EP3256008A1 - Production biosynthétique de rébaudioside m et procédés de récupération - Google Patents

Production biosynthétique de rébaudioside m et procédés de récupération

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Publication number
EP3256008A1
EP3256008A1 EP16749758.5A EP16749758A EP3256008A1 EP 3256008 A1 EP3256008 A1 EP 3256008A1 EP 16749758 A EP16749758 A EP 16749758A EP 3256008 A1 EP3256008 A1 EP 3256008A1
Authority
EP
European Patent Office
Prior art keywords
rebaudioside
mogroside
glycoside
terpenoid glycoside
terpenoid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16749758.5A
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German (de)
English (en)
Other versions
EP3256008A4 (fr
Inventor
Avetik Markosyan
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PureCircle USA Inc
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PureCircle USA Inc
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Publication date
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Publication of EP3256008A1 publication Critical patent/EP3256008A1/fr
Publication of EP3256008A4 publication Critical patent/EP3256008A4/fr
Withdrawn legal-status Critical Current

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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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/56—Flavouring or bittering agents
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation containing fruit or vegetable juices
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/54—Mixing with gases
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/60—Sweeteners
    • 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
    • A23L27/36—Terpene glycosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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

Definitions

  • the invention relates to a process for producing terpenoid glycosides by recombinant microorganisms and recovering the produced glycosides for use in various food products and beverages.
  • sweeteners such as dulcin, sodium cyclamate and saccharin were banned or restricted in some countries due to concerns on their safety. Therefore non- caloric sweeteners of natural origin are becoming increasingly popular.
  • the sweet herb Stevia rebaudiana Bertoni produces a number of diterpene glycosides which feature high intensity sweetness and sensory properties superior to those of many other high potency sweeteners.
  • the above-mentioned sweet glycosides have a common aglycon, steviol, and differ by the number and type of carbohydrate residues at the CI 3 and CI 9 positions.
  • the leaves of Stevia are able to accumulate up to 10-20% (on dry weight basis) steviol glycosides.
  • the major glycosides found in Stevia leaves are Rebaudioside A (2-10%), Stevioside (2-10%), and Rebaudioside C (1 -2%).
  • Other glycosides such as Rebaudioside B, D, E, F, G, H, I, J K, L, M, N, and O, Steviolbioside, Dulcoside A and Rubusoside are found at lower levels (approx. 0-0.5%).
  • Steviol glycosides differ from each other not only by molecular structure, but also by their taste properties. Usually stevioside is found to be 1 10-270 times sweeter than sucrose, Rebaudioside A between 150 and 320 times, and Rebaudioside C between 40-60 times sweeter than sucrose. Dulcoside A is 30 times sweeter than sucrose. Rebaudioside A has the least astringent, the least bitter, and the least persistent aftertaste thus possessing the most favorable sensory attributes in major steviol glycosides (Tanaka O. (1987) Improvement of taste of natural sweeteners. Pure Appl. Chem. 69:675-683; Phillips .C. (1989) Stevia: steps in developing a new sweetener. In: Grenby T.H. ed. Developments in sweeteners, vol. 3. Elsevier Applied Science, London. 1 -43.)
  • Rebaudioside M (also known as Rebaudioside X; CAS No: 1220616-44-3) is one of minor steviol glycosides found in Stevia rebaudicma plant. It was found to have superior taste properties and is a highly desirable natural high intensity sweetener (WO2013/096420 007748 incorporated herein as a reference, in its entirety).
