Process for preparing various fucosyl oligose and its use
阅读说明：本技术 各种新颖岩藻糖基低聚糖的制备方法和其用途 (Process for preparing various fucosyl oligose and its use ) 是由 金京宪 陈勇秀 尹银珠 俞素萝 李栽源 于 2018-11-15 设计创作，主要内容包括：本发明涉及一种用于各种新颖岩藻糖基低聚糖的制备方法和其用途。更具体地说,本发明允许通过使用GDP-L-岩藻糖供体和各种葡萄糖受体与α-1,2-岩藻糖基转移酶的酶反应来制备各种新颖岩藻糖基低聚糖并且建立其益生菌特性,且因此具有提供作为医药、食品、化妆产品等的材料的用途的效应。(The present invention relates to a preparation method for various novel fucosyl oligosaccharides and uses thereof. More specifically, the present invention allows the preparation of various novel fucosyl oligosaccharides and the establishment of their probiotic properties through enzymatic reactions with α -1,2-fucosyltransferase using GDP-L-fucose donors and various glucose acceptors, and thus has the effect of providing uses as materials for medicines, foods, cosmetic products, and the like.)
1. A probiotic pharmaceutical composition comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
2. The probiotic pharmaceutical composition of claim 1, wherein said probiotic pharmaceutical composition promotes proliferation of beneficial intestinal bacteria and inhibits growth of harmful intestinal bacteria.
3. The probiotic pharmaceutical composition of claim 2, wherein said beneficial gut bacteria is a strain belonging to the genus bifidobacterium, lactobacillus or streptococcus.
4. The probiotic pharmaceutical composition according to claim 3, wherein the strain belonging to the genus Bifidobacterium comprises Bifidobacterium longum subsp.
5. A probiotic cosmetic composition comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
6. A probiotic food composition comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
7. A method of making fucosyl oligosaccharides, comprising:
reacting a sugar acceptor (sugar acceptor) and a GDP-L-fucose (guanosine 5' -diphosphate-. beta. -L-fucose) donor with an alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) to produce a fucosyloligosaccharide,
wherein the sugar receptor comprises one or more selected from the group consisting of: glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose, and
the alpha-1, 2-fucosyltransferase includes any one selected from the amino acid sequences of SEQ ID NOS:1 to 3.
8. The method of making fucosyl oligosaccharide according to claim 7, wherein the reaction is performed at 20 ℃ to 40 ℃ for 3 hours to 24 hours.
9. A method of making fucosyl oligosaccharides, comprising:
culturing recombinant Escherichia coli or yeast, into which vectors for expressing ManB, ManC, Gmd and Wcag involved in De novo pathway (De novo pathway) for producing GDP-L-fucose (guanosine 5' -diphosphate-. beta. -L-fucose), and vectors for expressing alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) are introduced, in the presence of sugar acceptor (sugar acceptor) and glycerol, and culturing the recombinant Escherichia coli or yeast, into which vectors for expressing ManB, ManC, Gmd and Wcag involved in De novo pathway (De novo pathway) for producing GDP-L-fucose, and vectors for expressing alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) are introduced, and
isolating and purifying fucosyl oligosaccharide from the culture of Escherichia coli,
wherein the sugar receptor comprises one or more selected from the group consisting of: glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose, and
the alpha-1, 2-fucosyltransferase includes any one selected from the amino acid sequences of SEQ ID NOS:1 to 3.
10. The method of making fucosyl oligosaccharide according to claim 9, wherein the culturing comprises Fed-batch culturing (Fed-batch culture).
11. The method for preparing fucosyl oligosaccharide according to claim 10, wherein the fed-batch culture is performed at 25 ℃ to 37 ℃ for 2 hours to 60 hours, and IPTG (isopropyl β -D-1-thiogalactopyranoside) is added thereto, followed by fed-batch culture at 20 ℃ to 30 ℃ for 60 hours to 150 hours.
12. A method of improving gut health in an individual comprising:
administering to the individual a probiotic pharmaceutical composition,
wherein the probiotic pharmaceutical composition comprises one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
13. The method of improving gut health in an individual according to claim 12, wherein the probiotic pharmaceutical composition treats any one of heartburn, dyspepsia, bloating, diarrhea, constipation and flatulence.
Technical Field
The present invention relates to a method for producing various novel fucosyl oligosaccharides by using enzymatic reactions of GDP-L-fucose donors and various sugar acceptors with alpha-1, 2-fucosyltransferases and their use as probiotics.
Background
Probiotics refer to substances that are selectively fermented by beneficial intestinal bacteria to improve the intestinal flora and benefit human health. In recent years, studies on the correlation between human diseases and intestinal flora have been reported, and intestinal flora is considered as the second human genome, and thus studies in the field are rapidly progressing. In particular, according to the results of studies that have been reported, it is shown that obesity, diabetes, and immune function are improved according to the increase of the distribution of beneficial intestinal flora, and studies on intestinal flora are receiving more attention.
Fucosyl oligosaccharides are one of the main components of breast milk oligosaccharides and have the activity of probiotics selectively fermented by probiotic microorganisms and the physiological activity of preventing the sedimentation of intestinal pathogenic microorganisms. Besides intestinal health-related physiological activities, fucosyl oligosaccharides are known to enhance memory and are expected to help prevent dementia.
