WO2018124143A1 - 新規ステビオール配糖体およびその製造方法、ならびにそれを含む甘味料組成物 - Google Patents
新規ステビオール配糖体およびその製造方法、ならびにそれを含む甘味料組成物 Download PDFInfo
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L2/38—Other non-alcoholic beverages
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A23L2/52—Adding ingredients
- A23L2/60—Sweeteners
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/205—Heterocyclic compounds
- A23L27/2052—Heterocyclic compounds having oxygen or sulfur as the only hetero atoms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/30—Artificial sweetening agents
- A23L27/33—Artificial sweetening agents containing sugars or derivatives
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C12N9/1051—Hexosyltransferases (2.4.1)
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
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- C12N15/09—Recombinant DNA-technology
Definitions
- the present invention relates to a novel steviol glycoside, a method for producing the same, and a sweetener composition containing the same.
- the present invention further relates to foods and drinks containing novel steviol glycosides, plants and extracts thereof, and flavor modifiers.
- Stevia rebaudiana leaves contain a secondary metabolite called steviol, a diterpenoid, and steviol glycosides are about 300 times sweeter than sugar, making them calories. It is used in the food industry as a sweetener. Obesity has been developed internationally as a serious social problem, and the demand for calorie-free sweeteners is increasing day by day from the viewpoint of promoting health and reducing medical costs.
- Stevia's main steviol glycoside is finally modified with a sugar to a glycoside called Rebaudioside A (Reb.A) with four sugars ( Figure 1).
- Stevioloside the steviol trisaccharide glycoside, which is the precursor, is the most quantitatively, and these two are the central substances of Stevia's sweetness.
- Stevioside has the highest content in stevia leaves and is known to exhibit sweetness about 250 to 300 times that of sugar.
- Reb.A is a steviol tetrasaccharide glycoside with high sweetness (350 to 450 times that of sugar) and good taste, and these are attracting attention as calorie-less sweeteners.
- glycosides that are considered to be reaction intermediates and analogs with different types of sugars are known.
- Rebaudioside sugars of Reb.A are glucose, but among them, rebaudioside C (Reb.C) in which rhamnose is added instead of glucose at the 2nd position of glucose at the 13th position, Rebaudioside F (Reb.F) with xylose added at the position is known.
- Rebaudioside C Reb.C
- Rebaudioside F Reb.F
- Reb.A where all four glycoside sugars are glucose, has a good taste so far, it is said that Stevia plants with higher Reb.A content than wild type Stevia plants will be obtained by breeding etc. Attempts have been made (for example, Patent Document 1).
- some stevia varieties that have undergone breed improvement may contain trace amounts of steviol glycosides whose structure has not yet been specified. It may have a characteristic flavor.
- research on steviol glycosides in which glucose is further added to Reb.A and varieties containing the same have been progressed, but varieties containing many steviol glycosides containing rhamnose such as Reb.C
- an object of the present invention is to determine the structure of a trace amount of novel steviol glycosides that affect the taste quality and grasp the taste quality characteristics.
- a further object of the present invention is to provide a novel steviol glycoside, a process for producing the same, and a sweetener composition containing the same.
- the present inventors have succeeded in determining the structure of a small amount of a novel steviol glycoside that affects taste quality.
- the present invention is based on the above findings.
- a small amount of novel steviol glycosides that affect taste quality can be provided. Furthermore, according to this invention, the manufacturing method of a novel steviol glycoside, the sweetener composition containing the novel steviol glycoside, food-drinks, a plant, its extract, and a flavor regulator can be provided.
- (A) is a diagram showing the 1 H- 1 H cozy spectrum (800MHz, Pyr-d5) of Compound 11 is a diagram showing a (b) the HSQC spectrum of compound 11 (800MHz, Pyr-d5) .
- Reb. It is a graph which shows the evaluation result of the retrofit improvement effect with respect to A.
- Reb. It is a graph which shows the evaluation result of the backward improvement effect with respect to D. It is a graph which shows the evaluation result of the sweet taste enhancement effect with respect to sugar (sucrose) of the flavor regulator of this invention.
- rebaudioside In this specification, “rebaudioside”, “rebaudioside” and “Reb.” Have the same meaning, and all mean “rebaudioside”. Similarly, in the present specification, “zulcoside” has the same meaning as “zulcoside”, and both mean “dulcoside”.
- novel steviol glycosides The present inventors have identified for the first time the structure of a trace amount of a novel steviol glycoside that affects taste quality.
- the novel steviol glycoside of the present invention (hereinafter also referred to as “the glycoside of the present invention”) has the formula (1) Or a derivative, salt, or hydrate thereof.
- the glycoside of the present invention has a sugar chain containing 3 molecules of glucose at the 19th position of steviol and a sugar chain containing 2 molecules of glucose and 1 molecule of rhamnose at the 13th position. ing.
- the glycoside of the present invention may be not only a compound represented by the formula (1) but also a derivative, salt or hydrate thereof.
- the “derivative” means a compound formed by a structural change of a small part in the compound, for example, a compound in which a part of the hydroxyl group is substituted with another substituent. To do.
- a part of the hydroxyl group contained in the compound is selected from hydrogen, halogen, alkyl group, alkenyl group, alkynyl group, aryl group, amino group, cyano group and the like. And a compound substituted with a substituent.
- a salt of a compound of formula (1) means a physiologically acceptable salt of a compound of formula (1), such as a sodium salt.
- the “hydrate of the compound of the formula (1)” means a compound in which a water molecule is added to the compound of the formula (1).
- the glycoside of the present invention is not particularly limited, but may be a plant-derived product, a chemical synthesis product, or a biosynthesis product. For example, it may be isolated and purified from a plant body rich in Reb.C, but may be obtained by chemical synthesis or biosynthesis. The detail of the manufacturing method of the glycoside of this invention is mentioned later in this specification.
- the glycoside of the present invention has a sweetness higher than that of sugar (sucrose) and has a good taste after sweetening, and affects the taste quality only by being contained in a small amount in foods and drinks. be able to. Therefore, the glycoside of the present invention can be used as a novel sweetener.
- the novel steviol glycoside of the present invention has the formula (A) Or a derivative, salt, or hydrate thereof.
- sweetener composition comprising a novel steviol glycoside
- a sweetener composition comprising a compound represented by formula (1), or a derivative, salt or hydrate thereof (hereinafter referred to as Also referred to as “sweetener composition of the present invention”).
- the sweetener composition of the present invention is not particularly limited as long as it contains a compound represented by the formula (1) or a derivative, salt or hydrate thereof, and a compound represented by the formula (1) or a derivative thereof.
- a composition containing an extract containing a salt or hydrate is not particularly limited as long as it contains a compound represented by the formula (1) or a derivative, salt or hydrate thereof.
- the amount of the glycoside of the present invention contained in the sweetener composition of the present invention is not particularly limited.
- the sweetener composition of the present invention is preferably a composition comprising the glycoside of the present invention in an amount at least 0.01% greater than the amount present in wild-type stevia or stevia extract.
- the glycoside of the present invention was first detected from varieties rich in Reb.C, and was not contained or contained in wild type stevia or its extract. Even if it is the amount below the detection limit value.
- the sweetener composition of the present invention may further contain other steviol glycosides.
- the sweetener composition of the present invention comprises, in addition to the glycoside of the present invention, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, Rebaudio Side F, Rebaudio Side I, Rebaudio Side J, Rebaudio Side K, Rebaudio Side N, Rebaudio Side M, Rebaudio Side O, Rebaudio Side Q, Rebau
- steviol glycosides selected from the group consisting of Dioside R, Dulcoside A, Rubusoside, Steviol, Steviol Monoside, Steviol Bioside and Stevioside may be further included.
- the composition ratio of the glycoside of the present invention to other steviol glycosides is preferably 0.01: 9.99 to 6: 4 by mass ratio.
- the sweetener composition of the present invention may further contain a general sweetener.
- Such common sweeteners include fructose, sugar, fructose glucose liquid sugar, glucose, maltose, sucrose, high fructose liquid sugar, sugar alcohol, oligosaccharides, honey, sugarcane juice (brown molasses), starch syrup, Natural sweeteners such as Rahan fruit powder, Rahan fruit extract, licorice powder, licorice extract, Somatococcus danieri seed powder, Somatococcus danieri seed extract, and artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, saccharin Etc.
- natural sweeteners are preferably used from the viewpoints of refreshing, ease of drinking, natural taste, and imparting an appropriate richness, and fructose, glucose, maltose, sucrose, and sugar are particularly preferably used. Only one kind of these sweetening ingredients may be used, or a plurality of kinds may be used.
- a food / beverage product comprising the compound represented by formula (1) or a derivative, salt or hydrate thereof (hereinafter referred to as “the food / beverage product of the present invention”) Is also provided).
- the food or drink of the present invention is not particularly limited as long as it contains a compound represented by formula (1), or a derivative, salt, or hydrate thereof, and a compound represented by formula (1), or a derivative or salt thereof.
- the food / beverage products containing the extract containing a hydrate, and a sweetener composition may be sufficient.
- the food and drink means beverages and foods. Accordingly, in one embodiment, the present invention provides a novel beverage or food and provides a method for producing the beverage or food.
- the amount of the glycoside of the present invention contained in the food / beverage product of the present invention varies depending on the specific food / beverage product, but is preferably about 0.0004% to 0.8%, preferably 0.04% to 0.00%. 4% is particularly preferable. By setting the content within this range, there is an advantage that post-drawing can be suppressed.
- the food and drink of the present invention may further contain other steviol glycosides.
- the sweetener composition of the present invention comprises, in addition to the glycoside of the present invention, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, Rebaudio Side F, Rebaudio Side I, Rebaudio Side J, Rebaudio Side K, Rebaudio Side N, Rebaudio Side M, Rebaudio Side O, Rebaudio Side Q, Rebau
- steviol glycosides selected from the group consisting of Dioside R, Dulcoside A, Rubusoside, Steviol, Steviol Monoside, Steviol Bioside and Stevioside may be further included.
- the composition ratio of the glycoside of the present invention to other steviol glycosides is preferably 0.01: 9.99 to 6: 4 by mass ratio.
- the food and drink of the present invention may further contain a general sweetener.
- Such common sweeteners include fructose, sugar, fructose glucose liquid sugar, glucose, maltose, sucrose, high fructose liquid sugar, sugar alcohol, oligosaccharides, honey, sugarcane juice (brown molasses), starch syrup, Natural sweeteners such as Rahan fruit powder, Rahan fruit extract, licorice powder, licorice extract, Somatococcus danieri seed powder, Somatococcus danieri seed extract, and artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, saccharin Etc.
