KR101919129B1 - Method for preparing fructooligosaccharides - Google Patents

Abstract

The present invention relates to a method for producing a fructooligosaccharide in a high yield by treating an enzyme having high activity to a reaction substrate and a fructooligosaccharide produced by the method.

Description

Method for preparing fructooligosaccharides [

The present invention relates to a method for producing fructooligosaccharide in high yield by treating an enzyme having high activity and a fructooligosaccharide produced thereby.

Fructo-oligosaccharides are natural oligosaccharides of three or more sugars, and are composed of 1-Kestose (GF2), Nystose (GF3) , 1-F fructofuranosyl nystose. Fructo-oligosaccharides are found in vegetables, mushrooms and fruits such as bananas, onions, asparagus, burdocks, garlic, honey, chicory roots and the like. They are found in Agave vera curz (potato plants) and potatoes. Fructo-oligosaccharides show 60% sweetness of sugar, they are well dissolved in water and show refreshing sweetness similar to sugar. They have been used as low-calorie food for a long time because of difficulty in digestion and absorption in our bodies. Also, it contains more than 33% of dietary fiber, which helps to prevent the growth of non - petioles, enteric bacteria, and the growth of enteric bacteria in the lower intestine when ingested from our body. In addition, fructo-oligosaccharides have the function of increasing calcium solubility and promoting absorption in the colon by ingestion alone.

Fructo-oligosaccharides began to be produced using enzymes obtained from microorganisms in the 1980s. Β-Fructofuranosidase, an enzyme that produces fructo-oligosaccharides from sugar, is an enzyme producing fructo-oligosaccharides produced by microorganisms that produce fructo-oligosaccharides in an inverse proportion from sugar, And finally produces 1-F fructofuranosylnitose. This enzyme can be isolated from bacteria or fungi. The reported microorganisms include Aspergillus niger, Aureobasidium pullulans , Athrobacter sp . Penicillium and frequentens .

It is an object of the present invention to provide a method for producing fructooligosaccharide with high yield by treating an enzyme having high activity to a reaction substrate.

Yet another object of the present invention is to provide a fructooligosaccharide produced by treating an enzyme having high activity in a reaction substrate.

Yet another object of the present invention is to provide an Aspergillus strain which is derived from the wild type and has high activity per unit protein of the enzyme produced from the strain.

A novel Aspergillus strain or non-GMO strain derived from wild type which can produce a fructooligosaccharide converting enzyme having an excellent activity to convert sugar into fructooligosaccharide in an excellent yield was isolated and identified, The composition of the medium for optimization was confirmed. In addition, by confirming the optimal pH condition or reaction time of the cell reaction, which is excellent in the ability to convert sugar to fructooligosaccharide using the above-mentioned cells, conditions for efficiently producing fructooligosaccharide in a large quantity were established and the present invention was completed.

Hereinafter, the present invention will be described in more detail.

One example of the present invention is a method for producing fructooligosaccharide in a high yield by treating at least one enzyme selected from the group consisting of a highly active enzyme, a bacterial strain producing the enzyme, a culture, a supernatant of a lysate, and a lysate to a reaction substrate And a fructooligosaccharide produced by the method.

According to the production invention of the present invention, fructooligosaccharide can be produced with high yield, and the fructooligosaccharide can be produced by using 1-kesotse (GF2), Nystose (GF3) and 1-F- And 1-F-fructofuranosyl nystose (GF4).

In one preferred embodiment, the fructooligosaccharide of the present invention is prepared by reacting a fructooligosaccharide converting enzyme with a fructooligosaccharide converting enzyme at a temperature of 60 DEG C for 30 hours, %, Preferably 15 to 45 wt%, and more preferably 28 to 35 wt%.

In one preferred embodiment of the present invention, the fructooligosaccharide of the present invention is characterized in that when the fructooligosaccharide converting enzyme is reacted at a temperature of 60 ° C. for 30 hours, the content of the fructo-oligosaccharide converting enzyme is 0 to 50% by weight 0.01 to 50% by weight, preferably 0 to 35% by weight or 0.01 to 35% by weight.

In one preferred embodiment, the fructooligosaccharide of the present invention is a 1-F-fructofuranosyl-converting enzyme when the fructooligosaccharide converting enzyme is reacted at a temperature of 60 DEG C for 30 hours, based on the total solid content of fructooligosaccharide of 100% Can contain from 0 to 15% by weight or from 0.01 to 15% by weight, preferably from 0 to 10% by weight or from 0.01 to 10% by weight, more preferably from 0 to 5% by weight or from 0.01 to 5% by weight .

When the fructooligosaccharide converted by the fructooligosaccharide converting enzyme of the present invention is produced by reacting with sugar for 30 hours at a temperature of 60 ° C, the value measured in the area% measured by HPLC is 1 - And a content ratio of 1-F-fructofuranosylnitol (1-fecitose: nystose and 1-F-fructofuranosylnitol) is 0.5 to 2.0, preferably 0.8 to 1.5 %. ≪ / RTI >

In the production method of the present invention, sugar may be used as a reaction substrate, and one or more selected from the group consisting of fructose and glucose may be additionally produced using sugar as a reaction substrate, and the reaction product may include fructooligosaccharide, fructose, And the like.

In one preferred embodiment, the fructooligosaccharide of the present invention, when reacted with a fructooligosaccharide converting enzyme at a temperature of 60 DEG C for 30 hours using sugar as a reaction substrate, is characterized in that the fructooligosaccharide conversion enzyme Can be produced in an amount of 50 to 70% by weight, preferably 55 to 65% by weight, more preferably 60 to 63% by weight.

In one embodiment, the step of reacting or treating the strain with a substrate may be carried out by culturing a lysate of the strain, a supernatant of the lysate, or an enzyme derived from the bacterial strain in a culture medium containing sugar. In another embodiment, the step of reacting the substrate with the substrate comprises contacting the substrate with a substrate, such as a cell, a culture of the strain, a lysate of the strain, and / or a supernatant of the lysate, Or contacting the substrate with the carrier on which the strain is immobilized. By reacting the strain with the substrate, the fructooligosaccharide can be produced by converting the substrate, for example, sugar to fructooligosaccharide.