Among fucosyl oligosaccharides, fucosyl galactose (FGal), which is a disaccharide, is well known to promote the growth of nerve cells when it is used to treat hippocampal neurons. This suggests the possibility that FGal may be used as a medicament for the treatment of nervous system related diseases.
As the population aging progresses rapidly throughout the world, the number of patients with neurological diseases increases rapidly, and thus, the demand for the development of effective therapeutic agents is also increasing. For example, the global market for alzheimer's disease therapy is expected to extend to $ 133 billion in 2023, while the market for parkinson's disease therapy is expected to extend to $ 47 billion in 2022. However, to date, mainly symptomatic relief drugs have been developed, and thus there is no fundamental therapeutic drug so that the entire demand cannot be met, and therefore, it is expected to greatly expand the potential market. Furthermore, due to economic growth, it is expected that third world purchasing power expansion will contribute to future market growth.
The physiological activities of various fucosyl oligosaccharides described above, such as probiotic activity, memory enhancement, and promotion of growth of nerve cells, are expected to be produced from fucose units, and thus the present inventors produced different types of fucosyl oligosaccharides and examined their properties as probiotics using an enzymatic reaction of α -1,2-fucosyltransferase, and thus completed the present invention.
Disclosure of Invention
Technical problem
It is an object of the present invention to provide a method for producing various fucosyl oligosaccharides by enzymatic reaction of α -1,2-fucosyltransferase with GDP-L-fucose donor and various sugar acceptor.
It is another object of the present invention to provide the use of fucosyl oligosaccharides with probiotic properties.
Technical solution
According to one aspect of the present invention there is provided a probiotic pharmaceutical composition comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
The present invention also provides a method of improving gut health in an individual comprising administering to the individual a probiotic pharmaceutical composition.
The present invention also provides a probiotic cosmetic composition comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
The invention also relates to a probiotic food composition: comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
The present invention also provides a method for producing fucosyl oligosaccharide, comprising reacting a sugar acceptor (sugar acceptor) and a GDP-L-fucose (guanosine 5' -diphosphate-beta-L-fucose) donor with alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) to produce fucosyl oligosaccharide,
wherein the sugar receptor comprises one or more selected from the group consisting of: glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose, and
the alpha-1, 2-fucosyltransferase comprises any one selected from the amino acid sequences of SEQ ID NOS:1 to 3.
The present invention also provides a method of producing fucosyl oligosaccharides, comprising: culturing a recombinant escherichia coli or yeast into which vectors for expressing ManB, ManC, Gmd, and WcaG involved in De novo pathway (De novo pathway) for producing a GDP-L-fucose (guanosine 5' -diphosphate- β -L-fucose) donor are introduced in the presence of a sugar acceptor (succinicator) and glycerol; and a vector for expressing alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase); and isolating and purifying fucosyl oligosaccharide from the E.coli culture,
wherein the sugar receptor comprises one or more selected from the group consisting of: glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose, and
the alpha-1, 2-fucosyltransferase comprises any one selected from the amino acid sequences of SEQ ID NOS:1 to 3.
Advantageous effects of the invention
The invention has the following effects: provided is a method for producing fucosyl oligosaccharides using an enzymatic reaction of a GDP-L-fucose donor and various sugar acceptors with alpha-1, 2-fucosyltransferase or a metabolic engineering technique by applying the enzymatic reaction to recombinant Escherichia coli.
The present invention also provides the effect of fucosyl oligosaccharides as probiotic material in the medical, cosmetic and food fields based on their probiotic properties.
Drawings
Fig. 1 is a view showing the production of different types of fucosyl oligosaccharides by enzymatic reaction of α -1,2-fucosyltransferase using GDP-fucose donor and various sugar acceptors.
Figure 2 shows the results of tests demonstrating the reactivity between alpha-1, 2-fucosyltransferase and various sugar acceptors.
Fig. 3 to 5 show LC/MS analysis results of fucosyl oligosaccharides produced by an enzymatic reaction with α -1,2-fucosyltransferase using a GDP-L-fucose donor and a sugar acceptor such as glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose.
FIG. 6 shows views of batch fermentation results (B) and batch feed fermentation results (C) for production of 2-fucosylgalactose using recombinant Escherichia coli (A).
FIG. 7 shows the enzyme reaction results of 2-fucosylgalactose (B) separated and purified by size exclusion chromatography using a G-10 column in the fermentation broth of recombinant Escherichia coli (A) containing a blank vector without a gene encoding enzyme (C) as a control, using 2-fucosylgalactose as a substrate to confirm the alpha-1, 2-fucosyltransferase of the alpha-1, 2-glycosidic bond of 2-fucosylgalactose (D), and the enzyme reaction results of alpha-1, 2-fucosidase of the crude enzyme solution of recombinant Escherichia coli.
Fig. 8 shows the results of confirming the probiotic activity of 2-fucosylgalactose in the strain belonging to the genus bifidobacterium.