- natural sweeteners are preferably used from the viewpoints of refreshing, ease of drinking, natural taste, and imparting an appropriate richness, and fructose, glucose, maltose, sucrose, and sugar are particularly preferably used. Only one kind of these sweetening ingredients may be used, or a plurality of kinds may be used.
- Examples of the food of the present invention are not particularly limited, but foods include confectionery, bread making, flour, noodles, rice, agricultural and forestry processed foods, livestock processed products, processed fishery products, milk -Dairy products, oils and fats, processed oils and fats, seasonings or other food materials.
- beverage of the present invention are not particularly limited, and for example, carbonated beverages, non-carbonated beverages, alcoholic beverages, non-alcoholic beverages, coffee beverages, tea beverages, cocoa beverages, nutritional beverages, functional beverages, and the like. Can be mentioned.
- the beverage of the present invention may be prepared as a packaged beverage that has been sterilized by heating and packed in a container.
- the container is not particularly limited, and examples thereof include a PET bottle, an aluminum can, a steel can, a paper pack, a chilled cup, and a bottle.
- heat sterilization the kind is not specifically limited, For example, it can carry out using normal techniques, such as UHT sterilization and retort sterilization.
- the temperature of the heat sterilization step is not particularly limited, but is, for example, 65 to 130 ° C., preferably 85 to 120 ° C. and 10 to 40 minutes. However, if a sterilization value equivalent to the above conditions is obtained, sterilization at a suitable temperature for several seconds, for example, 5 to 30 seconds is not problematic.
- a plant containing a novel steviol glycoside and an extract thereof are provided.
- the food / beverage products Preferably a drink containing the plant body of this invention or the extract of a plant body is provided.
- the amount of the glycoside of the present invention contained in the plant of the present invention is not particularly limited, but is preferably 0.001% to 1.000%, and more preferably 0.01% to 0.80%.
- the plant of the present invention is preferably a plant containing the glycoside of the present invention in a higher content by 0.1% or more than that of the wild type Stevia species.
- the steviol glycoside of the present invention is not contained at all in the wild-type stevia, or even if it is contained, the amount is below the detection limit value.
- Constants 0.01% or more of the glycoside of the present invention compared to wild-type stevia species means fresh leaves of wild-type stevia plants (unit amount of extract obtained from non-dried leaves (eg 10 ml))
- unit amount of extract obtained from non-dried leaves eg 10 ml
- concentration the same unit amount of the extract obtained from the non-dried leaves of the plant of the present invention (the wild type stevia plant)
- the amount (concentration) of the glycoside of the present invention contained in the same amount as the extract obtained from the leaves of the leaves of the present invention is 0.01% or higher.
- the ratio of the glycoside of the present invention to the total steviol glycoside is 0.01% or more” is present in the extract obtained from fresh leaves (non-dried leaves) of the stevia plant of the present invention. It means that the glycoside of the present invention is present at a ratio of 0.01% or more with respect to the total amount of steviol glycoside.
- the total steviol glycoside does not include unknown steviol glycosides, nor does it include steviol glycosides that are present below the detection limit.
- the total steviol glycoside is rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudio.
- the content of the glycoside of the present invention in the plant of the present invention is as described above, but when the dried leaf is obtained from the plant of the present invention, the glycoside of the present invention relative to the weight of the dried leaf.
- the dry leaves of the plant body of the present invention means that the fresh water content of the plant body of the present invention is dried, so that the water content is 10 wt% or less, 7 wt% or less, 5 wt% or less, 4 wt% or less, 3
- the weight is reduced to not more than wt%, not more than 2 wt%, not more than 1 wt%.
- the moisture content of the dried leaves of the plant of the present invention is 3 to 4% by weight.
- Examples of the plant body of the present invention include, for example, a plant body containing a large amount of Reb.C.
- the steviol glycoside of the present invention is not contained at all in the wild-type stevia, or even if it is contained, the amount is below the detection limit value.
- the present inventors have found that the steviol glycosides of the present invention are contained more in plants containing a large amount of Reb.C. Therefore, novel steviol glycosides and extracts thereof include plants and extracts thereof that are rich in such Reb.C.
- Such a plant containing a large amount of Reb.C is not particularly limited.
- it is a high rebaudioside C-containing non-genetically modified stevia plant containing 20% or more of rebaudioside C compared to wild-type stevia species.
- a plant body in which the ratio of rebaudioside C in the total steviol glycoside is 40% or more (hereinafter also referred to as “high Reb.C plant body”) can be mentioned.
- Examples of such a high Reb.C plant include, for example, a high rebaudioside C-containing non-genetically modified stevia plant containing 20% or more of rebaudioside C compared to wild type stevia species, and total steviol glycosides. Examples include plants having a rebaudioside C ratio of 40% or more in the body.
- the high Reb.C plant is a species derived from a wild Stevia plant and has a gene mutated so that rebaudioside C is high.
- the mutation of the gene is not particularly limited, and examples thereof include those that occur under natural conditions, those that occur by non-genetic recombination techniques, and those that occur by genetic recombination.
- High Reb.C plants can be screened, for example, by detecting a gene polymorphism from the tissue of the plant.
- “screening” means identifying a high Reb.C plant body and other plant bodies and selecting a high Reb.C plant body.
- the high Reb.C plant can also be obtained by a screening method comprising the step of identifying a polymorphism in which the 60th base sequence of the base sequence shown in SEQ ID NO: 11 is mutated from wild type A to T in the genome of the test plant. Can be screened.
- the plant body of the present invention includes not only the whole plant body but also a plant organ (eg, leaf, petal, stem, root, seed, etc.), plant tissue (eg, epidermis, phloem, soft tissue, xylem, vascular bundle, fence-like shape) Tissue, spongy tissue, etc.) or various forms of plant cells (eg, suspension culture cells), protoplasts, leaf sections, callus, and the like.
- a plant organ eg, leaf, petal, stem, root, seed, etc.
- plant tissue eg, epidermis, phloem, soft tissue, xylem, vascular bundle, fence-like shape
- Tissue eg, spongy tissue, etc.
- various forms of plant cells eg, suspension culture cells
- protoplasts eg, leaf sections, callus, and the like.
- the plant body of the present invention may include a tissue culture or a plant culture cell. It is because a plant body can be regenerated by culturing such tissue culture or plant culture cells.
- tissue culture or plant culture cells of the plant of the present invention include embryos, meristem cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf sections, and callus. It is not limited.
- the plant extract of the present invention can be obtained by reacting the fresh or dried leaves of the plant of the present invention with an appropriate solvent (an aqueous solvent such as water or an organic solvent such as alcohol, ether and acetone). It can.
- an appropriate solvent an aqueous solvent such as water or an organic solvent such as alcohol, ether and acetone.
- the plant extract of the present invention preferably contains the glycoside of the present invention in a content higher by 0.01% or more than the wild type stevia species, and the ratio of the glycoside of the present invention to the total steviol glycosides Is 0.01% or more.
- “contains the glycoside of the present invention in a content higher by 0.01% or more than the wild type Stevia species” is as described above.
- “the ratio of the glycoside of the present invention to the total steviol glycoside is 0.01% or more” is as described above.
- Flavor modifier containing a novel steviol glycoside The novel steviol glycoside of the present invention is considered to have an influence on the flavor of the stevia extract, although only a trace amount is contained in the stevia extract. . Although not being bound by theory, it is considered that the flavor of food and drink can be adjusted by adding a small amount of the steviol glycoside of the present invention. Therefore, according to one aspect of the present invention, there is provided a flavor adjusting agent comprising a compound represented by the above formula (1) or a derivative, salt or hydrate thereof.
- the “flavor modifier” refers to a substance that, when added to a food or drink, adjusts the flavor of the food or drink.
- the flavor adjusting agent of the present invention when added to a food or drink, the flavor of the food or drink itself can be adjusted without the consumer recognizing the taste of the flavor adjuster itself.
- the steviol glycoside of the present invention has a feature that sweetness is better after that of conventional steviol glycosides, so that it is used as a flavor regulator to adjust the sweetness of food and drink. be able to.
- the flavor adjusting (enhancing) agent of the present invention preferably contains one or more other sweeteners in addition to the compound represented by the above formula (1) or a derivative, salt or hydrate thereof.
- Such sweeteners include rebaudioside A, rebaudioside D, rebaudioside B, rebaudioside M, rebaudioside N, rebaudioside O, rebaudioside E, One or more steviol glycosides selected from the group consisting of rebaudioside K and rebaudioside J, fructose, sugar, fructose glucose liquid sugar, glucose, maltose, sucrose, high fructose liquid sugar, sugar Natural sweeteners such as alcohol, oligosaccharides, honey, sugar cane juice (brown molasses), syrup, rakan fruit powder, rahan fruit extract, licorice powder, licorice extract, somatococcus danieli seed powder, somatococcus danieli seed extract And artificial sweeten
- the flavor adjusting agent of the present invention is a flavor adjusting agent that improves after-treatment, including a compound represented by the above formula (1) or a derivative, salt, or hydrate thereof.
- the Brix of foods and drinks that are usually marketed is up to about 15. However, it is currently being considered to reduce the amount of sugar in foods and drinks by increasing health consciousness and introducing sugar taxes. Yes. When the amount of sugar is reduced, attempts have been made to supplement the Brix reduced by the reduction with a sweetener other than sugar (for example, a non-calorie sweetener).
- the Brix becomes 5
- a sweetener other than sugar For example, if the amount of sugar in a food or drink originally having a Brix of 10 is halved, the Brix becomes 5, so it is necessary to add a sweetener other than sugar to ensure a sweetness equivalent to Brix5.
- many sweeteners other than sugar have a unique flavor different from that of sugar, and one of the characteristic flavors is that the sweetness is not good.
- the flavor adjusting agent containing the steviol glycoside of the present invention can be used as a flavor adjusting agent for improving the after-effect.
- “Brix” is an index of sweetness of food and drink, and is a value obtained by converting a soluble solid content concentration to a weight percentage concentration of a sucrose solution at 20 ° C. Therefore, it is indicated by the amount (g) of sucrose in 100 g of the sucrose aqueous solution.
- Brix5 indicates a sweetness level corresponding to the sweetness level of a solution containing 5 g of sucrose in 100 g of an aqueous sucrose solution.
- the flavor adjusting agent of the present invention is preferably added in an amount of 1% by mass to 15% by mass with respect to the sweetener other than sugar contained in the food or drink, based on the mass of the sweetener. More preferably, the addition amount of the flavor adjusting agent of the present invention is an amount of 1.5% by mass to 12% by mass based on the mass of the sweetener relative to other sweeteners contained in the food and drink, The amount is preferably 3.5% by mass to 11% by mass.