In the method for producing fructooligosaccharide, the concentration of sugar used as a substrate for efficient fructooligosaccharide production may be 30 to 90, preferably 40 to 80% (w / v) based on the total reactants. The sugar can be used in the form of a solution dissolved in a buffer solution or water (for example, distilled water).

The enzyme used in the production method of the present invention may be a fructooligosaccharide-forming enzyme, for example, beta-fructofuranosidase, and may be one produced from Aspergillus niger strain.

The Aspergillus sp. Strain is preferably an Aspergillus niger strain and has a strong starch saccharifying ability and is capable of producing a large amount of citric acid, gluconic acid and the like from sugar, and is used in an organic acid fermentation industry The strains of the present invention can be obtained by subjecting a wild-type Aspergillus or the like isolated from a fermented food to a random mutation such as N-methyl-N-nitroso-guanidine (NTG) (Non-GMO) that has undergone mutation by the first or more.

Preferably, the strain of the present invention is preferably a strain of Accession No. KCTC13140BP ( Aspergillus niger SYG-Neo1) deposited at KCTC in 2016.10.28.

As used in the specification of the present invention, the activity of the strain is the activity of the strain calculated by the following equation (1), and the amount of 1-cystose (mM) produced per minute of the reaction solution containing the supernatant and the substrate Quot;), < / RTI >

[Equation 1]

For example, the Aspergillus niger mutant used in the production method of the present invention is a mutant of wild type Aspergillus oryzae, which has been subjected to a random mutation for a second time or more. The strain activity calculated by the above formula (1) 600 U / ml, preferably 200 to 400 U / ml. The activity of the strain is characterized in that the relative activity of the strain is 150 to 800%, preferably 300 to 650% based on 100% of the wild type Aspergillus oryzae activity.

In the case of genetically modified organisms, GMOs, consumers are very reluctant to purchase them when used in foods, and genetically modified organisms are harmful to environmental ecology. In addition, when an enzyme of a genetically modified organism (GMO) is used, it is possible to produce high-efficiency fructooligosaccharide, but it has a disadvantage in that it takes much time and cost to prove safety for ingesting by a human. On the other hand, in the case of using the natural strain (non-GMO strain with food experience) which has been proven as safe, the natural strain (Non-GMO strain having edible experience) was modified to reduce the above- Lt; / RTI > is important.

The fermented food may be, but is not limited to, at least one selected from the group consisting of miso, chungkukjang, koji, kuchujang, natto, and meju.

The Aspergillus strain of the present invention is characterized in that it is capable of producing a fructooligosaccharide converting enzyme capable of converting a high transversal sugar into a fructooligosaccharide with high yield. The strain may comprise a nucleic acid sequence encoding the fructofuranosidase enzyme and / or a control sequence operably linked upstream thereof to regulate the expression of the fructofuranosidase enzyme in the strain have. Preferably, the strain of the present invention may have the 18S rRNA sequence of SEQ ID NO: 5.

The enzyme having the high fructooligosaccharide converting ability of the present invention may be at least one selected from the group consisting of the cells of the strain, the culture of the strain, the lysate of the strain and the supernatant of the lysate. Preferably, the enzyme may be a fructofuranosidase enzyme, preferably a beta-fructofuranosidase enzyme, contained in the supernatant of the lysate of the strain. For example, the beta-fructofurachidase enzyme of the present invention may have the gene sequence of SEQ ID NO: 7.

The excellent conversion of the enzyme produced by the strain of the present invention not only yields fructooligosaccharide but also shortens the time required for the production of the same amount of fructooligosaccharide as compared with the most widely used Meiji strain. Therefore, the Aspergillus niger strain of the present invention or the enzyme produced thereby is not only a useful substitute for fructooligosaccharides, especially conventional strains which can be usefully used for mass production thereof, but also enhances the yield of fructooligosaccharide production .

The enzymatic activity used in the specification of the present invention is the activity of converting fructooligosaccharide into a unit protein which is calculated by dividing the activity of the strain calculated by the following formula 2 by the weight of the protein solid content in the reaction solution containing the supernatant and the substrate of the strain culture Lt; / RTI >

&Quot; (2) "

For example, the enzyme used in the production method of the present invention has an enzyme activity per unit protein of 300 to 1500 U / mg, preferably 300 to 1400 U / mg, more preferably 300 to 500 U / mg, or 1200 to 1400 U / mg. The activity of the strain is characterized in that the relative enzyme activity is 150 to 1000%, preferably 600 to 1000% or 250 to 400% higher on the basis of the enzyme activity 100% produced from the wild-type Aspergillus niger strain.

In the case of preparing fructooligosaccharide as a strain of the present invention, 0.005 to 0.050 g, preferably 0.007 to 0.03 g, of the sugar substrate 1 kg of the reaction substrate is reacted at a concentration of 60 (w / v)% for 30 hours, Is characterized in that 0.009 to 0.02 g of enzyme is reacted.

The enzyme of the present invention is excellent in storage stability. When the enzyme activity measured by the above formula (2) is 100% at the initial storage, it is preferably 60 to 100% when stored at a temperature of 4, 25 or 37 캜 for 7 months Was maintained in the range of 70 to 100%, and more preferably, in the range of 75 to 95%, indicating that the long-term storage property was excellent. For example, 90 to 100%, preferably 90 to 95% when stored at a temperature of 4 DEG C for 7 months.

The published Japanese specification for the export of fructo-oligosaccharides contains sugar of 33% or less, sugar 10% ± 1.5, 1-Kestose (GF2) 26% ± 3, Nystose (GF3) 26% ± 3, 1-F-Fructofuranosyl nystose (GF4) 5% ± 3, the total fructooligosaccharide content should be 57% or more, but the water-soluble standard will pass the product if the fructooligosaccharide content is 55% or more regardless of the sugar composition. The fructooligosaccharide produced from the strain of the present invention may satisfy all the above specifications.

The temperature at which the fructooligosaccharide converting enzyme of the present invention is reacted with the substrate is excellent at an enzyme activity at a reaction temperature of 40 to 80 ° C, preferably 50 to 70 ° C, more preferably 55 to 65 ° C. In addition, the enzyme exhibits excellent enzyme activity when it is reacted at a temperature of 60 ° C in the range of pH 4 to 8, preferably pH 5 to 7, more preferably pH 5.5 to 6.5.

