Novel aureobasidium pullulans strain for efficiently producing oligosaccharide and application of novel aureobasidium pullulans strain

Abstract

The invention relates to an aureobasidium pullulans CJFSKY2201 strain which is preserved by a preservation number of KCCM13230P, and a preparation method of the aureobasidium pullulans A culture of the strain; the use of the strain in the production of oligosaccharides; compositions comprising the strains, lysates thereof, cultures thereof, concentrates thereof, or dried products thereof; and a method for producing an oligosaccharide, said method comprising a step of culturing said strain and a step of obtaining an oligosaccharide from the culture.

Description

Technical Field

[0001] The present disclosure relates to the Aureobasidium pullulans CJFSKY2201 strain deposited under the accession number KCCM13230P; a culture of the strain; use of the strain in producing oligosaccharides; a composition comprising the strain, a lysate thereof, a culture thereof, a concentrate thereof, or a dried product thereof; and a method for producing oligosaccharides, the method comprising the steps of culturing the strain and obtaining oligosaccharides from the culture. Background Art

[0002] Oligosaccharides play an important role in various biological processes and are used in dairy products, confectionery, beverages, and other industries to enhance functionality, improve sweetness, and enhance physical properties. As their functions become more widely known, interest in oligosaccharide synthesis is growing. Currently, fructooligosaccharides are the best-selling oligosaccharides in Korea and abroad. They are widely used as an ingredient in various health functional foods, and their sales are increasing annually. From this point forward, overall demand for oligosaccharides is expected to continue to increase.

[0003] Oligosaccharides are present in natural foods such as tomatoes, bananas, onions, mushrooms, etc., but it is difficult to extract oligosaccharides directly from natural foods, so they are mostly synthesized. In addition, for the research and practical application of oligosaccharides in medical and health functional foods, their large-scale synthesis is necessary, but the synthesis of oligosaccharides is very difficult. At present, enzymatic methods and whole-cell biocatalytic methods using microorganisms are used to produce oligosaccharides, and chromatography and crystallization are known existing methods for producing high-content fructooligosaccharides. However, due to the extremely low yield and the difficulty of large-scale production, both methods lack economic efficiency (Korean Patent No. KR 10-1994-0005660B1).

[0004] Demand for oligosaccharides continues to increase, and investment in equipment is ongoing, but the development of production strains has not yet begun. However, further physical investment has become impossible within the limited manufacturing space, and the rate of productivity improvement relative to the investment amount is insufficient.

[0005] Against this background, the present inventors have made efforts to increase the activity of the strain itself by developing the strain itself for oligosaccharide production, and as a result, they developed a strain that rapidly converts a sugar solution into oligosaccharides, thereby completing the present disclosure. Summary of the Invention

[0006] [Technical Issues]

[0007] The present disclosure aims to provide a strain of Aureobasidium pullulans CJFSKY2201 deposited under the accession number KCCM13230P and a method for producing oligosaccharides using the strain.

[0008] [Technical solution]

[0009] The object of the present disclosure is to provide the Aureobasidium pullulans CJFSKY2201 strain deposited under the accession number KCCM13230P.

[0010] Another object of the present disclosure is to provide a culture of the Aureobasidium pullulans CJFSKY2201 strain deposited under the accession number KCCM13230P.

[0011] Yet another object of the present disclosure is to provide a method for producing oligosaccharides, the method comprising the steps of culturing a strain and obtaining oligosaccharides from the culture.

[0012] [Beneficial Effects]

[0013] In the present disclosure, it is confirmed that the Aureobasidium pullulans CJFSKY2201 strain deposited with the deposit number KCCM13230P has a high ability to synthesize oligosaccharides, so it can be used to efficiently produce oligosaccharides, and the oligosaccharides produced by the production method of the present disclosure can be effectively applied to the production of food compositions, feed compositions, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Graphs showing flux and daily cumulative yield over 36 days of oligosaccharide production reactions for existing strains and selected mutant strains are shown. DETAILED DESCRIPTION

[0015] The present disclosure will be described in detail below. At the same time, each embodiment and embodiment disclosed in the present disclosure can also be applied to other embodiments and embodiments. In other words, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific embodiments described below.

[0016] One aspect of the present disclosure provides a strain of Aureobasidium pullulans CJFSKY2201 deposited under the accession number KCCM13230P.

[0017] As used herein, "Budding Aureobasidium" is a yeast-like fungus distributed in nature. Budding Aureobasidium is known to exist in various environments, such as forest soil, fresh water, and air, as well as on the surfaces of various plants, and is known to have various enzyme activities, such as oligosaccharide synthase, siderophore, and pullulan.