- the content of sweeteners other than sugar in the food and drink to which the flavor adjusting agent of the present invention is added is preferably 5 to 13, more preferably 5 to 12, and further preferably 5 to 7 in terms of Brix.
- the content of sweeteners other than sugar is 5 in terms of Brix
- the content of sweeteners other than sugar is 5 in terms of Brix
- the amount that “the content of sweeteners other than sugar is 5 in terms of Brix” is 0.025 g in 100 g of an aqueous solution containing sweeteners other than sugar.
- the flavor adjusting agent of the present invention is a flavor adjusting agent added in an amount of 1% by mass to 15% by mass on the basis of the mass of the sweetener with respect to a sweetener other than sugar contained in food and drink.
- the content of sweeteners other than sugar is 5.5 to 12 in terms of Brix.
- the flavor modifier of the present invention is added in an amount of 1.5% by mass to 12% by mass based on the mass of the sweetener relative to the sweetener other than sugar contained in the food or drink.
- the content of the sweetener other than sugar is 5 to 13 in terms of Brix.
- Rebaudioside A Although it does not specifically limit as another sweetener contained in food-drinks, Rebaudioside A, Rebaudioside D, Rebaudioside B, Rebaudioside M, Rebaudioside N, Rebaudioside One or more steviol glycosides selected from the group consisting of O, rebaudioside E, rebaudioside K, and rebaudioside J, fructose, fructose glucose liquid sugar, glucose, maltose, high fructose liquid Sugar, sugar alcohol, oligosaccharide, honey, sugar cane juice (brown sugar nectar), syrup, arahan fruit powder, arahan fruit extract, licorice powder, licorice extract, somatococcus daniel seed powder, somatococcus daniel seed extract, etc. Natural sweeteners and artificial sweeteners such as acesulfame potassium, sucralose, neotame, aspartame, saccharin It is.
- the flavor adjusting agent of the present invention is a flavor adjusting agent that enhances sweetness, including a compound represented by the above formula (1) or a derivative, salt or hydrate thereof.
- a flavor modifier that enhances sweetness is a flavor modifier that, when added to a food or drink containing a sweetener, can provide the food or drink with a sweetness that is higher than the sweetness obtained by simply adding the sweetness of the flavor modifier. Means. For example, when a flavor adjuster equivalent to Brix 0.1 is added to a food or drink having sweetness equivalent to Brix 9, the sweetness of the food or drink can be set to a sweetness exceeding Brix 9.1 (eg, Brix 9.2).
- a flavor modifier By using such a flavor regulator that enhances sweetness, the total amount of sweeteners used can be reduced, and there is an advantage that calories can be reduced and costs can be reduced.
- the sweet taste enhancing flavor-adjusting agent of the present invention has a sweetness enhancement target sweetener having a sweetness degree of 1 to 10, and is 0.05 to 2% by weight based on the mass of the sweetener to be sweetened. It is preferably added in an amount of 0% by mass, more preferably in an amount of 0.1% by mass to 1.5% by mass, and in an amount of 0.2% by mass to 1.2% by mass. Is more preferable.
- the sweetener for which sweetness is to be enhanced is not particularly limited, but rebaudioside A, rebaudioside D, rebaudioside B, rebaudioside M, rebaudioside N, rebaudioside O , One or more steviol glycosides selected from the group consisting of Rebaudioside E, Rebaudioside K, and Rebaudioside J, fructose, sugar, fructose glucose liquid sugar, glucose, maltose, sucrose , High fructose liquid sugar, sugar alcohol, oligosaccharide, honey, sugar cane juice (brown molasses), syrup, rakan fruit powder, rakan fruit extract, licorice powder, licorice extract, somatococcus daniel seed powder, somatococcus daniel seed Natural sweeteners such as extracts and artificial artificial ingredients such as acesulfame potassium, sucralose, neotame, aspartame, saccharin Seasonings and the like.
- the steviol glycoside of the present invention is produced by (A) isolation / purification from a plant body, (B) chemical synthesis, or (C) biosynthesis. be able to. Each will be described below.
- the novel steviol glycoside can be isolated and purified from the plant body.
- the novel steviol glycoside is extracted in the form of an extract by reacting the fresh or dried leaves of the plant of the present invention with an appropriate solvent (aqueous solvent such as water or organic solvent such as alcohol, ether and acetone). can do.
- an appropriate solvent aqueous solvent such as water or organic solvent such as alcohol, ether and acetone.
- ethyl acetate and other organic solvents water gradient, high-performance liquid chromatography (High Performance Liquid Chromatography: HPLC), ultra-high performance liquid chromatography (Ultra (High) Performance)
- HPLC High Performance Liquid Chromatography
- Ultra (High) Performance Ultra-high performance liquid chromatography
- UPLC Liquid Chromatography
- novel steviol glycoside content in the plant can be measured by the method described in WO2016 / 090460 or the method described in the examples described later. Specifically, it can be measured by sampling fresh leaves from the plant of the present invention and performing LC-MS / MS.
- Steviol glycosides have a structure in which various sugar canes (glucose, rhamnose, xylose, etc.) are added to steviol of aglycone in various bond forms (bonding position and solid). Therefore, the target steviol glycoside can be obtained through various synthetic routes depending on the starting material selected. However, it is understood by those skilled in the art of the present invention that the time and yield for obtaining the target compound vary greatly depending on the synthesis route.
- the present inventors have now known a novel method for producing the steviol glycoside of the present invention with high selectivity and high yield by a specific synthetic route.
- a novel method for producing the steviol glycoside of the present invention with high selectivity and high yield by a specific synthetic route.
- Scheme 1 when chemical synthesis of steviol glycosides is performed, as shown in Scheme 1, steviol glycosides are divided into “steviol glycosides” and “sugar hemiacetal bodies”. And proceed with synthesis.
- Steviol glycoside can be prepared by deriving from existing natural products (rebaudioside, zulcoside, stevioside, steviolbioside, rubusoside, etc.).
- sugar hemiacetal bodies can be prepared from existing natural products, or can be prepared by chemical synthesis. The present inventors have found that when steviol glycoside and sugar hemiacetal are condensed using Mitsunobu reaction, the desired steviol glycoside can be obtained with good yield and extremely high ⁇ selectivity.
- a method for producing a compound represented by the formula (1) (A) The following formula (2) From the rebaudioside C shown by the following formula (3) (In the formula, each PG independently represents a protecting group.) And (B) from the glucopyranoside derivative (4) (In the formula, each PG independently represents a protecting group.)
- a step of synthesizing a compound represented by: A method is provided comprising:
- a process for producing a compound represented by the formula (1) wherein a compound represented by the above formula (3) and a compound represented by the above formula (4) are converted into a phosphine reagent. And in the presence of an azo compound, the following formula (5) (In the formula, each PG independently represents a protecting group.)
- a method is provided, further comprising the step of obtaining a compound represented by:
- examples of the protecting group include an acyl protecting group, a trisubstituted silyl group, an acetal protecting group, and an ether protecting group.
- Preferable examples include trisubstituted silyl groups (such as trimethylsilyl group, triethylsilyl group, and t-butyldimethylsilyl group) or acyl protecting groups (such as acetyl group and benzoyl group).
- Steviol glycoside can be obtained, for example, according to the following scheme 2 using naturally occurring rebaudioside C (zulcoside B) as a raw material.
- rebaudioside® C is dissolved in a solvent such as methanol and water, a strong base such as sodium hydroxide is added, and the mixture is refluxed at 60 ° C. to 120 ° C. for 2 hours or longer.
- a strong base such as sodium hydroxide
- the glucose molecule is eliminated from the position, and the compound 2 is obtained.
- the solvent may be evaporated after neutralizing the reaction solution with a cation exchange resin or the like.
- Compound 2 can be obtained by further dissolving Compound 2 in a solvent such as pyridine and adding acetic anhydride to protect the hydroxyl group contained in Compound 2.
- the trisaccharide hemiacetal can be obtained, for example, according to the following scheme 3 using a commercially available glucopyranoside derivative as a raw material.
- 4-Methoxyphenyl ⁇ -D-Glucopyranoside (4) is dissolved in a solvent such as acetonitrile, benzaldehyde dimethyl acetal and camphorsulfonic acid (acid catalyst) are added, and the mixture is stirred at 25-80 ° C for 2 hours or more.
- Compound 5 is obtained.
- compound 5 is dissolved in 2,3,4,6-Tetra-O-acetyl- ⁇ -D-glucosypyranosyl2-2,2,2-Trichloroacetimidate (6) and molecular sieves 4 ⁇ in a solvent such as dichloromethane, and trimethylsilyltrifluoromethane is obtained.
- the sulfonate is added at a low temperature (for example, 0 ° C.), and the mixture is stirred at room temperature for 2 hours or more to obtain the compound 7.
- Compound 7 is dissolved in a solvent such as ethanol, P-toluenesulfonic acid is added at room temperature, the mixture is stirred at 60 ° C. to 80 ° C. for 2 hours or longer to complete the reaction, neutralized with triethylamine, and concentrated under reduced pressure.
- the obtained syrup is dissolved in a solvent such as pyridine, and acetic anhydride or the like is added to obtain compound 8 in which the hydroxyl group is protected.
- Compound 9 is obtained by dissolving compound 8 in acetonitrile and water, adding an oxidizing agent such as cerium ammonium nitrate, and stirring for 5 minutes to 2 hours.
- the steviol glycoside of the present invention introduces a polynucleotide encoding a predetermined protein into host cells derived from bacteria, plants, insects, mammals other than humans, etc., and steviol or steviol glycosides.
- Glucose, UDP-glucose and / or UDP-rhamnose can also be produced as a substrate.
- the substrates steviol, steviol glycoside, UDP-glucose, and UDP-rhamnose may be given or biosynthesized in cells.
- Examples of the predetermined protein include stevia-derived UGT85C2 (amino acid sequence is SEQ ID NO: 2), UGT74G1 (amino acid sequence is SEQ ID NO: 4), UGT91D2 (amino acid sequence is SEQ ID NO: 6), UGT76G1 (amino acid sequence is SEQ ID NO: 8).
- UDP-rhamnose synthase AtRHM2 (amino acid sequence is SEQ ID NO: 10) derived from Arabidopsis thaliana, but are not limited thereto as long as they have equivalent activities.
- the above protein is an enzyme derived from Arabidopsis thaliana or stevia, and is expected to have a high activity even in an environment outside plant cells such as Arabidopsis thaliana or stevia (for example, extracellular, in host cells other than stevia).
- a polynucleotide encoding the above protein for example, UGT85C2 gene is SEQ ID NO: 1, UGT74G1 gene is SEQ ID NO: 3, UGT91D2 gene is SEQ ID NO: 5, UGT76G1 gene is SEQ ID NO: 7, and AtRHM2 gene is SEQ ID NO: 9). Introduced into host cells derived from bacteria, fungi, plants, insects, mammals other than humans, etc.