FIG. 9 shows the results of confirming the metabolism of 2-fucosylgalactose in pathogenic microorganisms.
Detailed Description
To date, the inventors of the present invention first produced fucosyl galactose (FGal) by an enzymatic reaction and microbial fermentation (U.S. patent No. 7,858,578), which has been produced only by chemical synthesis so far. Furthermore, the inventors first demonstrated that FGal is a probiotic fermented by bifidobacteria, which are probiotic microorganisms. Specifically, among various sugar receptors, the disaccharide base structure agarobiose, which constitutes the major carbohydrate agarose of red algae, is used first to produce 2-fucosylagarobiose. Agarobiose is known as a bioactive substance having an antioxidant effect, an antimicrobial effect, an anti-inflammatory effect, and an anticancer effect, and 2-fucosylagarobiose produced by a fucosylation reaction is a new material and at the same time, is expected to be a material having various physiological activities.
Accordingly, the present invention relates to a probiotic pharmaceutical composition comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
Fucosyl oligosaccharides are probiotics that promote the proliferation of beneficial intestinal bacteria, inhibit the growth of harmful intestinal bacteria, and are not metabolized by pathogenic microorganisms. In addition, the fucosyl oligosaccharide can also prevent pathogenic bacteria and viruses from infecting the human body and proliferating by binding to the adhesion site or receptor site of salmonella, helicobacter, pathogenic escherichia coli, norovirus, and the like. Among them, 2-fucosylgalactose can be used as a therapeutic agent for neurodegenerative diseases such as parkinson's disease and alzheimer's disease by promoting the growth of nerve cells, and 2-fucosylagarobiose can also be used as an antioxidant, an antibacterial agent, an anti-inflammatory agent, and an anticancer agent due to the characteristics of agarobiose itself such as antioxidant characteristics, antibacterial characteristics, anti-inflammatory characteristics, and anticancer characteristics.
According to one embodiment of the present invention, Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. infantis) ATCC 15697 decomposes 2-fucosylgalactose into galactose and fucose by intracellular fucosidase and uses both, and Bifidobacterium bifidum (Bifidobacterium bifidum) DSM 20082 extracellularly decomposes 2-fucosylgalactose into galactose and fucose, and then uses only galactose. Furthermore, it can be seen that escherichia coli (e.coli) causing urinary tract infection, neonatal meningitis and sepsis and Salmonella typhimurium (Salmonella enterica serovar typhimurium) causing diarrhea and bacteremia are not able to metabolize 2-fucosylgalactose and are probiotics selectively fermented by probiotic microorganisms.
Fucosyl oligosaccharides can be produced by reacting a sugar acceptor and a GDP-L-fucose (guanosine 5' -diphosphate- β -L-fucose) donor with an α -1,2-fucosyltransferase (α -1,2-fucosyltransferase) enzyme. Alternatively, recombinant E.coli or yeast into which vectors for expressing ManB, ManC, Gmd and Wcag involved in the De novo pathway for producing GDP-L-fucose are introduced may be cultured in the presence of a sugar acceptor and glycerol, respectively; and a vector for expressing the alpha-1, 2-fucosyltransferase, and fucosyl oligosaccharide can be obtained from the culture by isolation and purification.
As used herein, the term "beneficial intestinal bacteria" has the same meaning as "probiotics" and refers to strains that have a beneficial effect on the intestinal environment when ingested and reach the intestine, and refers to bacteria that survive in gastric acid and bile acids to reach the small intestine and thus proliferate and settle in the intestine, have a beneficial effect in the intestine, and meet conditions such as non-toxic and non-pathogenic. For example, beneficial intestinal bacteria include bacteria belonging to the genera Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), Enterococcus (Enterococcus), Streptococcus (Streptococcus), and Bifidobacterium (Bifidobacterium). In particular, the beneficial gut bacteria are strains belonging to the genus bifidobacterium, lactobacillus, streptococcus, more particularly to the genus bifidobacterium or streptococcus. Still more specifically, the strain belonging to the genus Bifidobacterium according to the invention comprises Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. infantis) ATCC 15697, Bifidobacterium bifidum (Bifidobacterium bifidum) DSM 20082 or Bifidobacterium infantis (Bifidobacterium kashiwanomicronense) DSM 21854. The strain belonging to the genus Lactobacillus comprises Lactobacillus reuteri (Lactobacillus reuteri), and the strain belonging to the genus Streptococcus comprises Streptococcus thermophilus (Streptococcus thermophilus).
As used herein, the term "harmful intestinal bacteria" refers to strains that have an adverse effect on the intestinal environment when ingested and reach the intestine. Examples of the harmful enteric bacteria include strains belonging to the genera Pseudomonas aeruginosa (Pseudomonas-aeruginosa), Vibrio (Vibrio), Staphylococcus (Staphylococcus), Clostridium perfringens (Clostridium perfringens), Eubacterium (Eubacterium), and Bacteroides (Bacteroides) and sulfate reductants (sulfate reducers). Specifically, the harmful enteric bacteria are strains belonging to the genus Clostridium perfringens, Eubacterium or Bacteroides. More specifically, the strain belonging to the genus Clostridium perfringens comprises Clostridium difficile (Clostridium difficile) or Clostridium perfringens (Clostridium perfringens). The strain belonging to the genus Eubacterium comprises Eubacterium mucosum (Eubacterium limosum), and the strain belonging to the genus Bacteroides comprises Bacteroides fragilis (Bacteroides fragilis).