The fructooligosaccharide production method of the present invention may further include an aeration step in culturing for conversion enzyme production, and may be carried out at 0.1 to 5 vvm, preferably 0.5 to 3 vvm. The aeration can further increase the conversion efficiency of the fructooligosaccharide of the strain.

The fructooligosaccharide converting enzyme of the present invention can be characterized in that it is reacted with the substrate under the conditions of 3 to 60 hours, preferably 12 to 48 hours.

Another example of the present invention provides a culture medium for culturing an Aspergillus strain having the property of converting a reaction substrate into a fructooligosaccharide, or a composition for producing a fructooligosaccharide containing the strain.

The above Aspergillus strain or the above-mentioned fructooligosaccharide converting enzyme can be equally applied to a culture medium for culturing Aspergillus strain or a composition for producing fructooligosaccharide.

The strain applied to the culture medium composition or the production composition of the present invention has excellent fructooligosaccharide conversion ability and the fructooligosaccharide conversion ability is determined by the enzyme which converts the sugar into the fructooligosaccharide, Preferably a beta-fructofuranosidase enzyme, wherein the composition may comprise a medium composition in which the fructooligosaccharide converting enzyme activity is optimized.

Accordingly, the medium composition of the present invention may have a C / N ratio ranging from 1: 3 to 1:10, preferably 1: 3 to 1: 7.

As another example of the present invention, there is provided an Aspergillus strain which is derived from a wild type strain, wherein a strain having high activity per unit protein of an enzyme produced from the strain or a fructooligosaccharide converting enzyme produced by the strain is provided do.

The method for producing the fructooligosaccharide may be applied equally to the strain and the enzyme.

As an example of the present invention, a functional food containing a fructooligosaccharide produced by treating an enzyme having high activity to a reaction substrate is provided.

The method for producing the fructooligosaccharide may be equally applied to the functional food.

The Aspergillus niger strain of the present invention can produce a fructooligosaccharide having an activity of converting fructooligosaccharide from a sugar at a high yield at a high yield in a large scale and can produce a fructooligosaccharide having a fructooligosaccharide And it is expected to be widely used in functional sugar-related health foods and medicines industry since it can produce enzymes with high storage stability.

Fig. 1 shows the results of contour line analysis of the Aspergillus niger strain of the present invention.
Fig. 2 shows the result of analysis of the reaction optimization tool of Aspergillus niger strain of the present invention.
FIG. 3 shows the results of comparing the activity changes when the parent strain Aspergillus niger and the strains 1 and 2 of the present invention were cultured in a 5 L fermentor.
Fig. 4 shows the result of SDS-PAGE of the enzyme protein by the purification step.

The present invention will be described in more detail with reference to the following examples. However, the scope of protection of the present invention is not intended to be limited to the following examples.

Example  One: Food origin  Microbial separation

1.1 Culture and isolation of a strain in a sample

To investigate microorganisms producing fructooligosaccharide converting enzyme, domestic miso, Chungkukjang, Nuruk, Kochujang, Natto and Meju (purchased from department store, Mart and traditional market: Daegu Lotte Department Store, Daegu Chilsung Market, Daejeon Dunsan- Lotte Mart). 1 g of the collected food sample was suspended in 10 mL of 0.85% NaCl, and 100 μl of the suspension was plated on a Wallerstein Laboratory Nutrient Agar (WLNA), a Potato Dextrose Agar (PDA) and an Inulin Agar (IA) medium and then cultured at 28 ° C for 3 days. The colonies grown in the solid medium were sorted and separated in different shapes and sizes, and cultured in the production medium at 30 ° C for 5 days with shaking. The composition of each of the above media is shown in Table 1 below.

Medium component WLNA (g / L) PDA (g / L) IA (g / L) Production badge Yeast extract 4 - One 35 Potato starch - 4 - - Inulin - - 20 - Bactocasitone 5 - - - Sucrose - - - 150 Dextrose 50 20 - - K ₂ HPO ₄ One - One - KCl 0.125 - - - MgSO ₄ -7H ₂ O 0.25 - 0.5 - FeCl ₃ 0.0025 - - - MnSO ₄ -4H ₂ O 0.0025 - - - NaNO ₃ - - 3 - Bromocresol green 0.022 - - - Agar 20 20 20 - CMC * - - - 5 pH pH6 - pH5 pH 6.5

* CMC: carboxymethylcellulose sodium salt

The supernatant was centrifuged and the supernatant was used as a crude enzyme. Crude enzyme was reacted for 24 hours at 40 ℃ using 100 g / L sugar as a substrate. The reaction products were analyzed by thin layer chromatography (TLC). The TLC plate was developed three times using TLC silica gel 60 F254 and 85% acetonitrile as an electric solvent and immersed in a mixed solution of methanol: sulfuric acid: N- (1-Naphthyl_ethylene diamine dihydrochloride = 95: And the color development was observed by heat treatment. Ten kinds of strains transformed from the 467 kinds of strains into fructo-oligosaccharides were screened by TLC.

Forty-seven species of strains screened in the solid medium were 48 in the Nuruk sample, 68 in the Chungkukjang sample, 13 in the Kochujang sample, 138 in the Miso sample, 19 in the Meju flour sample, 46 in the Meju sample, 4 in makkolli samples, 8 in makgeolli samples, and 123 in others.

1.2 Selection of parent strain

Ten strains selected in Example 1.1 were inoculated into a liquid medium containing 15% sucrose, 3.5% yeast extract and 0.5% carboxymethylcellulose-CMC (pH 6.5), and cultured at 30 ° C And cultured for 5 days with shaking. The culture broth was centrifuged to separate the supernatant and cells, and the supernatant was used as a crude enzyme. Crude enzyme was reacted with 100 g / L sugar at 40 ℃ for 30 min. The activity of fructooligosaccharide - forming enzyme was compared by HPLC RI analysis. Agilent HPLC was used. Specific analytical conditions were as follows.