- the substrate steviol, steviol glycosides, UDP-glucose, UDP-rhamnose to give the compounds of the present invention.
- the compound of the present invention can be produced by expressing the protein in a host cell and providing an appropriate substrate.
- a method for producing the novel steviol glycoside of the present invention wherein a non-human transformant introduced with at least one of the following polynucleotides (a) to (g) is used: A method characterized by this is provided.
- a polynucleotide encoding a protein having an activity of adding glucose to the hydroxyl group at position 13 of steviol glycosides (b) SEQ ID NO: 3 A polynucleotide containing a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 3, or 90% or more identity to the amino acid sequence of SEQ ID NO: 4, and steviol A polynucleotide encoding a protein having an activity of adding glucose to a carboxylic acid at the 19th position of a glycoside (c) the nucleotide sequence of SEQ ID NO: 5 A polynucleotide having a nucleotide sequence having a
- 91% or more, 92% or more, 93% or more, 94% or more independently of the base sequence of each SEQ ID NO described in the above (a) to (g), 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, Polynucleotides having a sequence identity of 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more may be used.
- amino acid sequence has a sequence identity of 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, and is described in (a) to (g) above.
- a protein having a predetermined activity may be used.
- the polynucleotide encoding the protein is preferably introduced into the host in a state of being inserted into an appropriate expression vector.
- the polynucleotides may be individually inserted into separate vectors.
- Suitable expression vectors are usually (I) a promoter capable of transcription in a host cell; (Ii) a polynucleotide of the present invention linked to the promoter; and (iii) an expression cassette comprising, as a component, a signal that functions in a host cell for transcription termination and polyadenylation of an RNA molecule. .
- the method for producing an expression vector includes, but is not limited to, a method using a plasmid, phage or cosmid, or a DNA molecule having a necessary component.
- the specific type of the vector is not particularly limited, and a vector that can be expressed in the host cell can be appropriately selected. That is, according to the type of the host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. Good.
- the expression vector of the present invention contains an expression control region (for example, promoter, terminator and / or origin of replication) depending on the type of host to be introduced.
- Conventional promoters for example, trc promoter, tac promoter, lac promoter, etc.
- yeast promoters include glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter,
- the promoter for filamentous fungi include amylase and trpC.
- promoters for expressing a target gene in plant cells include cauliflower mosaic virus 35S RNA promoter, rd29A gene promoter, rbcS promoter, and enhancer sequences of the cauliflower mosaic virus 35S RNA promoter derived from Agrobacterium. And the mac-1 promoter added to the 5 'side of the mannopine synthase promoter sequence.
- animal cell host promoters include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.).
- promoters that are inducibly activated by external stimuli include mouse mammary tumor virus (MMTV) promoter, tetracycline responsive promoter, metallothionein promoter, heat shock protein promoter, and the like.
- the expression vector preferably contains at least one selectable marker.
- auxotrophic markers LEU2, URA3, HIS3, TRP1, ura5, niaD
- drug resistance markers hygromycin, zeocin
- geneticin resistance gene G418r
- copper resistance gene CUP1
- cerulenin resistance gene fas2m, PDR4
- Minoru Ogura et al., Biochemistry, vol. 64, p. 660, 1992; Hussain et al., Gene, vol. 101, p. 149, 1991 can be used.
- a host cell transformation method As a host cell transformation method, a commonly known method can be used. For example, electroporation method (Mackenxie, D. A. et al., Appl. Environ. Microbiol., Vol. 66, p. 4655-4661, 2000), particle delivery method (JP 2005-287403), sphero Plast method (Proc. Natl. Acad. Sci. USA, vol. 75, p. 1929, 1978), lithium acetate method (J. Bacteriology, vol. 153, p. 163, 1983), Methods in yeast genetics, 2000 Edition : A) Cold Spring Harbor Laboratory Course Manual)), but is not limited thereto.
- electroporation method Mackenxie, D. A. et al., Appl. Environ. Microbiol., Vol. 66, p. 4655-4661, 2000
- particle delivery method JP 2005-287403
- sphero Plast method Proc. Natl. Aca
- non-human transformant By culturing the non-human transformant thus obtained, it is possible to cause the non-human transformant to produce steviol glycosides.
- a non-human transformant is preferably yeast.
- sample 1 is a high Reb.C plant having a polymorphism in which the 60th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 11 is mutated from A to T in the genome.
- the polymorphism had a statistical correlation with the phenotype of high RebC concentration. became.
- test solution was prepared by weighing 10.0 mg of each dried stevia leaf that had been lyophilized into a glass vial, adding 1.0 ⁇ mL of water / methanol (1/1 vol / vol) as an extraction solvent, and then ultrasonically washing.
- a steviol glycoside extract was obtained from stevia leaves by irradiating ultrasonic waves at a set temperature of 25 ° C. for 20 minutes with a vessel (AS ONE, AS52GTU). Further, for use in HPLC-MS, it was diluted 10-fold with water / methanol and filtered through a filter (Nacalai Tesque, Cosmonis filter S (solvent system)) having a pore size of 0.45 ⁇ m.
- LC mobile phase mobile phase A is 0.2% acetic acid-containing milli-Q water
- mobile phase B is methanol
- the binary gradient is kept constant at 10% mobile phase B concentration for 0 ⁇ 5 minutes, then for 15 minutes.
- the mobile phase B concentration was changed from 10% to 70%, and the B concentration was further changed from 70% to 100% in 5 minutes.
- the mobile phase B concentration was maintained at 100% for 5 minutes.
- the flow rate of the mobile phase was 0.4 ⁇ mL / min, and 5 ⁇ L of stevia leaf extract that had been diluted and filtered was injected.
- MS was a triple quadrupole mass spectrometer LCMS-8030 (Shimadzu Corporation) equipped with an electrospray ionization (ESI) ion source.
- the mass spectrometry measurement was performed in a negative ion measurement mode and a selected ion monitoring (SIM) mode in which measurement is performed by selecting an m / z value.
- the m / z value to be selected is the m / z value calculated based on the molecular weight of steviol glycosides composed of D-glucopyranosyl (glc), L-rhamnopyranosyl (rha), and xylopyranosyl (xyl) in the sugar chain. Selected.
- m / z 641.2 (glc (2)), 773.2 (glc (2), xyl (1)), 787.2 (glc (2), rha (1)), 803.2 (glc (3)), 935.3 ( glc (3), xyl (1)), 949.3 (glc (3), rha (1)), 965.3 (glc (4)), 1095.4 (glc (3), rha (2)), 1097.4 (glc (4 ), Xyl (1)), 1111.4 (glc (4), rha (1)), 1127.4 (glc (5)), 1257.5 (glc (4), rha (2)), 1259.5 (glc (5), xyl (1)), 1273.5 (glc (5), rha (1)), 1289.5 (glc (6)), 1435.6 (glc (6), rha (1)) were selected.
- FIG. 1 shows a selected ion chromatogram of sample 1 (EM3-4) at m / z 1273.5.
- the peak observed at Retention Time (Rt) 28.23 minutes is the same as the rebaudioside N sample in mass and retention time.
- Rt227.73 is an unknown substance.
- the peak value of Rt 27.73 min from the sample 4 in which the content of rebaudioside C is lower than that of rebaudioside A and the sugar chain elongation is lower than the other samples was below the detection limit.
- HPLC-ESI-HRMS high-performance liquid chromatography-electrospray ionization accurate mass spectrometry
- the HPLC instrument configuration uses a Prominence LC-20AD (Shimadzu Corporation) for the liquid-feed unit LC pump, and the separation column SM-C18 mm (4.6 mm x 250 mm) (manufactured by Intact) was used.
- the LC mobile phase is pumped using 0.2% acetic acid-containing milli-Q water as mobile phase A, methanol as mobile phase B, and the binary gradient gradient is constant at 10% mobile phase B concentration for 0-5 minutes. Thereafter, the mobile phase B concentration was changed from 10% to 70% in 15 minutes, and the B concentration was changed from 70% to 100% in 5 minutes.
- the mobile phase B concentration was maintained at 100% for 5 minutes.
- the flow rate of the mobile phase was 0.4 ⁇ mL / min, and 5 ⁇ L of stevia leaf extract that had been diluted and filtered was injected.
- Orbitrap® Elite® MS manufactured by Thermo Fisher Scientific
- the mass spectrometry measurement was performed in the negative ion measurement mode, in the range of m / z 150 to 2000, with a setting resolution of 60,000.
- MS / MS measurement was performed in the CID mode in which the target m / z 1273.5 was selected and fragmentation was performed by collision with an inert gas. Irradiation of energy required for fragmentation was performed at 35, which is a collision energy standard specific to the device.
- MS / MS and MS3 fragmentation pattern analysis was performed on standard rebaudiosides A and D, M, which have known structures.
- MS / MS of the new steviol glycoside data was obtained in which the ionic strength of the peak from which all of the sugar chain ester-bonded at position 19 was eliminated was the strongest. This result shows that the total molecular weight of the sugar chain is found by ester bonding to the 19th carbon.
- FIG. 3 shows MS / MS and MS 3 fragmented mass spectra of rebaudioside N and a novel steviol glycoside.
- rebaudioside N was equivalent to the elimination of 2 glc and 1 rha.
- the peak at z 803.37 was the main peak.
- a peak was detected as a main peak at m / z 787.38 corresponding to the elimination of 3 Glc.
- trisaccharide hemiacetal (9) a trisaccharide skeleton is formed by the condensation reaction of appropriately protected glucose acceptor (5) and glucose donor (6) ⁇ , and the protecting group at the 1-position of the reducing end is deprotected. As a result, a trisaccharide hemiacetal body (9) was obtained.
- the reaction proceeded with 47% ( ⁇ form only) with good and complete ⁇ selectivity.
- a novel steviol glycoside (11) was obtained.
- Stevia leaf cDNA was obtained by extracting total RNA from Stevia leaves using the RNeasy Plant Mini kit (QIAGEN), and 0.5 ⁇ g of the RNA was subjected to reverse transcription (RT) reaction with Random Oligo-dT primer.
- the PCR reaction solution (50 ⁇ l) was composed of 1 ⁇ l of stevia leaf-derived cDNA, 1 ⁇ KOD plus buffer (TOYOBO), 0.2 mM dNTPs, each primer 0.4 pmol / ⁇ l, 1 mM MgSO 4 , 1 U heat-resistant KOD plus polymerase.