The probiotic pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical arts, and include, inter alia, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., various phosphates, glycine, sorbic acid, potassium sorbate, and partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based bases, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, waxes, polyethylene glycol, or lanolin, although the invention is not limited thereto.
Furthermore, the probiotic pharmaceutical composition of the present invention may further comprise a lubricant, a wetting agent, an emulsifier, a suspending agent, a preservative, and the like, in addition to the ingredients described above.
In one embodiment, the probiotic pharmaceutical composition of the present invention may be formulated and used in various preparation forms suitable for oral administration or parenteral administration.
Non-limiting examples of formulations for oral administration include sugar-coated tablets (troches), buccal tablets (lozenge), tablets, aqueous suspensions, oily suspensions, prepared powders, emulsions, hard capsules, soft capsules, syrups, and elixirs.
To formulate the probiotic pharmaceutical composition of the invention for oral administration: binders such as lactose, sucrose, sorbitol, mannitol, starch, colloidal starch (Amylopectin), Cellulose (Cellulose), Gelatin (Gelatin), etc., excipients such as Dicalcium phosphate (Dicalcium phosphate), etc., disintegrants such as corn starch, sweet potato starch, etc., lubricants such as Magnesium stearate (Magnesium stearate), Calcium stearate (Calcium stearate), Sodium stearyl fumarate (Sodium stearyl fumarate), Polyethylene glycol wax (Polyethylene glycol wax), etc.; or the like, and a sweetener, an aromatic, a syrup, etc. may also be used.
Further, in the case of capsules, in addition to the above-mentioned materials, liquid carriers such as fatty oils and the like may be used.
Non-limiting examples of formulations for parenteral administration may include injections, suppositories, respiratory inhalation powders, spray aerosols, oral sprays, mouthwashes, toothpastes, ointments, coating powders, oils and creams.
To formulate the probiotic pharmaceutical composition of the present invention to be used for parenteral administration, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, a medicament for external application, etc. may be used, and propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension.
Further, more specifically, when the probiotic pharmaceutical composition of the present invention is formulated into an injection, the probiotic pharmaceutical composition of the present invention is mixed with a stabilizer or a buffer in water to prepare a solution or a suspension, followed by preparing into an ampoule (ampoule) or vial (visual) unit dosage form. Further, when the pharmaceutical composition of the present invention is formulated into an aerosol, a propellant or the like for dispersing an aqueous dispersion concentrate or a wet powder agent may be blended with an additive.
In addition, when the probiotic pharmaceutical composition of the present invention is formulated into ointments, creams, and the like, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, and the like may be used as a carrier.
The pharmaceutically effective amount and effective dosage level of the probiotic pharmaceutical composition of the present invention may vary depending on the formulation method, administration time and/or administration route of the composition, and may depend on the type of reaction and the extent of the reaction to be achieved including via administration of the probiotic pharmaceutical composition of the present invention; the type, age and weight of the individual to whom the composition is administered, general health, symptoms or severity of the disease, sex, diet, excretion, drugs used by the individual at the same time or at different times, components of other compositions, and the like, and similar factors well known in the medical arts, and an effective dose for targeted therapy can be determined and prescribed by one of ordinary skill in the art. The probiotic pharmaceutical composition of the present invention may be administered once a day or in multiple doses. Thus, an effective dose is not intended to limit the scope of the present invention in any way.
The administration route and the administration method of the pharmaceutical composition of the present invention may be independent of each other, the administration method is not particularly limited, and the administration route and the administration method may be any as long as the probiotic pharmaceutical composition can reach the corresponding site. The probiotic pharmaceutical composition may be administered orally or parenterally.
The parenteral administration may be, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, etc., and the probiotic medical composition may be coated or sprayed on the affected site or inhaled, but the present invention is not limited thereto.
The probiotic pharmaceutical composition of the present invention may preferably be administered orally or by injection.
The present invention also provides a method of improving gut health in an individual comprising administering to the individual a probiotic pharmaceutical composition.
Methods of improving intestinal health according to the present invention include treating heartburn, dyspepsia, abdominal distension, diarrhea, constipation, flatulence, and the like.
As used herein, the term "subject" refers to all animals including mammals, including mice, livestock, humans, and the like.
In the method for improving intestinal health according to the present invention, detailed descriptions of the dose, administration route, administration method, and the like of the probiotic pharmaceutical composition are the same as those described above with respect to the pharmaceutical composition.
The present invention also provides a probiotic cosmetic composition comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
The fucose-containing oligosaccharides may exhibit functionality to thicken the epidermal layer of the skin to relieve wrinkles. Therefore, the fucosyl oligosaccharide can exhibit excellent anti-wrinkle activity in consideration of skin permeability and absorption rate. Among fucosyl oligosaccharides, 2' -fucosyl agarobiose can be effectively used for inhibiting or treating specific reactions, acne, dandruff, etc. by the antioxidant activity and antibacterial activity of agarobiose itself.
