing cloudiness compared to the existing isomaltooligosaccharide, and improving sweetness since there is no already (異味) or off-flavor. In addition, it has an excellent body feeling based on the same Bx%, and has the effect of improving sweet taste strength and sweet taste persistence.

In addition, the oligosaccharide composition of the present application has a relatively high viscosity, so it is suitable for dairy products and cereal bars that require physical properties such as mouthfeel.

1 is a diagram showing an HPLC chromatogram of an oligosaccharide composition prepared according to Example 1 of the present application.

2 is a diagram showing an HPLC chromatogram of an oligosaccharide composition prepared according to Example 2 of the present application.

3 is a diagram showing an HPLC chromatogram of an oligosaccharide composition 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 oligosaccharide composition of Example 2 and the isomaltooligosaccharide (IMO) according to Experimental Example 4 of the present application.

5 is a view showing the result of comparing the sensory profile of the oligosaccharide composition of Example 2 and starch sugar (F55) according to Experimental Example 5 of the present application.

6 is a view showing a result of comparing the sensory profile of the oligosaccharide composition of Example 2 and the isomaltooligosaccharide (IMO) according to Experimental Example 5 of the present application.

7 is a view showing the results 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 an oligosaccharide composition.

The oligosaccharide composition includes a saccharide including an oligosaccharide composed of glucose, and the content of the indigestible oligosaccharide is 6.2 to 30 parts by weight based on 100 parts by weight of the saccharide.

The oligosaccharide composed of glucose may include oligosaccharides linked by α glycosidic bonds. 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.

The oligosaccharide composed of glucose may include indigestible oligosaccharide.

The oligosaccharide composition may be one that substantially does not contain isomaltooligosaccharide. That is, the oligosaccharide composed of glucose may be an oligosaccharide substantially not containing isomaltooligosaccharide, and the saccharide may also substantially not contain isomaltooligosaccharide.

The meaning that the oligosaccharide composition does not contain isomaltooligosaccharide substantially means the degree to which the oligosaccharide composition does not contain isomaltooligosaccharide at all or contains impurities (less than about 0.1% by weight).

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 oligosaccharide composition 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 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 lower limit 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 saccharide. 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. It may be included in an amount of 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, as described above, there is an effect such as preventing colon cancer, improving constipation, reducing blood cholesterol, helping diabetes, and reducing calories.

In the oligosaccharide composition, the moisture content is selected from one lower limit selected from 18% by weight, 19% by weight, or 20% by weight based on the total weight of the oligosaccharide composition and/or from 25% by weight, 24% by weight, or 23% by weight. It may be a range consisting of one upper limit, for example, it may be 18 to 25% by weight, 19 to 24% by weight, or 20 to 23% by weight. When the moisture content satisfies the above range, the softness and body feeling of the oligosaccharide composition may be improved, and sweetness may be improved.

The viscosity of the oligosaccharide composition 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, 800 at 25°C. It may be a range consisting of one upper limit selected from 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 oligosaccharide composition satisfies the above range at 25° C., it has excellent viscosity and is suitable for dairy products and cereal bars that require a mouthfeel.

The oligosaccharide composition 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, It may be a range consisting of one upper limit selected from 400 cP, for example, the viscosity at 30° C. may be 240 to 1,000 cP, 260 to 800 cP, 280 to 600 cP, or 300 to 500 cP.

The viscosity may be measured by measuring the viscosity of the oligosaccharide composition 70 Brix at 25° C. and 30° C. with a viscometer (Rapid visco analyzer, PerkinElmer) at a rotation speed of 160 rpm.

When the viscosity of the oligosaccharide composition satisfies the above range at 30° C., it has excellent viscosity and is suitable for dairy products, cereal bars, etc. requiring a mouthfeel.

The oligosaccharide composition 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 sugar and/or 50 parts by weight, 45 parts by weight, It may be included in a range consisting of one upper limit selected from 40 parts by weight, 38 parts by weight, and 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 It may be included in an amount of 27 to 40 parts by weight.

When the fructose satisfies the above range, the sweetness of the oligosaccharide composition 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 oligosaccharide composition may substantially contain no glucose. The meaning that the oligosaccharide composition does not contain glucose substantially means that the oligosaccharide composition does not contain glucose at all or is contained as an impurity (less than about 0.1% by weight).

The oligosaccharide composition contains 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, 7 parts by weight, based on 100 parts by weight of the saccharide with a degree of polymerization of 6 (DP6) or higher. , 8 parts by weight, 9 parts by weight, one lower limit selected from 10 parts by weight and/or one upper limit selected from 40 parts by weight, 39 parts by weight, 38 parts by weight, 35 parts by weight, 30 parts by weight, and 25 parts by weight It may be included in an amount in the range consisting of, 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 oligosaccharide composition from becoming cloudy, and the off-flavor is improved, so that sweetness and persistence of sweetness may be improved.