- the PCR reaction was carried out at 95 ° C. for 5 minutes, and then amplified at 30 ° C. for 0.5 minutes, at 50 ° C. for 0.5 minute, and at 68 ° C. for 2 minutes for a total of 30 cycles of amplification.
- Each PCR product was electrophoresed on a 0.8% agarose gel and stained with ethidium bromide.
- a combination of SrUGT85C2 using UGT85C2 as a template, SrUGT91D2 using UGT91D2 as a template, SrUGT74G1 using UGT74G1 as a template, SrUGT76G1 using UGT76G1 as a template, AtAHM2 as a template and primer combination of AtAHM2 and heat-resistant KOD DNA polymerase And restriction enzyme sites were added to both ends of each ORF.
- the obtained DNA fragment was subcloned using the Zero Blunt-TOPO PCR Cloning Kit (Invitrogen), and sequenced by the primer walking method using a synthetic oligonucleotide primer using DNA®Sequencer® model 3100 (Applied® Biosystems). It was confirmed that each UGT gene was cloned.
- Transformation of yeast Saccharomyces cerevisiae YPH499 strain (ura3-52 lys2-801 amber ade2-101 ochre trp1- ⁇ 63 his3- ⁇ 200 leu2- ⁇ 1 a) was used as a host, and the plasmids shown in Table 2 were introduced by the lithium acetate method.
- a strain that grows on SC-Trp & Ura & His agar medium (6.7 g of Yeast nitrogen baase without amino acids, glucose 20 g, amino acid mixed powder-Trp & Ura & His 1.3 g, Bacto agar 20 g per liter) was selected.
- the amino acid mix powder-Trp & Ura & His is adenine sulfate 2.5 g, L-arginine hydrochloride 1.2 g, L-aspartic acid 6.0 g, L-glutamic acid 6.0 g, L-leucine 3.6 g, L-lysine 1.8 g, L- It was prepared by mixing 1.2 g of methionine, 3.0 g of L-phenylalanine, 22.5 g of L-serine, 12 g of L-threonine, 1.8 g of L-tyrosine, and 9.0 g of L-valine.
- each transformant was inoculated into 10 ml of SC-Trp & Ura & His liquid medium (excluding Bacto agar of SC-Trp & Ura & His agar medium) and cultured with shaking at 30 ° C. for 1 day.
- 1 ml of the pre-culture solution is inoculated into 10 ml of SG-Trp & Ura & His liquid medium (6.7 g of Yeast nitrogen baase without amino acids, 20 g of galactose, 1.3 g of amino acid mix powder-Trp & Ura & His per liter) And cultured with shaking at 30 ° C. for 2 days.
- SG-Trp & Ura & His liquid medium 6.7 g of Yeast nitrogen baase without amino acids, 20 g of galactose, 1.3 g of amino acid mix powder-Trp & Ura & His per liter
- the bacterial cells were collected from the culture solution, and total RNA was purified using RNeasy Mini Mini Kit.
- RNA 1 ⁇ g of total RNA was taken, and cDNA was synthesized using Superscript II reverse transcriptase (Thermo Fisher Scientific) and random hexamer as primers.
- steviol glycosides Culture was performed under the same conditions as in the above examples, except that 0.5 ⁇ g or 2 ⁇ g of steviol (ChromaDex Inc.) was added to 1 ml of the medium in the liquid medium for main culture. After completion of the culture, the culture solution was separated into a supernatant and cells by centrifugation. The culture supernatant is washed with acetonitrile, applied to a Sep-Pak C18 column equilibrated with water, washed with 20% acetonitrile, eluted with 80% acetonitrile, dried and dissolved in a small amount of 80% acetonitrile. A glycoside sample was prepared. This glycoside sample was subjected to the following analysis.
- the evaluation was performed by selecting a sucrose-added sample having a sweetness intensity equivalent to that of the sample to which the new steviol glycoside was added, and 5 persons trained in the sweetener sensuality became panelists. Sensory evaluation was performed. As a result, it was found that the prepared sample to which the novel glycoside was added had sweetness equivalent to the Brix1 sucrose-added sample. Therefore, it was found that the novel steviol glycoside has a sweetness of 24 with respect to sucrose.
- Reb In order to evaluate the taste quality of various steviol glycosides, Reb.
- a beverage sample was prepared by adding A and a novel steviol glycoside to pure water. All the beverage samples were adjusted so that the sweetness was RebA: 300 and the novel glycoside was 24, and the Brix in terms of sucrose (sucrose) was finally 2.
- the obtained beverage sample was subjected to sensory evaluation with indices of the degree of sweetness rise, bitterness, and sweetness after-effect.
- Persons (5 persons) who were trained regarding the sensuality of sweeteners were evaluated as panelists, and the evaluation criteria were as follows. Very weak (-3), weak (-2), slightly weak (-1), normal (0), slightly strong (+1), strong (+2), and very strong (+3).
- the new steviol glycoside is a conventional sweetener, Reb. It was found to have an equivalent bitter taste and a shorter sweet aftertaste compared to A.
- Reb. Evaluation of the effect of improving the pull-back effect on A Using the three-level aqueous solutions of Brix 5, 7 and 11, the Reb. Evaluation of the effect of improving the pull-back for A was performed. First, Reb. A sweetness degree of A was 237 times, and three-level aqueous solutions of Brix 5, 7 and 11 were prepared. The added amount of the flavor adjusting agent comprising the novel steviol glycoside of the present invention is Reb. Based on the added mass of A, the ratio was 1, 3.5, 5 and 10 mass%. A control sample (Cont) was obtained from Reb. Only A was added, and the flavor modifier of the present invention was not added.
- Reb. Evaluation of the effect of improving the pull-down effect on D Using the three levels of aqueous solutions of Brix 5, 7, and 11, the Reb. Evaluation of the effect of improving the pull-down with respect to D was performed.
- Reb. Three-level aqueous solutions of Brix 5, 7 and 11 were prepared with the sweetness level of D being 213 times.
- the added amount of the flavor adjusting agent comprising the novel steviol glycoside of the present invention is Reb.
- the ratio was 1, 3.5, 5 and 10% by mass based on the added mass of D, respectively.
- a control sample (Cont) was obtained from Reb. Only D was added, and the flavor modifier of the present invention was not added.
- a sweet taste-enhancing flavor modifier containing a novel steviol glycoside Using two aqueous solutions of Brix 5 and 7, the sweet taste enhancing effect of the flavor modifier of the present invention on sugar (sucrose) was evaluated.
- a two-level aqueous solution of Brix 5 and 7 was prepared using sugar.
- the addition amount of the flavor adjusting agent comprising the novel steviol glycoside of the present invention is 0.10, 0.44 and 0.80 mass% at the Brix 5 level based on the added mass of the sugar, and the Brix 7 In the level, the ratios were 0.10, 0.44, and 0.57% by mass.