[HPLC R1 analysis conditions]

Column: NH2-P50 4E (shodex) column

Mobile phase solvent: 70% acetonitrile

Analysis temperature: 30 ° C

Flow rate: 1 ml / min

Injection volume: 10l

Detector: RI detector

Identification was also performed on 10 isolates that were compared for activity. PCR was performed for identification of strains. PCR was carried out using universal primers NL1 and NL4, followed by sequencing. After the nucleotide sequence analysis, the strain was identified by referring to the NCBI database.

The results are shown in Table 2. The identified strains were Pichia farinose , Yarrowia lipolytica , Millerozyma farinose , Aspergillus oryzae, and Aspergillus niger , and the nucleotide sequences of the 18S ribosomal RNAs thereof are the sequences of SEQ ID NOS: 1 to 5, respectively. Among them, Aspergillus niger, which has the highest activity, was selected as the final best isolate.

Identified strain name 18S rRNA SEQ ID NO Food type Count Strain activity (U / mL) pichia farinose SEQ ID NO: 1 Miso One 3.3 Yarrowia liplytica SEQ ID NO: 2 Cheonggukjang One 1.71 Milerozyma farinose SEQ ID NO: 3 Cheonggukjang One 2.47 Aspergillus oryzae SEQ ID NO: 4 Meju 6 20-25 Aspergillus  niger SEQ ID NO: 5 Meju One 30 sum 10

Example  2. Fructooligosaccharide  Selection of high production primary mutants

2.1 Selection of first mutant

The parent strain isolated in Example 1 was plated on a Potato dextrose agar (PDA) solid substrate and incubated at 28 ° C for 3 days. Glycerol stock was prepared and stored in the solid medium. The glycerol stock was used to mutagenize glycerol stock and washed 3 times. Mutants were mutagenized using washed spores, and NTG (N-methyl-N-nitroso-guanidine) and UV 254 nm were used in combination. The washed spores were mixed with 1 ml of NTG solution dissolved at 2 mg / ml and left at room temperature for 10 minutes. The NTG solution was removed by centrifugation and then washed three times. The washed spores were suspended in 1 ml of sterilized water, appropriately diluted and plated on a solid medium. The solid medium composition is shown in Table 3 below.

Medium component g / L Glycerol 30 2-Deoxyglucose 1, 5 MgSO ₄ .7H ₂ O One Sodium glutamate 2 KCl 0.5 K ₂ HPO ₄ One FeSO ₄ .7H ₂ O 0.01 Agar 20 pH pH 6

2-deoxyglucose was used as a maker and irradiated for 1 min at 254 nm in a solid medium containing 2-deoxyglucose. The colonies selected through the mutation proceeded to an activity measurement by liquid culture (production medium of Table 1). 100 g / L of sugar was reacted at pH 40 (40 mM McIlvain buffer, pH 5) for 30 minutes at a reaction temperature of 40 ° C, and then treated with boiling water for 10 minutes to inactivate the enzyme. Were measured. At this time, the first mutants having excellent conversion activity were selected.

2.2 Optimum C / N ratio setting

Experiments were performed to set the optimal C / N ratio using the first-order mutant selected in Example 2.1. Using the experimental design method, activity values of 150 ~ 350 g / L of sugar and 30 ~ 60 g / L of yeast extract were incubated for 5 days and analyzed by Minitab 's reaction optimization tool. The culture medium was prepared by removing CMC from the existing production medium and adding 100 mM MES.

The results of the contour line analysis are shown in FIG. 1, and it was confirmed that when the contour line was checked, the higher the sugar concentration and the higher the yeast extract concentration, the higher the activity. Table 4 shows the results of comparing the activities by the media conditions, and FIG. 2 shows the result of analyzing the reaction optimization tool. 250 g / L of sugar and 53 g / L of CMC (100 mM of yeast extract) showed the maximum activity. Therefore, the medium C / N ratio, which can increase the fructooligosaccharide activity, was found to be 4.7. The activity of the strain was calculated by the following equation.

[Equation 1]

The strain (experimental group) Sugar (g / L) Yeast extract (g / L) C / N ratio The strain activity (U / ml) The first mutant (control) 150 35 4.3 81 Primary mutant (optimal condition) 250 53 4.7 140

Example  3. Fructooligosaccharide  High Production Secondary Mutant Selection

The cells were cultured in the same manner as in Example 2.1, except that the primary mutant of Example 2.1 was used as the parent strain, and then mutagenized using NTG (N-methyl-N-nitroso-guanidine) and UV 254 nm as a mutant, The mutant strains were tested for their ability to convert fructooligosaccharides and the second most active mutants were selected.

The strain was deposited with KCTC on October 28, 2016, and was granted accession number KCTC13140BP.

Example  4. Fructooligosaccharide  Evaluation of activity of high production strain

Colonies selected through mutation in Examples 2 and 3 measured the activity of liquid culture (production medium of Table 1). In the measurement of activity, GF3 or higher is difficult to quantify when GF3 is produced. Therefore, the enzyme should be diluted so that only GF2 is produced. 100 g / L of sugar is reacted in a pH 5 (40 mM McIlvain buffer) for 30 minutes at a reaction temperature of 40 ° C, and then treated with boiling water for 10 minutes to inactivate the enzyme. The enzyme-inactivated reaction solution was diluted 2-fold, subjected to HPLC analysis, and assayed for activity at the converted value. .

The activity of the strain was expressed by the following equation (1), and the activity of the enzyme was expressed as a strain activity per unit protein derived from the following equation (2).

[Equation 1]

&Quot; (2) "

The result of the above-mentioned activity of the strain and the result of measuring the enzyme activity as a strain activity per unit protein are shown in Table 5 below.

Strain The strain activity (U / ml) Protein (mg / ml) Enzyme activity (U / mg) Parent strain 35 15 47 Primary mutant 81 25 65 Secondary mutant 221 43 409

Example  5. Culture of strain by fermentation tank

The second mutant of Example 3 was cultured in a 5 L fermenter with a medium composition having a C / N ratio of 4.7. The culture conditions were maintained at pH 5.5 ~ 6.5 using ammonia water, cultured for 144 hours at a temperature of 30 ℃, rpm 300 ~ 650, and aerated with 1 vvm. The seed culture was inoculated to 3 ~ 5% of the fermenter culture. Activity and protein patterns were analyzed by sampling during culture.