In addition, the present application provides a method for preparing an oligosaccharide composition.

Method for producing the oligosaccharide composition, it may include contacting with sucrose (sucrose) and a flow of a raw material containing maltose Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) can glucan of in microbial Klein Kinase .

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). Specifically, the glucan sucrase may be dextransucrase (EC number 2.4.1.5), or alternative sucrase (Alternansucrases, EC number 2.4.1.140).

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 Leuconostoc It could be Leuconostoc kimchii. Also manufactured through the flow Pocono stock (Leuconostoc) in the microorganism glucan can may be a two sucrase activity increased microorganism compared to the current Pocono stock (Leuconostoc) in the microorganism derived from a natural, physical and / or chemical methods, for example It can be a microorganism.

The microorganisms in the leukonostock genus have an activity of glycotransferase including glucan sucrase of 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 higher, 4 IU/mL or higher, 4.5 IU/mL or higher, 5 IU/mL or higher, 5.5 IU/mL or higher, 6 IU/mL or higher, 1 IU/mL or higher, 1.5 IU/mL or higher, 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 One lower limit selected from /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, 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, 7 IU/mL or less It may be a microorganism in the genus Leukonostock, which is a range consisting of an upper limit.

The mixing weight ratio of the dry solid content of sucrose and 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. When the mixing weight ratio of the dry solid content of sucrose and maltose satisfies the above range, the cloudiness of the oligosaccharide composition may be reduced and sweetness may be increased.

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

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. This is the maltose starch β - and also generated when the decomposition by using a-amylase (β-amylase), is found in the seeds are germinated.

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.

The oligosaccharide composition may comprise oligosaccharides consisting of glucose, oligosaccharide consisting of the glucose, the raw material containing the sucrose and maltose of the flow Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) in Microbial It can be obtained by enzymatic conversion by glucan sucrase.

Kono stock flow (Leuconostoc) in microorganisms or flow Pocono stock dangjeon oligosaccharide is composed of glucose obtained is converted by an enzyme, such as enzyme glucan sucrase kinase of (Leuconostoc) in microbial origin as described above, α-1,3-glycoside It may include an oligosaccharide containing a bond and/or an α-1,6 glycosidic bond. Specifically, it may include oligosaccharides in which α-1,3 glycosidic bonds and α-1,6 glycosidic bonds are alternately linked.

Since the sugar transfer enzyme as described above is used, the oligosaccharide composed of glucose may be one that does not contain isomaltooligosaccharide substantially as described above.

The isomaltooligosaccharide is an oligosaccharide produced by enzymatic conversion in which a 1,2-glycosidic bond between glucose and fructose is rearranged into a 1,6-glycosidic bond, whereas the oligosaccharide composed of the glucose of the present application is sucrose and maltose is converted into stock flow Pocono (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) can glucan derived from microorganisms of the genus Klein azepin fructose (fructose, fructose) as the oligosaccharides may be obtained by. The oligosaccharides formed at this time may include oligosaccharides including α-1,3 glycosidic bonds and/or α-1,6 glycosidic bonds as described above.

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.

In addition, for the oligosaccharide, saccharide, and/or oligosaccharide composition composed of glucose, the above-described contents may be applied in the same manner.

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 at 28°C and 180 rpm for 24 hours, 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. (DP3+ means that the degree of polymerization is 3 or more)

JY164 selected in Table 1 is judged to be relatively excellent in the 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 transformation method that treats alkylating agents such as EMS (Ethyl Methane Sulfonate). Was sequentially applied to induce mutations and repeat selection of mutant strains, and a 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 (1 IU/ml) 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 citium ( Leuconostoc citreum ).

In the present application, the strain is referred to as leukonostock citium. It was named CJ-2B11 ( 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 oligosaccharide composition

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 samples of the oligosaccharide composition were obtained from each tube.

<Example 6>-Preparation of oligosaccharide composition

In Examples 1 to 5, the mixture was mixed so that the weight ratio of the dry solids of sucrose: the dry solids of maltose was 2:1, and instead of the enzyme solution according to Preparation Example 2, dextran sucrase derived from the genus Leukonstock (D9909) Except for using -10UN, Sigma-Aldrich Co.), an oligosaccharide composition was prepared in the same manner as in Examples 1 to 5.

<Example 7>-Preparation of oligosaccharide composition

Mixing so that the weight ratio of the dry solids of sucrose: dry solids of maltose in Examples 1 to 5 is 2:1, and instead of the enzyme solution according to Preparation Example 2, a known leuconostoc mesenteroides derived from NRRL B-1355 An oligosaccharide composition was prepared in the same manner as in Examples 1 to 5, except that the alternative sucrase was used.

<Experimental Example 1>

The degree of polymerization of the oligosaccharide composition 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 chromatograms for Examples 1 to 3 (chromatogram) is shown in FIGS. 1 to 3.