  • Rebaudioside M When producing Rebaudioside M in recombinant microorganism, one will be aiming to achieve the highest concentration/titer of Rebaudioside M (Reb M). For recombinant microorganism production of different compounds the commercial feasibility generally starts at titers above l Og/L. On the other hand it is known that Reb has limited solubility in the water of about l g/L (US2015/0017284 incorporated herein as a reference, in its entirety). Thus in recombinant microorganism production of Reb at concentrations above lg/L the Reb will precipitate/crystallize from media.
  • one of the first steps of downstream processing is the removal of microbial cells or cell debris.
  • This can be achieved by any method known to art, including, but not limited to, centrifugation, decanting, filtration etc.
  • a sludge of microbial cells (cell debris) and a clear solution of dissolved product are obtained.
  • the solution is processed further downstream for product recovery while the sludge is disposed off after sterilization. Due to limited solubility of Reb M, it crystallizes and during downstream processing significant amount of Reb M crystals are lost with the separated biomass/sludge during cell (cell debris) removal process. This reduces the efficiency of the entire process and makes it less commercially viable.
  • FIG. 1 shows a high-performance liquid chromatographic (HPLC) chromatogram of Rebaudioside M
  • FIG. 2 shows a HPLC chromatogram of -glycosylated Rebaudioside M containing -l ,4-glucosyl-derivatives of Rebaudioside M;
  • FIG. 3 shows a HPLC chromatogram of glucoamylase treated -l ,4-glucosyl- derivatives of Rebaudioside M.
  • the present invention is aimed to overcome the disadvantages of existing processes for producing steviol glycoside(s) found in Stevia rebaudiana plant by recombinant microorganism(s).
  • the invention relates to a process for producing steviol glycoside(s) found in Stevia rebaudiana plant by recombinant microorganism(s) and recovering the produced glycoside(s) for use in various food products and beverages as sweetener, sweetness enhancer, flavor, flavor modifier/enhancer.
  • the steviol glycosides are selected from the group consisting of stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside G, Rebaudioside H, Rebaudioside /, Rebaudioside J, Rebaudioside K, Rebaudioside L, Rebaudioside M, Rebaudioside N, Rebaudioside O, dulcoside A, steviolbioside, rubusoside, steviolmonoside, as well as any other steviol glycoside(s) found in Stevia rebaudiana plant, and mixtures thereof.
  • the invention in part, pertains to a process of producing steviol glycosides found in Stevia rebaudiana plant by fermentation of recombinant microorganism(s).
  • the process of invention may further include a step of adding alcohol to the fermentation media of recombinant microorganism(s) resulting in better dissolution of steviol glycosides.
  • the process of invention may include a step of adding solvent comprising alcohol to any media obtainable from downstream processing of fermentation media of recombinant host(s).
  • said media comprises separated recombinant microorganism(s) cells (cell debris) and crystalline steviol glycosides.
  • the alcohol may be selected from the group including but not limited to methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, and combinations thereof.
  • the alcohol may be in a form of aqueous solution or anhydrous.
  • the process of invention may include a step of producing -glycosylated derivatives of steviol glycosides found in Stevia rebaudiana plant wherein said a- glycosylated derivatives contain at least one -glycosyl residue in their molecule.
  • the process of invention may include a further step of selective hydrolysis of a- glycosydic bond(s) to convert -glycosylated derivatives of steviol glycosides to steviol glycosides found in Stevia rebaudiana plant.