The secondary mutant was cultured in a 5 L fermentor and supernatant was obtained. The fructooligosaccharide conversion activity and the strain activity over time were calculated in the same manner as in Example 4, and the conversion activity of fructooligosaccharide Protein concentrations in culture supernatants are shown in Table 6 below. As shown in Table 6, it was confirmed that the fructooligosaccharide converting enzyme was highly produced and the protein content was high in the second mutant strain, and the fructooligosaccharide producing enzyme was produced at the highest yield. As a comparative example, fructosylpuranosidase (Meiji) manufactured by Meiji Co., Ltd. was used to carry out the reaction by mixing with the substrate in the same manner as described above

Strain The strain activity (U / ml) Protein (mg / ml) Activity per unit protein (U / mg) Parent strain 150 30 400 Primary mutant 315 50 504 Secondary mutant 455 70 1300 Comparative Example - 740

When the reaction was carried out for 30 hours at a concentration of 60 (w / v)% of 1 kg of sugar as a reaction substrate in the production of fructooligosaccharide, 0.012 g of the enzyme was used for the second mutant and 0.008 g of enzyme Should be added.

Example  6. Fructooligosaccharide  Production enzyme separation and purification

In Example 5, MF (micro filtration) and UF (ultrafiltering) processes were first performed to obtain the enzyme from the culture of mutant strain 2 cultured in the 5 L fermenter. The supernatant obtained by centrifugation was subjected to filtration under reduced pressure using a 10 탆 filter paper, and the filtrate passed through the filter was recovered. The filtrate was passed through a semipermeable membrane of 30 KDa size using a filtrate from which cells were removed, and a substance larger than the semipermeable membrane size containing fructo-oligosaccharide producing enzyme was recovered and dialyzed with 20 mM sodium phosphate buffer (pH 7.2) The buffer was exchanged until it reached 6.8 ~ 7.2. UF treatment solution was centrifuged to recover the supernatant, and FPLC purification proceeded. Resin was Q Sepharose FF. Buffer A was 20 mM sodium phosphate (pH 7.2) and buffer B was buffer A and 1 M NaCl (pH 7.2). After resin filling, the buffer A was flowed at a flow rate of 10 ml / min about 2 to 3 times the volume of the resin to sufficiently stabilize the resin. When the resin was stabilized sufficiently, the supernatant treated with the impurity after the UF treatment was loaded at a flow rate of 10 ml / min. The eluate was detected with UV 260 nm and the fraction eluted was recovered to measure the enzyme activity. In the purification of fructooligosaccharide production enzyme from the culture supernatant, it was confirmed that the low molecular weight except for the target protein was removed step by step, and the pattern was similar to the enzyme band of Meiji which is currently used commercially.

The complete cds of beta-fructofuranosidase, an enzyme that produces fructooligosaccharide of the fructooligosaccharide-producing enzyme, was analyzed to show the sequence of SEQ ID NO: 7 (secondary mutant) (SEQ ID NO: 6) of the wild type (SEQ ID NO: 6) was compared with the secondary mutant sequence. As a result, it was confirmed that 1376 bp was changed from G to C in the parental gene sequence.

Example  7. Stability evaluation of enzyme long-term storage

In order to evaluate the long-term storage stability of the fructooligosaccharide-producing enzyme isolated in Example 6, the enzyme activity was measured at a temperature of 4, 25, and 37 ° C for 7 months. Enzyme activity was calculated in the same manner as in Example 4 above.

 As a result, it was confirmed that the activity was maintained at 92% at a temperature of 4 ° C, and that the long-term storage stability was excellent, and the activity was maintained at 80% and 79% at room temperature of 25 ° C or 37 ° C, respectively. Respectively.

Example  8. Fructooligosaccharide  Using production enzymes Fructooligosaccharide  production

The fructooligosaccharide production enzyme isolated in Example 6 was used to react with sugar to confirm that fructooligosaccharide production meets the standard. The published Japanese specification for the export of fructo-oligosaccharides contains sugar of 33% or less, sugar 10% ± 1.5, 1-Kestose (GF2) 26% ± 3, Nystose (GF3) 26% ± 3, 1-F-Fructofuranosyl nystose (GF4) 5% ± 3, the total fructooligosaccharide content should be 57% or more, but the water-soluble standard will pass the product if the fructooligosaccharide content is 55% or more regardless of the sugar composition. In order to confirm that the sugar composition according to this standard is produced, 1.5 Kg of sugar and 1 Kg of distilled water were mixed to prepare a substrate having a final concentration of 60%.

The pH of the substrate was adjusted to about 6.2 to 6.5 by adding HCl or NaOH if necessary. There is no pH control during the reaction. The purified enzyme was added to the prepared substrate and the reaction was carried out at a reaction temperature of 60 ° C. For the first reaction, the mixture was stirred at 200 rpm for mixing the enzyme and the substrate. After that, the reaction was allowed to proceed. The reaction was analyzed by HPLC RI and the yield of each saccharide composition was assessed as Area% values. As a comparative example, fructosylpuranosidase (Meiji) manufactured by Meiji Co., Ltd. was used and the reaction was carried out by mixing with the substrate in the same manner as above.

When the fructooligosaccharide-producing enzyme isolated in Example 6 was used, the fructooligosaccharide was found to be 55% or more when reacted for 15 hours to 30 hours, and the fructooligosaccharide content was 61.39% Respectively. The composition of the saccharide produced in the reaction for 30 hours is shown in Table 7 together with the results of the comparative examples.

Kinds Fru Glu Suc GF2 GF3 GF4 FOS GF2 / (GF3 + GF4) Example 7 1.28% 26.88% 10.83% 31.66% 26.04% 3.31% 61.01% 1.1 Comparative Example 2.35% 27.80% 10.74% 25.86% 26.72
% 6.53% 59.11% 0.78

As can be seen in Table 7, fructooligosaccharide can be produced at a higher level than that of the commercially available strain of the comparative example, and the production rate of GF2, i.e., 1-cystose, in the fructooligosaccharide is higher As well as the Japanese export standard.