[0018] The present inventors developed a new strain belonging to Aureobasidium pullulans, which was named Aureobasidium pullulans CJFSKY2201 and deposited under the accession number KCCM13230P.

[0019] The term "strain" as used herein refers to a collection of individuals derived from a single cell, reproducing asexually, and sharing the same genetic characteristics. Within a species, there are numerous strains with varying genetic characteristics. In this disclosure, the strain refers to Aureobasidium pullulans, specifically, the Aureobasidium pullulans deposited under accession number KCCM13230P, but is not limited thereto.

[0020] The Aureobasidium pullulans CJFSKY2201 strain disclosed herein can be an isolated strain or a recombinant strain, wherein the recombinant strain can be achieved by genetic modification. In addition, it can be a non-naturally occurring strain, or a mutant strain. In addition, the strain can include cells, dried cells, etc. of the Aureobasidium pullulans strain. The dried cells can be dried cells, such as spray-dried cells, freeze-dried cells, vacuum-dried cells, or drum-dried cells, but are not limited thereto.

[0021] In the present disclosure, in order to stably preserve the strain for a long time, the strain may be suspended in a storage solution prepared by mixing a predetermined amount of a glycerol component in water and stored at -70°C, or the strain may be suspended in 10% sterilized skim milk and freeze-dried, but is not limited thereto. The strain may be in a long-term storage form by various known methods.

[0022] Compared to the wild type, the strain may have an increased ability to synthesize oligosaccharides and may have increased oligosaccharide synthase activity, and the oligosaccharide synthase may be, but is not limited to, a fructosyltransferase. Fructosyltransferase is an enzyme that produces fructo-oligosaccharides using sugar or raw sugar as a raw material. Fructosyltransferase is known to hydrolyze raw sugar or sugar and then bind a fructose molecule to the raw sugar or sugar molecule to synthesize oligosaccharides.

[0023] Yet another aspect of the present disclosure provides a culture of the Aureobasidium pullulans CJFSKY2201 strain deposited under the accession number KCCM13230P.

[0024] As used herein, the term "culture" refers to growing a strain under appropriate artificially controlled environmental conditions. In the present disclosure, the "culture (cultured broth)" of a strain may be a culture broth containing cells, or may be cells from which the culture supernatant has been removed or concentrated. The composition of the culture may further include components that synergistically act on the growth of Aureobasidium pullulans, as well as common components required for culturing Aureobasidium pullulans, and therefore, a person skilled in the art can easily select the composition.

[0025] As for the culture medium and other culture conditions for culturing the strains of the present invention, any culture medium commonly used for culturing microorganisms of the genus Pullulan can be used without particular limitation. Specifically, the strains of the present invention can be cultured in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, inorganic salt, amino acid, vitamin and / or nucleic acid, etc., under aerobic or anaerobic conditions, while controlling the temperature, pH, etc.

[0026] As a culture medium, a natural culture medium or a synthetic culture medium can be used. As a carbon source of the culture medium, for example, glucose, sucrose, dextrin, glycerol, starch, etc. can be used. As a nitrogen source, peptone, meat extract, yeast extract, dry yeast, soybean, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds can be used, and as a phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or its corresponding sodium salt can be used, but are not limited to these components. As an inorganic compound, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. can be used, and as an inorganic salt, magnesium, manganese, calcium, iron, potassium, etc. can be used, but are not limited to this.

[0027] In one embodiment of the present disclosure, the Aureobasidium pullulans strain is seed cultured in a medium containing sucrose and yeast extract at pH 5 to pH 6, and main cultured in a medium containing sucrose, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate, an antifoaming agent, etc. at about pH 7, but is not limited thereto, and the components and input amounts of various materials can be appropriately adjusted.

[0028] Another aspect of the present disclosure provides a kind of bead on which a bacterial strain is immobilized. In the present disclosure, the beads are used for immobilized bacterial strains, and the preparation method thereof is not particularly limited. The immobilization refers to an operation in which cells are confined to a part of a certain space or object while retaining their catalytic activity and being able to be continuously reused. Usually, when selecting an immobilization carrier and an immobilization method, the characteristics of the microorganism should be considered, and the immobilization method includes an adsorption method, an embedding method, etc. As an immobilization carrier, calcium alginate, carrageenan, gelatin, chitosan, polyacrylamide gel, etc. can be used, but are not limited to this. In one embodiment of the present disclosure, an alginate solution is mixed with a cell suspension, the mixture is filtered, and then a bead manufacturing tank is filled with the mixture, which is immersed in a calcium chloride (CaCl2) solution to manufacture beads.