The cosmetic composition of the present invention comprises: water-soluble vitamins such as vitamin B1, vitamin B2, vitamin B6, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like; oil-soluble vitamins such as vitamin a, carotene, vitamin D2, vitamin D3, vitamin E (D1-alpha tocopherol, D-alpha tocopherol, etc.); polymer peptides such as collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin and the like; high-molecular polysaccharides such as hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or salts thereof (sodium salt, etc.); sphingolipids such as ceramide, phytosphingosine, sphingoglycolipid, etc.; or seaweed extract such as brown algae extract, red algae extract, green algae extract, etc.
The cosmetic composition of the present invention may contain, in addition to the above essential ingredients, other ingredients mixed in general cosmetics as needed. Other blending ingredients that may be added may include oil and fat ingredients: moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH regulators, alcohols, dyes, pigments, blood circulation promoters, cooling agents, antiperspirant (antiperspirant) agents, purified water, and the like. The oil and fat component may comprise ester-based oils and fats, hydrocarbon-based oils and fats, silicone-based oils and fats, fluorine-based oils and fats, animal oils and fats, vegetable oils and fats, and the like.
Further, other mixing ingredients that may be added are not limited to the above examples, and may be mixed within a range that does not adversely affect the objects and effects of the present invention.
The cosmetic composition of the present invention may be in the form of a solution, emulsion, viscous mixture, or the like.
The ingredients included in the cosmetic composition of the present invention may include ingredients commonly used as active ingredients in cosmetic compositions, and include, for example, general adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments, and perfumes.
The cosmetic composition of the present invention may be prepared in any form conventionally prepared in the art, and examples thereof include skin lotions, skin softeners, skin lotions, emulsions, astringents, emulsions, moisturizing milks, nourishing milks, massage creams, nourishing creams, moisturizing creams, hand creams, foundations, essences, nourishing essences, face masks, soaps, cleansing foams, cleansing emulsions, cleansing creams, shampoos, hair conditioners, hair essences, shampoos, hair dyes, hair treatment creams, body lotions, and body cleansers.
The present invention also provides a probiotic food composition comprising one or more fucosyl oligosaccharides selected from the group consisting of: 2' -fucosylglucose, 2' -fucosylgalactose, 2' -fucosylcellobiose, 2' -fucosylfructose, 2' -fucosylsucrose, 2' -fucosylmaltose, 2' -fucosylmannose, 2' -fucosylxylose, and 2' -fucosylagarobiose.
The food composition of the present invention can be used as a health functional food, a food additive or a dietary supplement.
When used as a food additive, the fucosyl oligosaccharide can be suitably used according to a general method, for example, directly added or mixed in combination with other foods or food ingredients.
Further, the amount of fucosyl oligosaccharide to be mixed may be appropriately changed according to the purpose of use (preventive, health or therapeutic treatment), and is preferably in the range of 0.01 to 95% by weight, more preferably 0.1 to 80% by weight, relative to the total weight of the food composition. When the equivalent is less than 0.01 wt%, the dosage effectiveness may be reduced, and when the equivalent is more than 95 wt%, formulation difficulty may exist.
Specifically, when prepared into foods or drinks, the fucosyl oligosaccharide of the present invention is added in an amount of 15 wt% or less, preferably 10 wt% or less, relative to the total weight of the raw materials. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be lower than the above range, and since there is no problem in terms of safety, an amount of the active ingredient larger than the above range may also be used.
The type of food is not particularly limited, but examples of the food to which the fucosyl oligosaccharide of the present invention can be added include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, instant noodles, other noodles, soft candy, dairy products containing ice cream, various soups, drinks, tea, beverages, alcoholic beverages, vitamin complex, and the like, and include all health foods in the ordinary sense.
When the food composition of the present invention is prepared as a beverage, the food composition may comprise additional ingredients, such as various flavor enhancers or natural carbohydrates as in general beverages. As natural carbohydrates, monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; natural sweeteners such as dextrin and cyclodextrin; and synthetic sweeteners such as saccharin and aspartame. The natural carbohydrate is present in an amount of 0.01 to 10 wt.%, preferably 0.01 to 0.1 wt.%, relative to the total weight of the food composition of the present invention.
The food composition of the present invention may contain various nutritional supplements, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acids and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonizing agents used in carbonated drinks, and the like, and may contain pulp for preparing natural fruit juices, fruit drinks, and vegetable drinks, but the present invention is not limited thereto. These components may be used alone or in combination thereof. The proportion of these additives is not particularly limited, but may be in the range of 0.01 wt% to 0.1 wt% relative to the total weight of the food composition of the present invention.
In the case of long-term ingestion for health and hygiene purposes or for health control purposes, the food composition of the present invention can be ingested for a long period of time since there is no problem in terms of safety.
The present invention also provides a method of producing fucosyl oligosaccharides, comprising reacting a sugar acceptor (sugar acceptor) and a GDP-L-fucose (guanosine 5' -diphosphate- β -L-fucose) donor with α -1,2-fucosyltransferase (α -1,2-fucosyltransferase) to produce fucosyl oligosaccharides.