Example  9. Depending on the reaction conditions Fructooligosaccharide  production

9.1 Evaluation of activity according to pH condition

When the sugar was converted to fructooligosaccharide using the enzyme produced by the fructooligosaccharide isolated in Example 6, the enzyme activity was changed to pH 6, pH 7, pH 8 and pH 8.5 at a reaction temperature of 60 ° C Were measured after 0, 1, 2, 3, and 4 hours. The enzyme activity was calculated in the same manner as in Example 4, and the value converted to the relative activity value at 100% was shown in Table 8 below.

pH Before reaction 1 hours 2 hours 3 hours 4 hours 6 100% 37.76% 22.28% 4.18% 3.62% 7 100% 18.54% 3.87% 0.64% 0.49% 8 100% 5.35% 0.61% 0.37% 0.27% 8.5 100% 1.34% 0.36% 0.15% 0.12%

As described above, it was confirmed that the enzyme activity was the best in the condition of pH 6, and the activity could be maintained for a long time.

9.2 Evaluation of activity according to temperature condition

When the sugar was converted to fructooligosaccharide by using the fructooligosaccharide-producing enzyme isolated in Example 6, the reaction was carried out at a reaction temperature of 60, 70, 80 and 90 ° C under the condition of pH 6, The enzyme activity was measured at 0, 1, 2, 3, and 4 hours. The enzyme activity was calculated in the same manner as in Example 4, and the value converted to the relative activity value at 100% was shown in Table 9 below.

Temperature Before reaction 1 hours 2 hours 3 hours 4 hours 60 ° C 100 37.76 22.28 4.18 3.62 70 ℃ 100 0.26 0.04 0.01 0.00 80 ℃ 100 0.01 0.00 0.00 0.00 90 ° C 100 0.00 0.00 0.00 0.00

As described above, it was confirmed that most of the enzyme was inactivated at the start of the reaction when the temperature was 70 ° C or higher, and that the enzyme activity could be maintained for a long time at a reaction temperature of 60 ° C.