[0029] Yet another aspect of the present disclosure provides a method for producing oligosaccharides, comprising the steps of culturing a strain and obtaining oligosaccharides from the culture.

[0030] As used herein, the terms "culture" and "culture (culture fluid)" are as described above.

[0031] Oligosaccharides are either sugar solutions obtained by enzymatically combining sugar components into linear or branched chains of 10 or fewer sugar molecules, or liquid or powder products obtained by filtering, refining, and concentrating sugar solutions. There are two types of food products: oligosaccharides and processed oligosaccharide products.

[0032] As used herein, the term "oligosaccharide" refers to a saccharide having about 2 to about 10 saccharide units, and its types include isomalto-oligosaccharides, fructooligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, gentio-oligosaccharides, malto-oligosaccharides, mixed oligosaccharides, and the like.

[0033] The "fructose oligosaccharide" of the present disclosure is a mixture of sugars in which one or more fructose molecules, specifically, about 1 to about 5 fructose molecules, more specifically, about 1 to about 3 fructose molecules are bound to the fructose residue of the sugar, and includes 1-fructose triose (GF2), fructose tetraose (GF3) and 1-F fructofuranosyl fructose tetraose (GF4) and the like, but not limited thereto. Fructo-oligosaccharides are mainly produced by enzymatic degradation of raw sugar or sugar as a raw material, but not limited thereto. Fructo-oligosaccharides are used in various processed foods and are widely used in the manufacture of rice cakes, Nagasaki cakes, Korean traditional candies, cakes, jellies and the like, but not limited thereto.

[0034] The processed oligosaccharide product refers to a product processed by adding food or food additives to oligosaccharides, and refers to a product containing fructooligosaccharides, isomaltooligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides or gentio-oligosaccharides in a content of 10% or more, or maltooligosaccharides in a content of 40% or more.

[0035] In the present disclosure, the method for obtaining oligosaccharides using the strain may be a method known in the art, and may include a continuous process production method, a semi-batch production method, a batch production method, etc., but is not limited thereto. In addition, the method may preferably be a method in which the culture of the strain is mixed with sugar and reacted, and then oligosaccharides are obtained from the reaction product, or a method in which fructosyltransferase is separated from the culture of the strain, the separated enzyme is mixed with sugar and reacted, and then oligosaccharides are obtained from the reaction product, but is not limited thereto. As used herein, "sugar (GF)" is also called sucrose (sucrose or saccharose), and refers to a disaccharide in which one fructose molecule and one glucose molecule are linked by a glycosidic bond.

[0036] The steps of mixing and reacting with sugar may be performed at a temperature ranging from 40° C. to 60° C., but not limited thereto, and the reaction time may be continued until the desired target substance is produced.

[0037] The step of obtaining oligosaccharides can be carried out by common separation methods known in the art. Such separation methods may include, for example, centrifugation, filtration, chromatography, crystallization, and the like. For example, the supernatant obtained by removing the biomass from the reaction product by low-speed centrifugation can be separated by ion exchange chromatography, but is not limited thereto. Alternatively, a process of separating and filtering the strain from the reaction product can be performed, and the target substance can be recovered without the need for a separate purification process. Alternatively, the recovery step may further include a purification process.

[0038] In the present disclosure, random mutations were induced in a budding Aureobasidium pullulans strain for oligosaccharide production by ultraviolet irradiation, resulting in the development of a strain that exhibits high titers even under the same equipment and culture conditions. This makes it possible to convert sugars into oligosaccharides faster and in larger quantities than previously possible. This not only improves productivity but also enables emergency supply. Furthermore, oligosaccharides can be produced in a shorter timeframe than previously possible, significantly reducing associated fixed costs such as fuel and manpower, and enabling more stable production of oligosaccharides.

[0039] Another aspect of the present disclosure provides a composition comprising the Aureobasidium pullulans CJFSKY2201 strain deposited under accession number KCCM13230P, a lysate thereof, a culture thereof, a concentrate thereof, or a dried product thereof. For more details about the above strain, refer to the above content.

[0040] The composition comprising the Aureobasidium pullulans CJFSKY2201 strain, its lysate, its culture, its concentrate or its dried product can be used as a composition for producing oligosaccharides, and for producing a food composition or a feed composition.