  • the invention in part, pertains to a composition comprising a-glycosylated derivatives of steviol glycosides found in Stevia rebaudiana plant.
  • the invention in part, pertains to a process for producing a composition comprising ⁇ -glycosylated forms of stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside G, Rebaudioside H, Rebaudioside I, Rebaudioside J, Rebaudioside K, Rebaudioside L, Rebaudioside M, Rebaudioside N, Rebaudioside O, dulcoside A, steviolbioside, rubusoside, steviolmonoside as well as any other steviol glycoside(s) found in Stevia rebaudiana plant, and mixtures thereof.
  • the process comprises an enzymatic a-glycosylation step.
  • the a-glycosylation step can occur within the recombinant host cell, on the surface of the recombinant host cell, or outside the recombinant host cell.
  • the ⁇ -glycosylation is achieved by using transglycosidase(s) and carbohydrate donors.
  • carbohydrate donor include starch, maltodextrins, corn syrup solids, cyclodextrins, sucrose, maltose, maltooligosaccharides, fructooligosaccharides, inulin, inulooligosaccharides, xylooligosaccharides, coupling sugar, lactose and combinations thereof.
  • the ⁇ -glycosylation is achieved by a-glycosyltransferase(s) and nucleotide glycosyl donor.
  • the ⁇ -glycosylation is achieved by a- glycosyltransferase(s) and non-nucleotide glycosyl donor.
  • cyclomaltodextrin glucanotransferase (CGTase; EC 2.4.1.19) enzyme(s) and starch (as glucose donor) were used to produce a-l ,4-glucosyl-derivatives of steviol glycosides containing at least one a-l ,4-glucosyl residue in their molecules.
  • enzymes and glycosyl residue donors may be used to produce a-glycosyl- derivatives of steviol glycosides containing at least one a- 1 , 1 -glycosyl residue, at least one a-l ,2-glycosyl residue, at least one a-l ,3-glycosyl residue, at least one a-l ,4-glycosyl residue, at least one a-l ,5-glycosyl residue, at least one a-l ,6-glycosyl residue in their molecules.
  • the enzymes can be in a form of cell-free culture broth, concentrated liquid cell-free culture broth, spray dried or freeze dried cell-free culture broth, or high purity protein. Free and immobilized enzyme preparations can be used.
  • the enzyme(s) for a- glycosyl-derivatives synthesis may be also incorporated into the recombinant microorganism capable of producing steviol glycosides molecules found in Stevia rebaudiana plant.
  • the enzyme(s) for -glycosyl-derivatives synthesis may be also incorporated into any recombinant host capable of producing steviol glycosides molecules found in Stevia rebaudiana plant.
  • the obtained a-glycosyl-derivatives have higher water solubility and hence do not precipitate.
  • the microbial cells (cell debris) removal results in no or minimal loss of product.
  • the a- 1 ,4-glucosyl-derivatives dissolved in supernatant are hydrolyzed by glucoamylase for selective hydrolysis of a-l ,4-glucosidic bonds and conversion of a-l ,4-glucosylated derivatives of steviol glycosides to steviol glycosides molecules found in Stevia rebaudiana plant.
  • enzymes capable of hydrolyzing a-l ,4-glycosidic bonds can be used as well.
  • the enzyme can be in a form of cell-free culture broth, concentrated liquid cell-free culture broth, spray dried or freeze dried cell-free culture broth, or high purity protein. Free and immobilized enzyme preparations can be used.
  • transglucosylation was accomplished by CGTase of Bacillus stearothermophilus St- 100 (PureCircle Sdn Bhd Collection of Industrial Microorganisms - Malaysia).
  • CGTase preparations The activity of CGTase preparations was determined according to the procedure described in Hale W.S., Rawlins L.C. (1951 ) Amylase of Bacillus macerans. Cereal Chem. 28, 49-58.
  • Starches of different origin may be used as donors of glucosyl units such as, derived from wheat, corn, potato, tapioca, and sago.
  • donors of glucosyl residues such as maltodextrins, cyclodextrins etc may be used as well.