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC13140BP

Checked on: 20161028

<110> SAMYANG CORPORATION <120> Method for preparing fructooligosaccharides <130> DPP20164679 <160> 7 <170> Kopatentin 1.71 <210> 1 <211> 646 <212> DNA <213> 18s rRNA of pichia farinose <400> 1 gt; agaagtccat aagttcttac acacaggtgt tttttttgtt tgtgaaaaaa atttactttg 120 gtctggaact agaaatagtt ttgggccaga gggcaactta acttcaattt atattgaatt 180 gtttttaaat ttatttgtca aattattgat attaatcaaa aatcttcaaa actttcaaca 240 acggatctct tggttctcgc atcgatgaag aacgcagcga aatgcgataa gtaatatgaa 300 ttgcagattt tcgtgaatca tcgaatcttt gaacgcacat tgcgcccttt ggtattccaa 360 agggcatgcc tgtttgagcg tcatttctct ctcaaaccgc aaggtttggt gttgagcaat 420 atagatattt cggtatctat ttgcttgaaa tggattggca tgagtattta cagtagataa 480 atgccgtttg actcttcaat gtattaggtc taaccaactc gttgaaacag ttagcggtag 540 tatctgtgta aaagaggctc ggccttacaa caatctacaa agtttgacct caaatcaggt 600 aggaataccc gctgaactta agcatatcaa aagcccggag gaaang 646 <210> 2 <211> 354 <212> DNA <213> 18s rRNA of Yarrowia liplytica <400> 2 ggtttccgta ggtgaacctg cggaaggatc attattgatt ttatctattt ctgtggattt 60 ctggtwtatt acagcgtcat tttatctcaa ttataactat caacaacgga tctcttggct 120 ctcacatcga tgaagaacgc agcgaaccgc gatatttatt gtgacttgca gatgtgaatc 180 atcaatcttt gaacgcacat tgcgcggtat ggcattccgt accgcacgga tggaggagcg 240 tgttcgctct gggatcgcat tgctttcttg aaatggattt tttaaactct caattattac 300 gtcatttcac ctccttcatc cgagattacc cgctgaactt aagcatatca ataa 354 <210> 3 <211> 813 <212> DNA <213> 18s rRNA of Milerozyma farinose <400> 3 atgggaggga aaagaagcca aaactctgat aaagacggca gaaggaaacg acataaggtt 60 tcggggttca tagatcctaa tacaagcgga atatacgcta catgtaatag agggaaagaa 120 aaccaatgcc gcaatgaatt gataaatttc ttcagtgaaa aagcagaaga gtattatggt 180 gatcttgatc tagaaagcga caaggaagat aaacaggaat tgactataga agagcagatt 240 gctgcagagg taggtaacct caaggacaag ggaaagaata aaaaagaaac tttcaagcct 300 attgacctag gatgtgaatg cttgattttc ttcaagactc gaaaacccgt tcagcctgcc 360 gaatttgtgc aaaggatatg ccaggagtgc catgacagca aaagaaagac aactaggtac 420 acgcagaagt tgacgccgat ctccttttcg gtctctccgt ccatcgagga gttgaaaaaa 480 ttggccaaga tagtgcttgg gccccatttt cataaggaag aaggacaaga gccacataaa 540 tttgcgatca atgttaccag acgtaacttt aacaccttgc caaagagcga cattataaaa 600 acggtagctg aatgcgtggg cagggagcac ggccattcag ttgacttaaa ggcattcgat 660 aaattaatac tagttgagtg ctataaaagc aatataggca tgagtgtagt tgaaaattat 720 aaccaactag aacggttcaa cttgcagcaa atctttgaca agaaccaaga gggagccgaa 780 gtcgaagcca agtccgattc taacgtagct tag 813 <210> 4 <211> 544 <212> DNA <213> 18s rRNA of Aspergillus oryzae <400> 4 ggggacctgc ggaaggatca ttaccgagtg tagggttcct agcgagccca acctcccacc 60 cgtgtttact gtaccttagt tgcttcggcg ggcccgccat tcatggccgc cgggggctct 120 cagccccggg cccgcgcccg ccggagacac cacgaactct gtctgatcta gtgaagtctg 180 agttgattgt atcgcaatca gttaaaactt tcaacaatgg atctcttggt tccggcatcg 240 atgaagaacg cagcgaaatg cgataactag tgtgaattgc agaattccgt gaatcatcga 300 gtctttgaac gcacattgcg ccccctggta ttccgggggg catgcctgtc cgagcgtcat 360 tgctgcccat caagcacggc ttgtgtgttg ggtcgtcgtc ccctctccgg gggggacggg 420 ccccaaaggc agcggcggca ccgcgtccga tcctcgagcg tatggggctt tgtcacccgc 480 tctgtaggcc cggccggcgc ttgccgaacg caaatcaatc tttccaggtg acctcgatca 540 beak 544 <210> 5 <211> 571 <212> DNA <213> 18s rRNA of Aspergillus niger <400> 5 ggtttccgta ggtgaacctg cggaaggatc attaccgagt gctgggtcct tcggggccca 60 acctcccacc cgtgcttacc gtaccctgtt gcttcggcgg gcccgccttc gggcggcctg 120 gggcctgccc ccgggaccgc gcccgccgga gaccccaatg gaacactgtc tgaaagcgtg 180 cagtctgagt cgattgatac caatcagtca aaactttcaa caatggatct cttggttccg 240 gcatcgatga agaacgcagc gaaatgcgat aactaatgtg aattgcagaa ttcagtgaat 300 catcgagtct ttgaacgcac attgcgcccc ctggtattcc ggggggcatg cctgtccgag 360 cgtcatttct cccctccagc cccgctggtt gttgggccgc gcccccccgg gggcgggcct 420 cgagagaaac ggcggcaccg tccggtcctc gagcgtatgg ggctctgtca cccgctctat 480 gggcccggcc ggggcttgcc tcgaccccca atcttctcag attgacctcg gatcaggtag 540 ggatacccgc tgaacttaag catatcaata a 571 <210> 6 <211> 1965 <212> DNA <213> beta-fructofuranosidase from Aspergillus niger wild type <400> 6 atgaagctca ccactaccac cctggcgctc gccaccggcg cagcagcagc agaagcctca 60 taccacctgg acaccacggc cccgccgccg accaacctca gcaccctccc caacaacacc 120 ctcttccacg tgtggcggcc gcgcgcgcac atcctgcccg ccgagggcca gatcggcgac 180 ccctgcgcgc actacaccga cccatccacc ggcctcttcc acgtggggtt cctgcacgac 240 ggggacggca tcgcgggcgc caccacggcc aacctggcca cctacaccga tacctccgat 300 aacgggagct tcctgatcca gccgggcggg aagaacgacc ccgtcgccgt gttcgacggc 360 gccgtcatcc ccgtcggcgt caacaacacc cccaccttac tctacacctc cgtctccttc 420 ctgcccatcc actggtccat cccctacacc cgcggcagcg agacgcagtc gttggccgtc 480 gcgcgcgacg gcggccgccg cttcgacaag ctcgaccagg gccccgtcat cgccgaccac 540 cccttcgccg tcgacgtcac cgccttccgc gatccgtttg tcttccgcag tgccaagttg 600 gatgtgctgc tgtcgttgga tgaggaggtg gcgcggaatg agacggccgt gcagcaggcc 660 gtcgatggct ggaccgagaa gaacgccccc tggtatgtcg cggtctctgg cggggtgcac 720 ggcgtcgggc ccgcgcagtt cctctaccgc cagaacggcg ggaacgcttc cgagttccag 780 tactgggagt acctcgggga gtggtggcag gaggcgacca actccagctg gggcgacgag 840 ggcacctggg ccgggcgctg ggggttcaac ttcgagacgg ggaatgtgct cttcctcacc 900 gaggagggcc atgaccccca gacgggcgag gtgttcgtca ccctcggcac ggaggggtct 960 ggcctgccaa tcgtgccgca ggtctccagt atccacgata tgctgtgggc ggcgggtgag 1020 gtcggggtgg gcagtgagca ggagggtgcc aaggtcgagt tctccccctc catggccggg 1080 tttctggact gggggttcag cgcctacgct gcggcgggca aggtgctgcc ggccagctcg 1140 gcggtgtcga agaccagcgg cgtggaggtg gatcggtatg tctcgttcgt ctggttgacg 1200 ggcgaccagt acgagcaggc ggacgggttc cccacggccc agcaggggtg gacggggtcg 1260 ctgctgctgc cgcgcgagct gaaggtgcag acggtggaga acgtcgtcga caacgagctg 1320 gtgcgcgagg agggcgtgtc gtgggtggtg ggggagtcgg acaaccagac ggccaggctg 1380 cgcacgctgg ggatcacgat cgcccgggag accaaggcgg ccctgctggc caacggctcg 1440 gtgaccgcgg aggaggaccg cacgctgcag acggcggccg tcgtgccgtt cgcgcaatcg 1500 ccgagctcca agttcttcgt gctgacggcc cagctggagt tccccgcgag cgcgcgctcg 1560 tccccgctcc agtccgggtt cgaaatcctg gcgtcggagc tggagcgcac ggccatctac 1620 taccagttca gcaacgagtc gctggtcgtc gaccgcagcc agactagtgc ggcggcgccc 1680 acgaaccccg ggctggatag ctttactgag tccggcaagt tgcggttgtt cgacgtgatc 1740 gagaacggcc aggagcaggt cgagacgttg gatctcactg tcgtcgtgga taacgcggtt 1800 gtcgaggtgt atgccaacgg gcgctttgcg ttgagcacct gggcgagatc gtggtacgac 1860 aactccaccc agatccgctt cttccacaac ggcgagggcg aggtgcagtt caggaatgtc 1920 tccgtgtcgg aggggctcta taacgcctgg ccggagagaa attga 1965 <210> 7 <211> 1965 <212> DNA <213> beta-fructofuranosidase from Aspergillus niger 2nd mutant strain <400> 7 atgaagctca ccactaccac cctggcgctc gccaccggcg cagcagcagc agaagcctca 60 taccacctgg acaccacggc cccgccgccg accaacctca gcaccctccc caacaacacc 120 ctcttccacg tgtggcggcc gcgcgcgcac atcctgcccg ccgagggcca gatcggcgac 180 ccctgcgcgc actacaccga cccatccacc ggcctcttcc acgtggggtt cctgcacgac 240 ggggacggca tcgcgggcgc caccacggcc aacctggcca cctacaccga tacctccgat 300 aacgggagct tcctgatcca gccgggcggg aagaacgacc ccgtcgccgt gttcgacggc 360 gccgtcatcc ccgtcggcgt caacaacacc cccaccttac tctacacctc cgtctccttc 420 ctgcccatcc actggtccat cccctacacc cgcggcagcg agacgcagtc gttggccgtc 480 gcgcgcgacg gcggccgccg cttcgacaag ctcgaccagg gccccgtcat cgccgaccac 540 cccttcgccg tcgacgtcac cgccttccgc gatccgtttg tcttccgcag tgccaagttg 600 gatgtgctgc tgtcgttgga tgaggaggtg gcgcggaatg agacggccgt gcagcaggcc 660 gtcgatggct ggaccgagaa gaacgccccc tggtatgtcg cggtctctgg cggggtgcac 720 ggcgtcgggc ccgcgcagtt cctctaccgc cagaacggcg ggaacgcttc cgagttccag 780 tactgggagt acctcgggga gtggtggcag gaggcgacca actccagctg gggcgacgag 840 ggcacctggg ccgggcgctg ggggttcaac ttcgagacgg ggaatgtgct cttcctcacc 900 gaggagggcc atgaccccca gacgggcgag gtgttcgtca ccctcggcac ggaggggtct 960 ggcctgccaa tcgtgccgca ggtctccagt atccacgata tgctgtgggc ggcgggtgag 1020 gtcggggtgg gcagtgagca ggagggtgcc aaggtcgagt tctccccctc catggccggg 1080 tttctggact gggggttcag cgcctacgct gcggcgggca aggtgctgcc ggccagctcg 1140 gcggtgtcga agaccagcgg cgtggaggtg gatcggtatg tctcgttcgt ctggttgacg 1200 ggcgaccagt acgagcaggc ggacgggttc cccacggccc agcaggggtg gacggggtcg 1260 ctgctgctgc cgcgcgagct gaaggtgcag acggtggaga acgtcgtcga caacgagctg 1320 gtgcgcgagg agggcgtgtc gtgggtggtg ggggagtcgg acaaccagac ggccacgctg 1380 cgcacgctgg ggatcacgat cgcccgggag accaaggcgg ccctgctggc caacggctcg 1440 gtgaccgcgg aggaggaccg cacgctgcag acggcggccg tcgtgccgtt cgcgcaatcg 1500 ccgagctcca agttcttcgt gctgacggcc cagctggagt tccccgcgag cgcgcgctcg 1560 tccccgctcc agtccgggtt cgaaatcctg gcgtcggagc tggagcgcac ggccatctac 1620 taccagttca gcaacgagtc gctggtcgtc gaccgcagcc agactagtgc ggcggcgccc 1680 acgaaccccg ggctggatag ctttactgag tccggcaagt tgcggttgtt cgacgtgatc 1740 gagaacggcc aggagcaggt cgagacgttg gatctcactg tcgtcgtgga taacgcggtt 1800 gtcgaggtgt atgccaacgg gcgctttgcg ttgagcacct gggcgagatc gtggtacgac 1860 aactccaccc agatccgctt cttccacaac ggcgagggcg aggtgcagtt caggaatgtc 1920 tccgtgtcgg aggggctcta taacgcctgg ccggagagaa attga 1965