[0041] The strain may be in a liquid or dry state, and the drying method may include but is not limited to air drying, natural drying, spray drying, and freeze drying.

[0042] The composition may further comprise a cryoprotectant or excipient. The cryoprotectant or excipient may be a non-naturally occurring substance or a naturally occurring substance, but is not limited thereto. In another specific embodiment, the cryoprotectant or excipient may be a substance that is not naturally in contact with the Aureobasidium pullulans CJFSKY2201 strain, or a substance that is not naturally contained at the same time as the strain, but is not limited thereto. In another specific embodiment, the composition may further comprise one or more cryoprotectants selected from the group consisting of glycerol, trehalose, maltodextrin, skimmed milk powder and starch; and / or one or more excipients selected from the group consisting of glucose, dextrin and skimmed milk. Based on the total weight of the composition, the content of the cryoprotectant of the present disclosure may be 0.01 wt % to 20 wt %, 0.01 wt % to 10 wt %. Specifically, the composition may contain 5 to 20% by weight of glycerol, 2 to 10% by weight of trehalose, 2 to 10% by weight of maltodextrin, 0.5 to 2% by weight of skim milk powder, and 0.1 to 1% by weight of starch. Furthermore, the excipients may be present in an amount of 75 to 95% or 85 to 95% by weight, based on the total weight of the composition.

[0043] In addition, the method for preparing a composition comprising the Aureobasidium pullulans CJFSKY2201 strain, its lysate, its culture, its concentrate, or its dried product may include the step of mixing the Aureobasidium pullulans CJFSKY2201 strain, its lysate, its culture, its concentrate, or its dried product with an additive. The additive may be the above-mentioned cryoprotectant or excipient.

[0044] Yet another aspect of the present disclosure provides a food composition comprising the oligosaccharide produced by the above production method.

[0045] As used herein, the term "oligosaccharide" is as described above.

[0046] As used herein, the term "food" may include meat, sausages, bread, chocolate, candy, snacks, sweets, pizza, instant noodles, other noodles, gelatin, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods and health foods. Foods in the ordinary sense are all included.

[0047] Health functional food has the same meaning as "food for special health use (FoSHU)", which refers to food with high pharmaceutical and medical effects, which is processed to effectively exhibit bioregulatory functions in addition to nutritional supply. In this article, "function" refers to obtaining an effect useful for health care applications, such as nutritional control or physiological effects on human body structure and function. The food of the present disclosure can be prepared by methods commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the above-mentioned production process. In addition, any preparation of food can also be prepared without limitation, as long as it is acceptable as a food.

[0048] Health food refers to food that has a positive effect on maintaining or promoting health status compared to ordinary food, while health supplement food refers to food used to supplement health. If necessary, health functional food, health food and health supplement food can be used interchangeably.

[0049] Specifically, health functional foods are foods prepared by adding the composition of the present disclosure to food materials such as beverages, teas, spices, gums, sweets, etc., or are made into capsules, powders, or suspensions. Health functional foods are foods that have specific health effects when consumed, but unlike general medicines, health functional foods do not have the side effects that may occur with long-term use of medicines because food is used as the raw material.

[0050] In this regard, the amount of the oligosaccharide included in the food is not particularly limited, but the content of the oligosaccharide may be 0.01 to 100 weight %, specifically, 1 to 80 weight %, based on the total weight of the food composition.

[0051] The food composition may further comprise a physiologically acceptable carrier. The type of carrier is not particularly limited. Any carrier commonly used in the art may be used.

[0052] In addition, the food composition may further comprise additional ingredients commonly used in food compositions to improve smell, taste, vision, etc. For example, the food composition may comprise vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folic acid, pantothenic acid, etc. In addition, the food composition may further comprise minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc.; and amino acids such as lysine, tryptophan, cysteine, valine, etc.

[0053] In addition, the food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT)), colorants (tar pigments, etc.), color developers (sodium nitrite, nitrite / sodium salt, etc.), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG)), sweeteners (sweetness, sodium cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), expanders (alum, D-potassium hydrogen tartrate, etc.), enhancers, emulsifiers, thickeners (adhesive paste), film formers, gum bases, defoaming agents, solvents, improvers, etc. The additives may be selected and used in appropriate amounts according to the type of food.

[0054] Yet another aspect of the present disclosure provides a feed composition comprising the oligosaccharide produced by the above production method.

[0055] As used herein, the term "oligosaccharide" is as described above.

[0056] As used herein, the term "feed" refers to any natural or artificial ration, single meal, or the like, or component of such a single meal, that is consumed, ingested, and digested or is suitable for consumption, ingestion, and digestion by an animal.