Wherein the sugar receptor comprises one or more selected from the group consisting of: glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose, and
the alpha-1, 2-fucosyltransferase comprises any one selected from the amino acid sequences of SEQ ID NOS:1 to 3.
As a conventional method for producing fucosyl oligosaccharides, chemical synthesis of 2-fucosyl galactose is known, but this method has a complicated synthesis process and uses organic solvents such as pyridine, acetic acid, dichloromethane and toluene, and even after many processes, it may be synthesized only in a derivative form instead of 2-fucosyl galactose (Kalovidouris SA et al, J.S.Chem.127: 1340-1341 (2005)).
However, in the method of producing fucosyl oligosaccharide according to the present invention, fucosyl oligosaccharide that is not in a derivative form can be produced by reacting a sugar acceptor and a GDP-L-fucose donor with α -1, 2-fucosyltransferase. Furthermore, it is possible to produce different types of fucosyl oligosaccharides using various sugar receptors (see fig. 1).
The carbohydrate receptor may be selected from the group consisting of: glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose.
The α -1,2-fucosyltransferase may be derived from Helicobacter pylori (Helicobacter pylori), Bacteroides fragilis (Bacteroides fragilis), escherichia coli (e.coli) O126, etc., and may specifically include any one selected from the amino acid sequences of SEQ id nos:1 to 3.
According to one embodiment of the present invention, the alpha-1, 2-fucosyltransferase exhibits differences in reactivity according to the type of sugar acceptor. For example, Bacteroides fragilis (Bacteroides fragilis) derived WcfB exhibited high activity for cellobiose, galactose with glucose, escherichia coli (e.coli) O126 derived WbgL exhibited high activity for lactose and galactose, and Helicobacter pylori (Helicobacter pylori) derived FucT2 exhibited high activity for cellobiose and galactose.
The enzymatic reaction of the sugar acceptor and the GDP-L-fucose donor with the alpha-1, 2-fucosyltransferase may be performed at 20 ℃ to 40 ℃ for 3 hours to 24 hours, more specifically, at 25 ℃ to 35 ℃ for 6 hours to 12 hours.
The fucosyl oligosaccharide produced by the method of producing fucosyl oligosaccharide according to the present invention may be 2 '-fucosyl glucose, 2' -fucosyl galactose, 2 '-fucosyl cellobiose, 2' -fucosyl lactose, 2 '-fucosyl fructose, 2' -fucosyl sucrose, 2 '-fucosyl maltose, 2' -fucosyl mannose, 2 '-fucosyl xylose, or 2' -fucosyl agarobiose.
The present invention also provides a method of producing fucosyl oligosaccharides, comprising: culturing a recombinant E.coli or yeast into which vectors for expressing ManB, ManC, Gmd and Wcag involved in De novo pathway (De novo pathway) for producing GDP-L-fucose (guanosine 5' -diphosphate-. beta. -L-fucose) donors are introduced in the presence of a sugar acceptor (succinicator) and glycerol; and a vector for expressing alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase); and isolating and purifying fucosyl oligosaccharide from the E.coli culture,
wherein the sugar receptor comprises one or more selected from the group consisting of: glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose, and
the alpha-1, 2-fucosyltransferase comprises any one selected from the amino acid sequences of SEQ ID NOS:1 to 3.
According to a method of producing 2-fucosyllactose, which is fucosyloligosaccharide, 2-fucosyllactose is produced from lactose using helicobacter-derived fucosyltransferase by fermentation using a known microbial metabolic engineering technique, but this is disadvantageous because it is not possible to produce various fucosyloligosaccharides.
In contrast, according to the method of producing fucosyl oligosaccharides according to the present invention, different types of fucosyl oligosaccharides can be produced by microbial fermentation using microbial metabolic engineering techniques, and for this purpose, escherichia coli or yeast in which a metabolic pathway of a material used as a sugar acceptor is deleted is used as a host, a de novo synthetic pathway is introduced into the host to produce GDP-1-fucose as a donor, a sugar substance is supplied as an acceptor, glycerol is used as a carbon source, and α -1,2-fucosyltransferase is introduced, thereby producing fucosyl oligosaccharides by fermentation. Thus, the method of the invention may comprise:
constructing vectors for expressing ManB, ManC, Gmd, and WcaG involved in De novo pathway (De novo pathway) for producing GDP-L-fucose (guanosine 5' -diphosphate- β -L-fucose) as a donor in escherichia coli and vectors for expressing α -1,2-fucosyltransferase (α -1,2-fucosyltransferase) enzyme;
introducing the vector into escherichia coli or yeast in which a metabolic pathway of a material serving as a sugar receptor is deleted to transform the escherichia coli or yeast;
culturing the transformed E.coli or yeast using glycerol as a carbon source in the presence of a sugar acceptor; and
fucosyl oligosaccharides were isolated and purified from the culture.