Before injecting the enzyme reaction oligosaccharide composition sample to 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 oligosaccharide composition of Examples 1 to 3 had a relatively low mixing ratio of sucrose, so that the oligosaccharide content of the polymerization degree 6 or higher (DP6+) was small, and the content of leucrose was small. .

In Examples 6 and 7, the HPLC results showed that the total oligosaccharides of DP3 to DP10+ were 43.77 and 50.56, and the contents of fructose were 38.22 and 38.69, respectively.

<Experimental Example 2>

The results of measuring the turbidity of the samples of the oligosaccharide composition 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 (wt%)	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 oligosaccharide compositions of Examples 1 to 5 have low turbidity (absorbance, 720 nm), and in particular, in the case of the oligosaccharide compositions of Examples 1 to 3, the oligosaccharide content of polymerization degree 6 (DP6) or higher It can be seen that this 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>

The oligosaccharide compositions of Examples 1 to 5 and 1 mL of each of the conventional oligosaccharide IMO (Chungjungwon Oligosaccharide, Subject) sample 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 oligosaccharide compositions of Examples 1 to 5 contain a high content of indigestible oligosaccharides (dietary fiber) compared to the content (6.09% by weight) of indigestible oligosaccharides (dietary fiber) contained in IMO on sale. I confirmed that.

<Experimental Example 4>

For the oligosaccharide composition 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 resistance complex stability, and the results are shown in FIG. 4. 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 oligosaccharide composition of the present application has improved sweetness, excellent body feel, and improved sweet taste strength and sweet taste persistence.

According to FIG. 4, the oligosaccharide composition of the present application maintains a residual ratio of an oligosaccharide composition having a polymerization degree of 3 or higher (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 conventional. It can be confirmed that the level is equivalent to isomaltooligosaccharide (IMO). That is, it can be confirmed that it can be applied to various processed foods such as dairy products.

<Experimental Example 5>

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 having the same dry solid content of 15% by weight. Figs. 5 and 6 show the results of comparative analysis of sensory profiles for sweetness, body sensation, sweetness persistence, taste/off-flavor, thick taste, and bitter taste.

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 oligosaccharide composition 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 was larger, so that the body feeling was enriched, the sweetness persistence was improved, and the content of indigestible oligosaccharides was high so that the thickness was low, and the off-flavor was 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.

[Accession number]

Depositary institution name: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM12588P

Consignment Date: 20190902

Claims (13)

1. In the oligosaccharide composition comprising a saccharide comprising an oligosaccharide composed of glucose,

   An oligosaccharide composition in which the content of the indigestible oligosaccharide is 6.2 to 30 parts by weight based on 100 parts by weight of the saccharide.
2. The method according to claim 1,

   The indigestible oligosaccharide is an oligosaccharide composition comprising an oligosaccharide linked by an α-1,3 glycosidic bond.
3. The method according to claim 1,

   The indigestible oligosaccharide is an oligosaccharide composition that is not degraded by digestive enzymes among the oligosaccharides composed of glucose.
4. The method according to claim 1,

   The oligosaccharide composition further comprises fructose.
5. The method according to claim 1,

   The oligosaccharide composition in which the content of the oligosaccharide of polymerization degree 6 (DP6) or higher is 2 to 40 parts by weight based on 100 parts by weight of the saccharide.
6. The method according to claim 1,

   An oligosaccharide composition wherein the content of the oligosaccharide composed of glucose is 35 to 80 parts by weight based on 100 parts by weight of the saccharide.
7. The method according to claim 1,

   An oligosaccharide composition having a moisture content of 18 to 25% by weight based on the total weight of the oligosaccharide composition.
8. The method according to claim 1,

   An oligosaccharide composition that has a viscosity of 400 cP or more at 25°C.
9. A method for producing a would which comprises a raw material containing sucrose and maltose flow into contact with the Pocono stock (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) can glucan derived from microorganisms of the genus Klein azepin-oligosaccharide composition.
10. The method of claim 9,

    The maltose is a method for producing an oligosaccharide composition that is derived from at least one selected from starch syrup, corn syrup, and hymaltose.
11. The method of claim 9,

    The oligosaccharide composition is a method for producing an oligosaccharide composition comprising an oligosaccharide composed of glucose.
12. The method of claim 11,

    Oligosaccharide composed of the glucose,

    A method for producing a raw material that is containing the sucrose and maltose resulting enzyme is converted by the current stock Pocono (Leuconostoc) in microorganisms or flow Pocono stock (Leuconostoc) spp glucan sucrase azepin the oligosaccharide composition.
13. The method of claim 11,

    The oligosaccharide composed of glucose comprises an oligosaccharide in which α-1,3 glycosidic bonds and α-1,6 glycosidic bonds are alternately linked.

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