[0057] There are no particular limitations on the type of feed, and commonly used feeds in the art can be used. Non-limiting examples of feed include plant feeds such as grains, roots / fruits, food processing byproducts, algae, fiber, pharmaceutical byproducts, oils and fats, starch, cucurbits, and grain byproducts; and animal feeds such as proteins, inorganic substances, oils and fats, minerals, oils and fats, single-cell proteins, zooplankton, or food. These feeds can be used alone or in combination of two or more.

[0058] The oligosaccharides included in the feed composition of the present disclosure may vary depending on the purpose and conditions of use of the feed. For example, the content of the oligosaccharides may be 0.01 wt % to 100 wt %, more specifically 1 wt % to 80 wt %, based on the total weight of the livestock feed composition, but is not limited thereto.

[0059] Yet another aspect of the present disclosure provides use of the Aureobasidium pullulans CJFSKY2201 strain deposited under the accession number KCCM13230P in producing oligosaccharides.

[0060] [Modes for Carrying Out the Invention]

[0061] Hereinafter, the present disclosure will be described in more detail with reference to the following exemplary embodiments. However, the following exemplary embodiments are only used to illustrate the present disclosure, and the scope of the present disclosure is not limited thereto.

[0062] Example 1. Method for culturing strains

[0063] In the present disclosure, the seed culture of the Aureobasidium pullulans strain was performed in a medium containing sucrose and yeast extract at a pH of 5 to 6, with an average optical density (OD) of 10.

[0064] The main culture of this strain was carried out in a medium containing sucrose, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate, an antifoaming agent, etc. at a pH of about 7, and the average OD at this time was 160.

[0065] Example 2. Screening of mutant strains

[0066] Example 2-1. Primary screening

[0067] Take 1mL of the culture of the Aureobasidium pullulans strain, dilute it with 9mL of 3M sterile diluent (saline), and then distribute it to sterile plates (SPL Petri dishes) and dilute it 10 times. Thereafter, irradiate these plates with ultraviolet (UV) light for 20 seconds at a certain height in the clean bench for a predetermined period of time. After irradiation, take out 0.1mL from each plate and dilute it 100 times with 9.9mL of 3M sterile diluent (saline). Each 1mL of the diluted solution is distributed onto the plate, cultured in PDA (BD Difco) medium in a light-shielding manner using aluminum foil, and cultured at 28°C for 2 to 3 days until the bacteria are fully grown.

[0068] Each group was set up according to the characteristics of the culture strains, and 150 mL of culture from each group was placed in a 500 mL shaking flask (Shaking Incubator, Lab Tech) and pre-inoculated at 220 RPM. After 21 hours, the optical density (OD) and titer were measured to preliminarily screen for dominant strains.

[0069] Example 2-2. Second sieve

[0070] Purified MCB 1 capsules (Thermo 1.8 mL) were prepared by adding 0.9 mL of 40% glycerol to 0.9 mL of each culture of the mutant strains screened in Example 1-1. Twenty strains were randomly selected from these cultures, and 150 mL of culture was placed into a 500 mL shake flask and pre-inoculated at 240 RPM. After 21 hours, the OD and titer were measured to screen for dominant strains.

[0071] After purifying MCB1 capsules prepared with the selected mutant strains, purify the vials of strains showing high titers, randomly select 10 strains, and pre-inoculate 150 mL of culture into 500 mL shake flasks at 240 RPM. After 21 hours, measure the OD and titer to screen for dominant strains.

[0072] Example 2-3. Final Screening

[0073] Each MCB1 capsule prepared with the strains exhibiting high titers in Examples 1-2 was purified. Ten strains were then selected based on their characteristics and passaged on slant culture. Subsequently, 150 mL of culture was placed in a 500 mL shake flask and pre-inoculated at 240 RPM. After 21 hours, OD and titer were measured to screen for dominant strains.

[0074] [Table 1]

[0075]

[0076]

[0077] As a result of the culture, as shown in Table 1, it was confirmed that the strain 4-1-7-1 had an approximately 24% increase in titer compared to existing strains, and was therefore ultimately selected as the optimal strain.

[0078] The screened strain was named Aureobasidium pullulans CJFSKY2201 and deposited in the Korean Collection of Microorganisms (KCCM), an international depository under the Budapest Treaty, on August 24, 2022, and was assigned the deposit number KCCM13230P.