- the persons (7 persons) who were trained regarding the sensuality of the sweetener were evaluated as panelists, and the sweetness intensity was evaluated.
- the results are shown in the graph of FIG.
- the vertical axis (sweetness intensity) on the graph represents the magnification of the sweetness intensity actually felt by the panel relative to the total sweetness of sugar and the flavor modifier (the sweetness of the flavor modifier is calculated as a sweetness factor of 24).
- sweetness intensity is calculated as a sweetness factor of 24.
- a sweetness enhancing effect of about 2 to 7% was observed.
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Abstract
Description
本発明者らは、味質に影響を与える微量の新規ステビオール配糖体の構造を初めて特定した。本発明の新規ステビオール配糖体(以下、「本発明の配糖体」ともいう)は、式(1)
で示される化合物、またはその誘導体、塩、もしくは水和物である。
本発明の一態様によれば、式(1)で示される化合物、またはその誘導体、塩、もしくは水和物を含む甘味料組成物(以下、「本発明の甘味料組成物」ともいう)が提供される。本発明の甘味料組成物は、式(1)で示される化合物、またはその誘導体、塩、もしくは水和物を含んでいれば特に限定されず、式(1)で示される化合物、またはその誘導体、塩、もしくは水和物を含む抽出物を含む組成物であってもよい。
本発明の一態様によれば、式(1)で示される化合物、またはその誘導体、塩、もしくは水和物を含む飲食品(以下、「本発明の飲食品」ともいう)が提供される。本発明の飲食品は、式(1)で示される化合物、またはその誘導体、塩、もしくは水和物を含んでいれば特に限定されず、式(1)で示される化合物、またはその誘導体、塩、もしくは水和物を含む抽出物や甘味料組成物を含む飲食品であってもよい。ここで飲食品とは、飲料及び食品を意味する。したがって、ある実施態様では、本発明は新規な飲料又は食品を提供し、また、当該飲料又は食品の製造方法を提供する。
点がある。
本発明の一態様によれば、新規ステビオール配糖体を含む植物体およびその抽出物が提供される。また本発明の他の一態様によれば、本発明の植物体または植物体の抽出物を含む、飲食品、好ましくは飲料、が提供される。本発明の植物体に含まれる本発明の配糖体の量は、特に限定されないが、0.001%~1.000%であるのが好ましく、0.01%~0.80%であることがより好ましい。
本発明の新規ステビオール配糖体は、ステビア抽出物の中に微量しか含まれていないものの、該ステビア抽出物の風味に影響を与えているものと考えられる。理論に拘束されるものではないが、本発明のステビオール配糖体を少量添加することで、飲食品の風味を調整できるものと考えられる。したがって、本発明の一態様によれば、上記式(1)で示される化合物またはその誘導体、塩、もしくは水和物を含む風味調整剤が提供される。
上述のとおり、本発明のステビオール配糖体は、(A)植物体からの単離・精製、(B)化学合成、または(C)生合成によって製造することができる。それぞれについて以下に説明する。
本発明の植物体は本発明の新規ステビオール配糖体を含むものであるため、該植物体から新規ステビオール配糖体を単離・精製することができる。新規ステビオール配糖体は、本発明の植物体の新鮮葉または乾燥葉に適切な溶媒(水等の水性溶媒又はアルコール、エーテル及びアセトン等の有機溶媒)を反応させることにより抽出液の状態で抽出することができる。抽出条件等はWO2016/090460に記載の方法や、後述の実施例に記載の方法を参照することができる。
本発明のステビオール配糖体の合成方法について、以下に詳細に説明する。
(A)下記式(2)
で示されるレバウディオサイドCから、下記式(3)
(式中、PGはそれぞれ独立に保護基を表す。)
で示される化合物を合成する工程と
(B)グルコピラノシド誘導体から式(4)
(式中、PGはそれぞれ独立に保護基を表す。)
で示される化合物を合成する工程と、
を含む、方法が提供される。
(式中、PGはそれぞれ独立に保護基を表す。)
で示される化合物を得る工程をさらに含む、方法が提供される。
本発明のステビオール配糖体は、所定のタンパク質をコードするポリヌクレオチドを、細菌、植物、昆虫、ヒトを除く哺乳動物などに由来する宿主細胞に導入して、ステビオールやステビオール配糖体、UDP-グルコースおよび/またはUDP-ラムノースを基質として生成することもできる。基質であるステビオール、ステビオール配糖体、UDP-グルコース、UDP-ラムノースは、与えてもよいし、細胞内で生合成させてもよい。所定のタンパク質の例としては、ステビア由来のUGT85C2(アミノ酸配列は配列番号2)、UGT74G1(アミノ酸配列は配列番号4)、UGT91D2(アミノ酸配列は配列番号6)、UGT76G1(アミノ酸配列は配列番号8)、及びシロイヌナズナ由来のUDP-ラムノース合成酵素AtRHM2(アミノ酸配列は配列番号10)、が挙げられるが同等の活性を有するものであればこれに限定されない。
(a)配列番号1の塩基配列を含有するポリヌクレオチド、配列番号1の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号2のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の13位の水酸基にグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド(b)配列番号3の塩基配列を含有するポリヌクレオチド、配列番号3の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号4のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の19位のカルボン酸にグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(c)配列番号5の塩基配列を含有するポリヌクレオチド、配列番号5の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号6のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の13位に結合したグルコースに1→2結合でラムノースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(d)配列番号7の塩基配列を含有するポリヌクレオチド、配列番号7の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号8のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の13位のグルコースの3位に1→3結合でグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(e)配列番号5の塩基配列を含有するポリヌクレオチド、配列番号5の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号6のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の19位のグルコースに、1→2結合でグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(f)配列番号7の塩基配列を含有するポリヌクレオチド、配列番号7の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号8のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の19位のグルコースに、1→3結合でグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(g)配列番号9の塩基配列を含有するポリヌクレオチド、配列番号9の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号10のアミノ酸配列に対して90%以上の同一性を有し、かつ、UDP-グルコースからUDP-ラムノースを生成する活性を有するタンパク質をコードするポリヌクレオチド
(i)宿主細胞内で転写可能なプロモーター;
(ii)該プロモーターに結合した、本発明のポリヌクレオチド;および
(iii)RNA分子の転写終結およびポリアデニル化に関し、宿主細胞内で機能するシグナルを構成要素として含む発現カセット
を含むように構成される。
発現ベクターは、少なくとも1つの選択マーカーを含むことが好ましい。このようなマーカーとしては、栄養要求性マーカー(LEU2、URA3、HIS3、TRP1、ura5、niaD)、薬剤耐性マーカー(ハイグロマイシン、ゼオシン)、ジェネチシン耐性遺伝子(G418r)、銅耐性遺伝子(CUP1)(Marin et al.,Proc.Natl.Acad.Sci. USA,vol. 81,p.337,1984)、セルレニン耐性遺伝子(fas2m, PDR4)(それぞれ、猪腰淳嗣ら,生化学,vol.64,p.660,1992;Hussain et al.,Gene,vol.101,p.149,1991)などが利用可能である。
サントリーグローバルイノベーションセンター株式会社(SIC)で開発した、4系統の新規ステビア植物体(サンプル1(EM3-4)、サンプル2(EM2-27-8)、サンプル3(EM2-27-15)、およびサンプル4(EM2-11))の葉から得られた抽出物の高速液体クロマトグラフィー(HPLC)-質量分析(MS)を行い、D-グルコピラノシル(glc)、L-ラムノピラノシル(rha)、キシロピラノシル(xyl)が糖鎖に構成されたステビオール配糖体の分子量をもとに、含有されるステビオール配糖体のスクリーニング分析を行った。ここで、サンプル1はゲノムに配列番号11に示す塩基配列の第60番目の塩基配列が野生型のAからTに変異した多型を有する高Reb.C植物体である。なお、高RebC濃度の表現型と配列番号11の多型との相関関係について統計解析を行ったところ、当該多型は高RebC濃度の表現型と統計学上の相関関係を有することが明らかになった。
本発明において、レバウディオサイドC高含有率品種から検出された新規ステビオール配糖体の構造解析は次の手順で行った。
(i)高速液体クロマトグラフィー(HPLC)- 高分解能質量分析(MS)およびMS/MS、3段階までのイオンの断片化(MS3断片化)のフラグメント化解析による構造推定、
(ii)化学反応による推定ステビオール配糖体標準品の化学合成、
(iii)化学合成標準品のHPLC- 高分解能MSおよびMS3断片化の保持時間と断片化パターンの一致による構造確認
検液の調製は、凍結乾燥処理を行ったステビア乾燥葉それぞれ10.0mgをガラスバイアルに秤量し、抽出溶媒として水/メタノール(1/1 vol/vol) を1.0 mL添加し、その後超音波洗浄器(AS ONE, AS52GTU)にて20分間、25°Cの設定温度にて超音波を照射し、ステビア葉からステビオール配糖体の抽出液を得た。さらにHPLC-MSに供するために、水/メタノールで10倍希釈を行い、細孔サイズ0.45 μmのフィルター(ナカライテスク、コスモナイスフィルターS(溶媒系))にてろ過を行った。
[化合物3]
1H-NMR (CDCl3, 400 MHz) δ 0.81 (m, 2H), 0.83-1.45 (complex, 19H), 1.39-1.91 (complex, 24H), 1.91-2.35 (s, 30H), 3.58 (m, 1H), 3.71-3.81 (complex, 4H), 3.95-4.12 (complex, 7H), 4.34-4.46 (complex, 3H), 4.56-4.66 (complex, 4H), 4.69-4.92 (complex, 7H), 5.05-5.14 (complex, 5H), 5.23-5.38 (complex, 6H), 5.45 (s, 1H); 13C-NMR (CDCl3, 100 MHz) δ 15.9, 17.3, 19.1, 20.5, 20.7, 20.8, 20.9, 21.1, 21.5, 21.7, 29.1, 37.8, 38.0, 39.5, 40.7, 41.4, 42.2, 43.8, 48.4, 53.8, 56.8, 61.6, 63.0, 65.5, 66.8, 68.0, 68.6, 69.3, 69.6, 69.8, 70.5, 70.9, 71.6, 71.9, 72.4, 72.8, 73.9, 74.9, 81.3, 87.3, 96.6, 96.8, 99.2, 99.4, 125.4, 128.3, 129.1, 137.9, 151.9, 168.9, 169.2, 169.5, 169.6, 169.8, 170.1, 170.2, 170.3, 170.6, 170.9, 176.8, 183.4
[化合物5]
1H-NMR (CDCl3, 400 MHz) δ 3.58 (m, 1H, H-5), 3.65 (t, 1H, H-4), 3.78 (m, 5H, H-2, H-6, OMe), 3.92 (t, 1H, H-3), 4.38 (dd, 1H, H-6’), 4.92 (d, J = 7.6 Hz, 1H, H-1), 5.57 (s, 1H, CHPh), 6.84 (dd, 4H, OMePh), 7.49 (m, 5H, Ph); 13C-NMR (CDCl3, 100 MHz) δ 31.1, 55.8, 66.7, 68.8, 73.4, 74.6, 80.5, 102.2, 102.5, 114.8, 118.8, 126.4, 128.5, 129.5, 136.9, 150.9, 155.9