  • Starch was subjected to partial hydrolysis (liquefaction) prior to the transglycosylation reaction.
  • the dextrose equivalent of the partially hydrolyzed starch can be in the range of about 10-25, preferably about 12-16.
  • Any enzyme capable of starch hydrolysis may be used for liquefaction, such as -amylases, / ⁇ -amylases etc.
  • CGTase and a-amylase mixtures as liquefying enzymes are preferred.
  • Reb M transglucosylation reaction Upon completion of Reb M transglucosylation reaction, about 0.5- 1.0 units of glucoamylase (AMG300L, Novozymes), per gram of solids, was added and the reaction was continued for about 12-16 hours at about 45-65°C, preferably about 60°C.
  • AMG300L Novozymes
  • One unit of glucoamylase activity is defined as the amount of glucoamylase that will liberate 0.1 ⁇ / min of /j-nitrophenol from the PNPG Solution under the conditions of the assay described in Food Chemicals Codex 5 th ed., p. 907.
  • the reaction is stopped by heating at about 95°C for about 15 minutes to inactivate the enzyme(s), and the solution may be treated with activated carbon and/or desalted by passing through ion exchange resins.
  • ion exchange resins Other appropriate decolorizing and desalting methods, such as membrane filtration, or other methods known in the art can be used.
  • the reaction mixture may be further concentrated by vacuum evaporator and/or dried by means of a spray dryer.
  • Other appropriate concentrating and drying methods such as membrane filtration, freeze drying, or other methods known to art can be used.
  • the steviol glycosides from obtained reaction mixture may be recovered by any method or combination of methods known to art for extraction, separation, purification, isolation and production of steviol glycosides found in Stevia rebaudiana plant.
  • Non limiting examples of such methods include extraction by water and or organic solvents, treatment with flocculants, coagulants, treatment with macroporous adsorption resins, ion- exchange resin treatment, activated carbon treatment, membrane filtration, RO-membrane filtration, microfiltration, nanofiltration, ultrafiltration, chromatography, HPLC, SMB- chromatography, supercritical fluid (SF) chromatography, adsorption resin chromatography, multicolumn adsorption chromatography, ion-exchange chromatography, continuous chromatography, supercritical fluid extraction, ultrasound assisted extraction, microwave assisted extraction, enzyme assisted extraction, solid-liquid extraction, liquid- liquid extraction, crystallization, ultrasound assisted crystallization, gradient crystallization, solvent-antisolvent crystallization, co-crystallization, centrifugation,
  • the obtained purified steviol glycoside(s) can be used as sweeteners, sweetness enhancers, flavor enhancers and flavor modifiers in various food and beverage products.
  • food and beverage products include carbonated soft drinks, ready to drink beverages, energy drinks, isotonic drinks, low-calorie drinks, zero-calorie drinks, sports drinks, teas, fruit and vegetable juices, juice drinks, dairy drinks, yoghurt drinks, alcohol beverages, powdered beverages, bakery products, cookies, biscuits, baking mixes, cereals, confectioneries, candies, toffees, chewing gum, dairy products, flavored milk, yoghurts, flavored yoghurts, cultured milk, soy sauce and other soy base products, salad dressings, mayonnaise, vinegar, frozen-desserts, meat products, fish-meat products, bottled and canned foods, tabletop sweeteners, fruits and vegetables.
  • the obtained purified steviol glycoside(s) can be used in drug or pharmaceutical preparations and cosmetics, including but not limited to toothpaste, mouthwash, cough syrup, chewable tablets, lozenges, vitamin preparations, and the like.
  • the obtained purified steviol glycoside(s) can be used "as-is” or in combination with other sweeteners, flavors, flavor ingredients and food ingredients.