The de novo synthesis pathway for producing GDP-L-fucose is a pathway in which intermediates such as mannose 6P (mannose 6P), mannose 1P (mannose 1P), GDP-D-mannose-4-ketone (mannose-4-ketone-6-mannose) are sequentially produced by using fructose 6P as a starting material and mannose-6-phosphate isomerase (ManM), phosphomannose mutase (ManB), mannose-1-phosphate guanyltransferase (ManC), GDP-mannose-4,6-dehydratase (GDP-mannose-4,6-dehydratase, Gmd) and GDP-L-fucose synthase (GDP-L-fucose synthase, Wcag), by sequentially producing the intermediates such as mannose 6P (mannose 6P), mannose 1P (mannose 1P), GDP-D-mannose (GDP-D-mannose) and GDP-4-ketone-6-mannose (mannose-4-mannose-6-ketone-6-mannose deoxymanose) and ultimately produces GDP-L-fucose.
The culture of transformed E.coli may comprise a Fed-batch culture (Fed-batch culture).
The fed-batch culture may be performed at 25 ℃ to 37 ℃ for 2 hours to 60 hours, and IPTG (isopropyl β -D-1-thiogalactopyranoside) may be added and the fed-batch culture may be performed at 20 ℃ to 30 ℃ for 60 hours to 150 hours.
Hereinafter, the present invention will be described in further detail with reference to the following examples, but these examples are not intended to limit the scope of the present invention.
Modes of the invention
< example 1> Synthesis of fucosyl oligosaccharide
To produce various fucosyl oligosaccharides, enzymatic reactions of α -1, 2-fucosyltransferases were performed under conditions of GDP-fucose donor and various sugar acceptors (fig. 1).
As alpha-1, 2-fucosyltransferase, there were used WbgL (GenBank: ABE98421.1) derived from Escherichia coli (E.coli), FucT2 (GenBank: AAC99764.1) derived from Helicobacter pylori (Helicobacter pylori), and WcfB (GenBank: AAD40713.1) derived from Bacteroides fragilis (Bacteroides fragilis). The pET21a plasmid vector containing the genes encoding each enzyme was converted into e.coli (e.coli) BL21(DE3) star. Each recombinant E.coli was cultured at 37 ℃ and 250rpm until OD 0.5, and then enzyme expression was induced at 16 ℃ and 250rpm for 18 hours using 0.1 mmol of IPTG.
Fig. 1 shows a view for producing different types of fucosyl oligosaccharides by enzymatic reaction of α -1,2-fucosyltransferase using GDP fucose donor and various sugar acceptors, such as glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose, in which an experiment of their reactivity was performed using sugar acceptors.
To produce the agarobiose, acid hydrolysis of agarose was performed. 10% (w/w) agarose was subjected to an acid hydrolysis reaction using 2% phosphoric acid at 90 ℃ for 3 hours. After the acid hydrolysis, calcium hydroxide was added to the reaction solution to neutralize and remove phosphoric acid.
Experiments on enzymatic reactivity between alpha-1, 2-fucosyltransferase and various sugar acceptors were performed as follows. 2 mM GDP-fucose donor, 5 mM of each sugar acceptor, 1 mM of 1, 4-Dithiothreitol (DTT), 50 mM of Tris-HCl (pH 7.0), and 5 mg/ml of a crude enzyme solution in E.coli cells expressing WbgL, FucT2, or WcfB were allowed to react at 600rpm at 30 ℃ for 12 hours.
As shown in fig. 2, WcfB derived from Bacteroides fragilis (Bacteroides fragilis) exhibited high activity for cellobiose, galactose as glucose, WbgL derived from escherichia coli (e.coli) O126 exhibited high activity for lactose and galactose, and FucT2 derived from Helicobacter pylori (Helicobacter pylori) exhibited high activity for cellobiose and galactose.
< example 2> enzymatic reaction of alpha-1, 2-fucosyltransferase of various sugar acceptors
Fucosyl oligosaccharides are produced by enzymatic reaction of WbgL using GDP-fucose donor and galactose or glucose as acceptor, which is alpha-1, 2-fucosyltransferase. The sugar acceptor used in the enzymatic reaction is glucose, galactose, cellobiose, lactose, fructose, sucrose, maltose, mannose, xylose, and agarobiose. The enzyme reaction conditions were as follows: 1 mg/ml of each recombinant E.coli intracellular crude enzyme solution, 0.2 mmol of GDP-fucose donor, 0.5 mmol of sugar acceptor and 20 mmol of sodium phosphate buffer (pH 6.0) were used to allow the enzyme reaction to be maintained at 30 ℃ for 12 hours. LC/MS analysis of the reaction products was performed to confirm the exact mass values. In the LC/MS analysis, a siemer heschel porous graphitic carbon LC column (siemer heschel technology) was used and the positive ion mode was used. Two mobile phases, for example a 25 micromolar lithium chloride solution as mobile phase a and a 100% acetonitrile solution as mobile phase B, were flowed at a ratio of 0.2 ml/min and a gradient of 0% to 80% for 41 minutes. At this time, the temperature of the column was maintained at 70 ℃. The source-related parameters are as follows: a spray gas flow of 1.5 liters/min, an interface voltage of 4.5 kilovolts, a detector voltage of 1.65 kilovolts, a Curved Desolvation Line (CDL) temperature of 200 ℃, and a heating block temperature of 200 ℃. MS analysis was performed in the range of 100m/z to 700 m/z.