[化合物8]
1H-NMR (CDCl3, 400 MHz) δ 1.94-2.17 (complex, 30H, OAc), 2.91 (m, 1H), 3.33 (m, 1H), 3.71 (m, 1H), 3.76 (m, 5H), 3.94 (t, 1H), 4.09-4.17 (complex, 5H), 4.31 (dd, 1H), 4.88 (m, 3H), 4.96-5.08 (complex, 4H), 5.18 (m, 2H), 5.26 (t, 1H), 6.84 (dd, OMePh); 13C-NMR (CDCl3, 100 MHz) δ 20.6×2, 20.7×4, 20.8×2, 20.9, 21.1, 55.8, 60.3, 60.5, 61.8, 62.6, 67.6, 68.3, 71.4, 71.6, 71.9, 71.0, 72.1, 72.7, 73.1, 82.3, 98.5, 98.7×2, 114.9, 116.1, 150.1, 155.4, 168.9, 169.4×2, 169.5, 170.2, 170.3, 170.4, 170.5
[化合物9]
1H-NMR (CDCl3, 400 MHz) δ 1.95-2.33 (complex, 55H, OAc), 3.61 (m, 6H), 3.73 (m, 1H), 3.91-4.31 (complex, 12H), 4.40 (m, 2H), 4.61 (d, J = 7.6 Hz, 1H), 4.65 (d, J = 7.6 Hz, 2H), 4.73 (d, J = 8.0 Hz, 1H), 4.82 (d, J = 8.0 Hz, 1H), 4.85-4.98 (complex, 4H), 5.01-5.21 (complex, 9H), 5.41 (d, J = 3.2 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) δ 20.8, 20.9×3, 21.1, 21.2, 21.5, 29.4, 29.8, 61.6, 61.7×2, 61.9, 62.4, 62.5, 67.3, 67.5, 67.9, 68.1×2, 68.2, 68.3, 71.6, 71.8, 71.9×2, 71.1, 72.2, 72.3, 72.8, 73.0, 74.8, 77.4, 78.3, 81.7, 82.8, 92.2, 95.6, 98.9, 99.5, 100.1, 101.5, 125.4, 128.3, 129.1, 137.9, 168.9, 169.4, 169.5×2, 169.9, 170.0, 170.1×2, 170.3×2
[化合物10]
1H-NMR (CDCl3, 400 MHz) δ 0.50-1.05 (complex, 7H), 1.19 (d, 3H, H-6 of Rham), 1.23 (s, 3H), 1.35-2.30 (complex, 80H), 3.59 (m, 1H), 3.72 (m, 5H), 3.92-4.11 (complex, 10H), 4.21 (dd, 1H), 4.31 (dd, 1H), 4.42 (m, 3H), 4.60 (d, J = 7.6 Hz, 1H), 4.71-4.95 (complex, 9H), 5.07 (m, 6H), 5.19 (t, 1H), 5.29 (m, 4H), 5.59 (d, J = 7.6 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) δ 16.7, 17.4, 20.5, 20.7×3, 20.8, 20.9×4, 21.1×2, 21.6, 29.2, 39.5, 42.5, 44.2, 53.8, 57.4, 61.9, 66.7, 68.0, 68.3, 68.4, 68.5, 69.7, 71.1, 71.5, 71.8, 71.9, 72.0, 72.2, 72.3, 72.4, 72.9, 73.0, 75.0, 80.1, 86.8, 91.3, 96.4, 96.9, 99.2, 99.3, 99.5, 125.4, 128.3, 129.2, 152.8, 169.0, 169.1, 169.3, 169.5×2, 169.6, 170.1×2, 170.2×2, 170.5, 170.6, 170.9, 174.8
[化合物11]
1H-NMR (pyridine-d5, 800 MHz) δ 0.68 (m, 1H), 0.86 (m, 1H), 0.97 (m, 1H), 1.04 (m, 4H), 1.28 (m, 1H), 1.43 (m, 5H), 1.63 (s, 3H), 1.70 (s, 3H), 1.93-2.20 (complex, 8H), 2.40 (d, 1H), 2.84 (d, 1H), 3.64 (m, 1H), 3.82 (m, 1H), 3.94-4.15 (complex, 13H), 4.17-4.35 (complex, 14H), 4.45-4.59 (complex, 5H), 4.88 (m, 2H), 4.99 (d, J = 7.2 Hz,1H), 5.07 (s, 1H), 5.14 (d, J = 8.0 Hz, 1H), 5.32 (d, J = 8.0 Hz, 1H), 5.65 (s, 1H), 5.72 (d, J = 8.0 Hz, 1H), 6.20 (d, J = 8.0 Hz, 1H), 6.48 (s, 1H); 13C-NMR (pyridine-d5, 200 MHz) δ 17.0, 19.1, 20.2, 20.9, 22.3, 29.4, 37.7, 38.5, 39.9, 40.8, 41.9, 42.7, 43.4, 44.7, 48.4, 49.8, 54.1, 57.7, 61.9, 62.4, 62.5, 62.6, 63.6, 69.4, 69.8×2, 71.6, 71.7, 72.5, 72.8, 74.1, 75.2, 75.5, 76.0, 76.4, 77.5, 77.6, 78.5×2, 78.6×2, 78.8×2, 86.9, 88.5, 89.8, 93.5, 98.4, 101.9, 103.9, 104.4, 104.8, 105.2, 154.5, 176.1
(i)と同条件で、HPLC- 高分解能MS/MSおよびMS3断片化による化学合成品(化合物11)とステビア葉抽出液の比較を行った結果、保持時間28.19分のピークで化学合成品とステビア葉抽出液からのピークが一致した(図6)。この結果から、植物体の抽出液から得られた新規ステビオール配糖体は化合物11と同じ構造を有することが確認された。
酵母において、ステビオールから新規ステビオール配糖体を生成させた。まず、ステビア由来の配糖化酵素遺伝子UGT85C2、UGT91D2、UGT74G1、UGT76G1の4種と、シロイヌナズナ由来UDP-ラムノース合成酵素遺伝子AtRHM2を同時に発現させる酵母を育種した。
UGT85C2遺伝子増幅用プライマーセット
CACC-NdeI-SrUGT85C2-Fw(下線部NdeI認識部位):
5’-CACCCATATGGATGCAATGGCTACAACTGAGAA-3’(配列番号12)
BglII-SrUGT85C2-Rv(下線部BglII認識部位):
5’-AGATCTCTAGTTTCTTGCTAGCACGGTGATTT-3’(配列番号13)
UGT91D2遺伝子増幅用プライマーセット
SrUGT91D2-pET15b-FW
5’-TGCCGCGCGGCAGCCATATGTACAACGTTACTTATCATC-3’(配列番号35)
SrUGT91D2-pET15b-RV
5’-GTTAGCAGCCGGATCCTTAACTCTCATGATCGATGGCAA-3’(配列番号36)
UGT74G1遺伝子増幅用プライマーセット
CACC-NdeI-SrUGT74G1-Fw(下線部NdeI認識部位):
5’-CACCCATATGGCGGAACAACAAAAGATCAAGAAAT-3’ (配列番号14)
BamHI-SrUGT74G1-Rv(下線部BamHI認識部位):
5’-GGATCCTTAAGCCTTAATTAGCTCACTTACAAATT-3’ (配列番号15)
UGT76G1遺伝子増幅用プライマーセット
CACC-NdeI-SrUGT76G1-Fw(下線部NdeI認識部位):
5’-CACCCATATGGAAAATAAAACGGAGACCA-3’ (配列番号16)
BamHI-SrUGT76G1-Rv(下線部BamHI認識部位):
5’-GGATCCTTACAACGATGAAATGTAAGAAACTA-3’ (配列番号17)
このPCR産物はpENTR-TOPO Directionalベクター(Invitrogen)に製造業者が推奨する方法でサブクローニングした。DNA Sequencer model 3100(Applied Biosystems)を用い、合成オリゴヌクレオチドプライマーによるプライマーウォーキング法によって配列を決定し、目的のUGT遺伝子、すなわちUGT85C2、UGT91D2、UGT74G1、UGT76G1の全てのUGT遺伝子がクローニングできたことを確認した。
これらのUGT遺伝子、およびシロイヌナズナ由来のUDP-ラムノース合成酵素遺伝子AtRHM2(J Biol Chem 2007 Oka et. al)を酵母発現ベクターに組み込むために下記のプライマーセットを設計した。
SrUGT85C2セット
Bgl2-UGT85C2-F(下線部BglII認識部位):
5’-ACAGATCTATGGATGCAATGGCTACAACTGAGA-3’ (配列番号18)
Sal-UGT85C2-R(下線部SalI認識部位):
5’-TAGTCGACTAGTTTCTTGCTAGCACGGTGATTTC-3’ (配列番号19)
SrUGT91D2セット
NotI-UGT91DIL3-F(下線部NotI認識部位):
5’-AAGCGGCCGCATGTACAACGTTACTTATCATCAAAATTCAAA-3’ (配列番号20)
Pac-UGT91D1L3-R(下線部PacI認識部位):
5’-CGTTAATTAACTCTCATGATCGATGGCAACC-3’ (配列番号21)
SrUGT74G1セット
Not-UGT74G1-F(下線部NotI認識部位):
5’-AAGCGGCCGCATGGCGGAACAACAAAAGATCAAG-3’ (配列番号22)
Pac-UGT74G1-R(下線部PacI認識部位):
5’-CGTTAATTAAGCCTTAATTAGCTCACTTACAAATTCG-3’ (配列番号23)
SrUGT76G1セット
Bam-UGT76G1-F(下線部BamHI認識部位):
5’-AAGGATCCATGGAAAATAAAACGGAGACCACCG-3’ (配列番号24)
Sal-UGT76G1-R(下線部SalI認識部位):
5’-GCGTCGACTTACAACGATGAAATGTAAGAAACTAGAGACTCTAA-3’ (配列番号25)
AtRHM2セット
Bam-AtRHM2-F(下線部BamHI認識部位):
5’-GGATCCATGGATGATACTACGTATAAGCCAAAG-3’ (配列番号26)
Xho-AtRHM2-R(下線部XhoI認識部位):
5’-CTCGAGTTAGGTTCTCTTGTTTGGTTCAAAGA-3’ (配列番号27)
(1)プラスミドpESC-URA-UGT56の構築
UGT85C2を制限酵素BglIIと制限酵素SalIで切り出し、ベクターpESC-URA(ストラタジーン)を制限酵素BamHIと制限酵素SalIで切断したものと連結して、プラスミドpESC-URA-UGT-5を得た。このプラスミドpESC-URA-UGT-5を制限酵素NotIと制限酵素PacIで切断したものと、UGT91D2を制限酵素NotIと制限酵素PacIで切り出したものを連結し、pESC-URA-UGT56を得た。
(2)プラスミドpESC-HIS-UGT78の構築
UGT76G1を制限酵素BamHIと制限酵素SalIで切り出し、ベクターpESC-HIS(ストラタジーン)を同じ制限酵素で切断したものを連結し、プラスミドpESC-HIS-UGT-8を得た。このプラスミドpESC-HIS-UGT-8を制限酵素NotIと制限酵素PacIで切断したものと、UGT74G1をNotIとPacIで切り出したものを連結し、pESC-HIS-UGT78を得た。
(3)プラスミドpESC-TRP-AtRHM2の構築
AtAHM2を制限酵素BamHIと制限酵素XhoIで切り出し、ベクターpESC-TRP(ストラタジーン)を同じ制限酵素で切断したものを連結し、プラスミドpESC-TRP-AtAHM2を得た。
Saccharomyces cerevisiae YPH499株(ura3-52 lys2-801amberade2-101ochre trp1-Δ63 his3-Δ200 leu2-Δ1 a)を宿主として、酢酸リチウム法で、表2のプラスミドを導入した。形質転換株として、SC-Trp&Ura&His寒天培地(1Lあたり、Yeast nitrogen baase without amino acids 6.7g、グルコース 20g、アミノ酸ミックスパウダー-Trp&Ura&His 1.3g、Bacto agar 20g)で生育するものを選抜した。
得られた形質転換株を以下の通り培養した。
まず、前培養としてSC-Trp&Ura&His液体培地(SC- Trp&Ura&His寒天培地のBacto agarを除く)10 mlに、それぞれの形質転換株を植菌し、30℃で1日間振とう培養した。次に、本培養として前培養液のうち、1 mlを10 mlのSG-Trp&Ura&His液体培地(1Lあたり、Yeast nitrogen baase without amino acids 6.7g、ガラクトース 20g、アミノ酸ミックスパウダー-Trp&Ura&His 1.3g)に植菌し、30℃で2日間振とう培養した。
UGT85C2発現確認用
UGT85C2-r1:
5’-CAAGTCCCCAACCAAATTCCGT-3’ (配列番号28)
UGT91D2発現確認用
UGT91D1L3-r1:
5’-CACGAACCCGTCTGGCAACTC-3’ (配列番号29)
UGT74G1発現確認用
UGT74G1-r1:
5’-CCCGTGTGATTTCTTCCACTTGTTC-3’ (配列番号30)
UGT76G1発現確認用
UGT76G1-r1:
5’-CAAGAACCCATCTGGCAACGG-3’ (配列番号31)
AtAHM2 発現確認用
AtAHM2-r1
5’-GCTTTGTCACCAGAATCACCATT-3’ (配列番号32)
GAL10p領域 (プロモーター領域)