  • Non-limiting examples of sweeteners include steviol glycosides, stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside G, Rebaudioside H, Rebaudioside I, Rebaudioside J, Rebaudioside K, Rebaudioside L, Rebaudioside M, Rebaudioside N, Rebaudioside O, dulcoside A, steviolbioside, rubusoside, steviolmonoside as well as any other steviol glycoside(s) found in Stevia rebaudiana plant and mixtures thereof, stevia extract, glycosylated steviol glycosides, Luo Han Guo extract, mogrosides, high-fructose corn syrup, corn syrup, invert sugar, fructooligosaccharides, inulin, inulooligosaccharides, coupling sugar, maltooligosaccharides, maltodextrins, corn syrup solids, glucose, maltose, sucrose, lac
  • Non-limiting examples of flavors and flavor ingredients include glycosylated steviol glycosides, steviol glycoside(s), mogroside(s), lemon, orange, fruity, banana, grape, pear, pineapple, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla flavors, NSF-01 , NSF-02, NSF-03, NSF-04 (available from PureCircle).
  • Non-limiting examples of other food ingredients include flavors, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilisers, thickeners, gelling agents.
  • flavors, acidulants, organic and amino acids coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilisers, thickeners, gelling agents.
  • a strain of Bacillus stearothermophiliis St-100 was inoculated in 2,000 liters of sterilized culture medium containing 1.0% starch, 0.25% corn extract, 0.5% (NH 4 ) 2 S0 4 , and 0.2% CaC0 3 (pH 7.0-7.5) at 56°C for 24 hrs with continuous aeration (2,000 L/min) and agitation (150rpm).
  • the obtained culture broth was filtered using Kerasep 0.1 ⁇ ceramic membrane (Novasep, France) to separate the cells.
  • the cell-free permeate was further concentrated 2-fold on Persep l OkDa ultrafilters (Orelis, France).
  • the activity of the enzyme was determined according to Hale, Rawlins (1951 ). A crude enzyme preparation with activity of about 2 unit/mL was obtained.
  • Bhd. (Malaysia), having water solubility of 0.5g/L (at 25°C) and containing 96.97% Reb M and 3.03% Reb D, was dissolved by boiling in 9,000 mL of water (pH was adjusted to pH 6.0) and was added to liquefied starch and stirred until a homogeneous solution was obtained. 200 units of CGTase was added to the solution and the mixture was held at a temperature of 65°C for 24 hours under continuous agitation. The obtained reaction mixture was heated at 95°C for 15 minutes to inactivate the enzyme. 20 grams of activated carbon was added and the mixture was heated to 75°C and held during 30 min.
  • the column was washed with 5 volumes of water and 2 volumes of 20% (v/v) ethanol.
  • the adsorbed glycosides were eluted with 50% ethanol.
  • Obtained eluate was passed through columns packed with Amberlite FPC23 (H + ) and Amberlite FPA51 (OH " ) ion exchange resins.
  • the ethanol was evaporated and the desalted and decolorized water solution was concentrated at 60°C under vacuum until 10% solids content.
  • the concentrated solution was left for 24hrs to crystallize Reb M.
  • the crystals were separated by filtration and dried under vacuum to yield about 4.5 g of Reb M with 99% purity (wt/wt on dried basis).
  • Reb M prepared according to EXAMPLE 3, was carried using 600ppm aqueous solution, with 20 panelists. Reb M sample was assessed along with 10% sucrose and 600ppm commercially available Reb A and Stevioside samples. Based on overall acceptance the most desirable and most undesirable samples were chosen. The results are shown in Table 2.
  • the same method can be used to prepare juices and juice drinks from other fruits, such as apples, lemons, apricots, cherries, pineapples, mangoes, etc.
  • a carbonated beverage according to formula presented below was prepared.
  • the steviol glycosides were represented by Reb A, Stevioside and Reb M sample, obtained according to EXAMPLE 3.
  • the sensory properties were evaluated by 20 panelists. The best results were obtained in samples prepared with Reb M. The panelists noted rounded and complete flavor profile and mouthfeel in cookies prepared with Reb M.