Claims (17)

KCTC13140BP having a storage stability of 60 to 100% relative enzyme activity based on 100% of enzyme activity at the initial storage at a temperature of 4 DEG C for 7 months at a temperature of 4 to &lt; RTI ID = 0.0 &gt; , A fructooligosaccharide converting enzyme produced from an Aspergillus niger strain having the amino acid sequence of KCTC13140BP having the amino acid sequence of SEQ ID NO: 1, a culture supernatant of an Aspergillus niger strain having the accession number KCTC13140BP producing the enzyme, Reacting at least one selected with the sugar as a reaction substrate to produce a fructooligosaccharide,
The step of reacting with the sugar is carried out in a pH range of 5.5 to 6.5 and a temperature range of 55 to 65 ° C,
Wherein the fructooligosaccharide comprises 1-cystose, nystose, and 1-F-fructofuranosylnitose and is selected from the group consisting of 1-kes Wherein the weight ratio of 1-cystose / 1-F-fructofuranosylnitose to the total weight of 1-fructose / 1-F-fructofuranosylnitose is 0.5 to 2.0. The production method according to claim 1, wherein the fructooligosaccharide is produced as 50 to 70% by weight of fructooligosaccharide based on 100% by weight of the total sugar solid content as a reaction substrate. delete The method according to claim 1, wherein the ratio by weight of 1-cystose to the total weight of said nystatose and 1-F-fructofuranosylnitose (1-cystose / (1-F-fructofuranosyl nitrate, Lt; / RTI &gt; is from 0.8 to 1.5. delete delete The production method according to claim 1, wherein the enzyme is a beta-fructofuranosidase having the gene sequence of SEQ ID NO: 7. delete delete delete delete As the Aspergillus nasi mutant strain having the accession number KCTC13140BP having fructooligosaccharide production activity,
The enzyme activity of the enzyme produced from the strain is 300 to 1500 U / mg, and the relative enzyme activity is 60 to 100% based on 100% of the enzyme activity at the initial storage at a temperature of 4 ° C for 7 months. Enzymes,
Wherein the fructooligosaccharide comprises 1-cystose, nystose and 1-F-fructofuranosylnitose, and when incubated in a pH range of 5.5 to 6.5 and a temperature range of 55 to 65 ° C, (Total weight of 1-cystose / nystose and 1-F-fructofuranosylnitol) based on the total weight of 1-F-fructofuranosylnitose is 0.5 to 2.0 Wherein the strain produces a fructooligosaccharide. delete delete delete delete delete

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