[0079] Example 3. Pilot reactor experiments of screened mutant strains

[0080] The final mutant strain (4-1-7-1) was tested alongside the existing strain in a pilot reactor scaled down to a field-scale reactor. The reactor flow rates and productivity of the existing strain and the mutant strain were compared. The reactor jacket temperature was 49°C.

[0081] As shown in Table 2 below, as a result of comparing the titer of the oligosaccharide productivity of the mutant strain (4-1-7-1) that directly affects the final screening and the existing strain, the existing strain showed an average titer of 370, and the mutant strain showed an average titer of 450, confirming that the average titer was increased by about 20% compared with the existing strain.

[0082] [Table 2]

[0083] filter strain OD CW Average titer Improvement rate Existing strains WCB 150 2.41 370 - mutant strains M4-1-7-1 165 2.54 450 About 20%

[0084] As a result of the oligosaccharide production reaction carried out for a total of 36 days, the mutant strain produced 178 L of oligosaccharide, and the existing strain produced 144 L of oligosaccharide. As shown in Table 3 below, it was confirmed that the oligosaccharide productivity of the mutant strain was increased by about 23% compared to the existing strain.

[0085] [Table 3]

[0086] filter strain Oligosaccharide content Sucrose content Average flow Productivity improvement rate Existing strains WCB 54% or above 19.4％ 0.174 - mutant strains M4-1-7-1 54% or above 18.4％ 0.213 About 23%

[0087] In addition, if Figure 1As shown, the flux of the mutant strain increased by an average of +0.04 compared with the existing strain, and the cumulative oligosaccharide production over 36 days also increased by approximately 23%.

[0088] Example 4. Field reactor experiments of screened mutant strains

[0089] The final mutant strain screened was compared with the existing strain using a field reactor. Other culture conditions were the same as in the pilot reactor experiments, and the flow rate and yield in the actual reactor were compared. The results confirmed that the culture profile, including titer, pH, DO, OD, and C / W, was nearly identical to those in the pilot reactor experiments. Furthermore, the field culture results confirmed that the mutant strain exhibited approximately 10% higher oligosaccharide productivity than the existing strain, as shown in Table 4 below.

[0090] [Table 4]

[0091]

[0092] Based on the above description, it will be understood by those skilled in the art that the present disclosure can be implemented in different specific forms without changing its technical spirit or basic characteristics. In this regard, it should be understood that the above embodiments are not restrictive, but illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than the description thereafter, and all changes and modifications that fall within the boundaries and scope of the claims, or the equivalents of these boundaries and scopes, are intended to be included in the claims.

[0093] International Table of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure

[0094] To: CJ CheilJedang Co., Ltd. is registered by the International Depository Organization in accordance with Regulation 7.1

[0095] Collection certificate issued by Zheng Gu in Seoul, South Korea

[0096] CJ CheilJedang Center, 330 Dongho-ro

[0097] (Zip Code) 100-400

[0098]

[0099] The above translation proves the content and context of the original text

[0100] October 31, 2022, licensed legal person Haner (India) PCT / RO / 134 form

Claims

1. A budding mold CJFSKY2201 strain deposited under the accession number KCCM13230P.

2. The strain according to claim 1, wherein the strain has an increased ability to synthesize oligosaccharides compared to the wild type.

3. A culture of the Aureobasidium pullulans CJFSKY2201 strain deposited under the accession number KCCM13230P.

4. A method for producing oligosaccharides, the method comprising: (a) culturing the strain according to claim 1; and (b) A step of obtaining oligosaccharides from the culture.

5. The method according to claim 4, wherein step (b) comprises mixing and reacting the culture with sugar, and then obtaining oligosaccharides from the reaction product.

6. The method according to claim 4, wherein step (b) is to separate the fructosyltransferase from the culture, and mix and react the separated enzyme with sugar, and then obtain oligosaccharides from the reaction product.

7. The method according to any one of claims 4 to 6, wherein the oligosaccharide is a fructooligosaccharide.

8. The method according to claim 7, wherein the fructooligosaccharide comprises 1-kestose, kestose and 1-Fructofuranosylkestose.

9. The process according to any one of claims 5 and 6, wherein the reacting step is carried out at a temperature ranging from 40°C to 60°C.

10. A composition comprising the Aureobasidium pullulans CJFSKY2201 strain deposited under the accession number KCCM13230P, a lysate thereof, a culture thereof, a concentrate thereof or a dried product thereof.

11. Use of the Aureobasidium pullulans CJFSKY2201 strain deposited under the accession number KCCM13230P in the production of oligosaccharides.

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