As shown in fig. 3 to 5, it was confirmed that each sugar acceptor produced 2 '-fucosyl glucose, 2' -fucosyl galactose, 2 '-fucosyl cellobiose, 2' -fucosyl lactose, 2 '-fucosyl fructose, 2' -fucosyl sucrose, 2 '-fucosyl maltose, 2' -fucosyl mannose, 2 '-fucosyl xylose, or 2' -fucosyl agarobiose.
< example 3> production of fucosylgalactose Using Metabolic engineering techniques
FGal, which is a bioactive substance, is produced from recombinant e.coli using metabolic engineering and then fermented. FIG. 6A is a view of FGal production using recombinant E.coli, in which enzymes ManB, ManC, Gmd, and Wcag involved in de novo pathway (de novo pathway) are introduced and galactose is supplied as an acceptor in order to produce GDP-fucose donor in E.coli cells. In order to use galactose as a receptor, an escherichia coli (e.coli) BL21(DE3) strain incapable of metabolizing galactose was used as a host. Helicobacter pylori (Helicobacter pylori) -derived FucT2 was used as the alpha-1, 2-fucosyltransferase and glycerol was supplied as the carbon source. Recombinant E.coli was cultured in an LB medium containing 2 g/L of galactose and 5 g/L of glycerol at 37 ℃ for 6 hours, and IPTG was added thereto, followed by culturing after the temperature was lowered to 25 ℃.
As shown in B of fig. 6, 1.7 g/l FGal was produced after 90 hours of batch fermentation.
Fig. 6C shows the fed-batch fermentation results, where about 12 g/l of FGal was produced after 90 hours of fermentation and a maximum of 17.7 g/l of FGal was produced after 120 hours of fermentation.
< example 4> examination of the Structure of 2-fucosyl galactose produced by metabolic engineering technique
To confirm the structure of FGal produced via recombinant e.coli, FGal was purified from the culture. At this time, G-10 column resin and water were used as mobile phases to perform size exclusion chromatography, and HPLC analysis was performed to confirm purification of FGal fractions with high purity. In the HPLC analysis, a Rezex ROA-organic acid H + (8%) column (Feinumet) was used, and a 0.005N sulfuric acid solution was flowed at a rate of 0.6 ml/min at 50 ℃.
To confirm the α -1, 2-glycosidic bond of the purified FGal, FGal as a substrate was subjected to enzymatic reaction of α -1, 2-fucosidase derived from Xanthomonas manihot (Xanthomonas manihot).
For this purpose, the gene coding for the alpha-1, 2-fucosidase derived from Xanthomonas manihot (NCBI reference sequence: WP _017167782.1) was introduced into the pETduet vector and the vector was then transformed into the E.coli (E.coli) BL21(DE3) strain. During the enzyme reaction, 1 mg/ml of FGal was used as a substrate and 1 mg/ml of the recombinant escherichia coli intracellular crude enzyme was used and the enzyme reaction was performed in 20 mmol of Tris-HCl buffer (pH 7.0) at 30 ℃ for 6 hours. At this time, as a control, the enzyme reaction was performed using crude enzyme in cells of recombinant E.coli containing a blank vector not containing a gene encoding the enzyme.
As shown in fig. 7, the α -1, 2-glycosidic bond was examined by enzymatic reaction of purified 2FG α -1, 2-fucosidase.
< example 5> examination of probiotic characteristics of 2-fucosylgalactose
To examine the probiotic effect of FGal, the cell growth of strains such as Bifidobacterium longum subsp. In addition, to test for the ability of pathogenic microorganisms to ferment FGal, cell growth of strains such as E.coli (E.coli) O1: K1: H7 and Salmonella typhimurium (Salmonella enterica serovar typhimurium) was monitored. At this time, the medium contained 10 g/L of bactopeptone, 5 g/L of yeast extract, 2 g/L of anhydrous K2HPO45 g/l of anhydrous sodium acetate, 2 g/l of triammonium citrate, 0.2 g/l of magnesium sulfate heptahydrate, 0.05 g/l of manganese sulfate, 1 ml/l of Tween 80 (polysorbate 80), 0.5 g/l of cysteine, and 3 g/l of purified FGal, and culturing the strain at 37 ℃.
As shown in fig. 8, it was confirmed that Bifidobacterium bifidum (Bifidobacterium bifidum) DSM 20082 first extracellularly decomposes FGal into fucose and galactose and then metabolizes a small amount of galactose, and Bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp.
As shown in fig. 9, pathogenic microorganisms are not able to metabolize FGal.
INDUSTRIAL APPLICABILITY
According to the present invention, fucosyl oligosaccharides can be used as probiotic material in the medical, cosmetic and food fields based on their probiotic properties.
<110> university school labor cooperation group of Korean university
<120> preparation method and use of various novel fucosyl oligosaccharides
<130>X18U13C0183
<150>KR 10-2017-0154840
<151>2017-11-20
<160>3
<170>KopatentIn 2.0
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<213> Bacteroides fragilis
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<213> Escherichia coli O126
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<213> helicobacter pylori
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