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  • General Chemical & Material Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Seasonings (AREA)

Abstract

La présente invention concerne divers procédés de récupération pour la récupération complète de glycosides de stéviol peu solubles obtenus dans des micro-organismes de recombinaison. Des glycosides de stéviol α-glycosyl solubles ont été entièrement récupérés dans un traitement en aval puis convertis en glycosides de stéviol par des hydrolases. Les glycosides de stéviol obtenus ont été purifiés et utilisés en tant qu'édulcorants, exhausteurs de goût sucré, exhausteurs de goût et modificateurs de goût dans des aliments, des boissons, des produits cosmétiques et des produits pharmaceutiques.
EP16749758.5A 2015-02-10 2016-02-10 Production biosynthétique de rébaudioside m et procédés de récupération Withdrawn EP3256008A4 (fr)

Applications Claiming Priority (2)

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US201562114134P 2015-02-10 2015-02-10
PCT/US2016/017236 WO2016130609A1 (fr) 2015-02-10 2016-02-10 Production biosynthétique de rébaudioside m et procédés de récupération

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EP3256008A1 true EP3256008A1 (fr) 2017-12-20
EP3256008A4 EP3256008A4 (fr) 2018-10-31

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CN (1) CN107404911A (fr)
BR (1) BR112017017207B1 (fr)
MX (1) MX2017010311A (fr)
WO (1) WO2016130609A1 (fr)

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US10517321B2 (en) 2015-07-10 2019-12-31 Sweet Green Fields USA LLC Compositions of steviol multiglycosylated derivatives and stevia components
EP3320104B1 (fr) 2015-07-10 2021-11-10 DSM IP Assets B.V. Procédé de préparation d'une composition de glycoside de stéviol
BR112018008710B1 (pt) 2015-10-29 2022-04-12 Firmenich Incorporated Adoçantes de alta intensidade
JPWO2017171023A1 (ja) * 2016-03-31 2018-04-12 サントリーホールディングス株式会社 ヘスペリジンの沈殿が抑制された飲料
US10085472B2 (en) * 2016-08-29 2018-10-02 Pepsico, Inc. Compositions comprising rebaudioside J
CN106554983A (zh) * 2016-10-28 2017-04-05 江南大学 甜菊糖杜克苷a的制备方法
US11284577B2 (en) 2017-03-08 2022-03-29 Purecircle Usa Inc. High rebaudioside M stevia plant cultivars and methods of producing the same
CN106866757B (zh) * 2017-03-16 2020-06-26 诸城市浩天药业有限公司 甜菊糖m苷晶型及制备方法和用途
JP7285786B2 (ja) 2017-05-03 2023-06-02 フィルメニッヒ インコーポレイテッド 高強度の甘味料の製造方法
KR20200125927A (ko) * 2018-02-26 2020-11-05 피르메니히 에스아 글리코실화된 텔펜 배당체, 텔펜 배당체 및 시클로덱스트린을 포함하는 조성물
CN108707163B (zh) * 2018-06-11 2020-08-14 江西师范大学 一种甜菊醇甙元的制备方法
BR112022018080A2 (pt) * 2020-03-13 2022-10-25 Amyris Inc Composições de adoçante de rebaudiosídeo m

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US4219571A (en) 1978-06-15 1980-08-26 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Process for producing a sweetener
US20090162487A1 (en) * 2007-12-21 2009-06-25 The Concentrate Manufacturing Company Of Ireland Beverage products and flavor systems having a non-sweetening amount of rebaudioside a
KR100888694B1 (ko) * 2008-09-01 2009-03-16 김경재 감미질이 우수한 효소처리스테비아 제조방법
US20140030381A1 (en) * 2011-02-17 2014-01-30 Purecircle Usa Inc. Glucosyl stevia composition
US9795156B2 (en) * 2011-03-17 2017-10-24 E.P.C (Beijing) Plant Pharmaceutical Technology Co., Ltd Rebaudioside B and derivatives
EP3009508B1 (fr) * 2011-08-08 2020-11-25 Evolva SA Production recombinante de glycosides de stéviol
CA2899276C (fr) 2013-02-06 2022-11-01 Evolva Sa Procedes pour la production amelioree de rebaudioside d et de rebaudioside m
US10017804B2 (en) * 2013-02-11 2018-07-10 Evolva Sa Efficient production of steviol glycosides in recombinant hosts

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Publication number Publication date
WO2016130609A1 (fr) 2016-08-18
MX2017010311A (es) 2017-12-14
EP3256008A4 (fr) 2018-10-31
CN107404911A (zh) 2017-11-28
BR112017017207A2 (pt) 2018-04-03
BR112017017207B1 (pt) 2022-10-18

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