PGAL10-f3:
5’-GATTATTAAACTTCTTTGCGTCCATCCA-3’ (配列番号33)
GAL1p領域(プロモーター領域)
PGAL1-f3:
5’-CCTCTATACTTTAACGTCAAGGAGAAAAAACC-3’ (配列番号34)
UGT85C2:UGT85C2-r1(配列番号28)とPGAL1-f3(配列番号34)
UGT91D2またはUGT91D2L3:UGT91D1L3-r1(配列番号29)とPGAL10-f3(配列番号33)
UGT74G1:UGT74G1-r1(配列番号30)とPGAL1-f3(配列番号34)
UGT76G1:UGT76G1-r1(配列番号31)とPGAL10-f3(配列番号33)
AtAHM2:AtAHM2-r1(配列番号32)とPGAL10-f3(配列番号33)
これにより、形質転換株で、導入した遺伝子が発現していることが確認できた。
培養は、本培養用の液体培地に培地1 mlあたり0.5μgまたは2μgのステビオール(ChromaDex Inc.)を添加した以外は、上記実施例と同様の条件で行った。培養終了後、培養液を遠心分離により、上清と菌体に分離した。培養上清を、アセトニトリルで洗浄後、水で平衡化したSep-Pak C18カラムに供し、20% アセトニトリルで洗浄後、80%アセトニトリルで溶出し、乾固後、少量の80% アセトニトリルに溶解して配糖体サンプルを調製した。この配糖体サンプルを以下の分析に供した。
LC-MSによる分析は、「新規ステビオール配糖体の単離」についての実施例に記載のとおり分析した。
新規ステビオール配糖体の甘味度を評価するため、Brix0.5から3まで0.5刻となるようショ糖を純水に添加したサンプルを調製した。新規ステビオール配糖体を415ppmとなるように純水に添加してサンプルを調製した。
各種ステビオール配糖体の味質の評価を行うため、図8に示した添加量となるようにReb.Aおよび新規ステビオール配糖体を純水に添加して、飲料サンプルを調製した。飲料サンプルは全て、甘味度をRebA:300、新規配糖体:24として、ショ糖(スクロース)換算のBrixが最終的に2となるように調整した。
(1)後引きを改善する対象の甘味料の甘味度測定
風味調整剤の評価を行う前に、後引きを改善する対象の甘味料の甘味度を測定した。甘味料としてはReb.A(純度100%)とReb.D(純度97%)を使用した。Reb.AとReb.Dをそれぞれ下表に示す量で水に溶解して水溶液を作成した。別途スクロース(砂糖)を用いて作成したBrix5~7の標準水溶液を準備し、甘味料の官能に関して訓練を受けた者(6名)がパネラーとなって評価を行い、Reb.A水溶液とReb.D水溶液の甘味が、標準水溶液のどの甘味に対応するかを評価した。結果を下表に示す。
上記の結果から、以下の試験においてはReb.Aの甘味倍率を237倍、Reb.Dの甘味倍率を213倍として評価を行った。なお、評価に用いた新規ステビオール配糖体の甘味倍率は、上記のとおり24倍である。
Brix5、7および11の3水準の水溶液を用いて、本発明の風味調整剤のReb.Aに対する後引き改善効果の評価を行った。まず、Reb.Aの甘味度を237倍として、Brix5、7および11の3水準の水溶液を作成した。本発明の新規ステビオール配糖体からなる風味調整剤の添加量は、Reb.Aの添加質量を基準として1、3.5、5および10質量%の割合とした。対照サンプル(Cont)はReb.Aのみを添加し、本発明の風味調整剤は添加しなかった。甘味料の官能に関して訓練を受けた者(7名)がパネラーとなって評価を行い、Contを3点として後引き改善を上限を6点として数値で評価した。後引きが改善されている場合、点数は高くなる。得られた評点の平均値を図9のグラフに示した。
Brix5、7および11の3水準の水溶液を用いて、本発明の風味調整剤のReb.Dに対する後引き改善効果の評価を行った。まず、Reb.Dの甘味度を213倍として、Brix5、7および11の3水準の水溶液を作成した。本発明の新規ステビオール配糖体からなる風味調整剤の添加量は、Reb.Dの添加質量を基準としてそれぞれ1、3.5、5および10質量%の割合とした。対照サンプル(Cont)はReb.Dのみを添加し、本発明の風味調整剤は添加しなかった。甘味料の官能に関して訓練を受けた者(7名)がパネラーとなって評価を行い、Contを3点として後引き改善を上限を6点として数値で評価した。後引きが改善されている場合、点数は高くなる。得られた評点の平均値を図10のグラフに示した。
Brix5および7の2水準の水溶液を用いて、本発明の風味調整剤の砂糖(スクロース)に対する甘味増強効果の評価を行った。まず、砂糖を用いてBrix5および7の2水準の水溶液を作成した。本発明の新規ステビオール配糖体からなる風味調整剤の添加量は、砂糖の添加質量を基準として、Brix5の水準では、0.10、0.44および0.80質量%の割合とし、Brix7の水準では、0.10、0.44および0.57質量%の割合とした。甘味料の官能に関して訓練を受けた者(7名)がパネラーとなって評価を行い、甘味強度の倍率で評価した。結果を図11のグラフに示した。グラフ上の縦軸(甘味強度)は砂糖と風味調整剤の合計甘味度(風味調整剤の甘味度はは甘味倍率24として算出)に対し、実際パネラーが感じた甘味強度の倍率を示した。いずれの試料においても2~7%程度の甘味増強効果が観察された。
Claims (18)
- 植物由来物、化学合成物、または生合成物である、請求項1に記載の化合物、またはその誘導体、塩、もしくは水和物。
- 請求項1または2に記載の化合物またはその誘導体、塩、もしくは水和物を含む、甘味料組成物。
- レバウディオサイドA、レバウディオサイドB、レバウディオサイドC、レバウディオサイドD、レバウディオサイドE,レバウディオサイドF、レバウディオサイドI、レバウディオサイドJ、レバウディオサイドK、レバウディオサイドN、レバウディオサイドM、レバウディオサイドO、レバウディオサイドQ、レバウディオサイドR、ズルコサイドA、ルブソシド、ステビオール、ステビオールモノシド、ステビオールビオシド及びステビオシドからなる群から選択される一種以上のステビオール配糖体をさらに含む、請求項3に記載の甘味料組成物。
- 請求項1または2に記載の化合物またはその誘導体、塩、もしくは水和物を含む、飲食品。
- 飲料である、請求項5に記載の飲食品。
- 請求項1に記載の化合物またはその誘導体、塩、もしくは水和物を含む、植物体。
- 請求項7に記載の植物体の抽出物。
- 請求項7に記載の植物体または請求項8に記載の植物体の抽出物を含む、飲食品。
- 飲料である、請求項9に記載の飲食品。
- 前記式(5)で示される化合物を得る工程の収率が40%以上である、請求項12に記載の方法。
- 請求項1または2に記載の化合物またはその誘導体、塩、もしくは水和物の甘味料としての使用。
- 請求項1に記載の化合物の製造方法であって、下記の(a)~(g)の少なくとも1つのポリヌクレオチドを導入した非ヒト形質転換体を用いることを特徴とする方法。
(a)配列番号1の塩基配列を含有するポリヌクレオチド、配列番号1の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号2のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の13位の水酸基にグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド(b)配列番号3の塩基配列を含有するポリヌクレオチド、配列番号3の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号4のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の19位のカルボン酸にグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(c)配列番号5の塩基配列を含有するポリヌクレオチド、配列番号5の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号6のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の13位に結合したグルコースに1→2結合でラムノースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(d)配列番号7の塩基配列を含有するポリヌクレオチド、配列番号7の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号8のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の13位のグルコースの3位に1→3結合でグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(e)配列番号5の塩基配列を含有するポリヌクレオチド、配列番号5の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号6のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の19位のグルコースに、1→2結合でグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(f)配列番号7の塩基配列を含有するポリヌクレオチド、配列番号7の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号8のアミノ酸配列に対して90%以上の同一性を有し、かつ、ステビオール配糖体の19位のグルコースに、1→3結合でグルコースを付加する活性を有するタンパク質をコードするポリヌクレオチド
(g)配列番号9の塩基配列を含有するポリヌクレオチド、配列番号9の塩基配列に対して90%以上の同一性を有する塩基配列を含有するポリヌクレオチド、または、配列番号10のアミノ酸配列に対して90%以上の同一性を有し、かつ、UDP-グルコースからUDP-ラムノースを生成する活性を有するタンパク質をコードするポリヌクレオチド - 前記非ヒト形質転換体が酵母である、請求項15に記載の方法。
- ステビオールを含む培地で前記非ヒト形質転換体の培養を行うことを特徴とする、請求項15又は16に記載の方法。
- 請求項1に記載の化合物またはその誘導体、塩、もしくは水和物を含む、風味調整剤。
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EP17888291.6A EP3564251A4 (en) | 2016-12-27 | 2017-12-26 | NOVEL STEVIOL GLYCOSIDE, METHOD FOR MANUFACTURING THEREOF AND SWEETENING COMPOSITION THEREOF |
BR112019013208-2A BR112019013208B1 (pt) | 2016-12-27 | 2017-12-26 | Composto, composição de adoçante, alimento ou bebida, método para produzir o composto, uso do composto, e, agente de controle do sabor |
AU2017388706A AU2017388706B2 (en) | 2016-12-27 | 2017-12-26 | Novel steviol glycoside, method for producing same, and sweetener composition containing same |
CN201780080717.3A CN110167950B (zh) | 2016-12-27 | 2017-12-26 | 新型甜菊醇糖苷及其制造方法,以及含有该物质的甜味剂组合物 |
NZ754394A NZ754394A (en) | 2016-12-27 | 2017-12-26 | Novel steviol glycoside, method for producing same, and sweetener composition containing same |
JP2018559554A JP6970689B2 (ja) | 2016-12-27 | 2017-12-26 | 新規ステビオール配糖体およびその製造方法、ならびにそれを含む甘味料組成物 |
US16/473,081 US20190352324A1 (en) | 2016-12-27 | 2017-12-26 | Novel steviol glycoside, method for producing same, and sweetener composition containing same |
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CN112964812A (zh) * | 2021-02-05 | 2021-06-15 | 山东省产品质量检验研究院 | 一种检测乳制品中8种甜味剂的方法 |
JP2021526521A (ja) * | 2018-06-08 | 2021-10-07 | ピュアサークル ユーエスエー インコーポレイテッド | 高純度ステビオール配糖体 |
JPWO2021020516A1 (ja) * | 2019-07-31 | 2021-10-28 | サントリーホールディングス株式会社 | 新規ステビオール配糖体およびその製造方法、ならびにそれを含む甘味料組成物 |
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EP3528640A4 (en) * | 2016-10-20 | 2020-09-16 | The Coca-Cola Company | DITERPENIC GLYCOSIDES ISOLATED FROM STEVIA, COMPOSITIONS AND PROCESSES |
CN110818750B (zh) * | 2019-11-22 | 2020-11-06 | 江西师范大学 | 一种甜菊苷r的合成方法 |
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CN110167950B (zh) | 2023-03-10 |
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JP6970689B2 (ja) | 2021-11-24 |
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